Metrics details Inflammation has been proven to be associated with chronic diseases We hypothesized that higher diet-induced inflammation is associated with increased risk of fatty pancreas (FP) Among 278 patients with common bile duct (CBD) stones 89 patients were diagnosed with fatty pancreas (case group) during endoscopic ultrasonography and the other 189 patients were healthy in this regard (control group) empirical dietary inflammatory pattern (EDIP) and dietary inflammatory score (DIS) were calculated based on a 168-question valid food frequency questionnaire Dietary inflammatory scores were significantly higher in the case group than in the control group EDIP and DIS were significantly associated with higher risk of FP in the crude and adjusted models higher scores of DII (OR T2 vs T1 = 1.36; 95% CI 0.71–2.58 and OR T3 vs T1 = 3.3; 95% CI: 1.59–6.8; P for trend = 0.001) EDIP (OR T2 vs T1 = 1.7; 95% CI 0.89–3.3 and OR T3 vs T1 = 2.5; 95% CI 1.2–5.1; P for trend = 0.009) and DIS (OR T2 vs T1 = 1.48; 95% CI 0.74–2.97 and OR T3 vs T1 = 2; 95% CI 1.16–3.63; P for trend = 0.040) resulted in increased risk of FP development Diet-induced inflammation was associated with an increased propensity for developing fatty pancreas we hypothesized that higher dietary inflammatory indices is associated with increased risk of fatty pancreas in patients with common bile duct (CBD) stones this is the first study that investigated the relationship between FP and dietary inflammatory indices Demographic data and clinical characteristics were obtained via face-to-face interviews which were then cross-checked with patients’ medical records the body mass index (BMI) was computed by dividing the weight (kg) by the square of height (m2) Total energy and dietary nutrient intakes were then analyzed using Nutritionist 4 software (First Databank Inc. The disaggregated components were then added to their respective DIS food groups A higher score on all three indices implies an enhanced balance of pro- to anti-inflammatory dietary intakes The statistical package for social sciences version 21.0 for Windows (SPSS Inc. and P-values < 0.05 were considered statistically significant the normality of data was examined using a histogram chart and Kolmogorov–Smirnov test participants were categorized into 3 groups based on 33rd and 66th percentile values for the DII The qualitative variables between DII tertiles were compared using Chi-square or Fisher’s exact test and the results were expressed as count (percentage) For quantitative variables one way ANOVA was applied and the results were reported as mean ± SD Comparisons of dietary intakes across tertiles of DII and DIS were carried out by the use of one-way ANOVA and DIS with FP was evaluated using binary logistic regression Three models were synthesized to annihilate the effect of potential confounders for DII which have been provided with odds ratios (ORs) and 95% confidence intervals (CIs) BMI and energy intake were considered as confounders the first tertiles were considered as the reference category No difference was found in the demographic information of the participants Although there was a significant difference in weight and height between tertiles this significance was not found in the comparison of BMI The comparison of dietary intakes showed that with the increase in DII score energy intake and the percentage of carbohydrates increased significantly but no difference was observed in the percentage of protein and fat the EDIP score also increased significantly (P < 0.001) but the changes in the DIS score were close to the significant level (P = 0.05) The comparison of dietary components of EDIP and DIS are displayed in Table 3 energy drinks and some vegetables such as tomatoes increased significantly The analysis of DIS components also indicated a significant increase in intakes of processed meats as well as a significant decrease in intakes of some vegetables Table 4 describes the risk of developing FP based on scores of different indices of dietary inflammation The number of cases increased significantly with the increase in the scores of all three inflammatory indices EDIP and DIS were significantly associated with higher risk of FP in all three models of analysis it was found that this relationship was significant only between the third and first tertiles but the confidence interval indicated that this increase in risk of FP in the second tertile is not significant compared to the reference The association between dietary inflammatory indices and the risk of fatty pancreas EDIP empirical dietary inflammatory pattern The current case–control study yielded some fascinating findings regarding the association between diet-induced inflammation and the risk of fatty pancreas EDIP and DIS were significantly associated with higher risk of fatty pancreas which confirms the hypothesis of the study An important strength of this study is that the present study is the first to investigate the association between dietary inflammatory indices and FP Another important strength is the use of a valid and reproducible FFQ This allowed for a comprehensive assessment of the main sources of nutrients in the diet although some measurement errors may have occurred in FFQ Controls were carefully selected by ensuring none of them had diet-related diseases or other major risk factors of FP In addition to the positive aspects of this study it is important to acknowledge certain limitations that need to be considered the possibility of recall bias and selection bias in this study is inevitable but the use of reliable FFQs administered by trained interviewers might have decreased the risk of recall bias Other limitations of using an FFQ to assess dietary intake include potential inaccuracies in reporting and limited food choices the participants in this study were patients with CBD stones so the generalization of the results requires further studies our case–control study suggested that higher scores of EDIP and DIS are related to increased odds of FP in patients with CBD stones The datasets analyzed in the current study are available from the corresponding author on reasonable request Pancreatic steatosis and metabolic pancreatic disease: A new entity? Intra-pancreatic fat deposition: Bringing hidden fat to the fore Pancreatic steatosis is a new therapeutic problem in gastroenterology (Literature Review) Uniting epidemiology and experimental models: pancreatic steatosis and pancreatic cancer The association between pancreas steatosis and metabolic syndrome: A systematic review and meta-analysis The effect of lifestyle interventions on excess ectopic fat deposition measured by noninvasive techniques in overweight and obese adults: A systematic review and meta-analysis Non-alcoholic Fatty pancreas disease (NAFPD): An updated review Intrapancreatic fat deposition and nutritional treatment: the role of various dietary approaches Metabolic crosstalk between fatty pancreas and fatty liver: Effects on local inflammation and insulin secretion Associations between dietary inflammatory index and inflammatory markers in the Asklepios Study Effect of anti-inflammatory diets on inflammation markers in adult human populations: A systematic review of randomized controlled trials Inflammation and nutrition: Friend or foe? Therapeutic endoscopic ultrasound: European society of gastrointestinal endoscopy (ESGE) guideline Pancreatic steatosis: A new diagnosis and therapeutic challenge in gastroenterology Impact of endoscopic ultrasound procedures in various pancreatobiliary disorders in Indonesia based on a case series in a private hospital Krause’s Food and the Nutrition Care Process-e-Book (Elsevier Health Sciences Whole-grain intake and the prevalence of hypertriglyceridemic waist phenotype in Tehranian adults cooking yields factors and edible portion of foods An empirical dietary inflammatory pattern score enhances prediction of circulating inflammatory biomarkers in adults Designing and developing a literature-derived population-based dietary inflammatory index Development and validation of an empirical dietary inflammatory index Validation and adaptation of the empirical dietary inflammatory pattern across nations: A test case Development and validation of novel dietary and lifestyle inflammation scores Adjustment for total energy intake in epidemiologic studies 2003–2004: Documentation and User Guide (U.S Department of Agriculture The empirical dietary inflammatory pattern score and the risk of nonalcoholic fatty liver disease and cirrhosis Association between non-alcoholic fatty pancreatic disease (NAFPD) and the metabolic syndrome: Case-control retrospective study Liver biopsy may facilitate pancreatic graft evaluation: Positive association between liver steatosis and pancreatic graft adipose infiltration Inflammation initiates a vicious cycle between obesity and nonalcoholic fatty liver disease The association between dietary inflammation scores and non-alcoholic fatty liver diseases in Iranian adults fruit and risk of non-gallstone-related acute pancreatitis: A population-based prospective cohort study Association between dietary habits and pancreatitis among individuals of european ancestry: A two-sample Mendelian randomization study Dietary factors reduce risk of acute pancreatitis in a large multiethnic cohort Dietary inflammatory and insulinemic potential and risk of type 2 diabetes: Results from three prospective US cohort studies Empirical dietary inflammatory pattern and risk of metabolic syndrome and its components: Tehran Lipid and Glucose Study Association between the dietary inflammatory index food patterns and incidence of metabolic syndrome in a Mediterranean cohort: The SUN project Intensive therapy for diabetes through influence on innate immune system Association of pancreatic steatosis with chronic pancreatitis Is type 2 diabetes an autoimmune-inflammatory disorder of the innate immune system? A central role for JNK in obesity and insulin resistance Suppressor of cytokine signaling 3 (SOCS-3) protects β-cells against interleukin-1β-and interferon-γ-mediated toxicity Obesity is associated with macrophage accumulation in adipose tissue Circulating levels of MCP-1 and IL-8 are elevated in human obese subjects and associated with obesity-related parameters Association between elevated C-reactive protein levels and prediabetes in adults Associations of specific dietary protein with longitudinal insulin resistance prediabetes and type 2 diabetes: The Rotterdam study Acute dietary fat intake initiates alterations in energy metabolism and insulin resistance Prudent diet and the risk of insulin resistance Dietary inflammatory patterns are associated with serum TGs and insulin in adults: A community-based study in Taiwan Inflammation and lipid signaling in the etiology of insulin resistance Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus Download references Authors have no acknowledgments to declare Department of Clinical Nutrition and Dietetics Faculty of Nutrition Sciences and Food Technology National Nutrition and Food Technology Research Institute Shahid Beheshti University of Medical Sciences Mohammad Bahrizadeh & Azita Hekmatdoost Research Institute for Gastroenterology and Liver Diseases of Taleghani Hospital National Nutrition and Food Technology Research Institute and Faculty of Nutrition Sciences and Food Technology The authors declare no competing interests The study was approved by the Research Ethics Committee of Shahid Beheshti Medical University of Iran under protocol number IR.SBMU.RETECH.REC.1402.689 All participants provided written informed consent and were informed about the study All procedures performed in studies involving human participants adhered to the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-025-00092-5 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Michael Koren for this informative luncheon This informative luncheon is LIVE at WJCT Studios Click Here WJCT Studios100 Festival Park AvenueJacksonville Our top priority is providing value to members Your Member Services team is here to ensure you maximize your ACS member benefits Registration is now open for the Laparoscopic Common Bile Duct Exploration (LCBDE) Course a hybrid course with recorded didactic lectures and an in-person hands-on skills lab for surgeons who want an overview of pre- and intraoperative decision-making for patients with choledocholithiasis as well as performing and accurately interpreting intraoperative cholangiography The in-person skills lab will take place Friday 1:00–5:15 pm at the Boston Scientific Surgical Skills Training Center in Marlborough Registration is $1,575 for ACS members and $1,890 for non-members with an opportunity to claim up to 6.0 AMA PRA Category 1 Credits™ Sessions this year highlighted lifelong surgeon competency This surgical perspective provided ways to reduce the health burden of ovarian cancer Older patients with acute cholecystitis and multiple comorbid conditions who can safely undergo surgery should be managed with cholecystectomy Data showed that more than 40% of surgeons reported neck and back pain following abdominal operations Ritah Chumdermpadetsuk and David Lee discuss unique challenges that transplant surgeons have when it comes to retirement Catch up on communications between the ACS and the new HHS and CMS leadership The award will recognize an individual's contributions to the advancement of women in surgery Tune in on May 8 to hear medical professionals discuss improving outcomes and experiences for patients and families The American College of Surgeons has categories of membership that correspond with the varying stages of one’s surgical career FACS for Division of Advocacy and Health Policy leadership positions Robert Montgomery's improvements in surgical transplantation have landed him a spot on TIME magazine's annual list The American College of Surgeons is dedicated to improving the care of surgical patients and safeguarding standards of care in an optimal and ethical practice environment ACS/American College of Surgeons is a registered trademark of the American College of Surgeons RESTRICTED USE: Visitors to this website are strictly prohibited from using or information provided by the ACS into any third-party applications or websites without prior written authorization from the ACS the integration of ACS content into tools leveraging artificial intelligence (AI) or generative AI technologies and infrastructures Copyright © 1996-2025 American College of Surgeons Bacteria naturally present in the human intestine (known as the gut microbiota) can transform cholesterol-derived bile acids into powerful metabolites that strengthen anticancer immunity by blocking androgen signaling according to a preclinical study led by Weill Cornell Medicine investigators The study was published April 15 in Cell “I was very surprised by our findings. As far as I know, no one has previously discovered molecules like these bile acids that can interact with the androgen receptor in this way,” said co-senior author Chun-Jun Guo an associate professor of immunology in medicine in the Division of Gastroenterology and Hepatology and a scientist at the Jill Roberts Institute for Research in Inflammatory Bowel Disease at Weill Cornell Medicine David Artis, director of the Jill Roberts Institute and the Friedman Center for Nutrition and Inflammation and the Michael Kors Professor in Immunology, and Nicholas Collins assistant professor of immunology in medicine a current postdoctoral associate in Guo’s lab Primary bile acids are produced by the liver and released into the gut where diverse groups of bacteria work together to modify their chemical structures Researchers suspected these gut microbial modifications could affect how bile acids function and interact with human signaling pathways the investigators set out to explore the full extent of bacterial modifications to bile acids and understand how these changes affect their biological roles It turns out that gut bacteria have remarkable potential to transform bile acids “We discovered more than 50 different bile acid molecules modified by the microbiota – many of which had never been identified before,” said Guo who’s also the Halvorsen Family Research Scholar in Metabolic Health at Weill Cornell Medicine These newly uncovered structures could open the door to new biological insights – particularly in how they interact with human receptors that sense bile acids Given that bile acids share the same steroid backbone as sex hormones like testosterone and estrogen the structural resemblance raised an intriguing question for the researchers: Could these microbially modified bile acids also interact with sex hormone receptors in the body “It seemed like a wild idea at the time,” Guo said the answer appears to be “yes.” When the investigators tested the 56 altered bile acids that they discovered they found one that antagonizes the androgen receptor – a molecule that interacts with sex hormones to regulate many aspects of human development When they tested an additional 44 microbiota-modified bile acids that had previously been characterized the team found three more that act similarly This unexpected finding raised exciting new questions for the team regarding which specific cells were affected by the altered bile acids and what biological functions these modified molecules might influence the androgen receptor is also found in certain immune cells Previous studies have shown that blocking this receptor can enhance the ability of these immune cells to fight tumors The investigators wondered whether the bile acids could replicate this effect by binding to and inactivating the androgen receptor they treated mice with bladder cancer using these compounds – and observed a potent antitumor response Further analysis revealed that the modified bile acids specifically boosted the activity of T cells – the immune cells best equipped to kill cancer “Our results suggest that these altered bile acids help shrink tumors by enhancing T cells’ ability to survive within the tumor and destroy cancer cells,” Collins said “This study highlights the profound and evolving partnership between the human host and its gut microbiota emphasizing the importance of integrating microbial activity into the design of future cancer therapies,” Artis said “It also exemplifies the power of multidisciplinary collaboration in driving microbiome science toward deeper molecular understanding of host-microbe interactions.” This discovery opens up exciting new possibilities for boosting tumor-killing immune response Potential approaches include introducing targeted gut microbes to cancer patients before therapy or directly administering the anticancer bile acids as part of treatment Although these compounds still need to be tested in humans the team is optimistic that bile acids could eventually become a key component of effective cancer therapies – especially when combined with existing treatments for a more powerful impact For example: How might diet – which is known to influence microbiota composition – affect the production of these bile acids what physiological effects might these androgen receptor–blocking bile acids have in healthy individuals The team is now focused on precisely controlling the synthesis and release of these beneficial molecules using advanced techniques to genetically engineer gut commensal bacteria aiming to understand the broader physiological impact in the host initiated by these androgen blocking This work was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases the National Institute of Allergy and Infectious Diseases the National Institute of Arthritis and Musculoskeletal and Skin Diseases all part of the National Institutes of Health Many Weill Cornell Medicine physicians and scientists maintain relationships and collaborate with external organizations to foster scientific innovation and provide expert guidance. The institution makes these disclosures public to ensure transparency. For this information, see profile for David Artis Saima Sidik is a freelance writer for Weill Cornell Medicine Get Cornell news delivered right to your inbox Bile duct cancer is a rare and aggressive cancer that typically affects adults over the age of 50 Metrics details Conjugated bile acids (BAs) are multi-functional detergents in the gastrointestinal (GI) tract produced by the liver enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT) and by the microbiome from the acyltransferase activity of bile salt hydrolase (BSH) Humans with inflammatory bowel disease (IBD) have an enrichment in both host and microbially conjugated BAs (MCBAs) but their impacts on GI inflammation are not well understood We investigated the role of host-conjugated BAs in a mouse model of colitis using a BAAT knockout background Baat−/− KO mice have severe phenotypes in the colitis model that were rescued by supplementation with taurocholate (TCA) Gene expression and histology showed that this rescue was due to an improved epithelial barrier integrity and goblet cell function metabolomics also showed that TCA supplementation resulted in extensive metabolism to secondary BAs We therefore investigated the BSH activity of diverse gut bacteria on a panel of conjugated BAs and found broad hydrolytic capacity depending on the bacterium and the amino acid conjugate The complexity of this microbial BA hydrolysis led to the exploration of bsh genes in metagenomic data from human IBD patients Certain bsh sequences were enriched in people with Crohn’s disease particularly that from Ruminococcus gnavus This study shows that both host and microbially conjugated BAs may provide benefits to those with IBD but this is dictated by a delicate balance between BA conjugation/deconjugation based on the bsh genes present The recent expansion of conjugated BA diversity from the microbiome brings into question the role these compounds may have in gut inflammation and IBD the balance between conjugation and deconjugation of host and microbial enzymes is complex and there is a knowledge gap surrounding the role of BSHs and conjugated BAs in IBD Filling that gap may lead to the development of novel therapeutics using conjugated BAs or specific BSHs for treatment of this chronic inflammatory disease we investigated the hydrolytic capacity of 17 bacteria on diverse BAs and explored the BSH sequence space in publicly available metagenomic datasets from subjects with IBD Our results show that conjugated BAs are important for reducing pathology in an inflamed mammalian gut but that the balance between hydrolysis and conjugation is complex and dependent on the BSH sequences present in the microbiome a Experimental animal model in the Baat−/− KO mice versus wild-type mice under DSS treatment for 7 days (n = 4–5) d Example images of the colons from each group of mice f Representative photomicrographs of H&E-stained (original magnification g Histology scores with representative H&E staining c) expressed as the mean ± SEM; n = 4–5 animals per group; all boxplots are the interquartile range with the whiskers denoting minima and maxima; statistics analyzed by one-way ANOVA with Tukey’s post hoc a Experimental animal design using Baat−/− KO mice fed with 0.3% TCA under DSS-induced colitis (n = 6-8) (b) Weight changes during the experiment (Data expressed as the mean ± SEM) c Example images of the colons from each group of mice e Spleen index (spleen weight: body weight ratio) g Representative photomicrographs of alcian blue-periodic acid-Schiff (AB-PAS)-stained colon sections (original magnification h Goblet cell counts from each group of mice i Representative photomicrographs of MUC-2 staining colon sections (scale bar = 50 μm) with the whiskers denoting minima and maxima; n = 6–8 animals per group and mRNA expression data were tested by one-way ANOVA with Tukey’s post hoc MUC-2 staining data analyzed by Kruskal-Wallis test followed by Dunn’s test a Microbiome alpha diversity determined by the Shannon index; n = 4-5 animals per group (b) The principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity shows the microbiome β-diversity; n = 4–5 animals per group d–g The principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity of liver Heatmap values (Z-score by rows) represent mean abundance of each BA in each group Grey color indicates values that were not detected The number of mice in the microbiome and metabolome results was n = 4–5 mice per group with the whiskers denoting minima and maxima Microbiome data analyzed by one-way ANOVA with Tukey’s post hoc Metabolites data analyzed by Kruskal-Wallis followed by Dunn’s post hoc # indicated the significant differences between HC and TCA groups (p < 0.05) × indicated the significant differences between HC and DSS groups (p < 0.05) and * indicated the significant differences between DSS and DSS-TCA groups (p < 0.05) TCA decreased MCBAs under DSS-induced colitis though this did not reach statistical significance between the DSS and DSS-TCA groups a The abundances of conjugated cholic acids are represented as colors corresponding to log10­transformed peak areas (area under the curve Values represent the mean from n = 3 replicates of log10 areas changes compare to medium after 48 h in vitro culture Phylogenetic tree of 17 Bacteroidetes strains using GTDB-tk based on whole bacteria genome b Experimental animal design in wild-type C57BL/6 J mice fed with a final dose of 50 mg/kg TCA or GluCA peanut butter pellets under DSS-induced colitis (n = 10 each group) Fecal metabolites data was analyzed by Kruskal-Wallis followed by Dunn’s post hoc This suggests that TCA was more readily hydrolyzed by the gut microbiome to produce secondary BAs than GluCA treatment mirroring the effects shown from bacterial cultures in vitro b The alpha diversity of BSHs in IBD patients and healthy controls which was measured using the Shannon index and Observed BSHs c The β-diversity of BSHs was measured by principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity d The cumulative relative abundance of BSHs in IBD patients and healthy controls e–g Taxonomic characterization of BSHs at phylum h–k Examples of significant differing BSHs in IBD compared to healthy controls Data was analyzed by two-sided Wilcox rank-sum test followed by BH correction the causal relationships between BA dysregulation and IBD the role of the ever-expanding diversity of these molecules and the mechanisms by which they impact disease are poorly understood The pathological and multi-omics analysis of Baat−/− KO mice in the DSS model demonstrates that conjugated BAs are important for reducing pathology from inflammation and the likely mechanism is through their maintenance of the mucus barrier the effect of conjugated BAs on gut microbial diversity in an inflamed environment is an important area for further research especially considering this study shows that conjugated BAs can alter outcomes of GI inflammation who showed that conjugated BAs improve barrier integrity in gut epithelial cells and that inhibiting BSH has a similar effect in vivo Both this study and ours points to the potential to manipulate the conjugated BA profile and gut bacteria that shape it to benefit patients with chronic inflammatory GI disease The need to parse out these mechanisms is heightened by the principal finding of this study that at least one prevalent conjugated BA is important for improving pathology in an inflamed GI tract It is possible that other forms of conjugated BAs but the novelty and lack of commercial availability of these molecules limits current ability to experiment GCA and the diverse other forms of conjugated BAs could be promising therapeutics for subjects with gut inflammation particularly those with lower levels of these molecules natively which can be readily assayed by mass spectrometry To translate new knowledge of these molecules to medical use for any disease state the balance between their conjugation and deconjugation in the gut must be better understood and WT and Baat−/− KO mice were randomly assigned to four groups (n = 4 WT-healthy control (HC) The mice in the DSS group were administered 2.5% dextran sodium sulfate (DSS USA) daily in drinking water for 7 days to induce colitis Mice were weighed daily and on day 7 all mice were euthanized through anesthesia using isoflurane followed by cervical dislocation and spleens were weighed for comparisons across treatment groups In a follow-up taurocholic acid (TCA) supplementation experiment Baat−/− KO mice were subjected to a treatment with TCA or mock control 27 Baat−/− KO mice (6-8 weeks of age) were randomly assigned to four treatment groups (n = 6 HC 2.5% DSS was administered in drinking water for 6 days HC and TCA group mice were given regular water throughout the experiment Mice in TCA treatment groups were fed with food containing 0.3% TCA (ENVIGO while mice in the HC and DSS groups were given the same food but without TCA and fecal samples were collected by temporarily placing the mice in clean plastic cups on day 13 prior to sacrifice On day 13 all mice were euthanized through anesthesia using isoflurane followed by cervical dislocation and cecal content were then harvested after mice were euthanized All mice that passed the training were selected to the next stage of training (non-fasted training) where they were given a plain pure peanut butter pellet under normal chow feed supply for three consecutive days The mice that successfully completed training were included in subsequent study Forty male mice were randomly assigned to four groups (HC and DSS-GluCA groups) with 10 mice per group Mice in HC and DSS groups were treated with two 10 mg peanut butter pellets every day for 17 days and mice in DSS-TCA and DSS-GluCA groups were weighed and treated with two 10 mg peanut butter pellets with 25 mg/kg TCA or GluCA every day for 17 days 2.5% DSS was added to the drinking water for 7 days after the 10 days normal water Mice were weighed every day and fecal samples were collected before sacrifice On day 17 all mice were euthanized through anesthesia using isoflurane followed by cervical dislocation Spleen and colon were then harvested after mice were euthanized and feces and were frozen immediately by liquid nitrogen and stored at −80 °C until they were thawed for use Assessment of spleen inflammation index was performed by the spleen weight (g) normalized by its body weight (g) Disease activity index (DAI) was assessed by measuring the following: body weight and 4 = gross bleeding; diarrhea: 0 = well-formed stools Colon specimens were fixed overnight in 10% formalin at room temperature and stained with hematoxylin and eosin (H&E) A subset was stained with alcian blue-periodic acid-Schiff (AB-PAS) reaction to highlight mucin-containing goblet cells An independent board-certified veterinary anatomic pathologist who was blinded to the treatment evaluated the slides A modified scoring system was used to determine a histologic score including degree of inflammatory cell infiltration (normal=0 dense inflammatory infiltrate= 3) and changes to crypt architecture (normal=0 severe crypt distortion with loss of entire crypts=3) epithelial injury was determined by % of surface erosions goblet cell depletion was determined (depleted=0 and goblet cell counts were obtained for the subset stained with AB-PAS Colon sections were deparaffinized followed by heat induced epitope retrieval utilizing TRIS/EDTA buffer (pH 9.0; Scytek labs and treated with hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity Immunohistochemistry was performed on the Biocare intelliPath FlexTM automated platform (Biocare Medical Non-Specific Proteins were blocked with Rodent Block M (Biocare Medical by incubation in primary antibodies (MUC-2 antibody (1:500 and Rabbit-on-Rodent HRP-Polymer (Biocare Medical The reaction was developed with AEC (Biocare Medical USA) for 5 min followed by counter stain with CATHE hematoxylin (Biocare Medical Post staining slides were rinsed in distilled water cleared in Xylene and cover slipped with Optic Mount 1 media (Mercedes Scientific The intensity of MUC-2 and ZO-1 staining was analyzed using ImageJ software The quantitative intensity of MUC-2 and ZO-1 staining was calculated using the Deconvolution2 plugin in ImageJ software The relative mRNA expression was calculated using the comparative cycle method (2−ΔΔCt) β-actin served as an internal reference gene Sequences were rarefied to 9000 reads per sample and core diversity metrics Phosphate buffered saline (PBS) was added to liver v:w) and homogenized via bead bashing at 20 Hz for 30 s with 1 min of rest three times using a Bead Ruptor (Omni International cecal or fecal homogenate and 50 μL of serum was added to 150 μL ice cold methanol at a final concentration of 60% methanol followed by overnight incubation at 4 °C Extracted metabolites were stored at −80°C prior to mass spectrometry analysis All strains used in this study are listed in supplementary data S1 in the supplementary material All bacterial strains were cultured in Reinforced Clostridial Medium (RCM) and 0.5 g of L-cysteine in 1 L distilled water The medium was adjusted to a final pH of 7.0 and autoclaved at 121 °C for 15 min 200 μL of culture was added to 300 μL ice cold methanol at a final concentration of 60% methanol followed by overnight incubation at 4 °C Extracted metabolites were stored at −80 °C prior to mass spectrometry analysis The query BSH protein sequences were collected from the National Center for Biotechnology Information (NCBI) using the keywords “bile salt hydrolase” and “choloylglycine hydrolase” The source was limited to bacteria and archaea and the amino acid sequence length was 300 to 400 A total 3058 BSH sequences were obtained and then filtered for non-redundancy using CD-HIT (version 4.8.1 default parameter) at a 95% sequence identity threshold and the final output 778 BSHs sequences were used as a representative set of BSHs for further analysis The taxonomy information of BSHs was obtained from the NCBI taxonomy browser The metadata information was obtained from the materials provided in corresponding papers only the first time point per individual was selected A total of 774 samples were included in the analysis Data from PRJNA0072 were acquired from two different cohorts and 51 fecal samples were analyzed from Healthy subjects (HC) The BSHs were identified by taking the referenced 778 BSHs as query sequences and using DIAMOND (version 0.9.36) BLASTP with an e-value = 1e−5 and 40% sequence identity as the cutoff values To ensure the accuracy and reliability of the statistical analysis the putative BSHs present in less than 10% of all samples were excluded and a pseudo-count of 1×10-10 was added to avoid nonfinite values The cumulative BSHs relative abundance was calculated by summing the relative abundance of all BSHs in each sample Data was expressed as means ± standard errors of the means (SEM) and boxplots show the median minimum and maximum with individual data points visualized statistical significance between groups for weight changes mRNA expression and microbiome diversity metrics among groups were analyzed by one-way ANOVA followed by Tukey’s post-hoc test The statistical significance differences of the IHC staining and metabolome data were calculated using Kruskal-Wallis followed by Dunn’s post-hoc test Statistically significant differences of BSHs between IBD patients and healthy control were determined by Wilcox rank-sum test followed by Benjamini-Hochberg (BH) correction metabolome and BSHs data was quantified using principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity PERMANOVA testing was used to determine if there were significant differences among the groups All analyses were performed in R (version 4.3.0) and p values less than 0.05 (p < 0.05) was considered statically significant as specified in the text and figure legends Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Mass spectrometry data are publicly available within the MassIVE database (gnps.ucsd.edu) under MassIVE ID MSV000095705. 16S rRNA gene amplicon data were deposited in the NCBI under project number PRJNA1152983. Source data are provided with this paper Inflammatory bowel disease presentation and diagnosis and racial and ethnic distribution of inflammatory bowel disease in the United States The cost of inflammatory bowel disease in high-income settings: a lancet gastroenterology & hepatology commission Host–microbiota interactions in inflammatory bowel disease Gut microbial metabolome in inflammatory bowel disease: from association to therapeutic perspectives Reverse metabolomics for the discovery of chemical structures from humans Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases Gut microbiome structure and metabolic activity in inflammatory bowel disease The gut–liver axis and gut microbiota in health and liver disease Review: microbial transformations of human bile acids Bile acids and the gut microbiota: metabolic interactions and impacts on disease Another renaissance for bile acid gastrointestinal microbiology Bile acid metabolism and signaling in health and disease: molecular mechanisms and therapeutic targets Taxonomic profiling and populational patterns of bacterial bile salt hydrolase (BSH) genes based on worldwide human gut microbiome Mohanty I. et al. The changing metabolic landscape of bile acids – keys to metabolism and immune regulation. Nat. Rev. Gastroenterol. Hepatol. https://doi.org/10.1038/s41575-024-00914-3 (2024) A selective gut bacterial bile salt hydrolase alters host metabolism Bile salt hydrolase acyltransferase activity expands bile acid diversity Bile salt hydrolase catalyses formation of amine-conjugated bile acids Global chemical effects of the microbiome include new bile-acid conjugations Novel approaches in IBD therapy: targeting the gut microbiota-bile acid axis Noninvasive imaging and quantification of bile salt hydrolase activity: from bacteria to humans Dysbiosis-induced secondary bile acid deficiency promotes intestinal inflammation Altered profiles of fecal bile acids correlate with gut microbiota and inflammatory responses in patients with ulcerative colitis Baat Gene knockout alters post-natal development Bile acid conjugation deficiency causes hypercholanemia Host metabolism balances microbial regulation of bile acid signalling The gut microbial bile acid modulation and its relevance to digestive health and diseases The underappreciated diversity of bile acid modifications The role and function of mucins and its relationship to inflammatory bowel disease Microbiota and mucosal defense in IBD: an update Intestinal mucus and their glycans: a habitat for thriving microbiota Inhibition of microbial deconjugation of micellar bile acids protects against intestinal permeability and liver injury The tight junction protein ZO-1 Is dispensable for barrier function but critical for effective mucosal repair Gut barrier dysfunction—a primary defect in twins with crohn’s disease predominantly caused by genetic predisposition Bile acids signal via TGR5 to activate intestinal stem cells and epithelial regeneration Bile acids and their receptors: potential therapeutic targets in inflammatory bowel disease Intestinal microbiota: a source of novel biomarkers in inflammatory bowel diseases Gut microbiota-mediated secondary bile acids regulate dendritic cells to attenuate autoimmune uveitis through TGR5 signaling Differences in gut microbiota in patients with vs without inflammatory bowel diseases: a systematic review Akkermansia muciniphila alleviates dextran sulfate sodium (DSS)-induced acute colitis by NLRP3 activation Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut Self-administration of drugs in mouse models of feeding and obesity scalable and extensible microbiome data science using QIIME 2 DADA2: High-resolution sample inference from Illumina amplicon data Greengenes2 enables a shared data universe for microbiome studies Sharing and community curation of mass spectrometry data with global natural products social molecular networking Two distinct metacommunities characterize the gut microbiota in Crohn’s disease patients microbiota and metabolite networks in inflammatory bowel disease Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes Dynamics of metatranscription in the inflammatory bowel disease gut microbiome MetaWRAP—a flexible pipeline for genome-resolved metagenomic data analysis BBMerge – accurate paired shotgun read merging via overlap Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation Download references The authors would like to thank MSU mass spectrometry and metabolomics core for their help This work was funded by the Biochemistry and Molecular Biology Department Team-Up Grant and the Tetrad Grant Program of Michigan State University and the National Institute of Diabetes and Digestive and Kidney Diseases grant #1R01DK140854 awarded to PI Quinn Department of Biochemistry and Molecular Biology Department of Pathobiology and Diagnostic Investigation Nature Communications thanks Ipsita Mohanty reviewer for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-025-58649-x The image depicts gut microbes producing secondary bile acids to support the host’s immune defense against cancer Microbiota-derived bile acids (shown in light green) blocked the androgen receptor (depicted as a door) from binding with androgen (in light blue) enhancing the ability of CD8+ T cells to combat cancer cells Bacteria naturally present in the human intestine, known as the gut microbiota, can transform cholesterol-derived bile acids into powerful metabolites that strengthen anti-cancer immunity by blocking androgen signaling, according to a preclinical study led by Weill Cornell Medicine investigators. The study was published on April 15 in Cell Dr. David Artis, director of the Jill Roberts Institute and the Friedman Center for Nutrition and Inflammation and the Michael Kors Professor in Immunology, and Dr. Nicholas Collins “We discovered more than 50 different bile acid molecules modified by the microbiota—many of which had never been identified before,” said Dr who is also the Halvorsen Family Research Scholar in Metabolic Health at Weill Cornell Medicine These newly uncovered structures could open the door to new biological insights-particularly in how they interact with human receptors that sense bile acids the structural resemblance raised an intriguing question for the researchers: could these microbially modified bile acids also interact with sex hormone receptors in the body “It seemed like a wild idea at the time,” Dr When the investigators tested the 56 altered bile acids that they discovered they found one that antagonizes the androgen receptor — a molecule that interacts with sex hormones to regulate many aspects of human development This unexpected finding raised exciting new questions for the team: which specific cells were affected by the altered bile acids—and what biological functions these modified molecules might influence they treated mice with bladder cancer using these compounds—and observed a potent anti-tumor response Further analysis revealed that the modified bile acids specifically boosted the activity of T cells—the immune cells best equipped to kill cancer “Our results suggest that these altered bile acids help shrink tumors by enhancing T cells’ ability to survive within the tumor and destroy cancer cells,” Dr emphasizing the importance of integrating microbial activity into the design of future cancer therapies.” Dr “It also exemplifies the power of multidisciplinary collaboration in driving microbiome science toward deeper molecular understanding of host–microbe interactions.” or directly administering the anti-cancer bile acids as part of treatment the team is optimistic that bile acids could eventually become a key component of effective cancer therapies—especially when combined with existing treatments for a more powerful impact how might diet—which is known to influence microbiota composition—affect the production of these bile acids Many Weill Cornell Medicine physicians and scientists maintain relationships and collaborate with external organizations to foster scientific innovation and provide expert guidance. The institution makes these disclosures public to ensure transparency. For this information, see profile for Dr. David Artis Additional funding came from the Crohn’s & Colitis Foundation the Jill Roberts Institute for Research in IBD and the Allen Discovery Center Program—an initiative of the Paul G Allen Frontiers Group advised by the Paul G Further support was provided by the Feldstein Medical Foundation Back to News Glenn Center for Biology of Aging Research Prices may be subject to local taxes which are calculated during checkout doi: https://doi.org/10.1038/d41586-024-04062-1 Nature https://doi.org/10.1038/s41586-024-08329-5 (2024) Nature https://doi.org/10.1038/s41586-024-08348-2 (2024) Download references Reprints and permissions D.A.S. is a co-founder, consultant, board member and equity owner of Galilei Biosciences and MetroBiotech, companies working to treat diseases with SIRT6 activators and NAD boosters, respectively. He is also an equity owner of Tally Health, a DNA biomarker company. Other activities are listed at sinclair.hms.harvard.edu Microbial molecule of ageing gut nudges blood stem cells towards cancer Age-related blood condition counteracted with a common diabetes drug The best foods for healthy ageing ― and the worst Fungus from the human gut slows liver disease in mice Diet outperforms microbial transplant to drive microbiome recovery in mice Parkinson’s gut-microbiota links raise treatment possibilities Memories of a cold place trigger bodily responses to warm up HT is an interdisciplinary research institute created and supported by the Italian government whose aim is to develop innovative strategies to pr.. UNIL is a leading international teaching and research institution with over 5,000 employees and 17,000 students split between its Dorigny campus Department of Energy and Environmental Materials and advance cancer research in a leading translational institute Olivia Newton-John Cancer Research Institute We are seeking a tenure-track associate professor to promote interdisciplinary research in nanoprobe life sciences or related interdisciplinary field Metrics details Bacterial dysbiosis coincides with the carcinogenesis in malignancies such as lung and colon cancer and has recently been suggested to involve in the pathogenesis of hepatocellular carcinoma (HCC) the mycobiome has not yet been definitively linked to liver tumorigenesis Here we showed that the microbiota composition of HCC tumors was distinct from that of the normal adjacent to tumor (NAT) on the basis of richness and beta-diversity indices the fungal community that infiltrated HCC tumors was markedly enriched for Malassezia spp and genus Malassezia in tumors was substantially more abundant than that in NAT We also discovered that the relative abundance of genus Malassezia was strongly correlated with the tumor microenvironment (TME) signatures tumor-resident Malassezia could inhibit bile acid synthesis by downregulating the expression level of CYP7 A1 and CYP27 A1 we developed a set of Malassezia-related genes which could accurately predict patient survival our work shows that tumor-resident Malasseiza may promote HCC progression by downregulating bile acid synthesis and modulating the TME these studies have clarified the association between the microbial composition in the tumor microenvironment (TME) and the progression of liver cancer from different perspectives and using different techniques only focusing on the bacterial composition in the tumor is a common shortcoming of these studies The potential role of other microbial kingdoms and there is an urgent need for clear biomarkers to predict the efficacy of HCC immunotherapy due to the spatial structure of microorganisms located in the TME it can more directly influence various activities in the liver elucidating the relationship between the microbiome in the tumor and HCC is helpful to offer new treatment options using the paired intratumoral microbiome and host transcriptome samples from The Cancer Genome Atlas (TCGA) HCC cohort we characterized the intratumoral bacteriome and mycobiome composition and their interactions in HCC we identified high abundance of genus Malassezia in tumors and correlated the abundance of Malassezia with the TME signatures and bile acid synthesis we developed a set of genes correlated with Malassezia to predict the patient survival These results provide novel insights into the potential role of intratumoral mycobiome in HCC and offer guidance to improve the current landscape of HCC treatment Dataset GSE76427 was used to independently validate the performance our prognostic model All statistical analyses in this study were carried out using R software (version 4.2.1; http://www.R-project.org) The statistical analysis of continuous variables was conducted using a two-tailed Wilcoxon rank-sum test Nonmetric multidimensional scaling (NMDS) based on Bray-Curtis distance matrices was implemented in the R project and the visualization of the results was achieved using the “ggplot2” package in R a permutational multivariate analysis of variance (PERMANOVA) test was executed utilizing the “vegan” package a P-value of below 0.05 was considered statistically significant Microbial community composition in tumor and NAT of HCC (a) NMDS plot showing the difference in the intratumoral microbiome composition between tumor and NAT The p value was generated by PERMANOVA test Boxplot showing the difference in the (b) richness and (d) Simpson index between tumor and NAT Microbial community composition at the genus level in (e) liver tumor and (f) NAT (g) Species belonging to the genus Malassezia The top of the figure represents the three species the bottom represents the genus Malassezia and the width of the lines connecting the top and bottom represents the number of sequences aligned to the species (h) Procrustes analysis and Mantel analysis showing the correlation between bacteriome and mycobiome in liver tumors (i) Heatmap showing the Spearman correlation coefficient between individual dominant bacteria and individual fungus The column annotation indicates the relative abundance of bacteria these results demonstrate that HCC tumors create a microenvironment conducive to a bacteria-fungus synergistic relationship Malassezia plays a regulatory role in the TME of HCC patients Boxplot showing the differences in (a) stromal score and (c) tumor purity between tumor and NAT (d) Boxplot showing the differences in the Malassezia load between tumor and NAT *** indicate p < 0.001 and **** indicate p < 0.0001 Linear regression analysis showing the correlation between the relative abundance of Malassezia and the TME profiles (h) Boxplot showing the difference in the expression of gene SELE between tumor and NAT (i-l) Linear regression analysis showing the correlation between the expression of gene SELE and four HCC-related key genes (m) Linear regression analysis showing the correlation between the expression of gene SELE and the relative abundance of Malassezia suggesting a potential relationship between intratumoral Malassezia and local inflammation Linear regression analysis showing the correlation between the relative abundance of Malassezia and bile acid synthesis-related genes (f) Two main pathways of bile acid synthesis The red ovals represent the key enzymes that work Malassezia-related genes are strongly correlated with the TME profiles (a) Pipeline of the construction of Malassezia.sig Venn plot showing the intersection of DEGs and genes significantly correlated with Malassezia and (d) GO enrichment results of Malassezia.sig Linear regression showing the correlation between the GSVA score of Malassezia.sig and (e) stromal score (i) Heatmap showing the normalized abundance of various immune cell populations in groups of high- and low GSVA score Row annotation represents six approaches used to calculated the abundance of cells Column annotation represents the two groups (j) Boxplot showing the difference in the abundance of M1 macrophage and CD4+ T cell between high GSVA score and low GSVA score the Malasseiza.Sig is closely related with the TME in HCC Malassezia.sig is closely related with prognosis in HCC patients. (a) Pie chart showing the proportion of prognostic genes in Malassezia.sig. The prognostic genes were obtained by univariate Cox regression with a threshold of p < 0.05. (b) Forest plot showing the result of multivariate Cox regression. (c-g) Survival analysis of five genes in Fig. 5b Samples were divided into two groups (high and low expression) based on the median expression of each gene The p values were generated by log-rank test Performance of our prognostic model in (h) TCGA and (i) GSE76427 (j) Representative immunohistochemical staining images of five genes in tumor and NAT Our prognostic genes are significantly correlated with the TME signatures Heatmap showing the Spearman correlation coefficient between the five prognostic genes and the TME profiles in (a) TCGA (e-l) Linear regression plot showing the correlation between specific prognostic genes and the TME profiles We found that intratumoral Malassezia could inhibit bile acid synthesis by down-regulating the expression of CYP7 A1 and CYP27 A1 we developed a Malassezia-related gene signatures and prognostic model was built based on these signatures to predict the survival Although more in-depth mechanistic studies are needed to clarify the intrinsic link between Malassezia and the development of HCC our study provides new insights into the association of intratumoral mycobiome with HCC intratumoral Malassezia may indirectly affect the development and prognosis of HCC by regulating the TME We found that the relative abundance of Malassezia was negatively correlated with the expression of both CYP7 A1 and CYP27 A1 indicating the inhibitory effect of intratumoral Malassezia on bile acid synthesis Future studies should focus on the functional mechanisms by which Malassezia inhibits bile acid synthesis thereby contributing to the development of tests and treatments related to bile acid synthesis factors the development of more sensitive and effective markers for early diagnosis and prognostic prediction of HCC microbiome preparations for remodeling intratumoral microbiota and specific bile acid receptor agonists have important clinical application value and need to be further studied Inhibition of ENTPD2 is a promising strategy to enhance the efficacy of ICIs targeting PD-1/CTLA-4 We found that SUPT7L and COG1 genes were risk factors for survival in HCC patients but their underlying roles in HCC remains unclear and needs to be further explored The limitations of our study encompass a comparatively modest sample size and a cross-sectional study design while enabling the identification of associations falls short of establishing causal relationships we were unable to comprehensively adjust the data for all potential confounding variables Future endeavors necessitate the conduction of studies with expanded sample sizes and employing longitudinal designs to further elaborate upon our present findings the biological functions of tumor-resident mycobiome in HCC especially the relationship between Malassezia and the TME of HCC Conducting further experimental studies is imperative to elucidate these mechanisms which could significantly contribute to our understanding of HCC pathogenesis and facilitate the identification of potential therapeutic targets the etiology of HCC has far-reaching implications for gene expression based analyses such as metabolic dysfunction-associated steatotic liver disease (MASLD)-associated liver cancer and hepatitis B Due to the lack of relevant etiological data we were not able to stratify patients and perform comparative analyses to provide insight into the heterogeneity of HCC we showed that Malassezia is enriched in HCC tumor tissues and that the levels of Malassezia are correlated with the TME and bile acid synthesis elucidations of the underlying mechanisms by which intratumoral Malassezia regulates HCC behavior will contribute to the development of new prognostic and predictive biomarkers for HCC These relationships in the hepatic microenvironment suggest that the intratumoral mycobiome may emerge as a novel therapeutic target as well as a promising area for biomarker discovery The datasets generated and analysed during the current study are available in TCGA database (https://portal.gdc.cancer.gov/), and GEO database (https://www.ncbi.nlm.nih.gov/geo/) The human tumor Microbiome is composed of tumor type-specific intracellular bacteria Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions A pan-cancer analysis of the Microbiome in metastatic cancer Intratumor Microbiome features reveal antitumor potentials of intrahepatic cholangiocarcinoma Intratumoural Microbiome can predict the prognosis of hepatocellular carcinoma after surgery The intratumoral bacterial metataxonomic signature of hepatocellular carcinoma Intratumoral bacteria interact with metabolites and genetic alterations in hepatocellular carcinoma Association of intratumoral Microbiome diversity with hepatocellular carcinoma prognosis The mortality and overall survival trends of primary liver Cancer in the united States Real-world data on EGFR/ALK gene status and first-line targeted therapy rate in newly diagnosed advanced non-small cell lung cancer patients in Northern China: A prospective observational study LBA39Randomised efficacy and safety results for Atezolizumab (Atezo) + bevacizumab (Bev) in patients (pts) with previously untreated unresectable hepatocellular carcinoma (HCC) Immunotherapies for hepatocellular carcinoma The current landscape of immune checkpoint Blockade in hepatocellular carcinoma: A review Immune-Related adverse events associated with immune checkpoint Blockade and cytokines in intrahepatic cholangiocarcinoma Inferring tumour purity and stromal and immune cell admixture from expression data Signatures of T cell dysfunction and exclusion predict cancer immunotherapy response The advantages of a procrustean superimposition approach over the mantel test Antibodies for profiling the human proteome-The human protein atlas as a resource for cancer research Multi-kingdom microbiota analyses identify bacterial-fungal interactions and biomarkers of colorectal cancer across cohorts The evolving tumor microenvironment: from cancer initiation to metastatic outgrowth Targeting the gut and tumor microbiota in cancer Targeting PRMT3 impairs methylation and oligomerization of HSP60 to boost anti-tumor immunity by activating cGAS/STING signaling Targeting bile-acid signalling for metabolic diseases The tumour microenvironment shapes innate lymphoid cells in patients with hepatocellular carcinoma Development of a TMErisk model based on immune infiltration in tumour microenvironment to predict prognosis of immune checkpoint inhibitor treatment in hepatocellular carcinoma Gut microbial structural variation associates with immune checkpoint inhibitor response A pan-cancer mycobiome analysis reveals fungal involvement in Gastrointestinal and lung tumors Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor Metabolism as a new avenue for hepatocellular carcinoma therapy The fungal mycobiome promotes pancreatic oncogenesis via activation of MBL Breast cancer colonization by malassezia globosa accelerates tumor growth Identification of a tumour immune barrier in the HCC microenvironment that determines the efficacy of immunotherapy Semaphorin 3 C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression Neoadjuvant Tislelizumab plus stereotactic body radiotherapy and adjuvant Tislelizumab in early-stage resectable hepatocellular carcinoma: the Notable-HCC phase 1b trial Glycochenodeoxycholate promotes hepatocellular carcinoma invasion and migration by AMPK/mTOR dependent autophagy activation Dysregulated hepatic bile acids collaboratively promote liver carcinogenesis Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of inflammation during obstructive cholestasis which promote activation of receptor tyrosine kinases and signaling pathways in rat hepatocytes Prognostic roles of metabolic reprogramming-associated genes in patients with hepatocellular carcinoma Development and validation of a prognostic classifier based on lipid Metabolism-Related genes in gastric Cancer Targeting ACYP1-mediated Glycolysis reverses lenvatinib resistance and restricts hepatocellular carcinoma progression Deficiency of mitochondrial glycerol 3-Phosphate dehydrogenase contributes to hepatic steatosis Hypoxia inducible factor HIF-1 promotes myeloid-derived suppressor cells accumulation through ENTPD2/CD39L1 in hepatocellular carcinoma KEGG: Kyoto encyclopedia of genes and genomes Download references This work was supported by National Natural Science Foundation of China (grant no 62162025) and Hainan Provincial Natural Science Foundation of China (grant no These authors contributed equally: Weixi Shen and Zhihong Li The Second Affiliated Hospital of Harbin Medical University Ganzi Tibetan Autonomous Prefecture People’s Hospital The Fourth Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine All authors have read and approved the final manuscript The authors declare that they have no known competing financial interests or personal relationships Below is the link to the electronic supplementary material Download citation DOI: https://doi.org/10.1038/s41598-025-99973-y Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research Metrics details using untargeted metabolomics of mouse tissues we identified a family of BA–methylcysteamine (BA–MCY) conjugates that are abundant in the intestine and dependent on vanin 1 (VNN1) a pantetheinase highly expressed in intestinal tissues This host-dependent MCY conjugation inverts BA function in the hepatobiliary system Whereas microbiota-derived free BAs function as agonists of the farnesoid X receptor (FXR) and negatively regulate BA production BA–MCYs act as potent antagonists of FXR and promote expression of BA biosynthesis genes in vivo Supplementation with stable-isotope-labelled BA–MCY increased BA production in an FXR-dependent manner and BA–MCY supplementation in a mouse model of hypercholesteraemia decreased lipid accumulation in the liver consistent with BA–MCYs acting as intestinal FXR antagonists The levels of BA–MCY were reduced in microbiota-deficient mice and restored by transplantation of human faecal microbiota Dietary intervention with inulin fibre further increased levels of both free BAs and BA–MCY levels indicating that BA–MCY production by the host is regulated by levels of microbiota-derived free BAs We further show that diverse BA–MCYs are also present in human serum our results indicate that BA–MCY conjugation by the host balances host-dependent and microbiota-dependent metabolic pathways that regulate FXR-dependent physiology The human microbiome and child growth — first 1000 days and beyond Role of bile acids and their receptors in gastrointestinal and hepatic pathophysiology Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism Role of the microbiota in immunity and inflammation Regulation of inflammation by microbiota interactions with the host Bile acid metabolites control TH17 and Treg cell differentiation Morais, L. H., Schreiber, H. L. T. & Mazmanian, S. K. The gut microbiota–brain axis in behaviour and brain disorders. Nat. Rev. Microbiol. https://doi.org/10.1038/s41579-020-00460-0 (2020) Gut microbiota in human metabolic health and disease Host–gut microbiota metabolic interactions The influence of the gut microbiome on host metabolism through the regulation of gut hormone release The role of the gut microbiota in bile acid metabolism Endogenous bile acids are ligands for the nuclear receptor FXR/BAR The farnesoid X receptor — a molecular link between bile acid and lipid and glucose metabolism Farnesoid X receptor (FXR): structures and ligands Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver A metabolic pathway for bile acid dehydroxylation by the gut microbiome Bile acid induction specificity of 7α-dehydroxylase activity in an intestinal Eubacterium species Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome Consequences of bile salt biotransformations by intestinal bacteria The acidic pathway of bile acid synthesis: not just an alternative pathway Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-β-muricholic acid Gut microbiota and intestinal FXR mediate the clinical benefits of metformin Identification of a nuclear receptor for bile acids Evolution of the bile salt nuclear receptor FXR in vertebrates Feedback loops shape cellular signals in space and time XCMS Online: a web-based platform to process untargeted metabolomic data Comparative metabolomics with Metaboseek reveals functions of a conserved fat metabolism pathway in C Arifuzzaman, M. et al. Inulin fibre promotes microbiota-derived bile acids and type 2 inflammation. Nature https://doi.org/10.1038/s41586-022-05380-y (2022) Dysregulated microbial fermentation of soluble fiber induces cholestatic liver cancer Regulation of coenzyme A levels by degradation: the ‘ins and outs’ The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism BAAT gene knockout alters early life development and the gut microbiome and reveals unusual bile acids in mice Vanin 1: its physiological function and role in diseases Diverse biological activities of the vascular non-inflammatory molecules — the vanin pantetheinases Vanin1 (VNN1) in chronic diseases: future directions for targeted therapy Yao, L. et al. A selective gut bacterial bile salt hydrolase alters host metabolism. eLife https://doi.org/10.7554/eLife.37182 (2018) Cyp3a11 is dispensable for the formation of murine bile acids Guzior, D. V. et al. Bile salt hydrolase acyltransferase activity expands bile acid diversity. Nature https://doi.org/10.1038/s41586-024-07017-8 (2024) a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors Chemical genomics: functional analysis of orphan nuclear receptors in the regulation of bile acid metabolism Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis Differential regulation of bile acid homeostasis by the farnesoid X receptor in liver and intestine Intestinal farnesoid X receptor signaling modulates metabolic disease and promotion of colon epithelial cell proliferation and neoplasia in fibroblast growth factor 15-deficient mice Impaired negative feedback suppression of bile acid synthesis in mice lacking betaKlotho Hyodeoxycholic acid alleviates non-alcoholic fatty liver disease through modulating the gut–liver axis Microbial bile acid metabolites modulate gut RORγ+ regulatory T cell homeostasis Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells Effects of treatment with deoxycholic acid and chenodeoxycholic acid on the hepatic synthesis of cholesterol and bile acids in healthy subjects Farnesoid X receptor agonists as therapeutic target for cardiometabolic diseases Recent advances in the development of farnesoid X receptor agonists Understanding bile acid signaling in diabetes: from pathophysiology to therapeutic targets Bile acid metabolism and signaling in cholestasis Comparative metabolomics reveals endogenous ligands of DAF-12 Pantetheinase activity of membrane-bound vanin-1: lack of free cysteamine in tissues of vanin-1 deficient mice Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells Ecological memory of prior nutrient exposure in the human gut microbiome Dysregulation of ILC3s unleashes progression and immunotherapy resistance in colon cancer Highly sensitive feature detection for high resolution LC/MS Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking Cytoscape: a software environment for integrated models of biomolecular interaction networks Acylspermidines are conserved mitochondrial sirtuin-dependent metabolites Parkhurst, C. Fiji oil red O macro. Zenodo https://doi.org/10.5281/zenodo.14031611 (2024) Download references All chemical structures were created with ChemDraw This work was supported by the Crohn’s & Colitis Foundation (to M.A.) the Sackler Brain and Spine Institute Research Grant and a Brain and Behavior Research Foundation (NARSAD) Young Investigator Award (all to C.N.P.) Office of Naval Research grant N00014-18-1-2616 (to L.A.D.) the Howard Hughes Medical Institute (to F.C.S.) The Global Grants for Gut Health co-supported by Yakult and Nature Research (to R.A.Q.) the Glenn Greenberg and Linda Vester Foundation Allen Frontiers Group advised program of the Paul G and the US National Institutes of Health (K99AI173660 to M.A. K08MH130773 and NIAID Mucosal Immunology Studies Team Young Investigator Award to C.N.P. Present address: College of Pharmacy and Institute of Pharmaceutical Sciences These authors contributed equally: Tae Hyung Won Department of Chemistry and Chemical Biology Jill Roberts Institute for Research in Inflammatory Bowel Disease Division of Gastroenterology and Hepatology Friedman Center for Nutrition and Inflammation Department of Molecular Genetics and Microbiology Program in Computational Biology and Bioinformatics Allen Discovery Center for Neuroimmune Interactions Kenny Joselin Castro Ochoa & Lily Barash carried out the metabolomics and chemical synthesis and analysed most of the data provided the bacterial strains and helped with the monocolonization experiments helped with the mouse experiments and various other assays assisted with the metabolomic analyses and conducted the VNN1 assays provided the Baat-knockout mouse metabolome samples and data analysis The JRI Live Cell Bank contributed to clinical sample acquisition analysed the data and wrote the manuscript with input from all co-authors has contributed to scientific advisory boards at Pfizer Nemagene and the Kenneth Rainin Foundation is a cofounder of Ascribe Bioscience and Holoclara Inc. and a member of the scientific advisory board of Hexagon Bio The other authors declare no competing interests Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available The listed BA-MCY conjugates in each panel produced MS2 spectra very similar to the shown examples Blue arrows indicate inferred fragmentation MS2 fragments and structure parts highlighted in red represent MCY groups Green fragments are derived from water loss Relative abundances of BA-MCYO conjugates of less abundant BAs in serum of SPF (n = 11) and GF (n = 12) the three donors are represented by triangles Relative abundances of CA-MCY conjugates (b) and βMCA-MCY conjugates (c) as well as the corresponding free BAs in feces of SPF mice (n = 3) Relative abundances of BA-MCYO conjugates of less abundant BAs in feces of SPF (n = 3) and GF (n = 3) Relative abundances of BA-taurine conjugates in serum or feces of SPF (n = 11 for serum and n = 3 for feces) and GF (n = 12 for serum and n = 3 for feces) Relative abundances of free BAs in serum (f) or feces (g) of SPF (n = 11 for serum and n = 3 for feces) and GF (n = 12 for serum and n = 3 for feces) P values were calculated using unpaired two-sided Student’s t-test with Welch’s correction Source Data Relative abundances of CA-MCY (a) and βMCA-MCY conjugates (b) as well as corresponding free BAs in serum of mice fed control (n = 8) or inulin fiber diet (n = 7) Relative abundances of BA-MCYO conjugates as well as corresponding free BAs in serum of mice fed control (n = 8) or inulin fiber diet (n = 7) Relationship between abundances of free BAs and BA-MCY conjugates in feces of SPF mice used as control for the FMT study (n = 25) SPF mice fed a control diet for the inulin fiber diet study (n = 6) and inulin fiber diet fed SPF mice (n = 8) Relationship between abundances of free BAs and BA-MCY conjugates in serum of SPF as control for the FMT study (n = 11) SPF mice fed a control diet for the inulin fiber diet study (n = 8) and inulin fiber diet fed SPF mice (n = 7) Relative abundances of free BAs (f) and corresponding BA-MCY conjugates (g) in serum of human (n = 19) Source Data EICs for molecular ion peaks (black) and deuterium isotope peaks (red) of taurine conjugates of BAs (c) and pantetheine (d) in serum of mouse fed L-cys-d2 Extracted ion chromatograms (EICs) of BA-MCY and BA-MCYO conjugates (a,b,c) and BA-MCYO2 conjugates (d) in liver of Baat−/− mice and comparison with synthetic standards analyzed in ESI +  Relative abundances of BA-MCY conjugates (e) and corresponding free BAs (f) in liver of WT (n = 4) or Baat−/− (n = 5) mice P values were calculated using unpaired two-sided Student’s t-test with Welch’s correction Source Data and cecum of SPF (n = 11) and GF (n = 12 for liver and n = 13 for small intestine and cecum) mice Abundances of UDCA-MCY conjugates and UDCA (c) and 7-KDCA-MCY conjugates and 7-KDCA (f) in liver Source Data Steady-state kinetic analysis of CA-pant and pantetheine hydrolysis catalyzed by recombinant human VNN1 (ΔN490aa truncated) revealed both reactions follow saturation kinetics The steady-state kinetic parameters Km and Vmax are determined by HPLC-HRMS for pantothenic acid formation to be 39.78 ± 20.31 μM and 1.53 ± 0.20 min−1 for CA-pant and 74.07 ± 41.52 μM and 2.13 ± 0.37 min−1 for pantetheine The reaction mixtures contain 0.01 μM VNN1 Number of independent assays using the same batch of enzyme (n = 3) extracts of in vitro reaction of VNN1 hydrolyses CA-pantetheine and comparison with a synthetic standard analyzed in ESI +  EICs of CA-pant in small intestine of Vnn1−/− mice and comparison with a synthetic standard analyzed in ESI +  Relative abundances of CA-pant in small intestine and serum of WT (n = 5) and Vnn1−/− (n = 5) mice Source Data P values were calculated using unpaired two-sided Student’s t-test Source Data Deconjugation of CA-MCY conjugates in fecal suspensions obtained from SPF mice (a) (n = 3) and cultured gut bacteria (b) (n = 3) Relative abundances of CDCA-d5-MCY conjugates and corresponding free BA in feces of GF monocolonized with WT (n = 3) or BSH-deficient B Source Data βMCA-MCY was tested against a cell-based protein-protein interaction assays in both agonist and antagonist modes βMCA-MCY showed strong FXR antagonistic effects to GW4604-mediated activation of FXR βMCA-MCY showed no FXR agonistic effects in the assay Assays were performed in duplicate for each concentration FXR agonistic effect of CDCA as measured in the protein-protein interaction assays Data were normalized to the maximal and minimal response observed in the presence of control compound (GW4064) and vehicle (DMSO) CDCA-MCY showed FXR antagonistic effects to obeticholic acid (25 μM) (c) or CDCA (25 μM) (d) mediated activation of FXR Data were normalized to the maximal and minimal response observed in the presence of control compound (DY268) and vehicle (DMSO) and CDCA (g) in a cell-based assay on human primary hepatocytes TβMCA did not show FXR antagonistic effects in protein-protein interaction assays at the tested concentrations DY268 a synthetic FXR antagonist was used as a positive control Source Data Abundances of endogenously produced BAs in feces of mice administered CDCA-MCY or CDCA-d5-MCY daily for 14 days Shown are individual amounts of CDCA-derived BAs (a) and CA-derived BAs (b) in feces with control (corn oil) (n = 7) and CDCA-MCY fed mice (n = 7 for CDCA-derived pathway and n = 3 for CA-derived pathway) Total endogenously produced BAs in feces of ABX mice administered CDCA-d5-MCY Shown are total amounts of BAs (n = 13 for control and n = 14 for CDCA-MCY fed mice) Abundances of CDCA-derived BAs (d) and CA-derived BAs (e) in liver of mice administered CDCA-MCY or CDCA-d5-MCY daily for 14 days with control (corn oil) (n = 6) and CDCA-MCY fed mice (n = 3 for CDCA-derived pathway and n = 7 for CA-derived pathway) Abundances of CDCA-derived BAs (f) and CA-derived BAs (g) in serum of mice administered CDCA-MCY or CDCA-d5-MCY daily for 14 days Source Data Abundances of total BAs in liver (a) and serum (b) of WT and Nr1h4−/− mice administered CDCA-d5-MCY daily for 14 days with control (corn oil) (n = 4) and CDCA-d5-MCY fed mice (n = 4) Representative photomicrographs of oil red O staining of liver sections of mice treated with the indicated conditions Mice were fed control (n = 4 for vehicle and n = 4 for CDCA-MCY) or high cholesterol diet (HCD) (n = 4 for vehicle and n = 4 for CDCA-MCY) CDCA-MCY was delivered by oral gavage at a rate of 5 mg/kg body weight per day for two weeks Average measured oil red O area of liver sections of mice in c P values were calculated using one-way ANOVA with Tukey’s correction Source Data and descriptions for Supplementary Tables 1 MS features whose production is microbiota-dependent in serum MS features that were derived from supplemented CDCA-d5-MCY in SPF and ABX mice a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law Download citation DOI: https://doi.org/10.1038/s41586-024-08379-9 Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology Metrics details Bile acids are trans-genomic molecules arising from the concerted metabolism of the human host and the intestinal microbiota and are important for digestion energy homeostasis and metabolic regulation While diurnal variation has been demonstrated in the enterohepatic circulation and the gut microbiota and the influence of the host circadian system has not been determined we demonstrate robust daily rhythms in the circulating bile acid pool in healthy male participants We identify temporal relationships between bile acids and plasma lipids and show that these relationships are lost following sleep deprivation We also highlight that bile acid rhythmicity is predominantly lost when environmental timing cues are held constant Here we show that the environment is a stronger determinant of these temporal dynamics than the intrinsic circadian system of the host This has significance for the intimate relationship between circadian timing and metabolism little is known about the drivers of these modulations or their cross-talk and impact on the host bile acids may represent an important mechanism in the holobiont for the reciprocal tuning of host and microbial daily fluctuations Dysregulation of this pan-kingdom communication may using both entrained and constant routine protocols we demonstrate a robust daily rhythm in the overall circulating bile acid pool of healthy male participants with phase variation in the host-derived conjugated and microbiota-derived unconjugated bile acids We highlight relationships between daily time courses in bile acids and plasma lipids We show that sleep deprivation exerts a modest impact on the timings of these rhythms but results in the loss of synchronicity between the circulating bile acids and lipidome the rhythmicity of the bile acids is lost in constant routine conditions when the environmental timing cues are removed or kept constant These findings establish that the environment exerts a stronger influence on the circulating bile acids key trans-genomic signaling molecules that impact both the host and microbiota than the intrinsic circadian timing system of the host A Study design for the entrained human study (24 h sleep/wake) followed by 24 h of wakefulness B Daily patterns in the abundance of circulating plasma bile acids Cosine cycles were observed in Z scored mean time courses of tauro- and unconjugated forms of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) deoxycholic acid (DCA) and lithocholic acid (LCA) following a cosine pattern across the 36-hour period during the sleep/wake phase Values are the mean Z scores (shaded area indicates standard deviation) from all DLMO-corrected individual time courses C Cross-correlation analysis indicating phase differences between all bile acid species Values and red background intensity indicate the magnitude of phase difference in decimal hours Substantial phase differences can be observed between conjugated and unconjugated bile acids Source data are provided as a Source Data file These are calculated as the mean minimum and mean maximum concentration of each bile acid across the 24 h period from all participants TCDCA and GCDCA had the highest maximum circulating concentrations with 9.06 and 2.38 μM followed by the unconjugated CA (1.35 μM) and CDCA (1.31 μM) The rhythmic bile acids included primary and secondary bile acids as well as their conjugated and unconjugated forms The circulating conjugated bile acids (GCA peaked early in the evening (acrophase range: −1.42 to −5.93 h from DLMO; equivalent to ~16:00–20:00 h:min) and the unconjugated bile acids peaked several hours later in the early morning (acrophase range: 6.16–8.36 h from DLMO; ~04:00−06:00 h:min) Unconjugated LCA peaked in the plasma at a comparable time to its conjugated forms (17.29 h from DLMO; ~15:00) Cholecystokinin is a gut hormone released after a meal that stimulates gall bladder contraction to secrete bile into the intestine with conjugated bile acids increasing in the circulation prior to lunch and peaking after dinner and the balanced macronutrient and caloric density across the three daily meals these patterns are unlikely to be driven exclusively by food patterns Cross-correlation analysis identified strong similarities between the time courses of the bile acid profiles (mean ± SEM, R2 = 0.82 ± 0.01 between 16 × 16 comparisons), when time-aligned on maximal similarity (Fig. 1C) The strongest correlations were observed within the conjugated bile acids and within the unconjugated bile acids (R2 = 0.90 ± 0.01 and 0.89 ± 0.02 Lower correlations were observed between conjugated and unconjugated bile acids (R2 = 0.75 ± 0.01) minimal phase differences in time course profiles were seen within the conjugated bile acids or within the unconjugated bile acids (51 ± 10 mins and 31 ± 9 mins the average circular phase difference between conjugated and unconjugated bile acids was substantial (8:32 ± 00:24 h:min) This likely reflects the different sites of absorption with most gallbladder-secreted bile acids (i.e conjugated bile acids) being absorbed in the small intestine while the unconjugated forms are produced from bacterial deconjugation and absorbed in the colon and symbol size indicates correlation strength The distance of the edge indicates the phase difference between peak bile acid concentration and peak metabolite concentration A Phase differences in individual bile acids under entrained and sleep deprivation conditions B Phase differences in the cross-correlations between bile acid pairs under entrained and sleep deprivation conditions All pairs shown including rhythmic and non-rhythmic bile acids Red dashed line indicates no difference in phase C Phase differences in the relationships between bile acids and metabolites under both conditions All pairs shown including rhythmic and non-rhythmic bile acids with metabolites and lipids Blue lines and symbol color indicates no difference and red lines and symbols indicate a 12-hour shift in the relationship between conditions D Histogram indicating the frequency of bile acid-metabolite changes by circular hours between the two study conditions A peak can be observed in 0–2 hours phase shift between the two conditions (i.e. Attributing the changes to specific lipids (thus those species that exhibit a large phase shift in relation to bile acids) exposes a top-quartile contribution of 42 molecules The four most attributable bile acids were the unconjugated bile acids CA CDCA Primary and secondary bile acids in their conjugated (glycine- and taurine-) and unconjugated forms are displayed for all individuals from the entrained wake/sleep entrained with 24-hour wakefulness (sleep deprivation) Values are the mean Z scores (shaded area indicates standard error of the mean) from all DLMO-corrected individual time courses using a combination of highly controlled entrained and constant routine protocols we demonstrate for the first time in humans that bile acids host-microbial co-metabolites with the potential to influence the genome and metagenome follow diurnal rhythms that are primarily driven by the environment The rhythms in these pan-kingdom metabolites were significantly associated with daily patterns in plasma lipids emphasizing the influence of bile acids on the digestion and processing of lipids The constant routine protocol provides a gold-standard mechanism in humans to assess endogenous circadian rhythms in variables (i.e those that persist when external cues are removed or kept constant) Reduced rhythmicity of bile acids in the plasma during the constant routine protocol confirmed that circulating bile acids are while data in mice suggest that the synthesis and transport of bile acids may be regulated by the host circadian system the observations from this study indicate that the circulating levels which are partially influenced by gut microbial metabolism this results in consistent cycles in the release and circulation of bile acids throughout the day but these dynamics are lost when environmental cues are removed The implications of misalignment between feeding times and BSH activity on lipid processing and weight gain warrant further investigation It remains unclear how the potency of this activation varies across different bile acid species or how this activation changes throughout the day but these findings further support the role of these molecules in tuning the metabolic readiness of the holobiont for receiving and processing energy intake A limitation of the current study was an inability to sample the GI tract at the same temporal resolution as the blood This prevented the daily variation of the gut microbiota and BSH from being studied Given the known antimicrobial properties of the bile acid pool it would be interesting to investigate if the observed bile acid dynamics mapped to daily changes in microbial composition and whether these were lost under constant routine conditions such as smart capsules that can sample throughout the gut This study characterizes the daily rhythms of bile acids and pan-kingdom metabolites that serve as a major line of communication linking the intestinal microbiota with the human host genome These trans-genomic molecules provide a mechanism to prime the host biochemical system to process anticipated nutrients and energy intake and are strongly influenced by environmental cues The studies were approved by the University of Surrey Ethics Committee and were conducted at the Surrey Clinical Research Centre according to the Declaration of Helsinki and with regard to good clinical practice Organic solvents (HPLC grade) were used for the sulfation and precipitation and sodium sulfate was obtained from Sigma Aldrich (Dorset All mobile phases were prepared with LC-MS grade solvents and ammonium formate from Sigma Aldrich (Dorset BA standards and deuterated internal standards were obtained from Steraloids (Newport Written and oral consent was obtained from the participants prior to any procedures being performed Participants were allowed to withdraw from the study at any time participants had to meet defined inclusion/exclusion criteria to be deemed eligible for the study (see Extended Methodology in Supplementary Information) All participant information was coded and held in strictest confidence according to the Data Protection Act (United Kingdom For 7 days prior to the in-laboratory session for both studies individuals maintained regular sleep/wake schedules aligned with their habitual sleep patterns participants maintained a 2300–0700 h schedule individuals selected an 8-h sleep period going to bed between 2200 and 0100 h and waking up between 0600 and 0900 h and activity/light monitors (Actiwatch; CamNtech During the final 72 h of this baseline period participants were requested to refrain from alcohol and caffeine consumption This baseline period ensured that participants were not sleep-deprived and that their circadian phase was stabilized prior to entering the clinical study The constant routine protocol (Supplementary Fig. 2) included 15 healthy male participants the participants were admitted into the laboratory The in-laboratory session included an adaptation night with habitual sleep times (N1) followed by continual wakefulness until 2300 h on day 3 Electroencephalography monitoring occurred from 1200 h on day 2 until 2300 h on day 3 to ensure the participants remained awake throughout the protocol The participants were subjected to strictly controlled constant routine conditions including dim lighting (<5 lux in the direction of gaze) and hourly intake of isocaloric snacks with 100 ml of water Participants were unaware of clock time throughout the study period Hourly blood samples were collected via an intravenous catheter Bile acids were measured in 2-hourly blood samples collected from 1500 h on day 2 until 2300 h on day 3 The calculated DLMO was used to phase-adjust the bile acid and metabolite data Plasma samples were combined with ice-cold methanol (1:3 v:v) and centrifuged at 14,000 × g for 15 min at 4 °C The supernatant was then withdrawn into a glass vial and stored at −80 °C for LC-MS analysis Bile acid quantification was performed on the plasma samples from the entrained study deuterated internal standards were also added to the sample prior to the addition of methanol Chromatographic separation of bile acids was performed using Waters ACQUITY ultra-performance liquid chromatography (UPLC) (Waters Ltd Eluent A consisted of ultra-pure H2O:acetonitrile 100:0.1 (v:v) with ammonium acetate (1 mM) and acetic acid to adjust the pH to 4.15 Eluent B consisted of acetonitrile:2-propanol 1:1 (v:v) mixture a flow rate of 0.6 μL/min was used with 90% A which was then decreased to 65% A at 9.25 mins until 11.5 min The flow rate was then increased to 0.65 μL/min until 11.8 min and a final washing step was applied until 15 min For the quantification of bile acids in plasma from the entrained protocol the UPLC system was hyphenated with a Waters Xevo TQ-S mass spectrometer (MS) (Waters For the profiling approach used to assess bile acids in plasma from the constant routine study the UPLC system was coupled to a Xevo G2-S Q-ToF MS Both MS were equipped with an electrospray ionization source operating in negative ion mode (ESI-) MS settings were as follows: capillary voltage at 1.5 kV Plasma metabolic profiles were measured on a Waters Acquity UPLC system coupled to a Xevo TQ-S MS using the Biocrates AbsoluteIDQ p180 targeted metabolomics kit (Biocrates Life Sciences Plasma samples (10 µl) were prepared according to the manufacturer’s instructions adding several stable isotope-labeled standards to the samples prior to the derivatization and extraction steps Using either UPLC-MS/MS (liquid chromatography/mass spectrometry) or FIA-MS/MS (flow injection analysis/MS) up to 183 metabolites from five different compound classes (namely acylcarnitines Sample order was randomized and 3 levels of quality controls (QC) The levels of metabolites present in each QC were compared to the expected values and the percent coefficient of variation (CV%) was calculated Data were normalized between batches using the results of quality control level 2 (QC2) repeats across the plate (n = 4) and between plates using Biocrates METIDQ software (QC2 correction) two cosinor models with a level (horizontal) and sloping mesor were fitted using linear mixed-effect models accounting for individual variability as a random effect Each model was compared to a reduced linear model using a log-likelihood ratio test and only significant models in which the amplitude confidence interval did not include 0 (zero) were deemed periodic In instances where both level and sloping mesor cosinor models were periodic a further log-likelihood ratio test was used to determine whether the reduced model with a level mesor or the model with a sloping mesor should be used and mesor with variance measures for the population-level fit based on population fit adjusted for each participant’s random effect To identify time-delayed relationships across the bile acids and between bile acids and metabolites cross-correlation analysis was used we considered only rhythmic bile acids and their relationship to all metabolites/lipids detected As many statistical tests were performed between the rhythmic bile acids (n = 16) and the metabolites (n = 170) only those relationships with a significant R2 value above the 95% confidence interval of the mean (R2 cutoff = 0.832) were accepted Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article All data needed to reproduce the results presented here can be found in the manuscript, figures or supplementary materials (Source Data file). Source data are provided with this paper No code is available related to these analyses Flexible clock systems: adjusting the temporal programme The intestinal clock drives the microbiome to maintain gastrointestinal homeostasis Transkingdom control of microbiota diurnal oscillations promotes metabolic homeostasis Microbiota diurnal rhythmicity programs host transcriptome oscillations Hyocholic acid species improve glucose homeostasis through a distinct TGR5 and FXR signaling mechanism TGR5-mediated bile acid sensing controls glucose homeostasis Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation Bile acid reduces the secretion of very low density lipoprotein by repressing microsomal triglyceride transfer protein gene expression mediated by hepatocyte nuclear factor-4 Individual bile acids in portal venous and systemic blood serum of fasting man Diurnal changes in serum unconjugated bile acids in normal man Bile acid synthesis in humans has a rapid diurnal variation that is asynchronous with cholesterol synthesis Gut microbiome communication with bone marrow regulates susceptibility to amebiasis Systemic gut microbial modulation of bile acid metabolism in host tissue compartments Effect of acute total sleep deprivation on plasma melatonin cortisol and metabolite rhythms in females Effect of sleep deprivation on the human metabolome Meta-analysis on night shift work and risk of metabolic syndrome Age-related changes in acute and phase-advancing responses to monochromatic light Bile acid profiling and quantification in biofluids using ultra-performance liquid chromatography tandem mass spectrometry The impact of sleep disturbances on adipocyte function and lipid metabolism Actigraphic sleep duration and fragmentation are related to obesity in the elderly: the Rotterdam Study Complex interaction between circadian rhythm and diet on bile acid homeostasis in male rats Retinoic acid-related orphan receptor α regulates diurnal rhythm and fasting induction of sterol 12α-hydroxylase in bile acid synthesis Regulation of bile acid synthesis by the nuclear receptor Rev-Erb alpha Multiple mechanisms regulate circadian expression of the gene for cholesterol 7alpha-hydroxylase (Cyp7a) a key enzyme in hepatic bile acid biosynthesis Diurnal variations of mouse plasma and hepatic bile acid concentrations as well as expression of biosynthetic enzymes and transporters Bile acid metabolism and circadian rhythms Profiling rhythmicity of bile salt hydrolase activity in the gut lumen with a rapid fluorescence assay Regulation of host weight gain and lipid metabolism by bacterial bile acid modification in the gut Relationship of dietary antimicrobial drug administration with broiler performance decreased population levels of Lactobacillus salivarius and reduced bile salt deconjugation in the ileum of broiler chickens Guts and gall: bile acids in regulation of intestinal epithelial function in health and disease Farnesoid X receptor agonists: a promising therapeutic strategy for gastrointestinal diseases Bile acids induce the expression of the human peroxisome proliferator-activated receptor alpha gene via activation of the farnesoid X receptor The bile acid chenodeoxycholic acid increases human brown adipose tissue activity Associations between sleep loss and increased risk of obesity and diabetes Sleep disturbance induces increased cholesterol level by NR1D1 mediated CYP7A1 inhibition promote dyslipidaemia: a systematic review and meta-analysis Sleep duration and obesity among adults: a meta-analysis of prospective studies Shift work and poor mental health: a meta-analysis of longitudinal studies Gastrointestinal disorders among shift workers Prevalence of shift work disorder: a systematic review and meta-analysis Shift work and the risk of cardiovascular disease A systematic review and meta-analysis including dose-response relationship Associations between dietary patterns and bile acids—results from a cross-sectional study in vegans and omnivores Diurnal rhythms in blood cell populations and the effect of acute sleep deprivation in healthy young men Effect of sleep deprivation on rhythms of clock gene expression and melatonin in humans Comparing linear and nonlinear mixed model approaches to cosinor analysis cosinoRmixedeffects: an R package for mixed-effects cosinor models Download references The authors thank Professor Anne Skeldon (Department of Mathematics University of Surrey) for writing the cosinor analysis scripts for MatLab JRS is supported by the NIHR Southampton Biomedical Research Centre Biotechnology and Biological Sciences Research Council (BB/W00139X/1 and Medical Research Council (MR/W003597/1) Davies for data processing; and Daniel Barrett and the Surrey Clinical Research Centre medical and research teams for their help with the clinical study (supported by the Biotechnology and Biological Sciences Research Council grant (BB/I019405/1) to D.J.S. These authors contributed equally: Adesola T Bioaster Microbiology Technology Institute Nature Communications thanks Frédéric Gachon and the other reviewer(s) for their contribution to the peer review of this work Reprints and permissions Download citation DOI: https://doi.org/10.1038/s41467-024-53673-9 Metrics details an aqueous solution critical for fat absorption other components of bile are less well characterized Here we used global metabolomic analysis on bile from specific-pathogen-free Citrobacter rodentium-infected or Listeria monocytogenes-infected mice and identified a metabolome of 812 metabolites that were altered by both microbiota and enteric infection Hepatic transcriptomics identified enteric-infection-triggered pathways that probably underlie bile remodelling Enteric infection increased levels of four dicarboxylates in bile Analysis of Acod1−/− mice indicated that increased itaconate also increased tuft cell abundance altered microbiota composition and function as detected by metagenomic analysis leading to reduced Vibrio cholerae colonization Our data suggest that enteric-infection-associated signals are relayed between the intestine and liver and induce transcriptional programmes that shape the bile metabolome modifying the immunomodulatory and host defence functions of bile Unsupervised clustering (k-medoids) of the bile metabolites identified in four groups of mice (GF (n = 9) Lm (n = 7) and Cr (n = 6)) based on their patterns of relative abundance Each row represents a metabolite and each column represents one sample To obtain a minimal volume of 60 µl of bile for global metabolomic profiling samples were generated by pooling bile from 3 to 5 SPF mice and 2 or 3 GF mice; a total of 146 mice were used Violin plots of the distribution of the Z scores of the metabolites in each cluster Functional categories of bile metabolites in cluster 7 identified by unsupervised clustering Enriched pathways of metabolites with differential abundance between SPF and GF mice (f) SPF and Cr mice (g) and SPF and Lm mice (h) Pathways with the top 16 enrichment scores are listed for each comparison Differential abundance of bile metabolites in Cr (i) or Lm (j) animals compared with that in uninfected mice black and orange dots represent metabolites with reduced P values were generated using the two-tailed Wilcoxon rank test Structural formulas of infection-stimulated bile dicarboxylates Source data functions and regulation of other bile constituents have not been the subject of much experimental scrutiny changes in bile composition in response to the microbiota or enteric infection are largely unknown As bile is a key mediator of interorgan communication between the liver and gut we hypothesized that enteric infection may alter the composition and function of bile to facilitate intestinal defence Here we used global metabolomic analyses to characterize mouse bile and to investigate how its composition is altered by microbiota and enteric infection Our findings reveal that bile composition is highly complex responsive to the microbiota and infection and functions in an interorgan innate defence circuit that links the liver and intestine Differential abundance of bile lipids in GF (a) Lm (b) or Cr mice (c) compared with SPF mice Each dot represents one lipid; the categories of lipids are found at the top of the graph in a The y-axis represents the log2 fold change of the metabolite abundances in the experimental conditions (GF Lm and Cr in the numerator) relative to the SPF condition (denominator) Light grey dots indicate that statistically significant difference was not reached; colored dots indicate that statistically significant difference was reached (P < 0.05) Lipid sub-pathways with high enrichment scores by pathway enrichment analysis are indicated with dashed circles Source data reinforce the idea that the interplay between the microbiota and host has a profound impact on bile composition even in the absence of pathogen invasion of the gall bladder enteric infection leads to remodelling of the bile metabolome in a pathogen-specific manner rodentium replication in the intestine can impact the expression of this host lipid-synthesizing enzyme suggesting that these compounds were not pathogen derived these data reveal that the bile metabolome is markedly influenced by the microbiota and altered in shared as well as distinct ways by enteric infections we investigate potential pathways that generate these biliary compounds and phenotypes linked to itaconate consistent with the idea that enteric infection modulates hepatic transcriptional programmes even in the absence of detectable pathogen dissemination to the liver GCDH is a FAD-dependent enzyme that requires the ETF complex to transfer electrons to the ETC; Q electron transfer flavoprotein–ubiquinone oxidoreductase Etfa (i) and Sugct (j) in mouse liver in Lm versus SPF Cr liver positive versus SPF and Cr liver negative versus SPF conditions The relative expression levels of Acod1 in liver in Lm versus SPF Representative image of RNA-FISH analysis of Acod1 transcripts (red) in the liver; CK19 (green) Each experiment was repeated independently twice with similar results three animals per condition were used for analysis Adjusted P values were generated using DESeq2 (two-tailed Wald test P value adjusted for multiple testing using the procedure of Benjamini and Hochberg) All genes were statistically differentially expressed in at least two conditions (adjusted P < 0.05) Source data these data support the hypothesis that enteric infections shape the bile metabolome by modulating the expression of hepatic metabolic enzymes we focused our analyses on unveiling the functions of this dicarboxylate in intestinal homeostasis and defence Tuft cells were identified with anti-Dclk1 antibody (pink) Quantification of tuft cell numbers in the distal ileum (Data from 4 litters of mice in 3 independent experiments n = 6 for WT and n = 8 for Acod1−/− mice; lines connect littermates) Experimental scheme for studying the effects of Acod1 on faecal microbiota composition Littermates of mice from Acod1+/− heterozygote parents were separately housed according to genotype at 3 weeks of age and challenged with C Faecal samples were collected before and after clearance of C Relative abundance of faecal microbiota at the order level after clearance of C rodentium infection (n = 9 for WT and n = 11 for Acod1−/− mice) Abundance of Turicibacter sp002311155 in WT (n = 6) and Acod1−/− mice (n = 6) after C rodentium infection identified by shotgun metagenomics (n = 6 for WT and n = 6 for Acod1−/− mice) Relative abundance of chorismate mutase in Acod1−/− mice compared with WT mice after C rodentium infection (n = 6 for WT and n = 6 for Acod1−/− mice) Differentially abundant pathways in Acod1−/− mice (n = 6) compared with those of WT mice (n = 6) after C The four most enriched EC families in Acod1−/− mice compared with WT mice after C rodentium infection (n = 6 for WT and n = 6 for Acod1−/− mice): EC 1.3.99.22 (i) P values were generated using a two-tailed Mann–Whitney test (a Source data suggesting that itaconate influences gut microbiota composition at steady state suggesting that itaconate helps to maintain its abundance during and/or following perturbations such as C suggesting that itaconate inhibits specific functional pathways these data support the idea that itaconate modulates the composition and functional output of the gut microbiome following perturbations such as enteric infection cholerae in the proximal and distal small intestine of infected WT and Acod1−/− mice (data from four independent experiments Scheme for acetate and propionate utilization in V cholerae in M9 minimal medium supplemented with 10 mM acetate (c) propionate (d) or glucose (e) as the sole carbon source in the presence or absence of 10 mM of itaconate (n = 8 for acetate n = 6 for propionate and n = 4 for glucose) cholerae pHL_AceA strain in M9 minimal medium supplemented with 10 mM acetate and 0.5 mM itaconate data represent biological replicates from two independent experiments) cholerae ΔprpBΔaceA versus the WT strain in proximal (g) and distal (h) small intestine of infant mice CIs were calculated by dividing the ratio of white to blue (ΔaceAΔprpB/WT n = 11) colonies in the small intestine by the ratio of white to blue colonies in the inoculum and compared with the CI of Haiti WT lacZ− and Haiti WT lacZ+ strain (n = 6) P values were generated using the two-tailed Mann–Whitney test (a Source data supporting the idea that the pathogen relies on fatty acids for robust growth in the host intestine These data are consistent with the hypothesis that itaconate mediates host defence against V cholerae by impairing the pathogen’s capacity to consume fatty acids in the intestine these observations suggest that itaconate controls the composition of the intestinal epithelium and microbiota as well as intestinal defence suggesting that the hepatic sensing mechanisms are at least in part driven by host-derived factors along with microbial-associated products Identifying the signals and hepatic sensing and regulatory elements that govern the type magnitude and duration of hepatic-driven changes in bile makeup are key challenges for future studies our findings uncovered the complex and dynamic nature of bile composition and expands knowledge of the homeostatic functions of the liver and bile All animal experiments were conducted following the protocol (2016N000416) reviewed and approved by the Brigham and Women’s Hospital Institutional Animal Care and Use Committee SPF C57Bl/6J mice were purchased from the Jackson Laboratory (stock number 000664) The mice were kept in a Harvard Medical School animal facility for at least 72 h before the experiments GF C57Bl/6J mice were purchased from the Massachusetts Host-Microbiome Center Acod1−/− mice were purchased from the Jackson Laboratory (C57BL/6NJ-Acod1em1(IMPC)J/J stock number 029340) and bred in the Harvard Medical School animal facility C57Bl/6 mice with 3 day postnatal infants (P3) were purchased from the Charles River Laboratories (stock number 027) and kept in the Harvard Medical School animal facility until postnatal day 5 (P5) All mice were kept under 12 h light–dark cycles and temperature (68–75 °F) and humidity (50%) controlled lightly sedated with isoflurane inhalation and oro-gastrically inoculated with 3 × 109 CFU of L monocytogenes 10403S InlAm strain in a 300 µl mixture of 200 mM CaCO3 in PBS or with 1 × 109 CFU C rodentium ICC168 strain in 200 µl PBS using 18 G flexible feeding needles (DT 9928 Uninfected SPF mice were inoculated with 200 µl of PBS monocytogenes infection and 10 days after C Bile samples were collected from animals at approximately 3 p.m.; at this time of the day the bile volume of uninfected mice showed some variation but was not empty Bile samples were collected from the gall bladder using insulin syringes (BEC-309311 filtered with 0.22 µm centrifuge tube filters (8160 snap-frozen in liquid nitrogen and stored in −80 °C until analysis monocytogenes-infected mice and the liver and colon of C rodentium-infected mice were used for bacterial burden enumeration the compounds were identified by comparison with library entries of purified standards Metabolon uses a reference library based on authenticated standards which contains key information including mass-to-charge ratio (m/z) retention time index and chromatographic data (including MS/MS spectral data) on all molecules present in the library Metabolon’s Laboratory Information Management System maintains more than 3,300 commercially available purified standard compounds and these compounds were used for analysis on all platforms for determination of their analytical characteristics Biochemical identifications are based on three criteria: retention index within a narrow retention time index window of the proposed identification accurate mass match to the library ±10 ppm and the MS/MS forward and reverse scores between the experimental spectrum and authentic standards (ions present in the library spectrum) Biochemicals were distinguished and differentiated by the collective information of all three data points Area under the curve was used to quantify the peaks The peaks were quantified using area under the curve and reported as ‘original scale’ data and significance was determined using a Wilcoxon rank test ‘original scale’ data were used for calculating Z scores The clustering of the metabolites was performed using the partitioning around medoids (pam) function in the R package cluster (v2.1.4) with a parameter ‘k = 7’ We used the Metabolon in-house pathway database to identify enriched pathways representing differentially abundant metabolites The enrichment score is calculated using the following formula: where m = number of metabolites in the pathway, k = number of significant metabolites in the pathway, n = total number of significant metabolites and N = total number of metabolites. We arbitrarily set k > 4 to eliminate small pathways, which can confound these analyses. The enrichment scores for all the pathways are shown in Supplementary Table 3 SRM data for each metabolite were acquired in either negative or positive ionization modes and the Q1–Q3 transition for each metabolite was selected (itaconate (Q1 129.1 The Q3 peak areas in the SRM data were integrated using MultiQuant 3.0 for quantification across the sample set Serial diluted chemical standards of itaconate and 2-methylsuccinate were used to generate standard curves to infer the absolute concentration of these two compounds 1 ml of bacteria was collected and centrifuged at 8,000 g for 2 min; the supernatants were transferred to new tubes and centrifuged at 12,000 g for 2 min The supernatants were used for LC–MS/MS analysis rodentium following the protocols described above RNAs were extracted from the liver samples using Trizol (Thermo Fisher) and RNA-sequencing (RNA-seq) libraries were prepared using the KAPA RNA Hyperprep Kit (Roche) The libraries were sequenced on an Illumina NextSeq 550 instrument with paired-end runs of 2 × 75 bp and FASTQ files were generated by Illumina’s FASTQ generation pipeline (V1.0.0) Low-quality bases and the adaptors were trimmed using Trim Galore (v0.6.6) with a paired option The reads were mapped to the mouse reference genome (mm10) using STAR v2.7.3a with default parameters and the gene-level raw read count matrix was obtained using the featureCounts function in the subread (v2.0.0) Differentially expressed genes were identified using the R-package DESeq2 (v1.36.0) with an adjusted P < 0.05 and an absolute fold change of 1 and the clustering was performed using the pam function in the R-package cluster (v2.1.4) with a parameter ‘k = 7’ Pathway analysis was performed by the R-package clusterProfiler (v4.4.4) with gene sets from msigdbr R-package (v7.5.1) or KEGG metabolism pathways Metabolic network analysis (integrated analysis of genes and metabolites) was performed using Shiny GATOM (https://artyomovlab.wustl.edu/shiny/gatom/) with the following parameters: KEGG network scoring parameter for genes; P value threshold scoring parameter for metabolites with the thresholds set to −4 The fold changes in input data for genes were shrunk using the function lfcShrink (type is ‘apeglm’) in the R-package DESeq2 The fold changes for metabolites were ‘Fold of Change’ of L rodentium-infected mice compared with SPF mice reported by Metabolon Littermates of female mice (10–14 weeks old) from Acod1+/− parents were either infected with C rodentium following the protocol described above or maintained uninfected in a separate cage Bile samples were collected from uninfected animals (basal level) and infected mice at 10 dpi and prepared following the protocol described above (quantification of dicarboxylates) The abundance of succinate in samples was quantified using a polar metabolite detection pipeline on a 6500 QTRAP LC–MS/MS system at the mass spectrometry core facility of Beth Israel Deaconess Medical Center Sections of the distal ileum 1.5 cm long were dissected fixed with 4% paraformaldehyde for 2 h at room temperature and transferred to 30% sucrose in PBS at 4 °C overnight The samples were embedded in 1:2.5 of 30% sucrose and optimal cutting temperature compound solution and cut into 10 µm sections The slides were washed three times with PBS and blocked with 5% normal goat serum and 0.3% Triton X-100 for 1 h at room temperature The slides were incubated with rabbit anti-Doublecortin-like and CAM kinase-like 1 (DCAMK) polyclonal antibody (1:500 dilution Abcam) at 4 °C overnight and Alexa-594 goat anti-rabbit IgG secondary antibody (1:1,000 dilution Thermo Fisher) for 1 h at room temperature Images were captured using a Leica Stellaris Confocal Microscope at the Microscopy Resources on the North Quad (MicRoN) core of Harvard Medical School the number of Dclk1-positive cells was enumerated in a 2.5 mm-long representative tissue and presented as number of tuft cells per mm tissue cholerae in defined nutrient conditions was analysed using M9 minimal medium containing 1 mM MgSO4 0.3 mM CaCl2 and one carbon source as indicated: acetate (10 mM) or glucose (0.5%) When propionate (10 mM) was used as a carbon source To test the growth inhibitory effect of itaconate culture mediums were supplemented with a final concentration of 10 mM itaconate and the pH was adjusted to 7 using NaOH cholerae were grown in LB broth to OD60 ~1.2; 1 ml of V cholerae culture was pelleted by centrifugation at 8,000 g washed twice with M9 minimal medium and resuspended in 1 ml of M9 minimal medium and 5 µl was inoculated into 1 ml medium of the indicated conditions All cultures were grown at 37 °C with shaking OD600 was measured at 24 h (for growth in acetate or glucose) or 64 h (for growth in propionate) after inoculation cholerae pHL_AceA strain were prepared as described above and inoculated into M9 minimal medium supplemented with 10 mM acetate and 0.5 mM itaconate Pups were euthanized at 20 h postinfection The proximal and distal regions of the small intestine were dissected homogenized and plated on LB streptomycin (Sm) plates for CFU enumeration The donor and recipient strain were mixed at a 1:1 ratio and incubated at 37 °C for 4 h conjugation reactions were streaked on LB plates with Sm (200 µg ml−1) and carbenicillin (Cb; 50 µg ml−1) Double crossovers were isolated by restreaking single cross-over colonies on LB plates with 10% sucrose and incubating at room temperature for 2 days Duplicate patching was used to examine the Cb resistance of the colonies and the correct double cross-over was identified by screening Sm-resistant and Cb-sensitive colonies using colony PCR cholerae Haiti WT lacZ+ and ΔaceAΔprpB lacZ− strain were washed once with PBS and diluted 1:1,000 in PBS Pups at postnatal day 5 (P5) were orally inoculated with a 1:1 mixture (total 105 CFUs) of WT and the ΔaceAΔprpB V Animals were euthanized at 20 h postinfection Proximal and distal sections of the small intestine were dissected homogenized and plated on LB + Sm/X-gal for blue–white colony counting Competition indices (CIs) were calculated by dividing the ratio of white to blue (ΔaceAΔprpB /WT) colonies in the small intestine by the ratio of white to blue colonies in the inoculum and compared with the CIs of the Haiti WT lacZ− and Haiti WT lacZ+ strains rodentium-infected or PBS-treated animals were embedded in 1:2.5 of 30% sucrose and optimal cutting temperature compound solution cut into 10 µm sections and stored at −80 °C Staining followed the fresh frozen tissue protocol provided by ACDbio the slides were fixed with 4% paraformaldehyde that was pre-cooled to 4 °C and incubated at 4 °C for 10 min and washed three times with PBS The tissue was digested with protease IV and hybridized with an Acod1 probe (stock 450241 Slides were incubated with rabbit anti-CK19 monclonal antibody (1:500 dilution Abcam) at 4 °C overnight and Alexa-488 goat anti-rabbit IgG secondary antibody (1:1,000 dilution Images were captured using a Leica Stellaris Confocal Microscope at the MicRoN core of Harvard Medical School monocytogenes-infected and uninfected animals were homogenized in RIPA buffer (Thermo Fisher 89900) with Complete Mini Protease Inhibitor (Roche 11836170001) and centrifuged at 12,000 g for 5 min; supernatants were used for western blot analysis Proteins were separated using NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher NP0323BOX) and then transferred to nitrocellulose membranes (Thermo Fisher Western blot analysis was carried out using rabbit anti-Acod1 polyclonal antibody (1:1,000 dilution rabbit anti-HK2 polyclonal antibody (1:5,000 dilution rabbit anti-GCDH polyclonal antibody (1:1,000 dilution Sigma) and peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000 dilution Chemiluminescent signals were developed with West Pico PLUS Chemiluminescent Substrate (34580 Thermo Fisher) and imaged in the ChemiDoc Touch system (Bio-Rad) Details of the statistical methods used to analyse each experiment are presented in the figure legends GSEA function and enrichKEGG function of clusterProfiler were used in R; Mann–Whitney tests were performed in Prism; the Linear Discriminant Analysis Effect Size (LEfSe) was performed in the Conda environment P values generated by Mann–Whitney tests were not corrected for multiple testing leading to the use of a nonparametric analysis method animals were randomly assigned into groups Animal genotype blinding was used for the V cholerae intestinal colonization assay (Acod1−/− and WT animals) Histology analyses of slides stained with haematoxylin and eosin were performed by a pathologist blinded to the experimental scheme Sample collection and processing for other experiments were not performed in a blinded fashion No animals were excluded from the analysis Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The code for this paper is available via GitHub at https://github.com/yh766/NMICROBIOL_Bile_2024 Genome–microbiome interplay provides insight into the determinants of the human blood metabolome Global chemical effects of the microbiome include new bile–acid conjugations An online atlas of human plasma metabolite signatures of gut microbiome composition Acute-phase reactants in infections and inflammatory diseases: acute-phase reactants in infections and inflammatory diseases Humoral innate immunity and acute-phase proteins Boyer, J. L. Bile formation and secretion. Compr. Physiol. https://doi.org/10.1002/cphy.c120027 (2013) in Physiology of Membrane Disorders 2nd edn (eds Andreoli Intestinal bile acids induce a morphotype switch in vancomycin-resistant Enterococcus that facilitates intestinal colonization Infection trains the host for microbiota-enhanced resistance to pathogens Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites Moreau, F. et al. 1178-P: portal vein metabolites as intermediate regulators of the gut microbiome in insulin resistance. Diabetes https://doi.org/10.2337/db21-1178-p (2021) Influence of intestinal microorganisms on the metabolism of bile acids in mice Sulfated bile acids in germ‐free and conventional mice Characterization of bile acid homeostasis in germ‐free mice Gut microbiota promotes cholesterol gallstone formation by modulating bile acid composition and biliary cholesterol secretion Deciphering the landscape of host barriers to Listeria monocytogenes infection A multiorgan trafficking circuit provides purifying selection of Listeria monocytogenes virulence genes Dual roles of plcA in Listeria monocytogenes pathogenesis Intestinal epithelial cells and the microbiome undergo swift reprogramming at the inception of colonic Citrobacter rodentium infection Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis Metabolomic analyses reveal lipid abnormalities and hepatic dysfunction in non-human primate model for Yersinia pestis Knockout of the non-essential gene SUGCT creates diet-linked age-related microbiome disbalance with a diabetes-like metabolic syndrome phenotype Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production Glutaric aciduria type I: outcome following detection by newborn screening Deletion of 2-aminoadipic semialdehyde synthase limits metabolite accumulation in cell and mouse models for glutaric aciduria type 1 Improved assay of glutaryl-CoA dehydrogenase in cultured cells and liver: application to glutaric aciduria type I Electron transfer flavoprotein and its role in mitochondrial energy metabolism in health and disease C7orf10 encodes succinate-hydroxymethylglutarate CoA-transferase the enzyme that converts glutarate to glutaryl-CoA Dietary- and host-derived metabolites are used by diverse gut bacteria for anaerobic respiration Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1 Itaconate confers tolerance to late NLRP3 inflammasome activation The immunomodulatory metabolite itaconate modifies NLRP3 and inhibits inflammasome activation Itaconate links inhibition of succinate dehydrogenase with macrophage metabolic remodeling and regulation of inflammation Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine Bile acids drive the newborn’s gut microbiota maturation The microbiome modulating activity of bile acids Itaconate is an effector of a Rab GTPase cell-autonomous host defense pathway against Salmonella Insights into Vibrio cholerae intestinal colonization from monitoring fluorescently labeled bacteria Itaconate is a covalent inhibitor of the Mycobacterium tuberculosis isocitrate lyase Cholera toxin promotes pathogen acquisition of host-derived nutrients in Greek Medicine from Hippocrates to Galen (ed Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells Quantitative dose–response analysis untangles host bottlenecks to enteric infection Precision of a clinical metabolomics profiling platform for use in the identification of inborn errors of metabolism targeted mass spectrometry-based metabolomics platform for bodily fluids Host-associated niche metabolism controls enteric infection through fine-tuning the regulation of type 3 secretion Towards standards for human fecal sample processing in metagenomic studies Analysis of compositions of microbiomes with bias correction and strain-level profiling of diverse microbial communities with bioBakery 3 Improved metagenomic analysis with Kraken 2 Beresford-Jones, B. S. et al. The Mouse Gastrointestinal Bacteria Catalogue enables translation between the mouse and human gut microbiotas via functional mapping. Cell Host Microbe https://doi.org/10.1016/j.chom.2021.12.003 (2021) Bracken: estimating species abundance in metagenomics data Dissecting serotype-specific contributions to live oral cholera vaccine efficacy Mouse tissue harvest-induced hypoxia rapidly alters the in vivo metabolome between-genotype metabolite level differences Gut microbiota-produced succinate promotes C difficile infection after antibiotic treatment or motility disturbance Download references Gabriel Nunez and Robert Quinn for their contribution to the peer review of this work (a) The relative abundance of metabolites in benzoate metabolism (blue) and non-categorized compounds (black) in bile of GF mice compared to SPF mice p values were generated using two-tailed Wilcoxon rank test Dots labeled in light-grey do not reach statistical difference; dots labeled in other colors are statistically different (p<0.05) (b) Principal component analysis (PCA) of the bile metabolome in animals from the following four groups of mice: SPF Each dot represents one sample; percentage represents fraction of variance explained by each principal component (c–e) The functional categories of metabolites with differential abundance in the indicated comparison (f) The relative expression level of Ugcg in mouse liver in Lm vs SPF Adjusted p values were generated using DESeq2 (two-tailed Wald test p-value monocytogenes in bile from the gallbladder (g) and liver (h) at 4-day post orogastric infection rodentium in colon (i) and liver (j) at 10-day post orogastric infection The dotted line represents the limit of detection Source data (a) The functional categories of bile metabolites that were elevated in both Lm and Cr infection (b) The percentage of metabolites in each functional category that were elevated in both infection conditions (c–e) The differential abundance of metabolites in the indicated categories Dots labeled in light-grey do not reach statistical difference; dots labelled in other colours are statistically different (p < 0.05) (f-h) Changes in abundance of metabolites linked to amino acid metabolism pathways in GF vs SPF (f) Dots labelled in blue and orange were statistically significant and represent decreased and increased abundance respectively; the size of the dots varies according to the magnitude of the differ Source data (a) The absolute abundance of itaconate and 2-methylsuccinate and relative abundance of 2-oxoadipate in bile of uninfected animals (n = 6) and Cr infected mice (n = 6) and relative abundance of glutarate in bile of uninfected animals (n = 5) and Cr infected mice (n = 6) (b) The absolute abundance of itaconate and 2-methylsuccinate and relative abundance of glutarate and 2-oxoadipate in proximal small intestine contents of uninfected animals (n = 5) and Cr infected mice (n = 7) (c) The absolute abundance of itaconate and 2-methylsuccinate and relative abundance of glutarate and 2-oxoadipate in serum of uninfected animals (n = 6) and Cr infected mice (n = 6) glutarate and 2-oxoadipate in supernatant of Cr culture grown in a virulence inducing condition (n = 2) p-values were generated using the two-tailed Mann-Whitney test (a-c) Source data (a) Principal component analysis (PCA) analysis of liver transcriptomes in uninfected mice (SPF) rodentium infected animals were divided into two groups depending on whether or not they had detectable (Cr positive Integrated analysis of genes and metabolites regulated by L. monocytogenes (a) and C. rodentium (b) infection was carried out using ‘Shiny GATOM: integrated analysis of genes and metabolites’ (https://artyomovlab.wustl.edu/shiny/gatom/) The lines represent enzymes and the dots represent metabolites The size of the dots and the thickness of the lines correlates with statistical significance (bigger or wider represent lower p-values) and the color scale for the dots and lines represents fold change where red represents increased abundance and green represents decreased abundance (a–d) Differential transcript abundance of liver mitochondrial complex I (a) Cr infected liver positive (Cr pos) or liver negative (Cr neg) animals compared to SPF animals adjusted for multiple testing using the procedure of Benjamini and Hochberg Source data (a) Abundance of succinate in bile of WT (n = 6) and Acod1−/− mice (n = 6) of uninfected animals (b) Differential abundance of fecal microbiota at the OTU level between WT (n = 9) and Acod1−/− mice (n = 11) before C (c–e) The three most differentially abundant OTUs in Acod1−/− mice (n = 11) compared to WT mice (n = 9) before C (f) Differential abundance of fecal microbiota at the OTU level between WT (n = 9) and Acod1−/− mice (n = 11) post C (g–i) Three most differentially abundant OTUs in Acod1−/− mice (n = 11) compared to WT mice (n = 9) post C (j) Differential abundance of functions (Enzyme Commissions rodentium in the feces of infected WT (n = 6) and Acod1−/− mice (n = 5) p-values were generated by two-tailed Mann-Whitney test (a Source data (a) Hematoxylin and Eosin (H&E) stained small intestine of WT (top) and Acod1-/- (bottom) mice post V Images represent four independent animals in one experiment Proposed model of a gut-liver defense circuit in which intestinal infection stimulates modifications in bile composition that in turn modulate intestinal function Enteric infection (1) stimulates changes in hepatic transcriptional profiles (2) that alter bile composition (3) that in turn control microbiota and epithelial composition and host defense (4) A descriptive caption is given on top of each table Download citation DOI: https://doi.org/10.1038/s41564-024-01862-z Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research Metrics details Disruption of bile acid (BA) metabolism causes various liver diseases including hepatocellular carcinoma (HCC) the underlying molecular mechanism remains elusive we report that BA metabolism is directly controlled by a repressor function of YAP which induces cholestasis by altering BA levels and composition via inhibiting the transcription activity of Fxr Elevated BA levels further activate hepatic YAP resulting in a feedforward cycle leading to HCC Teads are found to bind Fxr in a DNA-binding-independent manner and recruit YAP to epigenetically suppress Fxr or alleviating YAP repressor function by pharmacologically activating Fxr and inhibiting HDAC1 or overexpressing an Fxr target gene Bsep to promote BA exportation alleviate cholestasis and HCC caused by YAP activation Our results identify YAP’s transcriptional repressor role in BA metabolism as a key driver of HCC and suggest its potential as a therapeutic target but the mechanism underlying Fxr regulation in the liver remains elusive it remains unclear whether and how YAP activation causes HCC by inducing cholestasis Here we identify a repressor function of YAP in regulating BA metabolism and export Activated YAP induce severe cholestasis and fibrosis YAP is recruited by TEAD transcription factors which interact with Fxr in a DNA-binding-independent manner to replace the transcription co-activator acetyltransferase activating Fxr weaken the YAP-TEAD canonical transcriptional activators’ function suggesting that Fxr plays an important role in the dynamic regulation of YAP transcriptional activation as well Our results show that YAP activation induces a repressive epigenetic reprogramming of Fxr and suggest activating Fxr or its targets in BA metabolism as therapeutic targets of YAP-induced HCC Cyp7b1 and Cyp27a1 was not further upregulated in the TetO-YAP; Sox9Δ liver because the hepatocytes were still dedifferentiated to a certain extent These results indicate that YAP activation in hepatocytes led to BA accumulation due to both BA overproduction and impaired exportation the reduction of Fxr transcriptional activity is an early event in YAP-induced cholestasis a–c qPCR analysis of Bsep and Shp expression (a) in the indicated liver samples ***P = 0.0003 in sequence; (b) in the indicated mouse MHOs ****P < 0.0001; (c) in MHOs were treated with 5 µM Fxr agonist GW4064 or OCA for 48 h d Total BA concentration in the medium of MHOs f Gene-set enrichment analyses (GSEA) for Fxr target gene expression by comparing TetO-YAP MHOs with and without Dox treatment (e) g qPCR analysis for BSEP and SHP expression in the human MHOs with 3 µM TDI-011536 (TDI) to activate YAP and 0.5 µM Verteporfin (VP) to inhibit YAP-TEAD binding h Total BA concentration in the medium of human MHOs with the indicated treatment i The predictions for HCC samples with ‘liver tumor YAP signature’ (red and blue) and ‘Hepatic FXR signature’ (orange and green) in LIRI-JP cohort (n = 236) Heatmap shows the expression of listed genes r values determined by Pearson correlation comparing YAP activity with gene expression j Expression of selected genes in YAP ‘high’ or ‘low’ groups of HCC samples from the cohort box extends from the 25th to 75th percentiles line in the middle of the box is plotted at the median whiskers down to the minimum and up to the maximum value and p-values were determined by two-sided unpaired t-test with Welch’s correction l GSEA for Fxr target gene expression by comparing YAPHigh HCC samples with all non-tumor samples (k) YAPlow HCC with all non-tumor samples in the cohort (l) p-values were determined by two-sided unpaired t-test (a) p-values were determined by 1-way ANOVA with Sidak’s test (b–d independently of Mst1/2 regulation of Fgf15 signaling YAP activation plays a pivotal role in BA metabolism These data show YAP may also inhibit FXR activities in human HCC as we found in mice a Venn diagrams showing the overlap of ChIP-seq peaks of Fxr and Tead4 in the indicated mouse liver b Heatmaps summarizing the signal intensity of the Tead4 and Yap ChIP-seq on Fxr binding sites in the indicated mouse liver c ChIP-seq signal tracks of the indicated gene locus in the indicated mouse liver and the blue-boxed region is the putative enhancer d ChIP-qPCR analysis of Fxr and Tead4 binding at the indicated gene locus in the freshly isolated hepatocytes **P = 0.0046 in sequence (Bsep promoter); **P = 0.0059 e Venn diagrams showing the total number of Tead4/Fxr co-binding of ChIP-seq peaks and the subset of peaks without TBE in mouse liver f qRT-PCR analysis of Bsep expression in the mouse MHOs for enhancer validation using CRISPRi p-values were determined by two-sided unpaired t-test ***P = 0.0006 g–i Luciferase assays in the Huh-7 cells with the indicated reporters All conditions used co-transfected with the Fxr expression plasmid p-values were determined by 1-way ANOVA with Sidak’s test (d indicating that DNA binding is not required for TEAD to inhibit FXR transcriptional activity b Immunoprecipitation of indicated proteins in Huh-7 cells c Immunoprecipitation of indicated proteins in the nuclear-enriched fraction of mouse hepatocytes d Immunoprecipitation of HA-FXR in the nuclear-enriched fraction of Huh-7 cells e Immunoprecipitation of HA-FXR in the nuclear-enriched fraction of Huh-7 cells Similar results were obtained from three independent experiments (a–e) while HDAC1 inhibition by Entinostat not only elevated the baseline level of GW4064-induced FXR transcriptional activity but also partially abolished FXR inhibition by YAP-TEAD These results suggest that HDAC1/2 mediates the YAP-TEAD inhibition of FXR transcriptional activity These data indicate that TEAD recruits YAP to inhibit FXR transcriptional activity by recruiting the NuRD complex to replace P300 a Luciferase activity assay in Huh-7 cells tracking the canonical YAP-TEAD transcription activator activity b Immunoprecipitation of HA-FXR in the nuclear-enriched fraction of Huh-7 cells c Immunoprecipitation of HA-FXR in the nuclear-enriched fraction of Huh-7 cells 8xGTII and 3xIR1 plasmids were co-transfected with FXR and TEAD4 plasmids YAP and pan-TEAD in the nuclear-enriched fraction of Huh-7 cells e ChIP-qPCR analysis of TEAD4 and HDAC1 binding at the 8xGTII TBE sites in the Huh-7 cells 8xGTII plasmid was co-transfected with GFP or Flag-YAP5SA plasmids f Immunoprecipitation of indicated proteins in the nuclear-enriched fraction of liver tissues g Representative images of liver from the indicated mice k–m qRT-PCR analysis of indicated genes in the liver tissue o Luciferase activity assay in primary mouse hepatocytes with indicated genotype tracking the canonical YAP-Tead transcription activity (n) 5 µM GW4064 and 3 µM TDI treatment was 24 h n = 5 biological replicates in each group (h–j) n = 3 biological replicates in each group (k–m) p-values were determined by 1-way ANOVA with Sidak’s test (a Similar results were obtained from three independent experiments (b–d This data suggests that the suppressive role of YAP on the IR1-dependent transcriptional activity is entirely dependent on the presence of Fxr Further inhibition of Fxr target gene expression by YAP in vivo is likely mediated by Fxr-independent mechanisms we showed that YAP directly controls a unique and key metabolic function of the liver—BA production and exportation—by inhibiting Fxr We further identified a transcriptional repressor function of YAP that is responsible for liver tumorigenesis transcriptional activation by YAP-Tead binding to TBE can also be reprogrammed by activated Fxr in hepatocytes All these findings show that YAP-induced liver tumor formation can be alleviated by pharmacological Fxr activation or suppression of HDAC1-mediated Fxr inhibition or genetically restoring Fxr-controlled BA export or reduction of serum BAs by feeding the mice with BA-absorbing resin Our results show BA metabolism is critically controlled by a reciprocal and repressive epigenetic reprogramming of YAP and Fxr BA metabolism can be therapeutically targeted for various liver diseases caused by YAP abnormality a YAP regulation of Fxr activity and BA metabolism and BAs forms a dynamic regulatory network YAP activation inhibits Fxr transcriptional activity causing BA accumulation in hepatocytes by downregulating Bsep expression Pharmacological activation of Fxr by GW4064 not only curtails BA production and promotes BA exportation but also directly inhibits YAP activity alleviating tumor formation caused by YAP activation b Dynamic regulation of YAP and FXR transcriptional activity by P300 and HDAC1 Pharmacological activation of FXR by GW4064 relieves YAP inhibition of FXR by recruiting P300 and inhibiting HDAC1 binding to FXR it promotes the repressor function of the DNA-binding YAP-TEAD complex converting it from a transcriptional activator to a transcriptional repressor while Fxr is only functional in hepatocytes The strong inhibitory role of Yap activation imposed on Fxr explains the need to keep YAP activity low in hepatocytes under normal conditions to allow Fxr to be promptly activated to regulate BA metabolism Mst1/2-independent role of YAP in regulating BA metabolism by reprogramming Fxr identified here suggests BA metabolism is regulated in multiple ways a combination of Fxr activation and HDAC1/2 inhibition may be a potent therapeutic strategy for this subtype of HCC Other mice were procured from the Jackson Laboratory: The Fxr−/− mice (JAX: 004144) Sox9fl/fl (JAX: 013106) and Yapfl/fl (JAX: 027929) 1 × 1011 genome copies (GC) of each AAV were administered to 3-week old mice via retro-orbital injection at a volume of 30 μl The pAAV-TBG-YAPS127A and pAAV-TBG-Bsep plasmids were generated in our lab For YAPS127A overexpression in the TetO-YAP mice 1 x 1011 GC of AAV-TBG-Cre were administered to 8-week old TetO-YAP mice YAPS127A expression was induced in the mice by Dox water (0.2 g/L) feeding Mouse liver was perfused with ice-cold PBS and cut into small pieces in ice-cold PBS the liver tissue was fixed in 4% paraformaldehyde for 12 h and then processed according to standard procedures and immunohistochemistry was performed according to standard procedures Sections were washed in 0.05% Triton/PBS (PBST) followed by incubation with the blocking buffer (5% Donkey serum/PBST) for 1 h at room temperature and then incubated overnight at 4 °C with diluted primary antibodies in the blocking buffer Then the sections washed with PBST and incubated with diluted secondary antibodies in the blocking buffer for 1 h at room temperature the sections were washed with PBST and mounted with Fluoroshield™ with DAPI (Sigma-Aldrich) mounting medium The following antibodies were used for immunostaining: F4/80 (MCA497 donkey anti-rat IgG Alexa Fluor 647 (A48272 Invitrogen) and donkey anti-rabbit IgG Alexa Fluor 568 (A10042 Invitrogen) Images were captured on a Leica DM6 confocal microscope and sectioned at 6μm for Hematoxylin and Eosin (H&E) staining and Sirius red staining Paraffin sections were rehydrated before staining tissue sections were stained with Mayer’s Hematoxylin solution (G-Biosciences) and Eosin staining solution (Thermo Fisher Scientific) subsequently Sections were incubated in Sirius staining solution (0.1% Direct Red 80 and 1.3% picric acid in distilled water) for 1 hour then washed in two changes of acidified water (0.5% acetic acid in distilled water) The stained sections were dehydrated and mounted for imaging Images were captured using a Keyence BZ-X710 microscope and media was measured using the Total Bile Acids Assay kit (Diazyme) according to the manufacturer’s instructions ALT Activity in the serum was measured using the Assay kit (Sigma-Aldrich) according to the manufacturer’s instructions BAs in the media were concentrated by mixing 100 µL of media with 1 mL of chloroform: methanol (1:1) The supernatant was dried and resolved in 10 µL of methanol To extract BAs in the HOs and liver tissues the samples were weighed and digested with proteinase K (1 mg/mL) for 3 h at 50 °C the samples were centrifuged at 8000 g for 10 min The BAs in the supernatant were extracted and partitioned by adding 3.3 volumes of chloroform: methanol (1:1) and resolved in 10-100 µL of methanol The TUNEL assay was performed using the Click-iT™ Plus TUNEL Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions the mouse heart and liver were surgically exposed after anesthesia a 25 G infusion set (Terumo) was immediately inserted into the right atrium and the portal vein was cut with scissors at the same time Mouse livers were then perfused sequentially with pre-warmed (37 °C) liver perfusion medium (Thermo Fisher Scientific) at a flow rate of 3 ml/min for 2 min and pre-warmed (37 °C) digestion solution (0.75 mg/ml collagenase type I in DMEM; Thermo Fisher Scientific) was perfused at the same flow rate for 3 min the portal vein was manually occluded by cotton tips every 30 s for 10 s the liver was surgically removed and cut into small pieces in ice-cold DMEM and hepatocytes from the digested tissue were released into the medium by gentle pipetting hepatocytes were passed through a 70μm cell strainer (Corning) and centrifuged at 50 g for 3 min The viable hepatocytes were purified using 40% Percoll in PBS (Cytiva) and centrifuged at 150 g for 5 min Mouse and human MHO culture was performed as previously described in ref. 31 freshly isolated mouse hepatocytes were embedded in the Cultrex BME Type R1 (R&D Systems for mouse HO generation Mouse HOs were maintained in the +GF medium The +GF medium for mouse HO culture contained Advanced DMEM/F12 (Thermo Fisher Scientific) with B27 minus vitamin A (Thermo Fisher Scientific) 15% RSPO1-conditioned medium (made in the lab using Cultrex HA-R-Spondin1-Fc 293 T cells (Bio-Techne) according to the manufacturer’s instructions) The cryopreserved human fetal HOs were retrieved The +GF medium for human HO culture contained Advanced DMEM/F12 (Thermo Fisher Scientific) with B27 minus vitamin A (Thermo Fisher Scientific) 2.5 mM nicotinamide (Sigma) and 1.25 mM N-acetylcysteine (Sigma) the HO cultures underwent one passage to enrich well-formed HOs HOs were retrieved from the BME using Cultrex Organoid Harvesting Solution (R&D Systems) Well-formed organoids were picked from the bottom of a well and further washed in Advanced DMEM-F12 and re-embedded into the BME for subsequent culture The cystic bile duct organoids were hand-picked or further removed by 36% Percoll (Cytiva) by centrifugation at 150 g for 5 min during passage The +GF medium was switched to the maturation medium (RGF medium) for the induction of MHOs The basal medium for human and mouse HO maturation contained William’s E Medium (Thermo Fisher Scientific) with B27 minus vitamin A (Thermo Fisher Scientific) penicillin-streptomycin (Thermo Fisher Scientific) 2.5 mM nicotinamide (Sigma) and 1.25 mM N-acetylcysteine (Sigma) the medium used was RGF with 10 nM T3 (Sigma) AAV-TBG-Cre was mixed with hepatocytes in BME at a concentration of 1 x 1010 genome copies (GC)/ml before seeding to induced conditional knockout in the HOs To induce YAPS127A expression in the TetO-YAP cells doxycycline (Sigma) was added to the culture medium at a concentration of 200 ng/ml The Huh-7 cells were obtained from JCRB cell bank (Cat: JCRB0403) and maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 100 U/ml penicillin-streptomycin (Thermo Fisher Scientific) Plasmid transfection was performed using the PEI MAX Reagent (Polysciences) according to the manufacturer’s instructions RIPA buffer containing Protease Inhibitor Cocktail (PIC) and PhosSTOP (Roche) was used to harvest whole tissue and cell lysates Protein concentrations were quantified by the BCA protein assay kit (Thermo Fisher Scientific) Equal amounts of protein were resolved by SDS-PAGE and further transferred to the Nitrocellulose Blotting Membrane (Cytiva) Target proteins were detected with the SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher Scientific) using the PXi4 Chemiluminescent Imaging System (Syngene) Reporter plasmids of Bsep enhancer (3xIR1) were generated by subcloning the genomic DNA PCR products into the pGL4.10 basic vector (Promega) 8xGTIIC-luciferase plasmid was from Addgene (#34615) Cells were transfected with pGL4.10 reporter plasmids with pTK-Renilla (Promega) and effector plasmids The luciferase activity was measured with a dual-luciferase reporter assay kit (Promega) according to the manufacturer’s instruction 48 h after transfection The Renilla luciferase activity was used to normalize the luciferase reporter activity DNA was removed from RNA using a RapidOut DNA Removal Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions 400–1000 ng RNA per sample reaction was used for mRNA isolation using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) Libraries were prepared using an xGen RNA Library Prep Kit (IDT) according to the manufacturer’s instructions Barcoded libraries were sequenced on an Illumina NextSeq 550/1000 at the Genomics Technology Laboratory of NCI Paired-end sequencing mode (2x45 bp) was used in the experiments GSEA was performed using the GSEA 4.0 software or ‘ClusterProfiler’ KEGG functional analysis was performed using ClusterProfiler’ Overexpressed or under-expressed genes in a signature set were assigned a value of 1 or −1 Samples predicted as having a signature with False Discovery Rate (FDR) of less than 0.05 were compared with the rest of the samples in the cohort Data were presented as mean ± standard deviation (SD) Data quantification and analyses were plotted using Prism 8 P-values were determined by 1-way ANOVA with Sidak’s test for comparison of more than two sample groups Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Bile Acids as Metabolic Regulators and Nutrient Sensors Three-dimensional spatially resolved geometrical and functional models of human liver tissue reveal new aspects of NAFLD progression The ascending pathophysiology of cholestatic liver disease Davit-Spraul, A., Gonzales, E., Baussan, C. & Jacquemin, E. Progressive familial intrahepatic cholestasis. Orphanet. J. Rare Dis. 4, 1. https://doi.org/10.1186/1750-1172-4-1 Intrahepatic cholestasis in common chronic liver diseases Pathogenesis of cholestatic liver disease and therapeutic approaches Hepatocellular carcinoma with obstructive jaundice: diagnosis The role of farnesoid X receptor in metabolic diseases A regulatory cascade of the nuclear receptors FXR and LRH-1 represses bile acid biosynthesis Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice Bile acids activate YAP to promote liver carcinogenesis Role of Farnesoid X Receptor and Bile Acids in Hepatic Tumor Development The Hippo Signaling Pathway in Development and Disease Hippo signaling interactions with Wnt/beta-catenin and Notch signaling repress liver tumorigenesis Hepatic Hippo signaling inhibits protumoural microenvironment to suppress hepatocellular carcinoma Yap-Sox9 signaling determines hepatocyte plasticity and lineage-specific hepatocarcinogenesis Hippo signalling in the liver: role in development Bile acid-mediated signaling in cholestatic liver diseases Hippo pathway activity influences liver cell fate Elucidation of a universal size-control mechanism in Drosophila and mammals YAP1 increases organ size and expands undifferentiated progenitor cells Expression of bile acid synthesis and detoxification enzymes and the alternative bile acid efflux pump MRP4 in patients with primary biliary cirrhosis High expression of the bile salt-homeostatic hormone fibroblast growth factor 19 in the liver of patients with extrahepatic cholestasis Gut microbiota depletion exacerbates cholestatic liver injury via loss of FXR signalling Conjugated Bile Acids Promote Invasive Growth of Esophageal Adenocarcinoma Cells and Cancer Stem Cell Expansion via Sphingosine 1-Phosphate Receptor 2-Mediated Yes-Associated Protein Activation Liu, Y. et al. Generation and characterization of mature hepatocyte organoids for liver metabolic studies. J Cell Sci. https://doi.org/10.1242/jcs.261961 Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid Identification of a chemical tool for the orphan nuclear receptor FXR The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver Hepatocellular carcinoma in ten children under five years of age with bile salt export pump deficiency Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis Targeted inactivation of sister of P-glycoprotein gene (spgp) in mice results in nonprogressive but persistent intrahepatic cholestasis In LiverTox: Clinical and Research Information on Drug-Induced Liver Injury (2012) Direct and Indirect Effects of Fibroblast Growth Factor (FGF) 15 and FGF19 on Liver Fibrosis Development 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA) a potent and selective FXR agonist endowed with anticholestatic activity Metformin interferes with bile acid homeostasis through AMPK-FXR crosstalk Mathur, B. et al. Nuclear receptors FXR and SHP regulate protein N-glycan modifications in the liver. Sci. Adv. 7. https://doi.org/10.1126/sciadv.abf4865 FGF15 Activates Hippo Signaling to Suppress Bile Acid Metabolism and Liver Tumorigenesis Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene Hippo signaling is a potent in vivo growth and tumor suppressor pathway in the mammalian liver Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer TEAD mediates YAP-dependent gene induction and growth control Switch enhancers interpret TGF-beta and Hippo signaling to control cell fate in human embryonic stem cells Transcriptional co-repressor function of the hippo pathway transducers YAP and TAZ A Regulation Loop between YAP and NR4A1 Balances Cell Proliferation and Apoptosis Decoding YAP dependent transcription in the liver FXR Isoforms Control Different Metabolic Functions in Liver Cells via Binding to Specific DNA Motifs Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements A peptide mimicking VGLL4 function acts as a YAP antagonist therapy against gastric cancer DNA-binding mechanism of the Hippo pathway transcription factor TEAD4 The p300 acetylase is critical for ligand-activated farnesoid X receptor (FXR) induction of SHP Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours Histone deacetylases and their inhibitors in cancer neurological diseases and immune disorders Mice with hepatocyte-specific FXR deficiency are resistant to spontaneous but susceptible to cholic acid-induced hepatocarcinogenesis Single-Cell Analysis of the Liver Epithelium Reveals Dynamic Heterogeneity and an Essential Role for YAP in Homeostasis and Regeneration Induction of AP-1 by YAP/TAZ contributes to cell proliferation and organ growth Nuclear receptor-dependent bile acid signaling is required for normal liver regeneration Conjugated bile acids promote cholangiocarcinoma cell invasive growth through activation of sphingosine 1-phosphate receptor 2 Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line Regeneration Defects in Yap and Taz Mutant Mouse Livers Are Caused by Bile Duct Disruption and Cholestasis YAP Drives Growth by Controlling Transcriptional Pause Release from Dynamic Enhancers YAP induces an oncogenic transcriptional program through TET1-mediated epigenetic remodeling in liver growth and tumorigenesis Single tumor-initiating cells evade immune clearance by recruiting type II macrophages Transcriptional repression of estrogen receptor alpha by YAP reveals the Hippo pathway as therapeutic target for ER(+) breast cancer YAP inhibits ERalpha and ER(+) breast cancer growth by disrupting a TEAD-ERalpha signaling axis Divergent WNT signaling and drug sensitivity profiles within hepatoblastoma tumors and organoids Wang, Y. et al. Berberine Prevents Disease Progression of Nonalcoholic Steatohepatitis through Modulating Multiple Pathways. Cells 10. https://doi.org/10.3390/cells10020210 clusterProfiler: an R package for comparing biological themes among gene clusters Nearest template prediction: a single-sample-based flexible class prediction with confidence assessment deepTools: a flexible platform for exploring deep-sequencing data Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities Download references We thank the Yang lab members for stimulating discussion This work was supported by National Institutes of Health grants R01CA222571 and R01AR070877 to Y.Y. and Y.J.; supported by VA Merit Award 5I01BX005730 the VA ShEEP grant (1 IS1 BX004777-01) and the Research Career Scientist Award from the Department of Veterans Affairs (IK6BX004477) to H.Z.; We thank the LC-MS Core of Virginia Commonwealth University for the technical support and thank Praju Vikas Anekal and Paula Montero-Llopis of the Microscopy Resources on the North Quad (MicRoN) core at Harvard Medical School for helpful discussions and training in confocal imaging Department of Microbiology & Immunology Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-025-58809-z Metrics details Bile acids (BAs) exert a profound influence on the body’s pathophysiology by intricately shaping the composition of gut bacteria the complex interplay between BAs and gut microbiota has impeded a systematic exploration of their impact on intestinal bacteria we investigated the effects of 21 BAs on the growth of 65 gut bacterial strains in vitro we examined the impact of BAs on the overall composition of intestinal bacteria The results unveiled distinct effects of various BAs on different intestinal strains and their diverse impacts on the composition of gut bacteria the inhibition of intestinal strains by BAs occurs through the accumulation of these acids within the strains The intracellular accumulation of deoxycholic acid (DCA) significantly influenced the growth of intestinal bacteria by impacting ribosome transcription and amino-acid metabolism The metabolomic analysis underscores the pronounced impact of DCA on amino-acid profiles in both in vivo and in vitro settings This study not only elucidates the effects of BAs on a diverse range of bacterial strains and their role in shaping the gut microbiota but also reveals underlying mechanisms essential for understanding and maintaining a healthy gut microbiota given its widespread impact on human health it is essential to comprehend the factors influencing the microbiome especially at the community and strain levels the role of BAs in shaping gut microbiota has important implications for human health We employed a comprehensive approach encompassing bacterial growth rate measurements (OD600) Our study has shed light on the intricate effects of BAs on intestinal bacteria demonstrating that different types of BAs exhibit varying effects on gut microbiota both in vivo and in vitro These effects are mediated in part by the effect of BAs on the ribosome as well as the bioaccumulation of BAs by bacteria These findings pave the way for future investigations into the role of BAs in host pathophysiology through their modulation of the intestinal microbiota A Heatmap showing the sensitivity or resistance of each bacterial strain to different concentrations of BAs B Number of bacterial strains exhibiting an inhibition rate >25% among 65 strains tested for each BAs and TUDCA on the growth of Bifidobacterium pseudocatenulatum Each group included six replicates (n = 6) and TUDCA on the growth of Bifidobacterium pseudolongum E Heatmap showing the inhibitory effect of 21 BAs on phyla D data with error bars represent mean ± SD D Data were analyzed by two-tailed unpaired Student’s t test These results indicate that different BAs have distinct effects on various bacterial phyla A The Venn diagram illustrated the overlapped OTUs between the group of control B Accumulation of bacterial phyla with their relative abundance in the gut microbiota between the group of control and LCA; C The abundance of Proteobacteria in the CDCA D Principal Coordinates Analysis (PCoA)showed β Diversity in CDCA E–G Linear discriminant analysis (LDA) coupled with effect size measurements identifies significant abundance Only taxa with LDA score greater than 4 are shown C Data with error bars represent min to max C Data were analyzed by two-tailed unpaired Student’s t test These findings provide further evidence that DCA and CDCA inhibit the growth of other bacteria and promote the relative abundance of Proteobacteria These findings suggest that BAs may modulate bacterial function through the regulation of these crucial pathways A Alpha diversity box plot (Ace) between the group of control B Principal coordinate analysis (PCoA) of beta diversity between the group of CDCA and control C PCoA of beta diversity between the group of DCA and control D–F Feces microbiome communities are significantly different between the group of control LDA score computed features differentially abundant between the control and CDCA (D) The criteria for feature selection was LDA Score >4 A Data with error bars represent min to max A Data were analyzed by two-tailed unpaired Student’s t test LCA and CDCA increased the abundance of Muribaculaceae in vivo DCA had the greatest impact on intestinal bacteria which is consistent with the trend of impact observed in vitro A Quantification of DCA concentrations in 22 strains by UHPLC-MS (n = 5–6) B Linear fitting analysis between DCA accumulation of 22 strains and inhibition rate of DCA (0.5 mM) to the 22 strains C Correlation between DCA inhibition rate and accumulation by Spearman analysis D–F Effects of different concentrations of PAβN and DCA on the growth curves of EDTE (D) PAβN(H):0.15 mg/ml; PAβN(M):0.1 mg/ml; PAβN(L):0.05 mg/ml (n = 6) G-I Effects of different concentrations of PAβN and DCA on the area under the curve of EDTE (G) D–I Data with error bars represent mean ± SD These results indicate that the accumulation of DCA within bacteria is positively correlated with their sensitivity to DCA inhibition B PCA of RNA-seq data from Bacteroides ovatus (A) and Escherichia coli K-12 (B) after 6 hours of incubation with solvent and DCA D Volcano plots showed significance change of genes in response to DCA in Bacteroides ovatus (C) and Escherichia coli K-12 (D) after 6 hours of exposure F Heatmaps of Gene Ontology (GO) pathway enrichments of differentially expressed genes in bacterial isolates treated with 0.4 mM of DCA compared to the vehicle control Colors represent false discovery rate (FDR) and size represents the number of genes in Bacteroides ovatus (E) and Escherichia coli K-12 (F) DCA may impact bacterial growth by affecting ribosome function and amino acid metabolism after entering the bacteria A Differential gene expression in Bacteroides ovatus treated with DCA C AAs Concentration in Bacteroides ovatus Treated with DCA D AAs concentration in Ecoli-K12 Treated with DCA E AAs in rat plasma treated with DCA or solvent A–E Data with error bars represent mean ± SD A–E data were analyzed by two-tailed unpaired Student’s t test C–E data were analyzed by Multiple t tests-one per row Given that bile acid significantly impacts both amino acid metabolism in various strains and the composition of intestinal bacteria the observed changes in amino acid composition within the body may result from a combination of these effects the effect of BAs on gut bacteria has not been systematically studied Our results indicated that BAs not only had different effects on 65 intestinal strains but also had important effects on the overall composition of intestinal bacteria in vitro and in vivo the more intestinal strains accumulated BAs the stronger the inhibitory effect of BAs on intestinal strains BAs affect ribosomal function and amino acid metabolism further contributing to the inhibition of bacteria The findings contribute to the current understanding of the structure and hierarchical organization of the gut microbiome as well as the development of BA-based antibiotics this work has the potential to deepen our understanding of the effects of BAs on the body’s pathophysiology by affecting gut bacteria Further research is needed to fully elucidate the molecular mechanisms underlying these effects and to explore potential clinical applications which shows that BAs may have the same effect on increasing the abundance of Akkermansia muciniphila bacteria the host’s metabolism may be affected by different BAs through their distinct impacts on the gut microbiota This manifestation of diarrhea is likely attributed to the distinct effects exerted by the two BAs on the composition and dynamics of the intestinal microbiota The strikingly different effects of structurally similar BAs on gut bacteria warrant further investigation This provides new therapeutic options for treating Clostridium difficile infection these BAs still induce significant changes in the 50S ribosomal protein DCA and CA increase the transcription of choloylglycine hydrolase whereas DCA reduces its transcription in Bacteroides ovatus These findings underscore the strain-specific and variable effects of BAs on bacterial transcriptomes While BAs do not inhibit the growth of some gut bacteria they can still affect the metabolism of intestinal bacteria highlighting the importance of considering the broader impacts of BAs on gut microbial physiology and metabolic activity even in the absence of marked changes in community structure or colonization level BAs have been shown to impact the metabolism of ribosomes and amino acids in bacteria the mechanism by which they act on ribosomes remains unclear and warrants further investigation studying whether BAs alter the composition of intestinal microbiota by modulating these metabolic pathways deserves further attention Our study highlights the significance of considering the unintended impacts of BAs on our associated microbial communities and the need for a broader pharmacological view Our findings demonstrate the usefulness of integrated in vitro and in vivo studies to elucidate the causality and mechanisms of complex interactions between BAs and microbiome Investigating the effects and mechanisms of BAs on intestinal microorganisms could lead to the discovery of the important role of BAs in pathophysiology Accurately weighing BAs of varying masses into 1.5 mL EP tubes DMSO was added for dissolution and then diluted with mGAM or BHI medium 50 μL of the resulting solution was transferred to pre-reduced 384-well plates that were placed in the anaerobic chamber overnight Each strain was screened at least three biological replicates Human stool-derived material was approved by the medical ethics committee of Xiangya Hospital Central South University (Approval Number: 2019040116) Informed consent was obtained from all donors who were 7 healthy male volunteers meeting specific inclusion criteria These criteria included being aged 18 and above having a body mass index between 18 and 25 kg/m2 the volunteers had not taken antibiotics or probiotics in the three months leading up to or during the sample collection period fresh fecal samples were promptly transferred to a temperature of −80 °C for the preparation of the gut microbiota reserve solution These procedures were carried out in compliance with the rigorous guidelines set by the medical ethics committee of Xiangya Hospital ensuring scientific integrity and ethical considerations in line with the standards required for publication in scientific journals the surface of the feces was carefully scraped and 1 gram of fecal matter from the central portion was weighed and suspended in a 50 mL sterile centrifuge tube containing 15 mL of pre-reduced phosphate-buffered saline (PBS) solution with 0.1% cysteine The suspension was thoroughly mixed by vortexing to ensure proper homogenization the suspension was promptly transferred to an anaerobic box with a controlled environment of 37 °C carbon dioxide allowing the suspension to stand for 5 minutes to facilitate the precipitation of insoluble particles The supernatant was then mixed with an appropriate quantity of 40% glycerol solution The suspension was dispensed into sterile cryovials in 1 mL aliquots and underwent a gradient-freezing process at −80 °C with a maximum storage duration of 7 months we first prepared the three BAs to a concentration of 100 mM using DMSO followed by the addition of 990 μL of mGAM medium take 100 µL of the prepared BAs solution and add it to 900 µL of cultured fecal bacteria solution for incubation Male Sprague-Dawley rats aged 7 weeks and weighing ~200 g (Changsha China.) were purchased and housed in a specific pathogen-free environment at the Experimental Animal Department of Central South University The ambient temperature was maintained at 22 ± 2 °C and the relative humidity was 60 ± 5% The rats were subjected to a 12-hour light/dark cycle Experimental Animal Department of Central South University approved under protocol CSU-2022-0087 the rats were randomly divided into control groups according to their weight The control groups were given an intragastric solvent (sodium CMC) while the experimental groups were given CDCA (150 mg/kg) with 6–7 rats in each group for a duration of 7 days Fecal samples were collected before and after gavage Genomic DNA was extracted from samples using the CTAB/SDS method and its concentration and purity were assessed on 1% agarose gels DNA was diluted to a concentration of 1 ng/μL using sterile water Specific primers with barcodes were used to amplify distinct regions of the 16S rRNA gene PCR reactions were performed with Phusion® High-Fidelity PCR Master Mix (New England Biolabs) The PCR products were analyzed by electrophoresis on a 2% agarose gel with 1× loading buffer (containing SYB green) and samples with a bright main strip between 400–450 bp (16S) were selected for further experiments The purified PCR products were subjected to sequencing libraries using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina USA) following the manufacturer’s instructions and index codes were added The quality of the library was evaluated using the Qubit® 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system the library was sequenced on an Illumina NovaSeq 6000 platform to generate 250 bp paired-end reads and UDCA) to the bacterial solution during the logarithmic growth phase The final concentration of each bile acid is 10 μg/mL prepare a stock solution with a concentration of 2.4 mg/mL use this stock solution to prepare a bacterial solution with a final volume of 1.2 mL and a concentration of 20 μg/mL and then 200 μL of the solution was used to measure the absorbance at OD600 The remaining bacterial solution was centrifuged at 14,000 rpm for 15 min at 4 °C The bacterial pellet was resuspended in PBS and centrifuged again under the same conditions and the pellet was washed again with PBS before being centrifuged to obtain the final pellet followed by the addition of 300 μL of methanol precipitation agent containing an internal standard The mixture was sonicated for 15 min and vortexed for 5 min before being centrifuged at 14,000 rpm for 15 min at 4 °C 100 μL of the supernatant was transferred to a sample injection vial for UPLC-MS analysis BAs analysis was conducted using an AB SCIEX UHPLC system coupled with a Triple Quad 6500+ mass spectrometer The flow rate of the mobile phase was 0.3 mL/min Separation of BAs was performed using a Waters T3 C18 column (1.7 μm The mobile phase A was water (containing 0.1% formic acid and 5 mM ammonium acetate) and phase B was acetonitrile The gradient program consisted of intervals: 30% to 50% B (first minute) Mass spectrometer capillary voltage was −4.5 kV Acquisition data were assessed with Analyst Software for peak integration Precisely weigh ~100 mg of cecal contents and add 1 mL of solution (methanol: water 1:1) extract 50 μL of the supernatant and combine it with 150 μL of methanol solution containing the internal standard Vortex the mixture thoroughly and centrifuge it at 14,000 rpm collect 80 μL of the supernatant and inject it into the sample vial for analysis The elution program employed a gradient with a flow rate of 0.35 mL/min as follows: starting with 30% B from 0 to 9 minutes transitioning to 35% B from 9 to 11 minutes ramping up to 100% B from 16 to 21 minutes decreasing to 30% B from 23 to 23.1 minutes and finally holding at 30% B from 23.1 to 25 minutes All other conditions remain consistent with the prior detection of bile acids An ultra-high-performance liquid chromatography (AB SCIEX UHPLC USA) coupled with a triple-quadrupole mass spectrometer (Triple Quad 5600 Amino acids were separated using a Waters T3 C18 column (1.7 μm Phase A is water and Phase B is acetonitrile The gradient program was structured as follows: 2% to 22% B (initial 4 min) Mass spectrometer capillary voltage was set at +5.5 kV Analyst Software was employed for acquisition data analysis Quantification of transcripts was performed by real-time PCR (QuantStudio 3) using the 2x SYBR Green qPCR Master Mix (biomake) The relative fold changes of gene expression were calculated using the cycle threshold (Ct) method Bacteroides ovatus and Escherichia coli K-12 underwent 6-hour treatment with 0.5 mM DCA or DMSO Total RNA extraction utilized Trizol Reagent (Invitrogen Life Technologies) followed by NanoDrop spectrophotometer (Thermo Scientific) and Bioanalyzer 2100 system (Agilent) assessment for quality and integrity Zymo-Seq RiboFree Total RNA Library Kit was employed to deplete ribosomal RNA from total RNA cDNA synthesis involved random oligonucleotides and SuperScript III followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H The resulting blunt-ended DNA fragments were hybridized with Illumina PE adapter oligonucleotides Library fragments were size-selected (400–500 bp) using the AMPure XP system (Beckman Coulter DNA fragments with adaptors on both ends were enriched using Illumina PCR Primer Cocktail in a 15-cycle PCR reaction Amplified products were purified with the AMPure XP system and quantified using the Agilent high-sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent) the sequencing library was sequenced on the NovaSeq 6000 platform (Illumina) Quantitative data were expressed as mean ± standard error of the mean The difference between the two groups was compared using a t test Statistical significance was determined at a two-tailed P value of less than 0.05 All statistical analyses and graphing were conducted using GraphPad Prism 8.0 and Origin 9.1 The 16S rDNA sequencing data and RNA sequencing had been deposited to the NCBI Sequence Read Archive database (Accession Number: PRJNA939937) The dataset regarding the metabolomics profiling analyzed during the current study is available from the corresponding author upon reasonable request The gut microbiome’s role in the development Bile acids in glucose metabolism and insulin signalling–mechanisms and research needs Rethinking healthy eating in light of the gut microbiome How gut microbes are joining the fight against cancer The gut microbiome in hypertension: recent advances and future perspectives The microbiome and risk for atherosclerosis The gut microbiota and obesity: from correlation to causality elegans caused by bacterial colonization of the intestine and subsequent activation of an innate immune response Molecular physiology of bile acid signaling in health Bile acid-microbiota crosstalk in gastrointestinal inflammation and carcinogenesis Bile acids and their receptors in metabolic disorders Intestinal bile acids induce a morphotype switch in vancomycin-resistant enterococcus that facilitates intestinal colonization Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians Interpersonal gut microbiome variation drives susceptibility and resistance to cholera infection Secondary bile acid-induced dysbiosis promotes intestinal carcinogenesis Interactions between gut bacteria and bile in health and disease Postprandial evolution in composition and characteristics of human duodenal fluids in different nutritional states Postprandial concentrations of free and conjugated bile acids down the length of the normal human small intestine Personalized mapping of drug metabolism by the human gut microbiome Bioaccumulation of therapeutic drugs by human gut bacteria Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization Antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump Which mechanisms of azithromycin resistance are selected when efflux pumps are inhibited Ribosome-targeting antibiotics and mechanisms of bacterial resistance 16S ribosomal RNA gene sequencing to evaluate the effects of 6 commonly prescribed antibiotics The mouse gastrointestinal bacteria catalogue enables translation between the mouse and human gut microbiotas via functional mapping Sequence variant analysis reveals poor correlations in microbial taxonomic abundance between humans and mice after gnotobiotic transfer Proteobacteria: microbial signature of dysbiosis in gut microbiota Elucidation of Proteus mirabilis as a key bacterium in Crohn’s disease inflammation Gut microbiome-mediated bile acid metabolism regulates liver cancer via NKT cells FXR regulates intestinal cancer stem cell proliferation Xylan alleviates dietary fiber deprivation-induced dysbiosis by selectively promoting Bifidobacterium pseudocatenulatum in pigs The landscape in the gut microbiome of long-lived families reveals new insights on longevity and aging-relevant neural and immune function Gut symbionts alleviate MASH through a secondary bile acid biosynthetic pathway Akkermansia muciniphila phospholipid induces homeostatic immune responses Akkermansia muciniphila: from its critical role in human health to strategies for promoting its abundance in human gut microbiome Akkermansia muciniphila mediates negative effects of IFNγ on glucose metabolism Franco-Belgian cooperative study of ursodeoxycholic acid in the medical dissolution of gallstones: a double-blind Chenodeoxycholic acid: a review of its pharmacological properties and therapeutic use Effects of tolC on tolerance to bile salts and biofilm formation in Cronobacter malonaticus Structure and origin of bile acids: an overview Recent advances toward a molecular mechanism of efflux pump inhibition Strain-dependent inhibition of clostridioides difficile by commensal clostridia carrying the bile acid-inducible (bai) operon Increase in ribosomal fidelity benefits salmonella upon bile salt exposure Twin-arginine translocation system is involved in citrobacter rodentium fitness in the intestinal tract Extensive impact of non-antibiotic drugs on human gut bacteria Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice Determination of amino acids in food and feed by microwave hydrolysis and UHPLC-MS/MS Download references This work was supported by the National Key Research and Development Program of China (no the National Natural Scientific Foundation of China (nos the Hunan Provincial Natural Science Foundation of China (2024JJ5585) and the scientific research project of Furong laboratory of Central South University (2023SK2083) Applied Technology of Pharmacogenomics (Ministry of Education) Hunan Key Laboratory of Pharmacomicrobiomics National Clinical Research Center for Geriatric Disorders Download citation DOI: https://doi.org/10.1038/s41522-024-00566-w Choose the health content that’s right for you Surgery is often the only treatment that can completely cure bile duct cancer But it can be complicated — and requires a team with a wealth of experience caring for patients with this rare cancer Fortunately, Iswanto Sucandy, MD a board-certified and fellowship-trained gastrointestinal and hepatopancreatobiliary surgeon at AdventHealth Tampa he and his team are making strides in bile duct cancer treatment What is Bile Duct Cancer?Bile is a substance that helps you digest food Your liver makes bile that is then stored in your gallbladder Bile duct cancer is a type of cancer that affects the thin tubes (ducts) that carry bile from your liver and gallbladder and through to your small intestines There are different types of bile duct cancer but one of the most common is perihilar cholangiocarcinoma (also called perihilar duct cancer or Klatskin tumor) These tumors start where the two bile ducts leaving the liver merge A New Surgical Approach Improves Recovery — and OutcomesBile duct cancer surgery is nothing new but it’s a complex procedure that must be done by a specially trained surgeon most cancer centers conduct bile duct surgery as an open procedure Since bile duct tumors are in a difficult part of the body where blood supply goes to the liver cancer can easily invade the artery and vein that bring blood to the liver And that means surgeons must be prepared to perform a procedure called vascular resection to remove the affected blood vessels “While we can usually see if blood vessels are invaded by cancer ahead of time we frequently find this cancer invasion while we’re performing the surgery,” says Dr we need to have a full team ready to go in the operating room for these reconstructions One of the other things that complicates this procedure is that surgeons must reconstruct the bile duct before finishing the surgery — a process that’s difficult to do with minimally invasive techniques Sucandy and his team have been using robotic surgery to complete this reconstruction with precision since 2019 “What’s unusual about our team is that we can offer this minimally invasive approach,” says Dr “And it makes such a difference to patients from across the country Successful Bile Duct Cancer Care Takes a TeamBecause of the complexity of treating bile duct cancer many patients need chemotherapy before their surgery to shrink tumors or after surgery to keep cancer at bay This complication makes the need for multidisciplinary coordination among the cancer team even greater Fortunately, even before beginning bile duct cancer treatment, the AdventHealth cancer team comes together to discuss each bile duct cancer patient From finding the right time to perform surgery so we can limit disruptions to their chemotherapy to finding the best treatment options for them we work in tandem to create a comprehensive and highly personalized care plan Benefits of Minimally Invasive Bile Duct Cancer SurgeryPatients with bile duct cancer can gain many benefits from this robotic procedure • Faster return to chemotherapy• Less blood loss• Quicker return to daily activities and work• Reduced wound complications• Shorter recovery time While this procedure is only performed in the U.S Sucandy has worked with expert surgeons in Italy and Portugal on research to study improve and share this technique so we can bring this procedure to more patients worldwide “We’re able to sync with other centers on research and put our experience in robotic bile duct cancer surgery together,” says Dr “Collaboration with other centers means we’re bringing a lot of experience of robotic techniques that can help patients.” Stopping Cancer with the Latest TechWith robotic surgery bile duct cancer surgery has gotten more precise more effective and less invasive — which means a faster recovery and better outcomes for you Take charge of your health. Learn more about robotic cancer surgery at AdventHealth Tampa Iswanto Sucandy is a highly experienced board-certified surgeon with dual fellowship training in Hepatobiliary and Pancreatic Surgery as well as Advanced Gastrointestinal Minimally Invasive Surgery He primarily specializes in minimally invasive and robotic surgery for disorders of the liver Our website uses cookies. Please review our privacy policy to find out more about the cookies we use Browsing our website means you accept these terms Metrics details Bile acids (BA) are supposed to cause metabolic alterations after bariatric surgery (BS) Here we report the longitudinal dynamics of the human BA metabolome by LC–MS/MS after BS versus low calory diet (LCD) in two obesity cohorts over 12 months Rapid and persistent oscillations of 23 BA subspecies could be identified with highly specific patterns in BS vs and TLCA represent most promising candidates for drug development According to the bilokine hypothesis7 each single BA should be measured by high end techniques such as LC–MS/MS upon BS in order to obtain reliable data and to identify single druggable BA with physiological impact The human BA metabolome has not yet been monitored systematically and longitudinally in obesity and during weight loss by LC–MS/MS.The present study deciphers the oscillations of 16 single BA species (and 7 classes of BA) by LC–MS/MS in obesity and longitudinally during weight loss induced by RYGB vs LCD in two large and deeply characterized cohorts of patients Specific patterns of BA alterations are presented as a basis and data resource for addressing single BA as potential drug targets in obesity and associated metabolic diseases These both cohorts comprise 91 patients undergoing RYGB and 88 patients undergoing LCD The multi-professional and standardized LCD program consisted of three phases over 52 weeks (months 1–3: only liquid and very low calory diet allowed by ingestion of a specified formula diet 5 times/day with a maximum of 800 kcal/day; months 4–5: transition phase with gradual replacement of liquid diet by mixed low calory meals together with ongoing life style modification; months 6–12: stabilization phase with low calory diet by normal meals and sustained life style modification) The study was approved by the local ethical committee at the University of Giessen All patients gave informed and written consent and were informed about the aim of the study Oscillations and dynamics of the human BA metabolome in obesity before and during weight lowering therapies by RYGB and LCD pie charts) and concentration (lower panels bar diagrams) of BA subspecies in n = 91 patients (75 females 16 males) before (V0) and after RYGB (V3: 3 months; V12: 12 months) is presented Percentage is relatively to the total BA pool that increases continuously after RYGB bar diagrams) of BA subspecies in n = 88 patients (59 females 29 males) before (V0) and during LCD (V3: 3 months; V12: 12 months) is presented patients were only allowed to ingest a very low calory and liquid formula diet patients switched to a normal hypo-caloric diet Percentage is relatively to the total BA pool that increases transiently at V3 The absolute concentrations of BA (nM) were measured by LC–MS/MS The Friedman`s two way analysis of variance by ranks was used for related samples and P values were corrected for multiple testing according to Bonferroni´s method B) Free and conjugated BA before and after RYGB versus LCD in obese patients secondary and tertiary BA before and after RYGB versus LCD in obese patients F) Percentage composition of 16 single BA before and after RYGB versus LCD in obese patients The concentration of each single BA (nM) was measured by LC–MS/MS and is given in the extended data files 1 and 2 During LCD, percentage (Fig. 1B, upper panel) and absolute concentration (Fig. 1B lower panel) of glycine-conjugated BA significantly increased from V0 to V3 and returned to pre-study levels at V12 The percentage of taurine-conjugated BA transiently increased at V3 and their absolute concentrations transiently increased at V3 as well alterations of BA during LCD are of transient nature Total and conjugated BA are increased at V3 lower panel) whereas tertiary BA remained unchanged in the context of rising total and free BA A stepwise and strong increase of primary and secondary but not tertiary BA characterizes the dynamics of BA after RYGB During LCD, percentage (Fig. 1D, upper panel) and concentrations of primary BA (Fig. 1D lower panel) were significantly lower at V12 when compared to pre-study levels Secondary BA had a higher percentage at V12 whereas their absolute concentrations remained unchanged during LCD over 12 months Relative percentage of tertiary BA showed only marginal variation the absolute concentration of tertiary BA significantly increased at V3 and returned to pre-study levels at V12 Primary BA are lower after 12 months of LCD whereas tertiary BA are transiently increased at V3 Nanomolar concentrations of 16 human bile acids (BA) by LC–MS/MS after Roux-en-Y gastric bypass (RYGB) (panel A) and during low calory diet (LCD) Mean ± SEM is presented by the bar diagrams These data indicate a similar extent of catabolism in the RYGB and LCD groups from V0 to V3 weight loss significantly continued in RYGB for 12 months cumulating in a loss of 36% of original body weight weight stabilized after a gradual switch back to regular diet from liquid formula diet continued catabolism might be responsible for persistent alterations of BA after RYGB diet-induced catabolism over three months is able to cause similar effects on bile acids catabolism per se induces BA alterations independent of the surgical procedure a systematic and longitudinal quantification by LC–MS/MS of all human bile acid subspecies with absolute concentrations and relative (percentage) changes has not yet been available so far Of major interest for potential drug development and GLCA were identified to increase rapidly after RYGB GLCA and TLCA remained increased over 12 months GLCA and TLCA fullfill the following three criteria simultaneously: a) rapid increase after RYGB c) rapid increase after LCD during the very low calory phase with ingestion of liquid diet only these three BA should be selected for evaluation of their potential pharmacological effects in obesity The present biostatistical data on single bile acids measured by LC–MS/MS might provide a basis for the future development of human reference ranges as typical for clinical cohorts we observed a high grade of inter-individual variability of bile acids This represents a limitation of the study and might lead to the definition of relatively wide normal ranges Characteristic patterns of BA metabolome oscillations during weight-lowering therapies in obese patients The large “Bilometer” scales summarize overall and longitudinal oscillations of BA main species (total secondary) during RYGB versus LCD in n = 179 obese patients RYGB causes rapid and persistent increases of BA concentrations a very low calory and strictly liquid diet over three months mimics the effects of RYGB at V3 but has no persistent effects on BA metabolome The small “Bilometer” scales indicate the identified and specific alterations of TLCA For identification of 16 single BA at each time point in both cohorts please refer to extended data files 1 and 2 and internal standard blank (water) were placed in a 1.5 mL Eppendorf tube 20 μL of internal standard mix containing stable isotope labelled BA for all analytes except for HDCA and its conjugates was added 30 µL of 1 mol/L HCl was added and proteins were precipitated with 1 mL of acetonitrile The dried supernatants were then redissolved in 100 μL of methanol/water (30/70 The LC–MS/MS system consisted of a 1200 series binary pump and a hybrid triple quadrupole linear ion trap mass spectrometer API 4000 Q-Trap (Applied Biosystems The LC analysis was performed using a NUCLEOSHELL RP18 core–shell column (Machery & Nagel Germany) with dimensions of 50 mm × 2 mm and a particle size of 2.7 μm The mobile phase consisted of methanol/water (1/9 v/v) as mobile phase A and methanol as mobile phase B both containing 0.1% ammonium hydroxide (25%) and 10 mmol/L ammonium acetate (pH 9) followed by a stepwise linear decrease to 53% A at 0.1 min A column wash was performed using 100% B for 0.5 min followed by re-equilibration using 100% A for 0.6 min at a flow rate of 0.8 mL/min BA were monitored by selected reaction monitoring using a product ion of m/z 74 for glyco-conjugated m/z 80 for tauro-conjugated and the precursor ion for free BA species Quantification is based on the ratio of area counts of the analyte to its respective stable isotope internal standard HDCA species were related to their respective UDCA Calibration was performed using six levels generated by BA addition to charcoal-stripped and pooled serum from healthy donors The abbreviations of bile acid subspecies are given here: Primary bile acids: CA glycohyodeoycholic acid) were under the detection limit of LC–MS/MS As described earlier38 the ROBS database was programmed with FileMaker Pro 13 a relational database management program which runs on Windows and Mac Systems as a multi-user system An additional web interface can be programmed for database applications on iOs- and android-compatible devices Pseudonymization runs over a 256 bit encoded database that is separated from the general network target appointments for the visits are fixed automatically A separate database with an individual input mask was programmed for data entry patient visits data were extracted into the computational software program IBM SPSS statistics The Friedman`s two way analysis of variance by ranks was used for related samples and P values were corrected for multiple testing according to Bonferroni´s method (Supplementary information in extended data files 1 and 2) Data availability and data on personal request Each single bile acid concentration at any time point in individual patients can be retrieved from extended data files 3 and 4 the authors are able to provide researches with yet unpublished data on correlations of 16 bile acid species with a huge variety of measured parameters in the study cohort Individual requests can be sent to andreas.schaeffler@innere.med.uni-giessen.de These parameters comprise 15 anthropometric items n = 50.400 possible correlations of interest can potentially be investigated 34 classified socio-economic variables were documented authors can provide associations of bile acid concentrations at V0 with yet unpublished gene expression data in subcutaneous and visceral adipose tissue of patients who underwent RYGB Bile acid control of metabolism and inflammation in obesity Browning of the white adipose tissue regulation: new insights into nutritional and metabolic relevance in health and diseases Bargut, T. C. L. et al. Browning of white adipose tissue: lessons from experimental models. Hormone Mol. Biol. Clin. Investing. https://doi.org/10.1515/hmbci-2016-0051 (2017) Regulation of body weight: Lessons learned from bariatric surgery The emerging role of bile acids in white adipose tissue Evidence of functional bile acid signaling pathways in adipocytes Schmid, A. et al. Downregulation of CTRP-3 by weight loss in vivo and by bile acids and incretins in adipocytes in vitro. Int. J. Mol. Sci. https://doi.org/10.3390/ijms21218168 (2020) Evidence-based German guidelines for surgery for obesity Does bariatric surgery improve adipose tissue function? Bariatric surgery and type 2 diabetes: are there weight loss-independent therapeutic effects of upper gastrointestinal bypass? Bariatric surgery and obesity: influence on the incretins Bariatric surgery - time to replace with GLP-1? The role of GLP-1 in postprandial glucose metabolism after bariatric surgery: a narrative review of human GLP-1 receptor antagonist studies Recent advances in the mechanisms underlying the beneficial effects of bariatric and metabolic surgery The role of bile acids in reducing the metabolic complications of obesity after bariatric surgery: a systematic review Weight loss induced by Roux-en-Y gastric bypass but not laparoscopic adjustable gastric banding increases circulating bile acids Roux-en-Y gastric bypass normalizes the blunted postprandial bile acid excursion associated with obesity Enhanced fasting and post-prandial plasma bile acid responses after Roux-en-Y gastric bypass surgery Bile acids increase independently from hypocaloric restriction after bariatric surgery Improvements in glucose metabolism early after gastric bypass surgery are not explained by increases in total bile acids and fibroblast growth factor 19 concentrations Serum bile acid along with plasma incretins and serum high-molecular weight adiponectin levels are increased after bariatric surgery Serum bile acids are higher in humans with prior gastric bypass: potential contribution to improved glucose and lipid metabolism Influence of Roux-en-Y gastric bypass on plasma bile acid profiles: a comparative study between rats The role of small heterodimer partner in nonalcoholic fatty liver disease improvement after sleeve gastrectomy in mice and FGF-19: Metabolic behavior patterns after Roux-en-Y gastric bypass and vertical sleeve gastrectomy High-throughput mediation analysis of human proteome and metabolome identifies mediators of post-bariatric surgical diabetes control Assessment of the role of FGF15 in mediating the metabolic outcomes of murine vertical sleeve gastrectomy (VSG) TGR5 contributes to glucoregulatory improvements after vertical sleeve gastrectomy in mice Targeting bile acid-activated receptors in bariatric surgery Metabolic effects of bile acids: potential role in bariatric surgery and signaling as central drivers for metabolic improvements after bariatric surgery Early increases in bile acids post Roux-en-Y gastric bypass are driven by insulin-sensitizing Progranulin serum levels and gene expression in subcutaneous vs visceral adipose tissue of severely obese patients undergoing bariatric surgery Chenodeoxycholic Acid as a Potential prognostic marker for Roux-En-Y gastric bypass in chinese obese patients Relevance in the use of appropriate internal standards for accurate Quantification using LC-MS/MS: Tauro-conjugated bile acids as an example Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry Download references This work was supported by grants of the von Behring-Roentgen foundation (69-0025) and of the German Research Association DFG (HO 6929/2-1 KA 1846/4-1).We would like to thank the patients for participating in this research project Open Access funding enabled and organized by Projekt DEAL Basic Research Laboratory of Molecular Endocrinology Institute of Clinical Chemistry and Laboratory Medicine Department of Internal Medicine – Endocrinology did the biometrics and was responsibel for biochemial measurements performed the quantification of bile acids by LC–MS/MS and R.B contributed to the study logistic procedures and did the data file entries JP was our advisor for data analysis and statistical calculations We confirm that all methods were performed in accordance with the relevant guidelines and regulations All patients gave informed consent and were informed about the aim of the study Data anonymization and privacy policy were accurately applied Download citation DOI: https://doi.org/10.1038/s41598-024-75831-1 In a surprising breakthrough, researchers at Weill Cornell Medicine have identified bile acid molecules—modified by the gut microbiota—that can block androgen receptor signaling and enhance the immune system’s ability to fight tumors. Published in Cell the study highlights the potential of gut-derived metabolites to influence hormone pathways and shape cancer therapy outcomes no one has previously discovered molecules like these bile acids that can interact with the androgen receptor in this way,” said Chun-Jun Guo co-senior author and associate professor of immunology in medicine undergo further chemical modification by intestinal bacteria “We discovered more than fifty different bile acid molecules modified by the microbiota—many of which had never been identified before,” said Guo Noting their shared steroid backbone with hormones like testosterone and estrogen researchers questioned whether the bile acids might interact with sex hormone receptors “It seemed like a wild idea at the time,” he added Four bile acids—one newly identified and three known compounds—were found to antagonize the androgen receptor This receptor plays a key role in development and immune cell regulation Previous studies had shown that blocking the androgen receptor could enhance these T cells’ ability to destroy cancer cells administration of the bile acids resulted in a “potent anti-tumor response.” Nicholas Collins co-senior author and assistant professor of immunology in medicine “Our results suggest that these altered bile acids help shrink tumors by enhancing T cells’ ability to survive within the tumor and destroy cancer cells.” co-senior author and director of the Jill Roberts Institute emphasized the clinical relevance: “This study highlights the profound and evolving partnership between the human host and its gut microbiota emphasizing the importance of integrating microbial activity into the design of future cancer therapies.” The team is now exploring approaches to therapeutically harness these metabolites including genetically engineering gut bacteria to produce them or administering them directly alongside conventional cancer treatments While the anticancer potential of these bile acids is promising researchers remain cautious about their systemic effects and the influence of variables like diet Salk scientists discover that removing bile acid-creating protein BAAT and adding bile acid UDCA controls tumor growth in mice with liver cancer; UDCA dietary supplements may be quick solution to improving liver cancer patient outcomes To understand why immunotherapy may be less effective in treating liver cancer scientists at the Salk Institute took a closer look at how the immune system and liver interact While studying mouse and human liver tumors they discovered that certain bile acids in the liver could affect the activity of cancer-fighting immune cells The researchers identified several liver bile acids associated with impaired T cell function and tumor growth and were able to successfully halt tumor growth and shrink existing tumors by blocking their production They also saw that one specific bile acid—ursodeoxycholic acid (UDCA) —had a positive effect on T cell activity in the liver boosting the levels of this bile acid through dietary supplementation was enough to control tumor growth in mice with liver cancer Because these supplements are already commercially available and used to help treat other liver diseases the researchers are hopeful that UDCA could be incorporated into liver cancer treatment plans to make immunotherapy more effective for these patients The findings, published in Science on January 9 help explain why immune cells behave differently in different tumor environments and offer several new molecular targets for improving liver cancer treatment and immunotherapy “How do organ-specific properties and processes influence the immune response?” asks Professor Susan Kaech senior author of the study and director of Salk’s NOMIS Center for Immunobiology and Microbial Pathogenesis “Livers have a particularly unique environment but we didn’t really understand how it was affecting the immune and cancer cells By investigating these liver-specific features we have identified several potential ways to regulate bile acids The liver produces more than 100 different bile acids which move through the intestines where they play important roles in digestion they must function around these bile acids Previous research has shown that excess bile acids can indicate poor health and exacerbate cancer but because most studies failed to separate the effects of each individual bile acid their specific roles in cancer remained unclear “Considering how T cell performance varies across different organs and tumors puts us at a great vantage point for looking at ways to optimize cancer treatment,” says Siva Karthik Varanasi former postdoctoral researcher in Kaech’s lab and current assistant professor at the University of Massachusetts Chan Medical School we’re able to see that bile acids in the liver are hugely influencing T cells’ ability to do their job and therefore may be a useful therapeutic target.” To explore the unique features of the liver tumor environment the Salk team first catalogued which bile acids were present in human liver cancer biopsies They discovered that the liver tumor samples had elevated levels of conjugated bile acids then asked whether this class of bile acids was directly contributing to cancer development After removing a protein called BAAT that makes conjugated bile acids they saw a reduction in tumor burden in their mice—a strong indicator that regulating BAAT levels in humans with liver cancer may improve their response to immunotherapy they separated out 20 different bile acids to see their individual impacts on T cell health which induced oxidative stress—a molecular imbalance that can lead to cell and tissue damage Secondary bile acids were much more influential with two showing especially significant effects: LCA and UDCA LCA impaired T cell function by causing endoplasmic reticulum stress wherein cells can no longer properly fold and modify proteins promoting the recruitment of immune cells to the liver Dietary supplementation of UDCA was enough to control tumor growth in mice with liver cancer offering an easily translatable approach to boosting immunotherapy efficacy in liver cancer patients These findings may shape the future of liver cancer treatment demonstrating that reducing BAAT and increasing UDCA can control tumor growth and improve T cell and immunotherapy efficacy “We’re already a huge step ahead when it comes to translating our findings to the clinic because UDCA supplementation is already used to treat liver disease and could easily be tested in liver cancer next,” says Kaech “We are really excited to also explore the role of the gut microbiome in all of this since bile acids are a huge part of that picture—how can we manipulate ‘good’ and ‘bad’ bacteria in the microbiome to further regulate bile acid levels How does the microbiome change during liver cancer Could probiotics be a therapeutic approach?” In addition to exploring dietary and microbiome manipulations that could help with liver cancer the team is curious to see if other conditions could be treated by targeting BAAT they believe chronic liver disease and obesity may benefit from the same reduction of conjugated bile acids and Gen-Sheng Feng of UC San Diego; Souradipta Ganguly and Debanjan Dhar of UC San Diego and Sanford Burnham Prebys Medical Discovery Institute; Marcos Teneche and Peter Adams of Sanford Burnham Prebys Medical Discovery Institute; Isaac Jensen and Donna Farber of Columbia University; Andrea Schietinger of Memorial Sloan Kettering Cancer Center Weill Cornell Graduate School of Medical Sciences and Parker Institute for Cancer Immunotherapy; and Mark Sundrud of Dartmouth College The work was supported by the National Institutes of Health (NCI CCSG: P30 014195 Audrey Geisel endowed Chair of Biomedical Science Altman Clinical Translational Research Institute (KL2TR001444) San Diego Digestive Diseases Research Center Unlocking the secrets of life itself is the driving force behind the Salk Institute award-winning scientists pushes the boundaries of knowledge in areas such as neuroscience developer of the first safe and effective polio vaccine nonprofit research organization and architectural landmark: small by choice By Brittany Phillips tissues or organisms – play an integral role in various diseases and studying the many metabolites (metabolomics) can teach us how the body works in ways that help researchers develop new treatments  As the prevalence of potentially life-threatening immunoglobulin E (IgE)-mediated food allergies continues to rise the development of therapies requires a comprehensive understanding of immune tolerance to allergens This includes how tolerance differs between individuals with and without food allergies associate professor of pediatrics and director of Bioinformatics at the UNC FAI in the Department of Pediatrics at the UNC School of Medicine is now identifying key metabolite pathways associated with food allergy and differential responses to one of the treatments for food allergy “Understanding how food allergies and their therapies (of which there are only two) work is key to discovering new treatments for food allergy,” said Virkud “We hope that using metabolomics as a tool to explore the immune system will help us learn more about immune tolerance to foods to help treat our patients with food allergies.” Published in the journal Pediatric Allergy and Immunology Virkud’s research studied children with food allergy compared to participants without food allergies and demonstrated that bile acids were higher in children with food allergies and lower in children without food allergies Researchers then looked at metabolite profiles in children receiving OIT and compared participants who were able to maintain the protection of OIT even after stopping OIT for a month (remission) to those who lost protection soon after stopping It was found that those who lost protection also had generally higher levels of bile acids those with remission had higher levels of two specific bile acids Virkud said that while the main job of bile acids is to help with digestion in the gut it turns out that bile acids are also important for controlling immune cells other studies demonstrated that lithocholates control the same immune cells that have been shown to be important for remission,” said Virkud “Connecting our data with these other studies suggests that certain bile acid profiles may be important for determining whether someone with food allergies has a better or worse outcome on OIT We also found differences in other metabolites (histidines and poly-unsaturated fatty acids) that have known roles in the immune system and we look forward to studying all of these connections further in the future.” The study examined metabolomic profiles of children with food allergy in multiethnic cohorts including: Genetics of Asthma in Costa Rica [GACRS] the Vitamin D Antenatal Asthma Reduction Trial [VDAART] infant cohort and the Peanut Oral Immunotherapy (PNOIT) trial Researchers aimed to determine key pathways of interest and associate the metabolomic profiles with therapeutic OIT outcomes Researchers also used repeated sampling on the same participants to define pathophysiologic differences between those who had transient protection versus sustained unresponsiveness while on OIT Virkud said her team was surprised by the findings “Clinical allergists and immunologists don’t generally spend a lot of time thinking about bile acids and many of the associations between the specific bile acids and immune cells we are interested in weren’t discovered until 2019-2020,” said Virkud “It was quite exciting to come across those papers that finally gave some biological significance to our findings.” The study concludes that further functional validation of these metabolic pathways in the context of allergic tolerance may both help identify patients most likely to benefit from OIT and guide the development of new pathways and potential therapies that can improve outcomes for patients with food allergies Written by Brittany Phillips Have a question? Need to reach the UNC Health News Team Call: (984) 974-1140 UNC Health Social Media Terms of Use Site Map Metrics details Evidence suggests that complex interactions among the gut microbiome and brain have important etiological and therapeutic implications in major depressive disorder (MDD) the influence of microbiome-gut-brain cross-talk on cognitive impairment in MDD remains poorly characterized We performed serum metabolomic profiling on 104 patients with MDD and 77 healthy controls (HCs) and also performed fecal metagenomic sequencing on a subset of these individuals The findings were validated in a separate cohort that included 40 patients with MDD and 40 HCs using serum-targeted metabolomics Abnormal bile acid metabolism was observed in patients with MDD The following gut microbiota corresponded to changes in bile acid metabolism and enzyme activities involved in the bile acid metabolic pathway including Lachnospiraceae (Blautia_massiliensis a combinatorial marker classifier that robustly differentiated patients with MDD from HCs was identified this study provides insights into the gut-brain interactions in the cognitive phenotype of MDD indicating a potential therapeutic strategy for MDD-associated cognitive impairment by targeting the gut microbiota and bile acid metabolism This study aims to address this knowledge gap We conducted a multi-omics analysis of the gut microbiota and serum metabolome using data from 104 patients with MDD and 77 healthy controls (HCs) The changes in metabolomics were validated in another cohort that included 40 patents with MDD and 40 HCs The multi-omics approach has the advantage of using multidimensional data which benefits the identification of biomarkers of cognitive deficits in MDD The current study contributes to our understanding of the role of the gut-brain axis in the pathogenesis of cognitive impairment in MDD The research also offers a crucial resource for future biomarker studies and the creation of innovative intervention methods for preventing and treating cognitive dysfunction in MDD The workflow diagram of this study systematically delineates the data collection and analytical pipeline for both the MDD (major depressive disorder) and HC (healthy Control) groups execution of differential and functional analyses THINC-it objective cognition was calculated as the average of the Z-scores from the four objective tests while composite cognition was determined by the average of all five tests in the THINC-it battery and Trials showed a positive correlation with the severity of cognitive impairment and Trails were multiplied by -1 to reverse the scoring (i.e. with higher scores indicating better cognitive performance All analyses were performed using SPSS (version 25) and R software (R-4.3.1) Continuous variables were compared using the Student’s t-test and Wilcoxon’s rank sum-test Categorical data were analyzed using the chi-square or Fisher’s exact tests Analysis of Covariance (ANCOVA) was used to compare cognitive function between the two groups VIP values were derived from the Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) and were visualized using the R package MetaboAnalystR The different metabolites were assessed using Student’s t-test and the false discovery rate (FDR) was adjusted using the Benjamini–Hochberg method Differential metabolites were determined based on VIP > 1 and FDR < 0.05 The relationship between the gut microbiota and cognitive function was conducted via Co-inertia analysis (CIA) The random 4.6-12 package was used to construct a classifier to discriminate between MDD and HCs based on a random forest model with the metabolites (bile acid and gut-derived neurotransmitter-like metabolites) and metagenomic species (species with LDA > 2.5 and P-value < 0.05) The variables with the highest predictive power were identified as those that yielded the maximum Area Under the Curve (AUC) Lasso penalized regression (LASSO) is a machine-learning method Feature selection was implemented by adding an L1 regularization term to the loss function We used LASSO to determine BAs that were related to cognitive function using the R package glmnet (version 2.0-13) and cognitive performance were analyzed using Spearman’s rank correlation coefficient and visualized via the Cytoscape software (version 3.10.0) A There is no significance alpha diversity differences between the two groups B PERMANOVA analysis reveals significant differences in bacterial signatures between the two groups (Bray-Curtis distance C Boxplots depict the differential abundances of species between patients with MDD and HCs (LDA > 2.5 and P-value < 0.05) Statistical analyses indicate significant variations in the abundance of species with P-values indicating levels of significance as follows: *P-value < 0.05 whereas the species enriched in MDD are shown in yellow D The boxplot demonstrates the significantly different relative abundance of functional pathways between MDD and HC groups (*P-value < 0.05 The above results suggest that it is essential to explore the influence of the gut microbiota on metabolic processes in conjunction with metabolomics we used untargeted LC-MS to analyze changes in the serum metabolic profile and its response to microbiota disturbances in patients with MDD A The OPLS-DA plot shows the discrimination between MDD and HCs The samples in green represent individuals with MDD whereas the samples in orange are from HCs (Q2 = 0.903 B Volcano plot illustrates the significant differences in metabolites between patients with MDD and HCs with 310 enriched in the patients with MDD (highlighted in red) and 926 enriched in the HCs (highlighted in green) Bubble plots illustrate the notably enriched KEGG pathways according to serum untargeted metabolomics (C) and targeted metabolomics profiling (D) The point size represents the number of differential metabolites involved in the respective pathways The rich factor is the ratio of the number of differential metabolites annotated within a given pathway to the total number of metabolites annotated for that pathway E Differences in global metabolic pathways in serum of MDD and HCs with red in the box indicating upregulated metabolites and green indicating downregulated metabolites Boxes with different colors represent the metabolic pathways the bile acid pathway was generally altered F Comparison of BAs and neurotransmitter-like metabolites in MDD vs Blue indicates HCs and yellow indicates patients with MDD (VIP > 1 and FDR < 0.05) we confirmed abnormal bile acid metabolism in patients with MDD Changes were observed in both metabolites and enzymes involved in these pathways These results partly explain the mechanism by which gut microbiota mediates metabolic abnormalities in MDD A Co-inertia analysis of serum metabolome versus fecal microbiota Fecal and serum samples are shown as squares and circles respectively; serum and fecal samples from the same individual are connected by lines Contributions of gut microbes to the five secondary BAs (B) and six neurotransmitter-like metabolites (C) we utilized correlation analysis and a best-fit multiple regression model to examine the correlation between gut microbiota and serum metabolites This approach allowed us to identify the key predictors of metabolite levels The size of the bubbles in the visualization represents the importance of each variable The bar chart on the right indicates the variance explained by the random forest model D Receiver operating characteristic (ROC) curve using secondary bile acids (red) and a combination with gut microbial (green) The right-hand plot displays the MDA score for the potential 20 markers in the model (yellow indicates enrichment in the MDD group Our findings suggest that microbes and gut-derived BAs may be potential diagnostic biomarkers of MDD and that the efficiency of diagnosis is comparable to that of widely known neurotransmitters A Schematic diagram of the main pathway of bile acid metabolism and involved enzymes Bile acids (BAs) enriched in MDD are shown in light red while those depleted in MDD are shown in light green Dark red and dark green represent elevated or reduced enzymes B Boxplot illustrating the differential of the abundance of three enzymes in the bile acid metabolism pathway from patients with MDD and HCs *P-value < 0.05; **P-value < 0.01; ns C Bar graph stacking the average relative abundance of the top 10 species contributing to EC3.5.1.24 in the MDD and HCs D The differential of relative abundance of the top ten species contribution to EC3.5.1.24 in the MDD and HCs E Stacked bar plot of the average relative abundance of the top 10 contributing species to EC1.1.1.159 in the MDD and HC groups F Bar graph stacking the average relative abundance of the top 10 species contributing to EC1.1.1.159 in the MDD and HCs G The boxplot illustrates the differential of peripheral white blood cell ratios **P-value < 0.01; ***P-value < 0.001 H Spearman correlation between BAs and inflammatory factors these results suggest a mechanism by which gut microbes mediate abnormal bile acid metabolism in patients with MDD This finding suggests that abnormal bile acid metabolism may be associated with changes in levels of inflammatory factors in patients with MDD A Co-inertia analysis (CIA) of serum metabolome and cognition are shown as circles and squares Yellow represents patients with MDD and blue represents HCs Connection lines reflect the metabolome and cognition function in one individual B CIA of fecal microbiome and cognition function C Lasso regression shows how bile acid coefficient values varied with THINC-it Composite Cognition performance in different regularization intensity ranges (lambda mini = 0.030) accompanied by a partial likelihood bias to determine the number of key predictors in cognitive assessment D Lasso regression of THINC-it Objective Cognition Gut microbes that were selected by the random forest regression model association between BAs and cognition function derived from LASSO regression Shapes of circles denoting species (yellow represents enrichment in the MDD and blue represents depletion) diamonds are BAs (light red indicates upregulation in the MDD and light green indicates downregulation) and octagons represent cognition function red and green edges denote positive correlation and negative correlation This suggests that cognitive impairment in patients with MDD may be similar to the pathological process of neurodegenerative disorders Additional studies are required to validate these findings Cognitive symptoms in patients with MDD respond poorly to widely used drugs that primarily act on 5-Hydroxytryptamine (5-HT) and norepinephrine (NE) receptors suggesting that there may be other mechanisms underlying cognitive impairment These results demonstrate that bile acid metabolism is significantly abnormal in patients with MDD which is linked to changes in the gut microbiome and cognitive impairment identified a network of interactions within the gut-bile acid-brain axis in MDD patients with cognitive impairment These findings provide a new therapeutic target for cognitive symptoms in MDD and suggest that the development of future probiotics or drugs to target bile acid metabolic pathways may be beneficial for cognition in patients with MDD Changes in gut microbes may contribute to cognitive impairment in MDD and a similar pathological process may occur in the above diseases with cognitive dysfunction To further assess the relationship between the metabolites mentioned above and the gut microbiota we used a random forest model to screen metabolite-associated gut microbes L-Glutamic acid is widely related to most gut microbes and Blautia_massiliensis were the major positive constituents of metabolites in the tryptophan pathway These gut-derived metabolites have neurotransmitter-like functions and suggest that the regulation of gut microbes may be beneficial for neurotransmitter imbalance in patients with MDD Our work contributes to the current evidence base by identifying the microbes and neurotransmitter-like metabolites related to MDD our results suggest that disturbances in the gut microbe-bile acid crosstalk may contribute to the pathophysiology of depression and are related to the inflammatory pathway These collective findings suggest that the disruption of microbiota-bile acid crosstalk may promote inflammation and MDD disease phenotypes we hypothesized that microbiota manipulation and supplementation with anti-inflammatory BAs may hold promise as a therapeutic avenue for cognitive impairment in MDD in which most of the metabolites could not be identified We focused only on metabolites known to be relevant to gut microbiota and the brain such as BAs and neurotransmitter-like metabolites and overlooking certain serum metabolites that might be vital in the pathophysiology of MDD Although we validated our results in another cohort using targeted metabolomics the metagenomic results could not be verified the verification of microbes should be performed in future studies with larger sample sizes The potential influence of low-dose intermittent medication use on our results deserves careful consideration; although reports suggest that such doses and frequencies are acceptable the results should still be interpreted with caution the cross-sectional design could only establish a correlation between gut microbiota longitudinal studies are required to clarify the roles of the microbiome and BAs in the cognitive symptoms of MDD limited by the operability of cognitive assessment tools we did not assess all dimensions of cognitive function More comprehensive tools should be selected in future studies the specific mechanisms underlying the role of gut-bile acid-brain axis disturbance in cognitive impairment related to MDD should be further clarified using fecal microbiota transplantation (FMT) in animal studies our study has made several important contributions namely identifying abnormalities of gut microbes and bile acid metabolism in patients with MDD elucidating the interaction between the gut microbiome and highlighting changes in microbial enzyme activity in the bile acid metabolic pathway These findings clarify that the gut-bile acid-brain axis network may be correlated with cognitive impairment in MDD providing new perspectives on the fundamental pathophysiological processes They identify potential targets for treating cognitive impairments in MDD and provide a basis for developing novel medications to improve cognitive symptoms in MDD patients The metagenomic sequencing data reported in this paper can be found in the Genome Sequence Archive (GSA) database. https://ngdc.cncb.ac.cn/gsa/s/BrtRjm6D (CRA019246) Smith K. Mental health: a world of depression. Nature. 2014;515:181. https://doi.org/10.1038/515180a Bortolato B, Miskowiak KW, Köhler CA, Maes M, Fernandes BS, Berk M, et al. Cognitive remission: a novel objective for the treatment of major depression? BMC Med. 2016;14:9. https://doi.org/10.1186/s12916-016-0560-3 Reppermund S, Ising M, Lucae S, Zihl J. Cognitive impairment in unipolar depression is persistent and non-specific: further evidence for the final common pathway disorder hypothesis. Psychol Med. 2009;39:603–14. https://doi.org/10.1017/S003329170800411X Semkovska M, Quinlivan L, O’Grady T, Johnson R, Collins A, O’Connor J, et al. Cognitive function following a major depressive episode: a systematic review and meta-analysis. Lancet Psychiatry. 2019;6:851–61. https://doi.org/10.1016/S2215-0366(19)30291-3 Amin N, Liu J, Bonnechere B, MahmoudianDehkordi S, Arnold M, Batra R, et al. Interplay of metabolome and gut microbiome in individuals with major depressive disorder vs control individuals. JAMA Psychiatry. 2023;80:597–609. https://doi.org/10.1001/jamapsychiatry.2023.0685 Michaudel C, Sokol H. The gut microbiota at the service of immunometabolism. Cell Metab. 2020;32:514–23. https://doi.org/10.1016/j.cmet.2020.09.004 Liu P, Liu Z, Wang J, Wang J, Gao M, Zhang Y, et al. Immunoregulatory role of the gut microbiota in inflammatory depression. Nat Commun. 2024;15:3003. https://doi.org/10.1038/s41467-024-47273-w Mayneris-Perxachs J, Castells-Nobau A, Arnoriaga-Rodríguez M, Martin M, de la Vega-Correa L, Zapata C, et al. Microbiota alterations in proline metabolism impact depression. Cell Metab. 2022;34:681–701.e610. https://doi.org/10.1016/j.cmet.2022.04.001 Boehme M, Guzzetta KE, Bastiaanssen TFS, van de Wouw M, Moloney GM, Gual-Grau A, et al. Microbiota from young mice counteracts selective age-associated behavioral deficits. Nat Aging. 2021;1:666–76. https://doi.org/10.1038/s43587-021-00093-9 Li Z, Zhu H, Guo Y, Du X, Qin C. Gut microbiota regulate cognitive deficits and amyloid deposition in a model of Alzheimer’s disease. J Neurochem. 2020;155:448–61. https://doi.org/10.1111/jnc.15031 Fröhlich EE, Farzi A, Mayerhofer R, Reichmann F, Jačan A, Wagner B, et al. Cognitive impairment by antibiotic-induced gut dysbiosis: analysis of gut microbiota-brain communication. Brain Behav Immun. 2016;56:140–55. https://doi.org/10.1016/j.bbi.2016.02.020 Cryan JF, O’Riordan KJ, Sandhu K, Peterson V, Dinan TG. The gut microbiome in neurological disorders. Lancet Neurol. 2020;19:179–94. https://doi.org/10.1016/S1474-4422(19)30356-4 Naomi R, Embong H, Othman F, Ghazi HF, Maruthey N, Bahari H. Probiotics for Alzheimer’s disease: a systematic review. Nutrients. 2021;14:20. https://doi.org/10.3390/nu14010020 Yang J, Zheng P, Li Y, Wu J, Tan X, Zhou J, et al. Landscapes of bacterial and metabolic signatures and their interaction in major depressive disorders. Sci Adv. 2020;6:eaba8555. https://doi.org/10.1126/sciadv.aba8555 Wang Y, Zhou J, Ye J, Sun Z, He Y, Zhao Y, et al. Multi-omics reveal microbial determinants impacting the treatment outcome of antidepressants in major depressive disorder. Microbiome. 2023;11:195. https://doi.org/10.1186/s40168-023-01635-6 Sun N, Zhang J, Wang J, Liu Z, Wang X, Kang P, et al. Abnormal gut microbiota and bile acids in patients with first-episode major depressive disorder and correlation analysis. Psychiatry Clin Neurosci. 2022;76:321–8. https://doi.org/10.1111/pcn.13368 Zheng P, Zeng B, Zhou C, Liu M, Fang Z, Xu X, et al. Gut microbiome remodeling induces depressive-like behaviors through a pathway mediated by the host’s metabolism. Mol Psychiatry. 2016;21:786–96. https://doi.org/10.1038/mp.2016.44 Skonieczna-Żydecka K, Grochans E, Maciejewska D, Szkup M, Schneider-Matyka D, Jurczak A, et al. Faecal short chain fatty acids profile is changed in Polish depressive women. Nutrients. 2018;10:1939. https://doi.org/10.3390/nu10121939 MahmoudianDehkordi S, Arnold M, Nho K, Ahmad S, Jia W, Xie G, et al. Altered bile acid profile associates with cognitive impairment in Alzheimer’s disease-An emerging role for gut microbiome. Alzheimers Dement. 2019;15:76–92. https://doi.org/10.1016/j.jalz.2018.07.217 Dalile B, Van Oudenhove L, Vervliet B, Verbeke K. The role of short-chain fatty acids in microbiota-gut-brain communication. Nat Rev Gastroenterol Hepatol. 2019;16:461–78. https://doi.org/10.1038/s41575-019-0157-3 Sun J, Zhang Y, Kong Y, Ye T, Yu Q, Kumaran Satyanarayanan S, et al. Microbiota-derived metabolite Indoles induced aryl hydrocarbon receptor activation and inhibited neuroinflammation in APP/PS1 mice. Brain Behav Immun. 2022;106:76–88. https://doi.org/10.1016/j.bbi.2022.08.003 Parker A, Fonseca S, Carding SR. Gut microbes and metabolites as modulators of blood-brain barrier integrity and brain health. Gut Microbes. 2020;11:135–57. https://doi.org/10.1080/19490976.2019.1638722 Pańczyszyn-Trzewik P, Czechowska E, Stachowicz K, Sowa-Kućma M. The importance of α-Klotho in depression and cognitive impairment and its connection to glutamate neurotransmission-an up-to-date review. Int J Mol Sci. 2023;24:15268. https://doi.org/10.3390/ijms242015268 Li Z, Lai J, Zhang P, Ding J, Jiang J, Liu C, et al. Multi-omics analyses of serum metabolome, gut microbiome and brain function reveal dysregulated microbiota-gut-brain axis in bipolar depression. Mol Psychiatry. 2022;27:4123–35. https://doi.org/10.1038/s41380-022-01569-9 Xie Z, Huang J, Sun G, He S, Luo Z, Zhang L, et al. Integrated multi-omics analysis reveals gut microbiota dysbiosis and systemic disturbance in major depressive disorder. Psychiatry Res. 2024;334:115804. https://doi.org/10.1016/j.psychres.2024.115804 Liu P, Gao M, Liu Z, Zhang Y, Tu H, Lei L, et al. Gut microbiome composition linked to inflammatory factors and cognitive functions in first-episode, drug-naive major depressive disorder patients. Front Neurosci. 2021;15:800764. https://doi.org/10.3389/fnins.2021.800764 Dong Z, Xie Q, Yuan Y, Shen X, Hao Y, Li J, et al. Strain-level structure of gut microbiome showed potential association with cognitive function in major depressive disorder: a pilot study. J Affect Disord. 2023;341:236–47. https://doi.org/10.1016/j.jad.2023.08.129 Hou Y, Yao S, Hu S, Zhou Q, Han H, Yu X, et al. PSYCHOMETRIC properties of the Chinese version of the THINC-it tool for cognitive symptoms in patients with major depressive disorder. J Affect Disord. 2020;273:586–91. https://doi.org/10.1016/j.jad.2020.03.146 Beghini F, McIver LJ, Blanco-Míguez A, Dubois L, Asnicar F, Maharjan S, et al. Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3. Elife. 2021;10:e65088. https://doi.org/10.7554/eLife.65088 Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, et al. Metagenomic biomarker discovery and explanation. Genome Biol. 2011;12:R60. https://doi.org/10.1186/gb-2011-12-6-r60 Zhu F, Guo R, Wang W, Ju Y, Wang Q, Ma Q, et al. Transplantation of microbiota from drug-free patients with schizophrenia causes schizophrenia-like abnormal behaviors and dysregulated kynurenine metabolism in mice. Mol Psychiatry. 2020;25:2905–18. https://doi.org/10.1038/s41380-019-0475-4 Hashimoto K. Gut-microbiota-brain axis by bile acids in depression. Psychiatry Clin Neurosci. 2022;76:281. https://doi.org/10.1111/pcn.13370 Li XY, Zhang SY, Hong YZ, Chen ZG, Long Y, Yuan DH, et al. TGR5-mediated lateral hypothalamus-dCA3-dorsolateral septum circuit regulates depressive-like behavior in male mice. Neuron. 2024;112:1795–1814. https://doi.org/10.1016/j.neuron.2024.02.019 Wang K, Zhang Z, Hang J, Liu J, Guo F, Ding Y, et al. Microbial-host-isozyme analyses reveal microbial DPP4 as a potential antidiabetic target. Science. 2023;381:eadd5787. https://doi.org/10.1126/science.add5787 Zimmermann M, Zimmermann-Kogadeeva M, Wegmann R, Goodman AL. Mapping human microbiome drug metabolism by gut bacteria and their genes. Nature. 2019;570:462–7. https://doi.org/10.1038/s41586-019-1291-3 Jia W, Xie G, Jia W. Bile acid-microbiota crosstalk in gastrointestinal inflammation and carcinogenesis. Nat Rev Gastroenterol Hepatol. 2018;15:111–28. https://doi.org/10.1038/nrgastro.2017.119 Guzior DV, Okros M, Shivel M, Armwald B, Bridges C, Fu Y, et al. Bile salt hydrolase acyltransferase activity expands bile acid diversity. Nature. 2024;626:852–8. https://doi.org/10.1038/s41586-024-07017-8 Lin S, Wang S, Wang P, Tang C, Wang Z, Chen L, et al. Bile acids and their receptors in regulation of gut health and diseases. Prog Lipid Res. 2023;89:101210. https://doi.org/10.1016/j.plipres.2022.101210 Wahlström A, Sayin SI, Marschall HU, Bäckhed F. Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism. Cell Metab. 2016;24:41–50. https://doi.org/10.1016/j.cmet.2016.05.005 Agirman G, Hsiao EY. SnapShot: the microbiota-gut-brain axis. Cell. 2021;184:2524. https://doi.org/10.1016/j.cell.2021.03.022 Radjabzadeh D, Bosch JA, Uitterlinden AG, Zwinderman AH, Ikram MA, van Meurs JBJ, et al. Gut microbiome-wide association study of depressive symptoms. Nat Commun. 2022;13:7128. https://doi.org/10.1038/s41467-022-34502-3 Li D, Sun T, Tong Y, Le J, Yao Q, Tao J, et al. Gut-microbiome-expressed 3β-hydroxysteroid dehydrogenase degrades estradiol and is linked to depression in premenopausal females. Cell Metab. 2023;35:685–694.e685. https://doi.org/10.1016/j.cmet.2023.02.017 Li D, Liu R, Wang M, Peng R, Fu S, Fu A, et al. 3β-Hydroxysteroid dehydrogenase expressed by gut microbes degrades testosterone and is linked to depression in males. Cell Host Microbe. 2022;30:329–39. https://doi.org/10.1016/j.chom.2022.01.001 Badal VD, Vaccariello ED, Murray ER, Yu KE, Knight R, Jeste DV, et al. The gut microbiome, aging, and longevity: a systematic review. Nutrients 2020;12:3579. https://doi.org/10.3390/nu12123759 Chen G, Zhou X, Zhu Y, Shi W, Kong L. Gut microbiome characteristics in subjective cognitive decline, mild cognitive impairment and Alzheimer’s disease: a systematic review and meta-analysis. Eur J Neurol. 2023;30:3568–80. https://doi.org/10.1111/ene.15961 Marizzoni M, Mirabelli P, Mombelli E, Coppola L, Festari C, Lopizzo N, et al. A peripheral signature of Alzheimer’s disease featuring microbiota-gut-brain axis markers. Alzheimers Res Ther. 2023;15:101. https://doi.org/10.1186/s13195-023-01218-5 Verhaar BJH, Hendriksen HMA, de Leeuw FA, Doorduijn AS, van Leeuwenstijn M, Teunissen CE, et al. Gut microbiota composition is related to AD pathology. Front Immunol. 2021;12:794519. https://doi.org/10.3389/fimmu.2021.794519 Morais LH, Schreiber HLT, Mazmanian SK. The gut microbiota-brain axis in behaviour and brain disorders. Nat Rev Microbiol. 2021;19:241–55. https://doi.org/10.1038/s41579-020-00460-0 Ahmed H, Leyrolle Q, Koistinen V, Kärkkäinen O, Layé S, Delzenne N, et al. Microbiota-derived metabolites as drivers of gut-brain communication. Gut Microbes. 2022;14:2102878. https://doi.org/10.1080/19490976.2022.2102878 Ho CSH, Tay GWN, Wee HN, Ching J. The utility of amino acid metabolites in the diagnosis of major depressive disorder and correlations with depression severity. Int J Mol Sci. 2023;24:2231. https://doi.org/10.3390/ijms24032231 Bian X, Zhou N, Zhao Y, Fang Y, Li N, Zhang X, et al. Identification of proline, 1-pyrroline-5-carboxylate and glutamic acid as biomarkers of depression reflecting brain metabolism using carboxylomics, a new metabolomics method. Psychiatry Clin Neurosci. 2023;77:196–204. https://doi.org/10.1111/pcn.13517 Hashimoto K, Sawa A, Iyo M. Increased levels of glutamate in brains from patients with mood disorders. Biol Psychiatry. 2007;62:1310–6. https://doi.org/10.1016/j.biopsych.2007.03.017 Lener MS, Niciu MJ, Ballard ED, Park M, Park LT, Nugent AC, et al. Glutamate and gamma-aminobutyric acid systems in the pathophysiology of major depression and antidepressant response to ketamine. Biol Psychiatry. 2017;81:886–97. https://doi.org/10.1016/j.biopsych.2016.05.005 Zanos P, Gould TD. Mechanisms of ketamine action as an antidepressant. Mol Psychiatry. 2018;23:801–11. https://doi.org/10.1038/mp.2017.255 Czapski GA, Strosznajder JB. Glutamate and GABA in microglia-neuron cross-talk in Alzheimer’s disease. Int J Mol Sci. 2021;22:11677. https://doi.org/10.3390/ijms222111677 Sinha SR, Haileselassie Y, Nguyen LP, Tropini C, Wang M, Becker LS, et al. Dysbiosis-induced secondary bile acid deficiency promotes intestinal inflammation. Cell Host Microbe. 2020;27:659–70. https://doi.org/10.1016/j.chom.2020.01.021 Beurel E, Toups M, Nemeroff CB. The bidirectional relationship of depression and inflammation: double trouble. Neuron. 2020;107:234–56. https://doi.org/10.1016/j.neuron.2020.06.002 Cai J, Rimal B, Jiang C, Chiang JYL, Patterson AD. Bile acid metabolism and signaling, the microbiota, and metabolic disease. Pharmacol Ther. 2022;237:108238. https://doi.org/10.1016/j.pharmthera.2022.108238 Ren Z, Zhao L, Zhao M, Bao T, Chen T, Zhao A, et al. Increased intestinal bile acid absorption contributes to age-related cognitive impairment. Cell Rep Med. 2024;5:101543. https://doi.org/10.1016/j.xcrm.2024.101543 Khalaf K, Tornese P, Cocco A, Albanese A. Tauroursodeoxycholic acid: a potential therapeutic tool in neurodegenerative diseases. Transl Neurodegener. 2022;11:33. https://doi.org/10.1186/s40035-022-00307-z Zangerolamo L, Vettorazzi JF, Rosa LRO, Carneiro EM, Barbosa HCL. The bile acid TUDCA and neurodegenerative disorders: an overview. Life Sci. 2021;272:119252. https://doi.org/10.1016/j.lfs.2021.119252 Shapiro H, Thaiss CA, Levy M, Elinav E. The cross-talk between microbiota and the immune system: metabolites take center stage. Curr Opin Immunol. 2014;30:54–62. https://doi.org/10.1016/j.coi.2014.07.003 Hurley MJ, Bates R, Macnaughtan J, Schapira AHV. Bile acids and neurological disease. Pharmacol Ther. 2022;240:108311. https://doi.org/10.1016/j.pharmthera.2022.108311 Joyce SA, O’Malley D. Bile acids, bioactive signalling molecules in interoceptive gut-to-brain communication. J Physiol. 2022;600:2565–78. https://doi.org/10.1113/JP281727 Download references The authors would like to thank all the participants and the Biobank of the First Affiliated Hospital of Xi’an Jiaotong University for banking biological samples This work was funded by the Key Program of National Natural Science of China (No the National Natural Science Foundation of China (No the Key Research and Development Program of Shaanxi (No and the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University (No These authors contributed equally: Min Jia The First Affiliated Hospital of Xi’an Jiaotong University Shaanxi Belt and Road Joint Laboratory of Precision Medicine in Psychiatry Center for Immunological and Metabolic Diseases and WW designed the project; Participants were recruited by QM and DY performed the data analyses; MJ interpreted the results and wrote the paper which was revised by XM and YG; All authors contributed to the revision of the paper Download citation DOI: https://doi.org/10.1038/s41398-024-03211-4 Metrics details accompanied by the hepatic accumulation of bile salts while underlying profibrogenic mechanisms remain incompletely understood we evaluated the role of extracellular pH (pHe) on bile salt entry and hepatic stellate cell (HSC) activation and proliferation various proton pump inhibitors (PPI) were tested for their ability to prevent bile salt entry and HSC activation the PPI pantoprazole was employed in the 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine (DDC)-diet model of cholestatic liver fibrosis that slightly acidic pHe (7.2–7.3) enhanced bile salt accumulation in HSC and was a prerequisite to bile salt-induced HSC activation Pantoprazole in the DDC model exhibited antifibrotic effects We conclude that bile salt-induced activation of HSC may depend on the slightly acidic microenvironment present in the perisinusoidal space and modulation of pHi in HSC may offer a novel pharmacological target in cholestatic disease mechanisms underlying bile salt-induced fibrogenesis remain scarcely characterized some of these results have hardly been reproduced since initial discovery and in vivo evidence for the contribution of these mechanisms to liver fibrosis remains scarce glycin-conjugated bile salts require a more acidic pH to become protonated and passively enter living cells the perisinusoidal space (or space of Disse) The pHe may thus be a component of the cellular microenvironment crucial for bile salt-induced signaling in HSC In the present study we have tested this hypothesis and were able to show that pHe crucially modulates activation of HSC by bile salts and that intracellular pH (pHi) in HSC can be targeted by proton pump inhibitors (PPIs) to prevent such activation we found that the proton pump inhibitor pantoprazole (PPZ) ameliorated liver fibrosis in a mouse model of cholestatic liver fibrosis indicating that modification of pHi in HSC by targeted therapies might be therapeutically employed LX2 cells were stimulated with CDC (A) or GCDC (B) for 24 h at the concentrations indicated and αSMA expression was quantified by western blotting in relation to GAPDH C Cell viability was assessed using WST-1 assays after 24 h of stimulation D LX2 cells were incubated with CLF at pHe 7.3 for 1 h at the indicated concentrations and intracellular fluorescence was quantified (n = 4–5; p < 0.05 seems unable to activate HSC at standard cell culture conditions A possible explanation for this difference is the lower pKa and reduced ability for passive cell entry of GCDC compared to CDC After allowing this standard culture medium (cell-free) to adjust in the CO2 atmosphere for 24 h pH was measured and a pH of 7.64 ± 0.1 (n = 5) was found Adjusting the bicarbonate buffer system in the cell culture medium 7.3 and 7.2 was applied for subsequent experiments While pH modification was overall well-tolerated cell viability started to decline from pH 7.2 (not shown) αSMA protein expression was determined by Western Blotting and representative images are shown as well as quantitative analysis of αSMA these results suggest that extracellular pH known to be slightly acidic in the natural microenvironment of HSCs crucially determines (passive) bile salt uptake and (G)CDC-induced activation and proliferation of HSC LX2 cells were pre-treated with PPZ (2.5–80 µM) for 24 h in buffered culture medium at pHe 7.3 and bile salt accumulation (C) were determined LX2 cells were simultaneously stimulated with GCDC (100 µM) and PPZ (5–80 µM) for 24 h in buffered culture medium at pHe 7.3 as well as αSMA and col1α1 protein expression (E representative blot and densitometric analysis) DNA amount as a surrogate of proliferation (F) and cell viability This observation supports our view that higher pHi is associated with an increase in bile salts entrapment in HSCs our results indicate that pHi critically determines bile salt accumulation and bile salt-induced HSC activation and that inhibition of intracellular proton pumps prevent bile salt accumulation and inhibit bile salt-induced HSC activation LX2 cells were stimulated with amiloride (10 and 100 µM) or bafilomycin A1 (1 and 10 nM) for 24 h in buffered culture medium at pHe 7.3. Based on results from Figs. 2 and 3 PPZ (80 µM) was used as a positive control for pHi alteration pHi values (A) and bile salt accumulation (B) were determined experiments were repeated in presence of GCDC (100 µM) and pHi was determined (C) αSMA and col1α1 protein expression were determined by Western Blotting and representative images are shown as well as quantitative analysis of αSMA and col1α1 DNA amount as a surrogate of proliferation was quantified by Picogreen assays (E) Cell viability was quantified by WST-1 assays (F) LX2 cells were stimulated with TGFβ (10 ng/ml) for 24 h in presence or absence of PPZ (80 µM) amiloride (10 and 100 µM) and bafilomycin A1 (1 and 10 nM) αSMA protein expression was determined by Western Blotting and representative images are shown as well as quantitative analysis of αSMA To further clarify the role of potential pleiotropic, antifibrotic effects of the inhibitors applied, as opposed to specific action via pHi alteration, the effects of PPZ, amiloride and bafilomycin A1 on TGFβ-induced LX2 activation were determined. We found that amiloride 10 and 100 µM did not affect TGFβ-induced LX2 activation (Fig. 4G) PPZ ameliorated TGFβ-induced activation to some extent although with lower efficacy compared to that seen in bile salt-induced LX2 activation ameliorated LX2 activation also in this setting hinting toward an additional pleiotropic effect pHi modification in LX2 by various PPIs may prevent bile salt-induced HSCs activation and proliferation Primary murine cells were stimulated with GCDC (100 µM) for 24 h after 1 day isolation in buffered culture medium to test various pHe values (7.6–7.2) mHSC cells were incubated in presence of CLF at various pHe (7.6–7.2) for 1 h and accumulation was determined fluorometrically (B) mHSC cells were stimulated with PPZ (80 µM) amiloride (10 and 100 µM) or bafilomycin A1 (1 and 10 nM) for 24 h in buffered culture medium at pHe 7.2 experiments were repeated in presence of GCDC (100 µM) an acidic (extracellular) microenvironment appears to be a prerequisite for bile salt-induced activation of primary murine HSCs inhibition of intracellular bile salt accumulation by use of various PPIs was able to prevent bile salt-induced activation of mHSCs C57BL/6 male mice aged 4–6 weeks were fed with control or DDC diet (0.1%) and were administered with H2O as control or PPZ at 5 mg/kg Representative images for general macroscopic appearance Sirius red staining and IHC for αSMA are given in (A) Liver inflammation was semi-quantitatively assessed (B) and quantitative assessment (% of total area) is given for Masson staining (C) Sirius red staining (D) and IHC for αSMA (E) is presented alkaline phosphatase (H) and total bilirubin (I) are given compared to control; #p < 0.05; ##p < 0.01 these in vivo data suggest that PPZ does not prevent cholestasis liver damage or liver inflammation in the DDC mouse model of chronic cholestasis but specifically ameliorates activation of profibrogenic pathways and liver fibrosis which are known to develop an increased pHi compared to quiescent HSCs showed an increase in CLF accumulation compared to quiescent HSCs Lastly, PPZ was tested in a mouse model of chronic cholestatic liver fibrosis. PPZ did not alter cholestasis, liver damage or inflammation in the DDC-mouse model but ameliorated liver fibrosis, indicative of a specific antifibrotic effect of PPZ (Fig. 6) who reported pro-proliferative effects of bile salts in rat HSC even at standard culturing conditions little consideration had then been given to the importance of the pH microenvironment and authors did not report the pH of their culturing condition we cannot exclude that these may have been more acidic than our standard culture conditions both before mentioned studies were performed in rat HSC while our experiments were performed in mouse and human HSCs which may also account for the different findings To further exclude a relevant contribution of active bile salt uptake to bile salt-induced activation in LX2 We found that inhibition of both bile salt uptake transporters was without effect on (G)CDC-induced LX2 activation suggesting a predominant role for passive bile acid entry in our system the role of TGR5 for (G)CDC-induced activation of dormant HSC seems negligible these pleiotropic effects of PPZ may have contributed to the observed effects on HSC activation To exclude a solely antifibrotic class effect of PPIs we tested additional proton pump inhibitors Both the NHE inhibitor amiloride and the v-H+-ATPase inhibitor bafilomycin A1 were demonstrated to also lower pHi was associated with reduced bile salt accumulation and activation of LX2 that proton pump-inhibition prevents LX2 activation specifically by pHi alteration and inhibition of intracellular bile salt accumulation amiloride and bafilomycin A1 on TGFβ-induced LX2 activation While bafilomycin A1 indeed was antifibrotic in this setting PPZ was less effective in lowering TGFβ-induced LX2 activation than in lowering bile salt-induced LX2 activation and amiloride was without antifibrotic properties upon TGFβ-stimulation while we cannot completely exclude additional part of their antifibrotic effects in our systems seem indeed to be attributable to pHi lowering and prevention of bile salt entry This possibly can be explained by an increased sensibility toward infections in this highly susceptible cohort and does not necessarily exclude beneficial effects on fibrogenesis when PPI are applied in early stages of disease our results suggest that other pHi modulating agents for specific uptake by HSCs might deserve further development which might come without the increased risk of infection that is associated with PPZ administration the relatively acidic pHe in the perisinusoidal space favors the passive entry of protonated bile salt and the relatively alkaline pHi leads to ionized bile salt accumulation in HSCs bile salt-induced HSCs activation is inhibited by preventing bile salt accumulation via acidifying pHi Cell medium at pH of 7.6–7.2 was generated by adjusting the bicarbonate buffer system through proportionately mixing of regular DMEM and DMEM without sodium bicarbonate Cells were cultivated in a humidified atmosphere with 5% CO2 and 21% O2 at 37 °C LX2 cells were stimulated with TGF-β (Peprotech glycochenodeoxycholate (GCDC Sigma-Aldrich Cell viability was measured by WST assay (Roche with the EASY READER SFplus (SLT-Labinstruments The pHi of cells was measured by using the cell-permeable probe 3′-O-acetyl-2′,7′-bis(carboxyethyl)-5,6-carboxyfluoresceinacetoxymethylester (BCECF-AM) Cells were cultured in a 96-well plate at 37 °C and then incubated with BCECF-AM for 30 min at 37 °C in full humidity with 5% CO2 Cells were washed twice with the HEPES buffer Fluorescence intensity was determined using a fluorescent plate reader with an excitation wavelength of 450 nm/490 nm and 535 nm emission wavelength (PerSeptive Biosystems pH calibration was made applying high K+ solutions (140 mM) at different pH values (7.8 buffered with HEPES or MOPS and 10 µM Nigericin (Invitrogen Bile salt accumulation in LX2 was evaluated by fluorometric quantification of Cholyl-Lysyl-Fluorescein (CLF) Following pre-incubation at indicated pHe and in presence or absence of indicated inhibitors cells were incubated with CLF at indicated concentrations at 37 °C for 30 min and washed twice with HBSS Fluorescence measurement was performed with 100 µL/well of HEPES buffer in a plate reader (Cyto Fluor 4000 PerSeptive Biosystems USA) at wavelength of excitation 485 nm and emission 530 nm Total DNA amount in cell culture as a surrogate of cell number was determined by the PicoGreen® dsDNA assay (Invitrogen USA) according to the manufacturer’s protocol with a CytoFluor Multi-Well Plate Reader Series 4000 (PerSeptive Biosystems mRNA Expression was calculated according to ΔΔCT method with β-actin as the housekeeping gene and normalized to the means of the controls separated by SDS-PAGE and transferred onto PVDF membrane (Merck-Millipore Membranes were blocked in 1% casein (Carl Roth Germany) for 30 min and incubated with primary antibodies against α-smooth muscle actin (αSMA 1:1000; Catalog number: ab6308) and GAPDH (Abcam 1:5000; Catalog number: ab8245) overnight at 4 °C followed by incubation with secondary goat anti-mouse IgG-HRP (Bio-Rad Visualization was performed with Clarity™ Western ECL Substrate (Super SignalTM West Femto Cells were divided into control and PPZ (2.5 80 μM) treatment groups when the cell confluency reached 60–80% cells were washed twice with Na+-containing HEPES buffer Cells were incubated at 37 °C for 30 min in Na+-containing HEPES with 5 μM BCECF-AM Glass slides were placed on the microscope stage of Ratio Photometry and Imaging Systems (PTI USA) and continuously perfused with Na+-free HEPES-buffered solutions by gravity (0–3 min: Na+-free HEPES buffer; 3–8 min: Na+-containing HEPES buffer with 10 μM Nigericin; 8–11 min: Na+-free HEPES buffer with 1% BSA; 11–14 min: Na+-free HEPES buffer; 14–20 min: Na+-containing HEPES buffer) BCECF-AM module of PTI was applied to measure pHi of cells with Na+-free HEPES buffer The fluorescent dye was excited at 440 and 485 nm alternatively and recorded every 30 s pHi alteration of cells during 14–17 min was calculated to represent Na+/H+ exchanger activity All animal protocols were approved by the Animal Ethics Committee of Zunyi Medical University and performed in accordance with the Good Laboratory Practice regulations for non-clinical laboratory studies of drugs issued by the National Scientific and Technological Committee of the People’s Republic of China Male C57BL/6J mice (8 weeks of age) were purchased from SPF (Beijing) Biotechnology Co. and they were maintained at 22 °C with a 12‐h:12‐h light/dark cycle and had free access to normal chow diet and water Mice were randomly allocated into three groups: control Mice in the control group were fed with control diet and mice in the DDC groups were fed with 0.1% DDC-containing diet by sterile H2O or 5 mg/Kg PPZ dissolved in sterile H2O twice per day for 4 weeks Mice were sacrificed after 4 weeks of treatment We have complied with all relevant ethical regulations for animal use alkaline phosphatase (ALP) and total bilirubin (TBIL) were measured by Beckman AU5800 Chemistry Analyzer using kits purchased from Beckman Coulter Inc Liver samples were fixed with 4% formaldehyde 3 μM sections were stained with hematoxylin and eosin (H&E) Sirius red and Masson staining according to standard protocols Liver inflammation was quantified via blinded scoring 0–4: 0: No inflammation; 1: Slightly chronic inflammation; 2: Mild chronic inflammation; 3: Mild inflammation; 4: Severe inflammation Paraffin-embedded sections (3 μM) of liver tissues were used for immunohistochemical staining 1:1000; Catalog number: ab124964) were applied as primary antibody Polyperoxidase-anti-Mouse/Rabbit IgG (Elabscience Catalog number: E-IR-R217B) was applied as second antibody Specific staining was visualized by light microscopy All in vitro experiments were repeated at least 3 times at least 5 samples were included in each group Statistical calculations were performed by using SPSS 25 (IBM USA) or GraphPad Prism 7 (USA) using analysis of t-test ANOVA with appropriate post hoc tests (Fisher’s LSD or Tukey’s) P-values lower than 0.05 were considered statistically significant All data are presented as mean ± standard deviation Further information on research design is available in the Nature Research Reporting Summary linked to this article All data supporting the results and conclusions of this paper are available upon request from the corresponding authors. All data behind the graphs and charts shown in the figures are presented in Supplementary Data Uncropped and unedited blot images are available in Supplementary Information 3′-O-acetyl-2′,7′-bis(carboxyethyl)-5,6-carboxyfuoresceinacetoxymethylester Sodium taurocholate co-transporting polypeptide Progressive familial intrahepatic cholestasis 2018 Annual Report of the European Liver Transplant Registry (ELTR)—50-year evolution of liver transplantation Bile acids in human livers with or without biliary obstruction Bile acids in normal rat livers and in those after bile duct ligation Hepatic toxicity in the rhesus monkey treated with chenodeoxycholic acid for 6 months: biochemical and ultrastructural studies Apoptotic body engulfment by a human stellate cell line is profibrogenic Phagocytosis of apoptotic bodies by hepatic stellate cells induces NADPH oxidase and is associated with liver fibrosis in vivo Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9 Bile acid-mediated thrombospondin-1 induction in hepatocytes leads to transforming growth factor-beta-dependent hepatic stellate cell activation Glycochenodeoxycholate promotes liver fibrosis in mice with hepatocellular cholestasis Hydrophobic bile salts induce pro-fibrogenic proliferation of hepatic stellate cells through PI3K p110 alpha signaling Bile acids induce hepatic stellate cell proliferation via activation of the epidermal growth factor receptor Bile acid-induced epidermal growth factor receptor activation in quiescent rat hepatic stellate cells can trigger both proliferation and apoptosis Secondary unconjugated bile acids induce hepatic stellate cell activation Unique inhibition of bile salt-induced apoptosis by lecithins and cytoprotective bile salts in immortalized mouse cholangiocytes Kinetic characterization of bile salt transport by human NTCP (SLC10A1) Sodium+/taurocholate cotransporting polypeptide as target therapy for liver fibrosis Insufficient evidence for NTCP activity in stellate cells A biliary HCO3− umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes The biliary HCO3− umbrella: a unifying hypothesis on pathogenetic and therapeutic aspects of fibrosing cholangiopathies Effect of perfusate pH on the influx of 5-5′-dimethyl-oxazolidine-2,4-dione and dissociation of epidermal growth factor from the cell-surface receptor: the existence of the proton diffusion barrier in the Disse space p70 ribosomal protein S6 kinase is a checkpoint of human hepatic stellate cell activation and liver fibrosis in mice cGMP-dependent protein kinase I (cGKI) modulates human hepatic stellate cell activation Cellular and molecular effects of Baccharis dracunculifolia D.C and Plectranthus barbatus Andrews medicinal plant extracts on retinoid metabolism in the human hepatic stellate cell LX-2 RNA sequencing of LX-2 cells treated with TGF-β1 identifies genes associated with hepatic stellate cell activation Mechanistic insights into the inhibition of NTCP by myrcludex B Classification of inhibitors of hepatic organic anion transporting polypeptides (OATPs): influence of protein expression on drug-drug interactions Intracellular pH regulation and Na+/H+ exchange activity in human hepatic stellate cells: effect of platelet-derived growth factor Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells Inhibition of the Na+/H+ exchanger reduces rat hepatic stellate cell activity and liver fibrosis: an in vitro and in vivo study Pantoprazole ameliorates liver fibrosis and suppresses hepatic stellate cell activation in bile duct ligation rats by promoting YAP degradation The role of transforming growth factor β1 in the regulation of blood pressure The adenosine monophosphate-activated protein kinase-vacuolar adenosine triphosphatase-pH axis: a key regulator of the profibrogenic phenotype of human hepatic stellate cells Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids Conjugated secondary 12α-hydroxylated bile acids promote liver fibrogenesis Expression and function of the bile acid receptor TGR5 in Kupffer cells Proton pump inhibitors induce apoptosis of human B-cell tumors through a caspase-independent mechanism involving reactive oxygen species Potential anti-inflammatory effects of proton pump inhibitors: a review and discussion of the clinical implications Proton pump inhibitor pantoprazole modulates intestinal microbiota and induces TLR4 signaling and fibrosis in mouse liver The association between proton pump inhibitor exposure and key liver-related outcomes in patients with cirrhosis: a veterans affairs cohort study Isolation and culture of hepatic lipocytes and sinusoidal endothelial cells by density gradient centrifugation with Stractan Aldosterone activates Na+/H+ exchange in vascular smooth muscle cells by nongenomic and genomic mechanisms Download references We are grateful for assistance in scientific writing provided by Dr This work was supported by the German Research Council (grant HO 4460/3-1 to S.H.) and National Natural Science Council of China (82073087) to B.T These authors contributed equally: Biguang Tuo Affiliated Hospital of Zunyi Medical University R.W.; analysis and interpretation of data: J.L. B.T.; drafting of the manuscript: J.L.; critical revision of the manuscript for important intellectual content: S.H. Communications Biology thanks Yuping Chen and the other anonymous reviewers for their contribution to the peer review of this work Primary handling editor: Christina Karlsson Rosenthal Download citation DOI: https://doi.org/10.1038/s42003-024-07192-4 Metrics details Diketopyrrolopyrrole-based blue dyes in dye-sensitized solar cells (DSCs) exhibit promise for building-integrated photovoltaics but their efficiency is compromised by dye aggregation-induced charge recombination Novel bile acid derivative co-adsorbents featuring bulky hydrophobic substituents at the 3-β position were synthesized to address this challenge designed to modulate intermolecular electronic interactions effectively altered the TiO2 surface coverage dynamics as evidenced by UV-Vis spectroscopy and dye-loading kinetics Systematic variation of hydrophilic substituents revealed structure-function relationships in dye separation efficacy Devices incorporating these co-adsorbers achieved power conversion efficiencies (PCE) of 7.6% surpassing reference devices (5.2%) and those using conventional chenodeoxycholic acid co-adsorbers (6.4%) The optimized devices exhibited a 30% increase in short-circuit current density and 60% peak external quantum efficiency at 550 nm Time-resolved photoluminescence spectroscopy confirmed suppressed non-radiative recombination while transient absorption spectroscopy revealed accelerated electron injection rates from 6.4 ps to 4.6 ps Electrochemical impedance spectroscopy elucidated the mechanism of reduced interfacial recombination These findings present a molecular engineering strategy for mitigating lateral charge transfer in planar dye systems advancing semi-transparent hybrid photovoltaics a Molecular structures of Dyenamo blue b Schematic representation of the adsorption process of the dye in the absence and presence of the bulky co-adsorbers (c) UV-Vis absorption spectra of the pristine dye and the dye with the co-adsorbers on mesoporous titania films d Plot of the loading amount of the dye on the titania films in the presence of the co-adsorbers as a function of increasing concentration the spectral absorption profiles of the dye are not affected by the co-adsorbers retaining the intense absorption peak at 580 nm and a shoulder around 400 nm This proves that replacing the hydroxyl group at the 3-β- position of CDCA with a bulky group retards the dye adsorption process by steric hindrance and separates the dye molecules more effectively a Current-voltage curves measured under AM1.5G simulated solar illumination b EQE spectra of the devices with the corresponding integrated photocurrents listed in the legend c Electron lifetime of the solar cells with the different co-adsorbers d Box plot showing the distribution of current densities of the devices based on the co-adsorbers e Box plot showing the distribution of power conversion efficiencies of the devices f Extracted charge in the solar cells as a function of open circuit potential Upon increasing the co-adsorber concentration We anticipate that the charge collection efficiency improves with the increase in co-adsorber concentration due to a more ordered coverage of the mesoporous titania layer This suggests that there are no significant changes in the conduction band edge position the reference cell demonstrates deviating behavior showing the charge collection efficiency is low in the absence of co-adsorbers a Current density-voltage (J–V) curves measured under AM1.5G simulated solar illumination b Box plot showing the distribution of the power conversion efficiencies for the co-sensitized devices c EQE spectra of the co-sensitized devices d Electron lifetime of the solar cells with the different co-adsorbers e Extracted charge in the solar cells as a function of open circuit potential f Photoluminescence spectra of the films on zirconium dioxide stained in the dye bath with co-adsorbers for 12 h The inset shows the time-resolved PL spectra of the co-sensitized films The photoluminescence lifetime at 12 h staining of the DB+ D35 co-sensitized ZrO2 films with and without the various co-adsorbers are shown in the inset histogram plot a Nyquist plots. b Bode plots. c Plots of charge transfer resistance and chemical capacitance as a function of voltage for DSCs based on the different co-adsorbers. d Electron recombination lifetime as a function of voltage for the cells with different co-adsorbers. a–c Solution state TA spectra of dyenamo blue, dyenamo blue with CDCA and dyenamo blue with BIAC03L co-adsorber in a 4-tert-butanol/acetonitrile solution (1:1 V/V) at different optical delays (d–f) TA spectra of dyenamo blue films on zirconium dioxide at optical delay of 1 ps plotted to compare against different concentrations of the co-adsorbers CDCA and BIAC03L Dye aggregation on the photoanode surface of DSCs can significantly impede their power conversion efficiency This effect is pronounced in diketopyrrolopyrrole-based dyes such as Dyenamo Blue where due to the large transient dipole moment in the structure lateral charge transfer between neighboring dye molecules occurs upon photo-excitation This affects the efficiency of the electron injection leading to lower PCE we investigated the use of bile acid derivatives as co-adsorbents to mitigate dye aggregation and enhance DSC performance A series of bile acid derivatives were synthesized systematically modifying the 3-β position with bulky hydrophobic groups and varying the number of hydrophilic substituents in the backbone We demonstrate that introducing bulky co-adsorbers is a good strategy to maintain good adsorption-injection characteristics of dyenamo blue dyes on mesoporous surfaces Results revealed that bulky hydrophobic groups at the 3-β position retards the uptake of dye molecules and effectively inhibits dye aggregation leading to a better packing density of the dye molecules on the TiO2 surface all the co-adsorbents with the bulky hydrophobic substituents and additional hydrophilic substituents showed a substantial improvement with a power conversion efficiency of >7% as compared to 5.17% in the reference device without co-adsorbers and 6.4% in the devices with a conventional co-adsorber that does not comprise the bulky hydrophobic substituent with a 30% increase in short-circuit current density (JSC) and a 30 mV increase in open-circuit voltage (VOC) was observed with the co-adsorber that had an expanded hydrophobic 3β region but no hydroxyl substituents on the hydrophilic side External quantum efficiency (EQE) measurements showed a maximum value of 60% at 550 nm for the optimized device compared to 45% for the reference device accompanied by a near 30% increase in integrated photocurrents This is supported by longer electron lifetime revealed from the transient photovoltage and charge collection measurements indicating a more effective suppression of the back reaction of the injected electron with the cobalt electrolyte the optimized co-adsorber continues to be the best-performing system with a PCE of 7.37% as compared to 5.87% in the reference device accompanied by an increase in the current density and a 30 mV increase in the open circuit voltage the devices with CDCA co-adsorber that do not include the bulky amide substituents at 3-β position showed a PCE of 7.19% It is noteworthy that the current densities reach 13.73 mA/cm2 in the BIAC03L devices as compared to 10.8 mA/cm2 and 12.82 mA/cm2 in the reference devices with no co-adsorber and the reference devices with the conventional co-adsorber This increase in photocurrent is also corroborated by the increased electron lifetime obtained by both transient photovoltage and impedance techniques re-iterating the role played by the bulky co-adsorbers in suppressed dye aggregation We hypothesized that the improved photocurrent arises from the suppression of the deactivation of the excited states via quenching processes between dye molecules To confirm this we conducted a series of ultrafast spectroscopic evaluations Time-resolved photoluminescence measurements revealed a prolonged lifetime indicating that staining the mesoporous layer in the presence of the bulky co-adsorbers leads to a less dense but more ordered packing of the dye molecules the non-radiative intermolecular recombinations are suppressed leading to substantial improvements in the photocurrents and power conversion efficiencies Our findings are further supported by femtosecond TAS data confirming that the co-adsorbents not only reduced dye aggregation but also accelerated electron injection rates with a decrease in injection time from 6.4 ps to 4.6 ps by highlighting the potential of bile acid derivatives with sterically demanding substituents as effective co-adsorbents our findings pave the way for facile access to high-performing blue-dye-based semi-transparent hybrid solar cells This offers promising prospects for dye solar cells in building-integrated photovoltaics we report a series of novel co-adsorbers with sterically demanding substituents offering unique supramolecular features By fine-tuning the spatial orientation of the substituents we have created co-adsorbers that have an expanded hydrophobic pocket as compared to the conventional CDCA co-adsorber used in the field of dye solar cells We have demonstrated that attaching bulky substituents at the 3-beta position is an effective strategy to mitigate the annihilation effects due to dye aggregation in hybrid solar cells The effect of increased power conversion is pronounced when using planar sensitizers such as dyenamo blue resulting in the highest current density The champion dyenamo blue devices based on our co-adsorbers achieved a remarkable PCE of 7.56% under AM1.5G simulated solar illumination with a significantly improved JSC of 13.1 mA cm2 a remarkable improvement from existing literature reports We have thoroughly investigated the effect of the co-adsorbers on the charge transfer characteristics and lateral intermolecular interactions using a combination of impedance and ultrafast spectroscopy techniques We have discovered that bulky co-adsorbers create an ordered molecular packing of the dye whilst extending the photoluminescence lifetime and enhancing the electron injection rates leading to improved photovoltaic performance The enhanced bulkiness of the co-adsorber leads to a less-dense monolayer formation of the dye on the mesoporous layer which decreases the area that is active for recombination of conduction band electrons back to the electrolyte Our findings offer a promising direction towards further improving performances of solar cells that can absorb the invisible near-UV and near-IR parts of the solar spectrum—a feature that can revolutionize building integrated photovoltaics Current-voltage measurements under AM 1.5G illumination were carried out in ambient air using a Sinus-70 solar simulator (Wavelabs Solar Metrology Systems GmbH) The irradiance (100 mW cm−2) was calibrated with a certified silicon diode (Fraunhofer) UK) was used to assess the solar cell performance (scan speed 25 mVs−1 A circular mask was employed to confine the active solar cell area to 0.196 cm2 Shown device performance metrics represent averages of four cells at all times pending reasonable error margins of the manual cell fabrication IPCE spectra were recorded with a Xenon light source (10 mW cm−2) and a CM110 monochromator (Bentham) The setup was calibrated with a certified silicon reference cell (Fraunhofer ISE Photocurrents were integrated based on the spectral distribution of sunlight AM 1.5G Electron recombination lifetimes were investigated using the dye-sensitized solar cell Toolbox (Dyenamo The solar cell was illuminated with a 1 W white LED Kinetics in the solar cell were probed by applying slow 10 Hz square-wave modulations on top of a base light intensity The solar cell voltage response was tracked in real time the measurement was repeated until a noise threshold was met; at which point the traces were fitted with first-order kinetic models and a single decay time constant from the compiled traces was extracted The measurements were run across a range of light intensities The accumulated charge in the photoanodes of the DSC devices was measured by illuminating the cells at different light intensities at an open circuit; then the potential simultaneously switched to short circuit The collected charge was converted to charge density assuming a 6 mm diameter of the circular aperture Photoluminescence lifetimes were recorded at the emission centre of 760 nm on an Edinburgh FLS980 spectrometer Samples were excited with an EPL-475 (472 nm and measured with a time-correlated single photon counting module and a Hamamatsu R928P photomultiplier tube Lifetime fits of the photoluminescence intensity I were extracted directly in the F980 software with numerical data reconvolution based on Marquardt–Levenberg algorithm with amplitude factors Bi and time constants τi the counts of photons before time zero were averaged to serve as a baseline the relative amplitudes in all scans were rescaled between the common baseline and the peak intensity normalized to 1 Electrochemical impedance spectra were recorded using a PGSTAT12 potentiostat (Autolab) in the frequency range from 100 kHz to 0.1 Hz at different DC bias potentials around the maximum power point The DSC devices were illuminated with a white LED for impedance analysis and fitted to Bisquert’s transmission-recombination line with an adapted version of the impedance fitting tool available on Z-view software The percentage of dye molecules adsorb on TiO2 surface relative to the reference device (blank) was obtained following the dye-desorption method After dipping the TiO2 substrates in the different co-adsorbate/dye solutions the molecules loaded on the semiconductor were then desorbed by dipping them in a solution of tetrabutylammonium hydrozide 0.1 mol L−1 followed by UV-Vis spectroscopic determination of dye concentration by Beer-Lambert law with the extinction coefficient of the peak absorption the dye TAS measurements were performed using an Ultrafast Systems TAS Spectrometer Samples were pumped with Ti:Sapphire laser at 1 kHz (800 nm source pump) Excitation wavelength was selected using an optical parametric amplifier a portion of the pump beam was routed through a sapphire crystal to achieve white light continuum to act a probe beam Excitation of 490 nm was used with a pump power of 100 μW The pump power was varied to check for linearity and minimized to limit dye photobleaching Four spectra were taken per sample and averaged to achieve the presented spectra Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Li, Y., Huang, X., Sheriff, H. K. M. & Forrest, S. R. Semitransparent organic photovoltaics for building-integrated photovoltaic applications. Nat. Rev. Mater. 8, 186–201. https://www.nature.com/articles/s41578-022-00514-0 (2023) Bing, J. et al. Total equivalent energy efficiency metric for building-integrated photovoltaic windows. Joule 7, 2668–2683. https://linkinghub.elsevier.com/retrieve/pii/S2542435123004841 (2023) Blue photosensitizer with copper(ii/i) redox mediator for efficient and stable dye-sensitized solar cells Transparent and colorless dye-sensitized solar cells based on pyrrolopyrrole cyanine sensitizers Hao, Y. et al. Novel blue organic dye for dye-sensitized solar cells achieving high efficiency in cobalt-based electrolytes and by co-sensitization. ACS. Appl. Mater. Interface 8, 32797–32804. https://pubs.acs.org/doi/10.1021/acsami.6b09671 (2016) Developments of diketopyrrolopyrrole-dye-based organic semiconductors for a wide range of applications in electronics A compact diketopyrrolopyrrole dye as efficient sensitizer in titanium dioxide dye-sensitized solar cells Lateral intermolecular electronic interactions of diketopyrrolopyrrole d–π–a solar dye sensitizers adsorbed on mesoporous alumina and n749 dye···tio2 interfaces that represent dye-sensitized solar cell working electrodes 2-(4-butoxyphenyl)-n-hydroxyacetamide: an efficient preadsorber for dye-sensitized solar cells First report of chenodeoxycholic acid–substituted dyes improving the dye monolayer quality in dye-sensitized solar cells Yum, J.-H. et al. Blue-coloured highly efficient dye-sensitized solar cells by implementing the diketopyrrolopyrrole chromophore. Sci. Rep. 3, 2446. https://www.nature.com/articles/srep02446 (2013) Holcombe, T. W., Yum, J.-H., Kim, Y., Rakstys, K. & Grätzel, M. Diketopyrrolopyrrole-based sensitizers for dye-sensitized solar cell applications: anchor engineering. J. Mater. Chem. A 1, 13978. https://xlink.rsc.org/?DOI=c3ta13643d (2013) Liu, P. et al. Molecular engineering of d-π-a type of blue-colored dyes for highly efficient solid-state dye-sensitized solar cells through co-sensitization. ACS Appl. Mater. Interfaces 10, 35946–35952. https://doi.org/10.1021/acsami.8b11405 (2018) Dye aggregation in dye-sensitized solar cells Self-assembly in aqueous bile salt solutions Characterization of carbon nanotube dispersions in solutions of bile salts and derivatives containing aromatic substituents New lamellar structure formed by an adamantyl derivative of cholic acid Co-adsorbing effect of bile acids containing bulky amide groups at 3β-position on the photovoltaic performance in dye-sensitized solar cells Shen, Z. et al. Molecular engineering of low-cost, efficient, and stable photosensitizers for dye-sensitized solar cells. Chem 9, 3637–3647. https://linkinghub.elsevier.com/retrieve/pii/S2451929423004126 (2023) Influence of electrolyte in transport and recombination in dye-sensitized solar cells studied by impedance spectroscopy Knorr, F. J., McHale, J. L., Clark, A. E., Marchioro, A. & Moser, J.-E. Dynamics of interfacial electron transfer from betanin to nanocrystalline TiO2 : the pursuit of two-electron injection. J. Phys. Chem. C 119, 19030–19041. https://doi.org/10.1021/acs.jpcc.5b05896 (2015) Synthesis of 3α- and 3β-dimers from selected bile acids One-pot mitsunobu-staudinger preparation of 3-aminocholan-24-oic acid esters from 3-hydroxycholan-24-oic acid esters Self-aggregation mechanism of a naphthylamide cationic derivative of cholic acid Benesperi, I. et al. Dynamic dimer copper coordination redox shuttles. Chem 8, 439–449. https://linkinghub.elsevier.com/retrieve/pii/S2451929421005234 (2021) Download references acknowledge the financial support from EPSRC UKRI for grants EP/W006340/1 (North East Ultrafast Transient Absorption Spectroscopy Facility) and EP/V035819/1 (Photocapacitors for Ambient Energy Applications) They also thank The Royal Society for grants IES313090 and URF191286 (University Research Fellowships) This support has been crucial for advancing our research in energy materials and devices We also gratefully acknowledge the influence of Costa Rican coffee a source of both inspiration and sustained energy Newcastle University for acquiring the femtosecond TAS measurements used in this work thank the School of Chemistry and the Center for Electrochemistry and Chemical Energy (CELEQ) of the Universidad de Costa Rica for financial support School of Natural and Environmental Science Centro de Investigación en Electroquímica y Energiá Química (CELEQ) conducted all the experiments pertaining to the solar cell fabrication and characterizations including current-voltage analysis electrochemical impedance spectroscopy and sample preparations for photoluminescence and TAS measurements; analyzed all the data and wrote the manuscript conducted all the structural characterizations and aided in writing the synthetic part of the methods section The authors declare no competing interests. Additional experimental details and supporting information can be found in the Supplementary Information Communications Chemistry thanks the anonymous reviewers for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s42004-025-01433-1 The government of Laos has for the first time shut down a farm where live bears were harvested for their bile after convincing the farm’s owner to voluntarily hand over three bears The rescued Asian black bears (Ursus thibetanus) are now being quarantined at the Luang Prabang Wildlife Sanctuary operated by Australia-based NGO Free the Bears “This is an important milestone for both Free the Bears and our government partners showing that it is possible to close a bear bile farm and signalling that Laos is increasing its capacity and commitment to take on those who are illegally exploiting wildlife for profit,” Rod Mabin usually hold Asian black and sun bears (Helarctos malayanus) in cages Bile is extracted from their gall bladders using a syringe for use in Asian traditional medicine as a supposed treatment for liver and kidney disease Mabin said that while the active compound Ursodeoxycholic acid found in bear bile is scientifically proven to address liver or bile duct diseases it can easily be synthesized in a laboratory “There is no legitimate reason to extract bile from bears or keep bears in bile farms.” possess or trade bears and their body parts in Laos under a 2007 wildlife law but Mabin said a loophole exempts bile farms established before the law’s enactment bile farms can only be closed when there are rescue facilities available to accept the bears “Rescued bears often have broken teeth and claws stunting and poor body condition with wasted muscles,” Mabin said “It is only in recent years that sufficient sanctuary space has become available to rescue more bears and close down farms.” The three recently rescued bears are also expected to suffer lifelong health issues due to a poor diet and regular bile extraction Veterinarians are monitoring their progress the bears will be released into the sanctuary’s natural outdoor habitat “We ‘enrich’ their environment to encourage them to explore and interact with their surroundings and give them choices to help them feel more in control of their environment This ultimately leads to healthier and more content bears,” Mabin said The NGO previously spotted 20-30 caged bears in the now-closed bear bile farm but Mabin said they don’t know what happened to the rest the Laos government and the charity visited the farm and convinced the owner to hand over the bears and close the farm The “fortress conservation” model is under pressure in East Africa as protected areas become battlegrounds over history and global efforts to halt biodiversity loss Mongabay’s Special Issue goes beyond the region’s world-renowned safaris to examine how rural communities and governments are reckoning with conservation’s colonial origins and trying to forge a path forward […] Metrics details tissue interaction such as the interplay between blood vessel (BV) and epithelial tissue is crucial for organogenesis Here we recapitulate the spatial arrangement between liver epithelial tissue and the portal vein to observe the formation of intrahepatic bile ducts (BDs) from human induced pluripotent stem cells (hiPSC) We co-culture hiPSC-liver progenitors on the artificial BV consisting of immature smooth muscle cells and endothelial cells liver progenitors within hiPSC-BV-incorporated liver organoids (BVLO) differentiate to cholangiocytes and acquire epithelial characteristics liver surface transplanted-BVLO temporarily attenuates cholestatic injury symptoms Single cell RNA sequence analysis suggests that BD interact with the BV in BVLO through TGFβ and Notch pathways Knocking out JAG1 in hiPSC-BV significantly attenuates bile duct formation highlighting BVLO potential as a model for Alagille syndrome we develop a novel 3D co-culture method that successfully establishes functional human BDs by emulating liver epithelial-BV interaction developing an approach to reconstruct human BDs around BV ex vivo is crucial to understand the etiology of aberrant formation of biliary structures in human newborns Here we analyzed the spatial relationship between the portal vein (PV) and the primitive IHBDs discovering that SM22+ immature smooth muscle cells (imSMCs) are in contact with CK19+SOX9+ cholangiocytes in the human fetal liver We hypothesized that imSMCs have pivotal roles in early BD development and thus By incorporating an artificial BV containing imSMCs and ECs we successfully generated BD structures in hiPSC-BV incorporated liver organoid (BVLO) Given that the molecular pathways regulating BV-BD interaction are emulated in BVLO this culture system offers a viable ex vivo model for human congenital biliary diseases A Wholemount immunofluorescence analysis of OPN+ cholangiocytes and PV-SMC at E15.5 SM22+ vascular SMCs delineates the PV thoroughly with a gradient of αSMA expression from high to low at the periphery suggesting variable SMC maturation stages in BD development Two-photon imaging was performed independently on biological replicates (n = 2) and the representative image is shown in this panel Confocal microscope imaging also performed (n = 3) B Immunotaining of PV-SMC and ductal plates in human fetal liver slice at GW15 Ductal plates highlighted with dotted lines Three to four PV areas per section (n = 2) were examined C scRNA-seq analysis of mesenchymal cell populations in human fetal liver at GW9 ~ 12 Dot plot shows cluster 1 contains ACTA2(gene coding αSMA)+TAGLN (gene coding SM22)+JAG1+ cells and JAG1+ cells are highlighted on each feature plot whereas TAGLN+JAG1+ and ACTA2low (expression level < 4) cells are highlighted in the lower right feature plot indicating TAGLN+ ACTA2low JAG1+ are enriched in cluster 1 D Schematic illustration of stepwise induction process of SMCs from hiPSCs E Differentiation of human iPSC-derived smooth muscle cells in-vitro visualized through bright field imaging and immunostaining for αSMA and SM22 Scale bars: 200 µm (phase contrast) and 100 µm (fluorescence) Experiments were repeated independently more than three times with similar result F qPCR analysis of the expression level of SM22 JAG1 of hiPSC-derived cells throughout different stages of SMCs in-vitro differentiation process The boxes indicate mean expression levels while error bars represent standard deviations (SD) P-value of 0.2066; αSMA expression of imSMC vs SMC Statistical significance: *P-value of 0.0148; JAG1 expression of imSMC vs SMC Statistical significance: **P-value of 0.0070) these data suggest that the immature status of PV-SMCs is correlated with the early stage of BD development when incorporated as the component of hiPSC-BV suggesting that imSMCs may have higher potential to induce cholangiocyte differentiation A Experimental workflow for generating iPSC-derived blood vessel on collagen-1 scaffold B Localization of Kusabira Orange (KO)-HE cells in optimized co-culture shown in merged phase-contrast (grayscale) Culture in each condition was repeated independently more than three times with similar results C Immunofluorescence of CK19 (cholangiocyte) and HNF4α (hepatoblast) at day 14 under different conditions Three to four fields of view from over five independent organoid samples were analyzed D H&E staining of co-cultured liver organoid with BV (BVLO) or without hiPSC-BV (LO) Three to four fields of view from over five independent organoids were analyzed (Error bars: SEM F FACS sorting of GFP-hiPSC-MC (GFP+CD31-) or GFP-hiPSC-EC (GFP+CD31+) populations post-dissociation followed by qPCR analysis of SMC markers (αSMA Living cells were identified by propidium iodide negativity and categorized by GFP and CD31 expression level (Statistical analysis using Two-tailed Mann-Whitney; n.s n.s; P-value of 0.1143; 0.4329; 0.3429); Endothelial markers (TGFB1 n.s; P-value of 0.7302; 0.7857; 0.3429; 0,3429) Cell isolation for LO and BVLO was performed at various days (LO day 1 (n = 4) Over three fields of view from over five independent organoid samples were analyzed We named this optimized condition as blood vessel incorporated liver organoid (BVLO) when SMCs instead of imSMCs were used to generate the hiPSC-BV Next, to better understand the gene expression changes during the cholangiocyte differentiation from HE cells, the KO+ cells in BVLO were sorted by flow cytometry, and expression of cholangiocyte lineage marker genes was analyzed by qPCR. The gene expressions related to the epithelial cytoskeleton (KRT7) and BD transporter (GPBAR1) were significantly upregulated (Fig. 2F) which supports our argument that fraction of HE cells differentiate into cholangiocytes in BVLO other BD transporters (AQP1 and CFTR) gene expression increase trend could also be observed these findings show that when co-cultured with hiPSC-BV containing JAG1 hiPSC-liver progenitors derived from HE differentiate into cholangiocytes forming BD-like structures A 3D imaging demonstrates BVLO tubular structure B TEM image of BVLO at varying magnifications Experiments were repeated independently two times with similar results D Rho123 transport in hiPSC-BD without and with hiPSC-BV and the Verapamil E Immunostaining after Rho123 incubation showing luminal accumulation of Rho123 in the absence of verapamil Experiments were repeated independently three times with similar results G Immunofluorescence pre- and post-forskolin treatment H Quantification of lumen area pre- and post-forskolin (Two-tailed Mann-Whitney test Statistical significance ****P-value < 0.0001) Lumen area for BVLO with Forskolin (n = 30) and without (n = 26) I GGT activity assay of BVLO (Two-tailed Mann-Whitney test Statistical significance **P-value of 0.0093) GGT activity of LO (n = 7) and BVLO (n = 8) was determined J Alkaline phosphatase activity of hiPSC-BD after BCIP/NBT staining Experiments were repeated independently more than three times with similar results K Schematic overview of liver transplantation procedure L Histological analysis of liver specimen rom NOG mice post-transplantation epithelial junction and polarity markers (βCAT human cell marker (Ku80) and a hepatocyte marker (HNF4A) N Schematic illustration of carbon ink injection process from EHBD post-transplantation O Bright field and fluorescence of carbon ink injection showing hCK7+ human and mOPN+ mouse BDs in the boundary transplanted area and the recipient area This responsiveness implies that tubular BVLO-BD possesses MDR1 dependent efflux function hiPSC-cholangiocytes within BVLO exhibit functional epithelial intercellular junctions and secretory functions in BVLO these results indicate that spatially organized hiPSC-BDs within BVLO establish BD-specific structural and functional features These results indicate that hiPSC-cholangiocytes in BVLO further maturated following in vivo incubation and that the transplanted BD is connected to the host IHBD A Schematic illustration of common bile duct (CBD) ligation procedure to induce obstructive cholestasis comparing non-transplanted or BVLO transplanted groups comparing non-transplanted and BVLO-transplanted groups Kaplan-Meier analysis with statistical significance assessed using the log-rank (Mantel-Cox) no adjustments were made for multiple comparisons D Serum marker in BDL-ligated NOG mice: Non-Transplanted vs E Percentage of body weight remaining in BDL-ligated NOG mice: Non-Transplanted vs Transplanted (Two-tailed Mann Whitney test day 5 (n = 3) and BVLO transplanted day 3 (n = 7) day 5 (n = 5); Error bars are represented as SEM F Wholemount immunofluorescence of thick liver slices post BDL and BVLO transplantation: BVLO non-transplanted vs BVLO-Transplanted group G Quantification of graft’ BD lumen area in BVLO-Transplanted NOG mice Statistical significance *p-value of 0.0101) Three biological replicates were prepared and from each three or four areas were selected in a thick section Lumen areas were quantified using imageJ analysis software and error bars representing SEM BVLO culture protocol was designed to emulate PV-BD interaction in developing liver by culturing the sheet-shaped hiPSC-liver organoid around hiPSC-BV Previous mouse studies demonstrated that TGFβ and Notch signals mediate the PV-BD interaction The latter signal is especially crucial for human BD development as evident by the BD paucity in Alagille syndrome attributed to JAG1 or NOTCH2 mutations the mechanism by which these signals are activated in human liver progenitor cells through PV-BD interaction during BD development is yet to be elucidated we analyzed gene expression profile of organoid component cells at single cell level to reveal signals transmitting BV-BD interaction in BVLO A Single cell RNA-seq analysis of dissociated BVLO identified four distinct cell clusters; Hepatocyte cluster contains ALB+ cells; Endothelial one contains PECAM-1+ cells; Cholangiocyte cluster contains SOX9+ NOTCH2+ cells; Mesenchymal cell cluster contains ACTA2+ part of BVLO component cells were annotated to immature hepatocytes or epithelial cells which show hybrid phenotypes of cholangiocytes and hepatocytes B Comparative clustering analysis of BVLO and human adult liver scRNA-seq data from “Human liver Cell Atlas” (GSE124395) This figure highlights the clustering proximity of cholangiocyte and hepatocyte within BVLO and human adult liver C Heat map visualization of cholangiocyte and hepatocyte markers expressed in BVLO-derived cells compared to those in primary human liver cells highlights the similarities in the corresponding marker expression D NicheNet analysis suggesting intercellular communication in BVLO This ligand–target matrixes denote the regulatory potential between 25 ligands in Mesenchymal Cell (MC: upper) or Endothelial cell (EC: lower) and target genes in Org-Cho (the highly expressed genes in Org-Cho against planar cultured HE cells) TGFβ1 in ECs and TGFβ1 as well as JAG1 in MCs potentially activate the corresponding downstream targets in cholangiocytes E Schematic illustration of possible EC and MC contribution to the BVLO signaling pathways that are suggested to be associated with the differentiation of cholangiocyte clusters from HE cells in BVLO and formation of BD A Representative phase-contrast and epifluorescence image of LO and BVLO containing KO-HE Each experiment was repeated independently three times with similar result (Scale bar: 500 µm) C Quantification of BD lumen number and CK19+ area ratio in LO or BVLO treated with A8301 Culture was independently repeated for LO (n = 4) (Kruskal-Wallis test followed by Steel multiple comparison test CK19+ area with statistical significance ** **P-value of 0.0095; 0.0043; 0.0022; 0.0022) lumen number with statistical significance **,**,**,**,**P-value of 0.0070; 0.0079; 0.0022; 0.0022 D Immunofluorescence of JAG1 from planar-cultured imSMCs (Scale bar: 50 µm) (Kruskal-Wallis with Steel multiple comparison test and TGFβ1:10 ng/ml vs TGFβ1:10 ng/ml+A8301:1 µM Statistical significance: *P-value of 0.0400; 0.0400) F Schematic illustration of BVLO generation using JAG1 knock out-imSMC G Bright field image of JAG1 knock-out iPSC-derived imSMCs (Scale bar 200 µm) epithelial junction and polarity markers (ZO1 two BVLOs incorporating the BV with JAG1 + / + Three to four fields from each organoid sample were analyzed Three to four fields in two different sections of each organoid sample were analyzed for quantification (Kruskal-Wallis with Steel multiple comparison test: *P-value of 0.0022 and 0.0022) J Schematic illustration of BVLO generation using JAG1 Knock out-HE cells K Immunofluorescence of BVLO containing JAG1 Knock-out HE cells (Scale bar: 100 µm) Culture was repeated independently two times with similar result the signaling pathways vital for mouse BD development are also instrumental in regulating human BD development and BVLO models that lacking JAG1 in BV-SMCs can specifically recapitulate the abnormal BD formation observed in human Alagille syndrome we generated BD structures within a hiPSC liver organoid by emulating inter-tissue interaction between PV and BD that occurs during fetal liver development this study is the first to establish a culture protocol to generate hiPSC-BD structures in the liver organoid by incorporating a large artificial BV Our mouse developmental observation suggests that immature SMCs play a crucial role at the initial stage of BD development governing the maturity of artificial BV cellular components is essential for efficient induction of 3D hiPSC-derived BD lumen structure that more effectively promote cholangiocyte differentiation and facilitate hiPSC-BD lumen generation suggesting that observed OPN expression marks a maturation process that happened after in vivo incubation acetylated tubulin was also only detected on the apical surface of BVLO-BD exclusively after transplantation the absence of primary cilia as indicated by the apical membrane localization of acetylated tubulin suggests incomplete maturation of hiPSC-BDs the BVLO culture protocol enables the generation of functional hiPSC-BD lumen structures in vitro which undergo further maturation post-transplantation future study to optimize the protocol is necessary to achieve full BD maturation Our culture system presents a unique aspect to model human biliary diseases over traditional hiPSC-biliary organoid by enabling the modulation of BV-BD interaction through gene editing. As shown in Fig. 6 we can recapitulate bile duct paucity seen in Alagille syndrome by knocking out JAG1 in imSMC composing the BV JAG1−/− hiPSCs-liver progenitors could still differentiate to cholangiocytes by interacting with JAG1 wild type hiPSC-imSMCs underscoring the versatility of the system Given the possibility to modify each component of BVLO this system offers a platform to scrutinize the etiology of other human BD aberrant formation hiPSC-BD holds promise for regenerative medicine application Transplanting BVLO onto the liver surface of a cholestatic mouse has shown potential to temporarily alleviate cholestatic symptoms The lumen continuity between hiPSC-BD and the host IHBD may enable BVLO to provide an additional reservoir to accommodate excess bile within the recipient’s liver Even though current benefit remains temporary and BVLO cannot completely resolve the obstruction in biliary diseases it represents a step forward in managing cholestatic conditions complementing current BVLO’s BD graft-host connection with hepatobiliary network continuity will enable the development of liver tissue on a dish complete with an integrated end-to-end bile drainage system Our method of co-culturing epithelial organoids with an artificial BV offers the ability to derive complex liver structures in vitro which are useful for modeling human biliary diseases Given the pivotal role of BV in facilitating blood circulation to deliver nutrients and oxygen to tissue/organ component cells the integration of BV into organoids has been a recent target for many researchers the current study shed light on another aspect of BV function: emulating BV-epithelial interaction could promote epithelial morphogenesis facilitating the development of 3D physiologically functional tissue structures in human epithelial organoids The use of hiPSCs was approved by the ethics committee of The University of Tokyo (2023-102-0305 The fetal liver tissue (the estimated age of the fetus was gestation week 15 (GW15)) used in this work came from clinical abortion The use of human fetal liver tissue by an opt-out consent was approved by the ethics committee of Asahikawa Medical University (22059) and that of the Institute of Medical Science Opt-out consent is a standard process in Japan to obtain human fetal tissue for research purposes Images were taken with a Leica SP8 confocal microscope (Leica Germany) and Olympus FVMPE-RS multiphoton microscope (Olympus The staining and imaging experiments were independently replicated more than three times (n = 3) using liver lobes from different embryos to account for any variability and MC harvested from planar culture were reseeded on a 24-well plate and MC (1 × 105) were seeded per well in an EZSPHERE® 24-well plate (Iwaki two wells of EZSPHERE® 24-well plate liver organoid sphere were collected and reseeded on top of 0.4 µm porous membrane THINCERT® cell culture insert (Greiner Germany) by using one well of silicone insert (ibidi AJI4/VEC-1 medium was used as a maintenance medium throughout the co-culture AJI4 media comprises of DMEM (high glucose) (Fujifilm Wako Gentamycin (50 ng/ml) mixed with 1:1 ratio of VEC-1 medium (KOHJIN BIO and then the smaller hole located in the head of the syringe was covered with Parafilm® a 1.5 ml tube containing 1 × 106 hiPSC-imSMC or hiPSC-mSMC cell pellet was prepared while 400 µl DMEM medium containing 5 µl 1 N NaOH was prepared in a separate tube TGFß1 (10 ng/ml) (R&D Systems) and 1 µM Jagged-1 Fc (R&D Systems were added to the tube containing DMEM medium (Fujifilm Wako 100 µl of rat collagen-1 (R&D System) was then added to DMEM-recombinant solution and mixed thoroughly DMEM-collagen mixed solution was then added to the cell pellet The final solution was transferred to a 1 ml syringe (Terumo Japan) was inserted to the middle of the solution to be incubated at 37 °C for 20 min—for gel solidification Excess water was removed with a hydrophilic nylon membrane 0.2 µm pore size (47 mm diameter) (Merck Millipore both sides of the SMC cell-containing vessel were attached to a soft catheter [Nipro safelet cath 26 G (Nipro Japan)] by creating ties using a 4 G surgical suture (Natsume 1 × 106 hiPSC-EC pellet was resuspended in a 15 µl DMEM medium and drawn into a 20 µl microsyringe (Trajan The cell suspension was then inserted slowly into the lumen of the SMC cell-containing vessel tube through the aperture of the soft catheter followed by incubation at 37 °C for 10 min the tube was reversed to ensure that the cell spread homogenously within the lumen followed by a second incubation at 37 °C for 10 min 100 µl of Miracell EC Culture medium (Takara The medium that was used to culture EC on day 1 after the first passage was StemPro 34-SFM (Gibco) followed by Miracell medium as aforementioned After aggregated liver organoid was overlaid surround hiPSC-derived BV ECM with the composition of 9:1 Matrigel matrix Growth Factor Reduce (Corning USA) and Rat collagen-1 are used to coat the outer layer of the liver organoid Laminin-511 5 µg/ml was added to the ECM mix for BVLO Macroscopical observation was carried out using Nikon eclipse Ti -S more than 3 organoids were generated for each combination of cells and recombinant proteins BVLO is an excellent system to emulate BD-BV interaction in vitro but it needs a bit more training and experience than culturing hiPSC Enzyme activity was stopped by the addition of 5% FBS in PBS The dissociated cell solution was filtered with 40 μm mesh and inserted into a 15 ml tube centrifugation (400 × g) was carried out for 5 min to pellet the cells 100 µl of 5% FBS in PBS was added to the tube Alexa647-conjugated anti-EpCAM antibody (1 µl) (Biolegend) was added to the tube and incubated at 4 °C in the dark for 15 min Cells were then washed with 5 ml 5% FBS in PBS Cell sorting was performed with the FACSAria TM III (BD biosciences We repeated cell isolation from 2 or 3 BVLOs three times at each time point Liver organoid-dissociated cells were distinguished from debris on the flow cytometric profile based on the Forward Scatter (FSC) and Side Scatter (SSC) Cell aggregates were gated out based on their properties displayed on the SSC width (SSC-W) versus height (SSC-H) dot plot Cells were then again gated in an FSC height (FSC-H) and FSC-area (FSC-A) dot plot to eliminate doublets Living HE-derived cells within the organoid was recognized by Kusabira Orange positive population (PE) Hoechst staining was omitted for cell sorting due to increased toxicity and causing a minimum number of cell recovery KO+/EpCAM+(APC+) cells or particular cells of interest like GFP+ BV cells were sorted on the basis of a IgG isotype control At least three independents total RNA was isolated for each group and qPCR was performed in duplicate for each RNA sample The results are presented as the average of three technical replicates (n = 3) with standard deviation as error bar RNA quality check was performed with 4150/4200 RNA screen tape (Agilent tech 5 µl of RNA sample buffer and 1 µl of RNA sample was added to each vial Vortex homogenization was performed for 1 min RNA integrity was evaluated using Agilent 4200 tapestation (Agilent Technologies and RNA samples with RNA integrity number > 8.8 were subjected for RNA-Seq analysis RNA-seq libraries were prepared using 30 ng of total RNA with an Ion AmpliSeq Transcriptome Human Gene Expression kit (Thermo Fisher Scientific United States) according to the manufacturer’s instructions The libraries were sequenced on the Ion Proton system using an Ion PI Hi-Q Sequencing 200 kit and Ion PI Chip v3 (Thermo Fisher Scientific) and the sequencing reads were aligned to hg19_AmpliSeq_Transcriptome_ERCC_v1 using Torrent Mapping Alignment Program the data were analyzed using AmpliSeqRNA plug-in v5.2.0.3 a Torrent Suite Software v5.2.2 (Thermo Fisher Scientific) which provides QC metrics and normalized read counts per gene Data were analyzed and visualized using the Subio platform (Subio We performed GO and KEGG pathway analysis using the DAVID Bioinformatics Resource ver Our data is deposited in GEO (accession # GSE198888) The raw read counts generated by cellranger were filtered based on mitochondrial gene feature number and RNA number (nCount_RNA ≤ 8000 & nCount_RNA ≥ 500 & nFeature_RNA ≤ 20000& nFeature_RNA  ≥ 200 & percent.mt < 10 & 0.8 > log10(nFeature_RNA/nCount_RNA)) and then they were normalized by the normalization method “NormalizeData” The top 2000 most variably expressed genes were used as the features of the dataset (FindVariableFeatures) The corrected data were used for UMAP (RunUMAP function; reduction = “PCA” Resolutions of 0.7 (FindClusters function) were used for clustering The clusters of interest were subset and compared for differential gene expression using the Wilcoxon rank-sum test (FindAllMarkers function) to identify marker genes or upregulated genes Gene ontology analysis was performed using the clusterprofiler package Genes upregulated in the selected clusters were obtained using the FindAllMarkers function and the genes enriched in these clusters were subsequently analyzed for the enrichment of biological processes (BP) using the compareCluster function (function = enrichGO The organoids were fixed with 2% PFA and washed one time with PBS Frozen blocks were made using OCT compound (Sakura Finetek Japan) and frozen using liquid nitrogen on cryomold number 2 (Sakura Finetek A thin frozen section (5–9 µm) was made using CryoStar NX70 Cryostat (Thermo Scientific) and incubated with primary antibody at 4 °C overnight the samples were washed three times with PBS and a secondary antibody was added and kept at room temperature for 1 h The nuclei were stained with DAPI solution (Nacalai) the organoid was mounted with Apathy’s Mounting Media Japan) and covered with cover glass (Matsunami Slides were prepared from more than three independent biological replicates Images were taken with Leica DMi8 microscope with the addition of Leica DFC 7000 T and Leica DFC 9000 GTC camera The liver organoid culture medium was removed and washed once with PBS The liver organoid was divided into two parts and the second part is treated with forskolin Fresh culture medium containing10 μM forskolin was added to the mold chamber and incubated at 37 °C for 90 min Macroscopical imaging was performed after 90 min and the liver organoid was fixed with 2% PFA Immunofluorescence analysis of both samples was then performed accordingly and the duct structures were identified on frozen sections More than 25 luminal structures were analyzed for their diameters Frozen sections were dried out and were circled with a hydrophobic marker Sections were washed three times with PBS containing 0.1% PBST For every 5 ml of alkaline phosphatase buffer (100 mM Tris-HCl [pH 9.0] 33 μl NBT color development substrate (Promega it was added to the slides and incubated for 5 min followed by dehydration with series of ethanol and xylene sections were mounted with a quick–mount solution (Daido Sangyo The ALP enzymatic activity staining was repeated using several sections prepared from more than three independent BVLOs BVLOs were generated in the presence of A8301 Medium containing them was replated every 2 days One JAG1 + /+ and two JAG1−/− -hiPSC clones (We acknowledge Dr Yohei Hayashi from Riken Bioresource research center for providing these valuable samples) were induced to differentiate to imSMCs and used for preparing the BVs which were cocultured with the sheet shape liver organoid to generate BVLOs cell differentiation and co-cultures were repeated three times independently and more than three BVLOs were generated for each clone and the mesothelium membrane from the left liver lobe was peeled off using a sterilized cotton bud Longitudinally unfolded liver organoid was transplanted to the peeled-off portion with liver organoid side facing bottom side and blood vessel side facing the upper side liver organoid was covered by returning the middle lobe on top of it and the peritoneum was closed using a 4 G suture The transplantation experiments were independently replicated more than three times (n > 5) to account for any variability Mice cholestatic model was performed by surgical ligation of the common bile duct mouse was anesthetized with 4% isoflurane at a flow rate of 1.3-2 L/ min for anesthesia induction Mouse abdominal fur was shaved with an electric shaver and plated on a 37 °C heated hot plate abdomen skin is sterilized and cut opened by midline laparotomy of ~3 cm The peritoneum was then cut to open peritoneal cavity Expose the bile duct by caudal movement of the gut Locate the pancreatic duct and place 6–0 suture around the common bile duct over the pancreatic duct Ligate common bile duct with two surgical knots without dissecting the bile duct in between Moisturize the peritoneal cavity with 0.9% NaCl and lift the liver over the Gut the liver organoid was transplanted in the surface of the liver as aforementioned A total of 16 mice were used for the cholestatic mice model transplantation experimented consisting of seven and nine mice were used for the No TP (BDL) and the BVLO group (BDL and BVLO transplantation) Tissue-Tek VIP 3 JR and Tissue-Tek®TECTM (Sakura Finetek CA) were used to embed the transplanted liver tissue in a paraffin block after being fixed with 10% formaldehyde Paraffin block was sliced at 7–9 µm (Thermo Scientific HM 340E USA) and was deparaffinized with xylene three times continued by washing with series of ethanol it was stained with hematoxylin (Muto Pure Chemicals Japan) for 3 min and washed with tap water for 10 min Counterstaining was done with eosin (Muto Pure Chemicals Japan) and then dehydrated with series of ethanol and xylene it was mounted with a quick–mount solution Each experimental group was sectioned and stained in triplicate resulting in a total of nine histological sections and representative picture is as shown in figure The results were expressed as means ± standard error (SEM) of independent experiments All experiments were performed at least three times independently and statistical significance was assessed by the nonparametric Mann–Whitney U test for gene expression analyses and forskolin quantification P values of ≤ 0.05 were statistically significant Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The bulk and scRNA sequencing data generated in this study have been deposited in the GEO database under accession code GSE240534 (subseries: GSE198888, GSE240019, and GSE270413). The source data generated in this study both for main figures and extended figures are provided in the source data file with this paper. Source data are provided with this paper Massive and reproducible production of liver buds entirely from human pluripotent stem cells Vascularized and functional human liver from an iPSC-derived organ bud transplant Takahashi, Y., Sekine, K., Kin, T., Takebe, T. & Taniguchi, H. Self-condensation culture enables vascularization of tissue fragments for efficient therapeutic transplantation. Cell Rep. https://doi.org/10.1016/j.celrep.2018.03.123 (2018) Ogawa, M. et al. Directed differentiation of cholangiocytes from human pluripotent stem cells. Nat. Biotechnol. https://doi.org/10.1038/nbt.3294 (2015) Sampaziotis, F. et al. Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation. Nat. Biotechnol. https://doi.org/10.1038/nbt.3275 (2015) Modeling human bile acid transport and synthesis in stem cell-derived hepatocytes with a patient-specific mutation Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells Efficient and controlled generation of 2D and 3D Bile duct tissue from human pluripotent stem cell-derived spheroids Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development: insights into Alagille syndrome Development of the bile ducts: essentials for the clinical hepatologist Biliary tract anatomy and its relationship with venous drainage Spatiotemporal expression of smooth muscle markers in developing zebrafish gut Tanimizu, N. et al. Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice. Hepatology https://doi.org/10.1002/hep.28521 (2016) Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9 Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells A tissue engineered blood vessel model of Hutchinson-Gilford progeria syndrome using human iPSC-derived smooth muscle cells Multilineage communication regulates human liver bud development from pluripotency Optimal hypoxia regulates human iPSC-derived liver bud differentiation through intercellular TGFB signaling Control of liver cell fate decision by a gradient of TGFβ signaling modulated by Onecut transcription factors Tanimizu, N., Kikkawa, Y., Mitaka, T. & Miyajima, A. α1- and α5-containing laminins regulate the development of bile ducts via β1 integrin signals. J. Biol. Chem. https://doi.org/10.1074/jbc.M112.350488 (2012) Takayama, K. et al. Laminin 411 and 511 promote the cholangiocyte differentiation of human induced pluripotent stem cells. Biochem. Biophys. Res. Commun. https://doi.org/10.1016/j.bbrc.2016.04.075 (2016) Individualized medicine using intestinal responses to CFTR potentiators and correctors A functional CFTR assay using primary cystic fibrosis intestinal organoids Bile duct epithelial tight junctions and barrier function Gamma-glutamyltransferase fractions in human plasma and bile: characteristic and biogenesis Adaptive remodeling of the biliary architecture underlies liver homeostasis NicheNet: modeling intercellular communication by linking ligands to target genes Osteopontin is a novel downstream target of SOX9 with diagnostic implications for progression of liver fibrosis in humans Expression of osteopontin correlates with portal biliary proliferation and fibrosis in biliary atresia Generation of human induced pluripotent stem cell lines carrying homozygous JAG1 deletions Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos Characterization of peribiliary gland–constituting cells based on differential expression of trophoblast cell surface protein 2 in biliary tract A human liver cell atlas reveals heterogeneity and epithelial progenitors Integrated analysis of multimodal single-cell data Effect of three types of mixed anesthetic agents alternate to ketamine in mice Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics Download references Department of Computational Biology and Medical Sciences Yokohama City University Graduate School of Medicine wrote the manuscript with the input from Y.K; E.C. acquired funding; All authors have read and agreed to the published version of the manuscript Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-024-51487-3 Metrics details Liquid biopsy using bile offers a promising non-invasive approach for molecular analysis in cholangiocarcinoma (CCA) such as proteins and circulating DNA (ctDNA) at room temperature has not been fully elucidated This study investigates the temporal stability of proteins and ctDNA in bile samples under room temperature conditions to optimize pre-analytical handling for molecular diagnostics Bile samples were collected from six patients diagnosed with CCA enzyme activity (E-Cadherin and N-Cadherin) and mutant KRAS ctDNA levels were assessed at 1- and 7-hour intervals using quantitative assays and droplet digital PCR (ddPCR) Proteins and enzyme activity demonstrated no significant degradation over the 7-hour room temperature storage period (P > 0.05) mutant KRAS ctDNA levels remained stable without significant changes (P > 0.05) confirming the preservation of molecular integrity in bile samples This study demonstrates that bile samples can maintain the stability of proteins and ctDNA for up to 7 h at room temperature These findings provide critical insights into bile sample handling supporting its application in liquid biopsy and molecular diagnostics for CCA Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, associated with poor prognosis and limited therapeutic options1 Early detection and precise molecular characterization are critical for improving clinical outcomes are invasive and often fail to capture the molecular heterogeneity of the tumor there is a growing need for less invasive and more comprehensive diagnostic methods These attributes position bile as a powerful tool for liquid biopsy in the context of CCA the absence of standardized protocols for bile sample preparation has hindered its integration into routine clinical practice and storage duration significantly impact the stability of bile-derived biomarkers potentially compromising the reliability of molecular testing Although previous studies have explored the utility of bile in molecular analysis All samples were stored for the analysis of circulating tumor DNA (ctDNA) at room temperature and analyzed at four-time intervals: 1 and 7 h after collection at − 80 °C for further analysis The following kits and reagents were used: NucleoSpin® cfDNA XS Kit (Macherey-Nagel) ddPCR™ KRAS Screening Multiplex Kit (Bio-Rad) Protein concentrations were determined using the Bradford assay kit (Thermo Scientific) and all other chemicals were sourced from Sigma (St DNA extraction was performed on 270 µL aliquots using the NucleoSpin® cfDNA XS Kit and ctDNA quality and quantity were analyzed using the Agilent 2100 Bioanalyzer Extracted ctDNA samples were stored at − 20 °C for subsequent use Amplification was conducted on a C1000 Touch Thermal Cycler (Bio-Rad) The fragment size distribution of bile ctDNA was analyzed using the Agilent 2100 Bioanalyzer Mutant KRAS DNA concentration was estimated using the Poisson distribution with a cut-off value of 1.7 copies/µL for KRAS mutations Protein concentrations in bile were determined using the Bradford method Absorbance was measured at 593 nm using an Infinite M 200 Pro microplate reader (TECAN The activities of E- and N-Cadherin in bile were quantified using enzyme-linked immunosorbent assay (ELISA) kits Diluted bile samples (100 µL) and standards were added to 96-well plates pre-coated with capture antibodies The reaction was developed using tetramethylbenzidine (TMB) substrate and absorbance was measured at 450 nm using an Infinite M 200 Pro microplate reader after the addition of stop solution Statistical analyses were conducted using R software (Version 4.4.2, https://web-r.org) and Sigma plot software (Version14.0, https://sigmaplot.softnic.kr) Continuous variables were presented as mean ± standard deviation (SD) and compared using independent t-tests Categorical variables were analyzed using χ² tests Time-dependent changes in variables were assessed using repeated measures ANOVA A p-value of < 0.05 was considered statistically significant To evaluate the clinical significance of the findings effect sizes were calculated alongside P-values Eta-squared (η²) was used to quantify the proportion of variance explained by the independent variable in repeated measures ANOVA Effect sizes were interpreted as small (η² ≤ 0.01) and large (η² ≥ 0.14) based on Cohen’s guidelines A schematic diagram illustrating the preparation process of bile fluid samples collected from patients with cholangiocarcinoma (CCA) The samples were stored for the analysis of circulating tumor DNA (ctDNA) at room temperature and analyzed at four-time intervals: 1 Protein concentration in bile samples from six CCA patients measured at 1 and 7 h after collection using the Bradford method No significant changes in protein concentration were observed over time (Repeated measures ANOVA; P = 0.828) Concentrations of ctDNA in bile samples from six patients with BTC (A) Four patients with wild-type KRAS had ctDNA concentrations below the cut-off value of 1.5 copies/µL (B) Two patients with KRAS mutations exhibited ctDNA concentrations above the cut-off value No significant changes in ctDNA concentrations were observed over time for either group (Repeated measures ANOVA; P = 0.926 and P = 0.399 Enzyme activities of E-Cadherin (A) and N-Cadherin (B) in bile from one BTC patient No significant changes in enzyme activity were observed over time (Repeated measures ANOVA; P = 0.650) One of the key findings of this study is the absence of significant changes in protein concentration and EMT marker activity over the 7-hour period While these results may be considered negative findings their implications are highly significant for the practical use of bile-based liquid biopsy The stability of ctDNA and protein biomarkers over time suggests that bile can serve as a reliable sample source for molecular diagnostics even in real-world clinical settings where immediate sample processing may not always be feasible This offers practical advantages for sample collection and transportation particularly in settings where immediate freezing or processing is not possible This result aligns with previous studies suggesting that bile proteins are relatively robust under similar conditions10 Protein stability is essential for downstream applications including proteomic profiling and biomarker discovery which are crucial for understanding CCA pathogenesis and prognosis These findings validate the practical utility of bile for protein-based assays in clinical and research settings The stability of ctDNA over several hours provides flexibility in sample handling facilitating its application in diagnostic and prognostic workflows E-Cadherin and N-Cadherin activities, markers of EMT, also showed no significant changes over the 7-hour observation period (P = 0.650, Fig. 4) We observed no significant changes in E-Cadherin and N-Cadherin enzyme activities in bile over a 7-hour period This stability suggests that these cadherin-associated enzymatic activities remain largely unaffected by short-term ex vivo conditions may provide a protective environment that minimizes enzymatic degradation such as cellular interactions or inflammatory mediators could contribute to the preservation of enzyme activity These findings indicate that short-term storage does not significantly impact cadherin enzyme activity in bile supporting its feasibility for biomarker analysis within this timeframe The stability of EMT markers in bile further supports its use as a medium for studying tumor biology and developing targeted therapies when bile samples were stored at 4 °C for two months in cfDNA-preserving tubes whereas slight degradation was observed in standard tubes The observed stability of ctDNA and protein levels in bile over the 7-hour period suggests that bile may provide a protective environment for these biomarkers Several biological and chemical factors may contribute to this stability where ctDNA is rapidly degraded by nucleases bile has a distinct composition that may protect ctDNA from enzymatic degradation which may form micelles or vesicle-like structures that encapsulate ctDNA and prevent nuclease-mediated degradation proteins in bile may be stabilized by hydrophobic interactions with bile acids or binding to other molecular carriers reducing susceptibility to proteolytic degradation this study provides initial evidence that bile can serve as a stable medium for biomarker preservation While overall KRAS mutant ctDNA levels remained stable This is presumed to be related to various factors as the variations observed were all within the cut-off value range they did not have a significant impact on the overall results including the small sample size (n = 6) and the lack of validation in larger Only one patient’s bile was analyzed for EMT markers and this restricts the generalizability of our findings the generalizability of these results may be limited by inter-individual variability in bile composition as the patient’s individual disease characteristics could influence enzyme activity measurements Larger cohorts are needed to validate our results and better understand the influence of patient-specific factors on bile composition and biomarker stability Although the primary aim of this study was to evaluate how long protein and ctDNA remain stable in bile at room temperature rather than to compare absolute biomarker levels between different patient groups the lack of a control group could be another limitation The application of bile biomarkers in the context of CCA diagnosis and prognosis holds significant promise and ongoing efforts should aim to expand the utility of bile in precision oncology this study demonstrates that ctDNA and protein biomarkers in bile remain stable for up to 7 h at room temperature supporting the feasibility of bile-based liquid biopsy in cholangiocarcinoma These findings suggest that bile could be a viable diagnostic medium offering practical advantages for clinical workflows where immediate sample processing is not always feasible Further studies with larger patient populations and long-term validation are needed to confirm these results our study provides a foundation for developing standardized protocols for bile sample handling and storage facilitating the integration of bile-based liquid biopsy into clinical practice Availability of data and materialsThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request Kirsten rat sarcoma viral oncogene homolog Percutaneous transhepatic biliary drainage DNA methylation markers for detection of cholangiocarcinoma: Discovery and clinical testing in biliary brushings and plasma Bile cell-free DNA as a novel and powerful liquid biopsy for detecting somatic variants in biliary tract cancer Cell-free DNA from bile outperformed plasma as a potential alternative to tissue biopsy in biliary tract cancer Liquid biopsy from bile-circulating tumor DNA in patients with biliary tract cancer Circulating microRNAs as biomarkers in bile-derived exosomes of cholangiocarcinoma A natural heme deficiency exists in biology that allows nitric oxide to control heme protein functions by regulating cellular heme distribution Role of palliative radiotherapy in unresectable intrahepatic cholangiocarcinoma: Population-based analysis with propensity score matching Elevated levels of neutrophil gelatinase-associated lipocalin in bile from patients with malignant pancreatobiliary disease Stability of bile proteome under varying storage conditions: Implications for biomarker studies Circulating tumor DNA in the diagnosis and monitoring of cholangiocarcinoma: A systematic review Stability and clinical significance of circulating tumor DNA in bile: A new avenue for cholangiocarcinoma diagnosis Expert consensus document: Cholangiocarcinoma: Current knowledge and future perspectives Signed in blood: Circulating tumor DNA in cancer diagnosis Stability of bile-derived cell-free DNA for molecular profiling of biliary tract cancer Diagnostic role of bile pigment components in biliary tract cancer A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories Download references the bile fluids were collected by Seo-Yeon Cha The biospecimen data used in this study was provided by the Keimyung University Dongsan Hospital Biobank which is a member of the Korea Biobank Network This work was supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education Science and Technology (NRF-2022 R1A2C4001769) Division of Hepatobiliary and Pancreatic Surgery Keimyung University Dongsan Medical Center Writing – original draft preparation: J.Y.H. This study was approved by the Institutional Review Board of Keimyung University Dongsan Medical Center under the approval number (DSMC 2022-07-034) All participants provided written informed consent to participate in the study The study was conducted in accordance with the Declaration of Helsinki Download citation DOI: https://doi.org/10.1038/s41598-025-96170-9 Metrics details Adenocarcinoma of the distal common bile duct (CBD) is a rare and aggressive malignancy that is often diagnosed at an advanced stage owing to nonspecific symptoms and delayed presentation This case report details the diagnostic and therapeutic challenges associated with distal CBD adenocarcinoma and highlights the need for an effective multidisciplinary approach A 54-year-old male with a significant smoking history presented with persistent right upper abdominal pain Imaging studies revealed intrahepatic bile duct dilatation Despite initial antibiotic therapy for suspected cholangitis the patient underwent endoscopic retrograde cholangiopancreatography (ERCP) and subsequently a surgical procedure The surgical resection of a common bile duct adenocarcinoma with lymphovascular invasion was successful with subsequent restoration of bile flow through Roux-en-Y hepaticojejunostomy Histopathological analysis confirmed tumor characteristics and clear surgical margins the patient demonstrated significant clinical improvement with normalized bilirubin levels and received appropriate management for his oncologic condition This case highlights the diagnostic complexity of distal CBD adenocarcinoma particularly in patients with delayed symptoms Multimodal imaging approaches and timely surgical intervention are crucial for effective management of this malignancy Enhanced awareness of atypical presentations and advancements in targeted therapies holds promise for improving outcomes in such challenging cases CT is valuable for staging and identifying metastases although it’s less sensitive in early malignancies Ultrasound is widely available and often used initially due to its accessibility and cost-effectiveness; however follow-up imaging with more advanced modalities such as MRCP or CT is frequently necessary to obtain detailed anatomical information and to confirm the diagnosis Combining these approaches optimizes diagnostic accuracy and reduces invasive biopsy needs, though histological confirmation remains essential [1] Immunotherapy may benefit patients with PD-1 or PD-L1 expression This case report describes a rare presentation of adenocarcinoma in the distal common bile duct with a delayed diagnosis highlighting the clinical and diagnostic challenges associated with atypical symptom progression By detailing the comprehensive diagnostic approach we sought to provide insights that can improve early detection and tailored therapeutic strategies for similar complex cases A 54-year-old married male with a 36-pack-year smoking history presented with complaints of persistent right upper abdominal pain that was unrelieved by analgesics accompanied by dark urine and scleral jaundice with a reported weight loss of 10 kg over the last two months The patient did not seek medical attention until the symptoms worsened and his medical history included hypertension for which he was on antihypertensive medication The patient had no significant surgical history Abdominal ultrasonography revealed intrahepatic bile duct dilatation and a distended common bile duct (CBD) measuring 20 mm with an intraluminal heterogeneous mass near its distal portion The gallbladder was filled with a biliary sludge The patient was admitted to the gastroenterology department for further treatment Initial blood tests showed elevated total bilirubin (17.8 mg/dL) and direct bilirubin (9.3 mg/dL) levels an empirical antibiotic therapy was initiated the patient did not show any significant improvement EUS was performed and revealed thickening of the bile duct was observed in the CBD at the mid-lower third obstructing flow through the mid-upper third and preventing the contrast material from passing toward the liver Multislice computed tomography (MSCT) revealed dilation of the biliary tract, with CBD measuring 17 mm, common hepatic duct (CHD) measuring 14 mm, and intrahepatic ductal dilation (see Fig. 1) and suspicious lesions were not observed within these structures The tissue specimens were sent for histopathological evaluation. Roux-en-Y hepaticojejunostomy was performed to restore bile flow and to connect the remaining portion of the CBD to the jejunal loop. The surgery concluded with jejunostomy creation and wound closure. It showed mild dilation of the CBD (17 mm), CHD (14 mm), and pancreatic duct (2.2 mm), with no visible stones or suspicious masses. The liver appeared homogenous with mild fatty infiltration and the gallbladder contained biliary sludge. No free fluid or suspicious lymphadenopathy were detected in the abdomen or pelvis. The cholecystectomy specimen revealed a gallbladder with dimensions of 9.8 × 4.5 × 1.6 cm and a surgical rupture measuring 1 cm with wall thickness ranging from 0.2 to 1 cm a septate cyst with wall thickness of 0.3 cm was observed the patient’s presentation with persistent right upper abdominal pain and significant weight loss over a three-month period before seeking medical care reflected a delayed diagnosis trajectory This delay in recognizing cancer likely contributed to the advanced stage of the disease upon diagnosis aligning with findings from similar studies on delayed presentations of bile duct cancers are crucial for improving survival outcomes underscoring the need for timely diagnostic and therapeutic efforts The combined use of multiple imaging modalities as observed in our patient’s diagnostic workup can improve diagnostic accuracy and help better evaluate complex cases of bile duct adenocarcinoma the patient underwent a successful RYHJ following resection of the distal CBD tumor which contributed to bile flow restoration and significant postoperative improvement These studies underscore the potential benefits of RYHJ particularly in complex biliary reconstructions and advanced cancers as observed in our patient’s favorable postoperative outcomes The presence of LVI in our case underscores the need for aggressive postoperative treatment strategies including adjuvant therapies to improve survival and reduce recurrence careful management of biliary drainage and minimization of postoperative complications are crucial for achieving a favorable outcome more timely screening could potentially lead to earlier intervention These advancements highlight the potential of personalized medicine to improve outcomes in patients with advanced CBD adenocarcinomas While these treatments are still in the early stages they offer optimism for more effective management of distal CBD adenocarcinomas in the future This case highlights the complexities of diagnosing and managing distal common bile duct CBD adenocarcinoma a rare and aggressive cancer often identified only in advanced stages owing to vague symptomatology and challenging imaging findings The successful surgical intervention and postoperative improvement in our patient underscores the value of a multidisciplinary approach that incorporates advanced imaging and targeted surgical techniques to achieve favorable outcomes as delayed diagnosis typically correlates with poor prognoses including biomarkers and molecular imaging offer hope for earlier detection and personalized treatment pathways for patients with distal CBD adenocarcinoma No datasets were generated or analysed during the current study Descriptive study of bile duct tumors in a Province and cause-specific survival analysis of ampullary carcinoma using the SEER database Ceruminous adenocarcinoma: A National Cancer database study of demographic characteristics Surgical outcomes and prognostic factors of distal common bile duct adenocarcinoma: chronological comparison in a single institutional experience of a tertiary Cancer center Incidence and demographics of bile duct cystadenocarcinoma from the National Cancer database Surgical strategy for bile duct cancer: advances and current limitations Current Targeted Therapy Options in the Treatment of Cholangiocarcinoma: A Literature Review The new era of immunotherapy in bile duct Cancer management Delay in diagnosis to treatment and impact on survival of gastric adenocarcinoma in a low income setting without screening facility The colorectal carcinoma prognosis factors: significance of diagnosis delay Revista Española De Enfermedades Digestivas Pancreatic adenocarcinoma masquerading as idiopathic chronic pancreatitis with delayed diagnosis Holzinger F, et al. Carcinoma of the cystic duct leading to obstructive jaundice. A case report and review of the literature. Dig Surg Vol. 1998;15(3):273–8. https://doi.org/10.1159/000018628 Advanced ultrasound diagnosis of extrahepatic bile duct lesions Fujita N et al. Endoscopic approach to early diagnosis of pancreatic cancer. Pancreas vol. 28,3 (2004): 279– 81. https://doi.org/10.1097/00006676-200404000-00012 Imaging of malignancies of the biliary tract- an update or endoscopic Ultrasound-Guided sampling for diagnosis of bile duct cancer: A Meta-Analysis Zhang X, Wang J, Wu W et al. Development and validation of a nomogram based on CT imaging features for differentiating pancreatic head Cancer in periampullary carcinomas, 09 August 2024, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-4694686/v1] Shao JH, Fang HX, Li GW, He JS, Wang BQ, Sun JH. Percutaneous transhepatic biliary drainage and stenting for malignant obstructive jaundice: A report of two cases. Exp Ther Med. 2015;10(4):1503–6. https://doi.org/10.3892/etm.2015.2701 Robotic extrahepatic biliary resection with Roux-en-Y hepaticojejunostomy for type 2 Klatskin tumor Number of positive lymph nodes and lymphatic invasion are significant prognostic factors after pancreaticoduodenectomy for distal cholangiocarcinoma The role of lymphovascular invasion and adjuvant therapy in patients with pancreatic adenocarcinoma Advances in the Early Detection of Hepatobiliary Cancers S37 the impact of early detection of malignant biliary obstruction by endoscopic ultrasound on clinical outcomes after surgical resection Cholangiocarcinoma: early detection and screening in high-risk population Are targeted therapies or immunotherapies effective in metastatic pancreatic adenocarcinoma Development of photodynamic and RNA medicine combination therapies for pancreatic ductal adenocarcinoma Biomarker-driven and molecularly targeted therapies for pancreatic adenocarcinoma Download references This research did not receive any specifc grant from funding agencies in the public Sakhr Alshwayyat & Tala Abdulsalam Alshwayyat Jordan University of Science and Technology Mohammad Shafa’a & Muhammad Fadi Alkurdi Muhammad Fadi Alkurdi conceived and supervised the conduct of the study All authors critically reviewed and revised the manuscript Ehical clearance was obtained from the ethical committee of Kalamoon University (Approval Number: 350) and consent was obtained from our patient to prepare the case for case report Written informed consent was obtained for the publication of this case report Download citation DOI: https://doi.org/10.1186/s12245-025-00859-7 Lipid accumulation in a murine model of fatty liver disease visualized by color-enhanced lipid droplets (pink) in liver tissue (green) Superimposed chemical structure of a newly discovered bile acid conjugate Beneficial gut microbes and the body work together to fine-tune fat metabolism and cholesterol levels, according to a new preclinical study by investigators from Weill Cornell Medicine and the Boyce Thompson Institute at Cornell University’s Ithaca campus The human body has co-evolved with the beneficial microbes that live in the gut (termed the microbiota) resulting in mutually favorable relationships that aid in the digestion of food and absorption of essential nutrients required for survival of the host and the gut microbes A central aspect of these relationships is the production of bioactive molecules that promote the breakdown of food One of the most important groups of such molecules are termed bile acids (also known as ‘bile’) which are produced from cholesterol in the liver and then delivered to the intestine where they promote fat digestion Scientists have known for some time that gut bacteria modify bile acids into a form that stimulates a receptor called FXR, which reduces bile production. The new study reveals that an enzyme produced by intestinal cells converts bile acids into a different form that has the opposite effect called bile acid-methylcysteamine (BA–MCY) inhibits FXR to promote bile production and help boost fat metabolism “Our study reveals there is a dialogue occurring between the gut microbes and the body that is vital for regulating bile acid production,” said co-corresponding author Dr. David Artis director of the Jill Roberts Institute for Research in Inflammatory Bowel Disease and the Friedman Center for Nutrition and Inflammation and the Michael Kors Professor in Immunology at Weill Cornell Medicine Bile acids help the digestive system break down fats into forms the body can take up and use. “But it now has become clear that bile acids are more than just digestive aids; they act as signaling molecules, regulating cholesterol levels, fat metabolism, and more,” said co-corresponding author Dr. Frank Schroeder a professor at the Boyce Thompson Institute and a professor in the Department of Chemistry and Chemical Biology in the College of Arts and Sciences at Cornell University controlling cholesterol metabolism and bile acid production to avoid excess buildup.” an assistant professor of immunology in medicine at Weill Cornell Medicine The multidisciplinary collaboration between Drs Artis and Schroeder has successfully merged the biomedical disciplines of immunology chemical biology and host-microbiota interactions they used a technique called untargeted metabolomics to identify all the molecules produced by mice with and without gut microbes they were able to distinguish which molecules were made by the gut microbes and which were produced by the body BA-MCYs stood out as molecules that were produced by the mice but were nevertheless dependent on the presence of gut microbes “The BA-MCYs demonstrate a new paradigm: molecules that are not produced by the gut microbes but are still dependent on their presence,” co-first author Dr the investigators then showed how the body makes the BA-MCYs and how these molecules provide a way for the body to counteract the microbe’s signals to produce less bile acid preventing the slowdown of cholesterol metabolism “When gut bacteria produce lots of bile acids that strongly activate FXR ensuring the bile acid system stays balanced.” The researchers also showed in their preclinical model that boosting BA-MCY levels helped reduce fat accumulation in the liver and that increasing intake of dietary fiber also enhanced BA-MCY production BA-MCYs were also detected in human blood samples indicating that a similar mechanism may occur in people,” Dr The results may suggest potential treatment targets for metabolic disorders high cholesterol and obesity-related disorders They also suggest that dietary approaches like boosting certain forms of fiber intake may help by supporting the body’s system of checks and balances The next steps for the collaborators are learning more about how these processes are regulated and studying this type of microbe-gut crosstalk in different disease states The investigators suggested their study approach may also help researchers study the role of the gut microbiota in a wide range of diseases from infection and chronic inflammation to obesity and cancer “Our paper is a roadmap to using untargeted metabolomics and chemistry to better understand how the dialogue between the gut microbiota and the body impacts a range of diseases,” Dr Many physicians and scientists at Weill Cornell Medicine maintain relationships and collaborate with external organizations to foster scientific innovation and provide expert guidance. The institution makes these disclosures public to ensure transparency. For this information, see profile for Dr. David Artis The research reported in this story was supported in part by the National Institute for General Medical Sciences National Institute of Diabetes and Digestive and Kidney Diseases the National Institute of Allergy and Infectious Diseases and the National Institute of Arthritis and Musculoskeletal and Skin Diseases Additional support was provided by the Allen Discovery Center program Allen Family Foundation; the Kenneth Rainin Foundation; Cure for IBD; Howard Hughes Medical Institute; the Sanders Family; the Rosanne H Silbermann Foundation; Glenn Greenberg and Linda Vester Foundation; and Weill Cornell Medicine Jill Roberts Institute Metrics details Bile acids are increasingly appearing in the spotlight owing to their novel impacts on various host processes there is growing attention on members of the microbiota that are responsible for bile acid modifications With recent advances in technology enabling the discovery and continued identification of microbially conjugated bile acids the chemical complexity of the bile acid landscape in the body is increasing at a rapid pace we summarize our current understanding of how bile acids and the gut microbiota interact to modulate immune responses during homeostasis and disease Bile acid–microbiota crosstalk in gastrointestinal inflammation and carcinogenesis The gut microbiome–bile acid axis in hepatocarcinogenesis Role of bile acids and bile acid receptors in metabolic regulation Bile salt biotransformations by human intestinal bacteria Influence of the amino acid moiety on deconjugation of bile acid amidates by cholylglycine hydrolase or human fecal cultures Intestinal transport and metabolism of bile acids Key discoveries in bile acid chemistry and biology and their clinical applications: history of the last eight decades Interactions between bacteria and bile salts in the gastrointestinal and hepatobiliary tracts Impairment of human lymphocyte function by bile salts Study of reticuloendothelial phagocytic capacity in patients with cholestasis Effect of cholestasis and bile acids on interferon-induced 2′,5′-adenylate synthetase and NK cell activities The identification of a new class of bile acid modifications: microbially conjugated bile acids Profiling the human intestinal environment under physiological conditions Alternating dual-collision energy scanning mass spectrometry approach: discovery of novel microbial bile-acid conjugates A strategy for screening and identification of new amino acid-conjugated bile acids with high coverage by liquid chromatography-mass spectrometry Neuroactive metabolites and bile acids are altered in extremely premature infants with brain injury Synthesis-based reverse metabolomics led to the identification of new microbial modifications of bile acids Identification of membrane-type receptor for bile acids (M-BAR) A G protein-coupled receptor responsive to bile acids Targeting farnesoid X receptor for liver and metabolic disorders Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis measurement and clinical implications in humans Leaky gut and autoimmunity: an intricate balance in individuals health and the diseased state Partners in leaky gut syndrome: intestinal dysbiosis and autoimmunity Isolation of proteins that interact specifically with the retinoid X receptor: two novel orphan receptors Identification of a nuclear receptor that is activated by farnesol metabolites Expression and activation of the farnesoid X receptor in the vasculature The bile acid receptor FXR is a modulator of intestinal innate immunity Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease Bile acids induce monocyte differentiation toward interleukin-12 hypo-producing dendritic cells via a TGR5-dependent pathway Bile acids activated receptors regulate innate immunity Bile salts of vertebrates: structural variation and possible evolutionary significance Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans A human-like bile acid pool induced by deletion of hepatic Cyp2c70 modulates effects of FXR activation in mice Is CYP2C70 the key to new mouse models to understand bile acids in humans Regulation of bile acid metabolism in mouse models with hydrophobic bile acid composition Glycine and taurine conjugation of bile acids by a single enzyme Molecular cloning and expression of human liver bile acid CoA:amino acid N-acyltransferase Evolutionary analysis of bile acid-conjugating enzymes reveals a complex duplication and reciprocal loss history The continuing importance of bile acids in liver and intestinal disease Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation Microbial hydroxysteroid dehydrogenases: from alpha to omega Clostridium scindens ATCC 35704: integration of nutritional requirements and global transcriptional responses to bile acids Lucas, L. N. et al. Dominant bacterial phyla from the human gut show widespread ability to transform and conjugate bile acids. mSystems https://doi.org/10.1128/msystems.00805-21 (2021) A biosynthetic pathway for the selective sulfonation of steroidal metabolites by human gut bacteria Gut commensal Christensenella minuta modulates host metabolism via acylated secondary bile acids A biosynthetic pathway for a prominent class of microbiota-derived bile acids Human gut bacteria produce ΤΗ17-modulating bile acid metabolites This study identifies human gut bacteria expressing hydroxysteroid dehydrogenases that convert lithocholic acid to 3-oxolithocholic acid and isolithocholic acid two bile acids that suppressed TH17 cell differentiation In search of sustainable chemical processes: cloning and functional characterization of the 7α- and 7β-hydroxysteroid dehydrogenases from Clostridium absonum Contribution of the 7β-hydroxysteroid dehydrogenase from Ruminococcus gnavus N53 to ursodeoxycholic acid formation in the human colon Biological synthesis of ursodeoxycholic acid Bile acids: natural ligands for an orphan nuclear receptor One of the two key studies demonstrating that bile salt hydrolase from members of the gut microbiota has acyltransferase activity and can conjugate amino acids to bile acids Production of new microbially conjugated bile acids by human gut microbiota Human metabolome variation along the upper intestinal tract Paired microbiome and metabolome analyses associate bile acid changes with colorectal cancer progression Natural and adaptive foxp3 + regulatory T cells: more of the same or a division of labor Genetic tracing reveals transcription factor Foxp3-dependent and Foxp3-independent functionality of peripherally induced Treg cells The microbiota regulates type 2 immunity through RORγt+ T cells Individual intestinal symbionts induce a distinct population of RORγ+ regulatory T cells This study shows an essential role for some primary and secondary bile acids and for Bacteroides expressing bile salt hydrolase in enhancing RORγt+ peripherally induced Treg cells in the colon Foxp3+ T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation This study demonstrated a key role for bile acids in modulating host immunity by promoting the differentiation of TH17 cells and of Treg cells A bacterial bile acid metabolite modulates Treg activity through the nuclear hormone receptor NR4A1 Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells Synthesis and identification of lithocholic acid 3-sulfate as RORγt ligand to inhibit TH17 cell differentiation This study demonstrates that 3β-hydroxydeoxycholic acid modulates antigen-presenting cells to induce colonic RORγt+ peripheral Treg cells The role of IL-22 in intestinal health and disease Gut microbiota–bile acid–interleukin-22 axis orchestrates polycystic ovary syndrome This manuscript describes a role for glycodeoxycholic acid and tauroursodeoxycholic acid in promoting IL-22 production by ileal RORγt+ ILC3s An elevated deoxycholic acid level induced by high-fat feeding damages intestinal stem cells by reducing the ileal IL-22 Inulin fibre promotes microbiota-derived bile acids and type 2 inflammation This study links cholic acid and chenodeoxycholic acid to induction of IL-33 increased IL-5 production by ILC2s and enhanced eosinophilia Role of farnesoid X receptor and bile acids in hepatic tumor development Mechanisms of inflammation-driven bacterial dysbiosis in the gut Diet rapidly and reproducibly alters the human gut microbiome Influence of bile acids on colorectal cancer risk: potential mechanisms mediated by diet–gut microbiota interactions Dysbiosis of the gut microbiota and colorectal cancer: the key target of molecular pathological epidemiology Short chain fatty acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases Microbiota-nourishing immunity: a guide to understanding our microbial self Microbiota-nourishing immunity and its relevance for ulcerative colitis The habitat filters of microbiota-nourishing immunity Crosstalk between microbiota-derived short-chain fatty acids and intestinal epithelial HIF augments tissue barrier function Bifidobacteria and butyrate-producing colon bacteria: importance and strategies for their stimulation in the human gut Short-chain fatty acids stimulate angiopoietin-like 4 synthesis in human colon adenocarcinoma cells by activating peroxisome proliferator-activated receptor γ Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion Colonocyte metabolism shapes the gut microbiota Cross-talk between farnesoid-X-receptor (FXR) and peroxisome proliferator-activated receptor gamma contributes to the antifibrotic activity of FXR ligands in rodent models of liver cirrhosis Promotion of lipogenesis by PPARγ-activated FXR expression in adipocytes The farnesoid X receptor regulates adipocyte differentiation and function by promoting peroxisome proliferator-activated receptor-gamma and interfering with the Wnt/beta-catenin pathways No vacancy: how beneficial microbes cooperate with immunity to provide colonization resistance to pathogens The Vat-AIEC protease promotes crossing of the intestinal mucus layer by Crohn’s disease-associated Escherichia coli Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile and toxin activity of clinically relevant C difficile strains by gut microbiota derived secondary bile acids Inhibiting the initiation of Clostridium difficile spore germination using analogs of chenodeoxycholic acid Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile Intestinal bile acids directly modulate the structure and function of C Ursodeoxycholic acid inhibits Clostridium difficile spore germination and vegetative growth and prevents the recurrence of ileal pouchitis associated with the infection Very long O-antigen chains enhance fitness during Salmonella-induced colitis by increasing bile resistance Survival of the fittest: how bacterial pathogens utilize bile to enhance infection Salmonella enterica serovar Typhimurium resistance to bile: identification and characterization of the tolQRA cluster Bile acids function synergistically to repress invasion gene expression in Salmonella by destabilizing the invasion regulator HilD Anti-infective bile acids bind and inactivate a Salmonella virulence regulator Type 6 secretion dynamics within and between bacterial cells Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut Expression of a yersinia pseudotuberculosis type VI secretion system is responsive to envelope stresses through the OmpR transcriptional activator Bile salts modulate the mucin-activated type VI secretion system of pandemic Vibrio cholerae Campylobacter jejuni type VI secretion system: roles in adaptation to deoxycholic acid Micelle formation of sodium deoxycholate and sodium ursodeoxycholate (part 1) Eggerthella lenta DSM 2243 alleviates bile acid stress response in Clostridium ramosum and Anaerostipes caccae by transformation of bile acids Linking long-term dietary patterns with gut microbial enterotypes Host lifestyle affects human microbiota on daily timescales Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10−/− mice Dysbiosis of the gut microbiota in disease Escherichia coli limits Salmonella Typhimurium infections after diet shifts and fat-mediated microbiota perturbation in mice Commensal Enterobacteriaceae protect against Salmonella colonization through oxygen competition Bile acid malabsorption is associated with diarrhea in acute phase of colitis PPARα-UGT axis activation represses intestinal FXR-FGF15 feedback signalling and exacerbates experimental colitis The bile acid sensor FXR is required for immune-regulatory activities of TLR-9 in intestinal inflammation Immunostimulatory DNA ameliorates experimental and spontaneous murine colitis Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis Splenic dendritic cell involvement in FXR-mediated amelioration of DSS colitis FXR mediates ILC-intrinsic responses to intestinal inflammation Mechanism of tissue-specific farnesoid X receptor in suppressing the expression of genes in bile-acid synthesis in mice Fucose ameliorate intestinal inflammation through modulating the crosstalk between bile acids and gut microbiota in a chronic colitis murine model Bile acid malabsorption in inflammatory bowel disease The emerging role of bile acids in the pathogenesis of inflammatory bowel disease The Hylemon–Björkhem pathway of bile acid 7-dehydroxylation: history Identification and characterization of major bile acid 7α-dehydroxylating bacteria in the human gut Completion of the gut microbial epi-bile acid pathway AM58-8 produces 7-dehydroxy-3β bile acids from primary bile acids Bile salt hydrolases: structure and function Download references was supported by T32 DK007202 and F32 AI169989 were funded by National Institutes of Health (NIH) grants AI126277 and DK136117 were supported by AMED grant JP233fa627003 by the Chiba University-University of California-San Diego (UCSD) Center for Mucosal Immunology Division of Host–Microbe Systems and Therapeutics Skaggs School of Pharmacy and Pharmaceutical Sciences Collaborative Mass Spectrometry Innovation Center Chiba University-UC San Diego Center for Mucosal Immunology All authors contributed to the conception and discussion of the article wrote the first version of the manuscript with contributions from L.R.H designed the original concepts for the figures reviewed and edited the manuscript pre-submission and post-submission All authors approved the submitted version of the article bileOmix and Sirenas and a Scientific co-founder Enveda and Arome with prior approval by UC San Diego Download citation DOI: https://doi.org/10.1038/s41577-024-01057-x Eliza Loukou TWO toilet related symptoms could be the first signs of a hard-to-spot cancer that's on the rise in the UK Bile duct cancer - which doctors call cholangiocarcinoma - affects the network of small tubes that connect the liver According to Cancer Research UK around 3,100 people in the UK are diagnosed with bile duct cancer each year Meanwhile, researchers from University College London Hospitals (UCLH) and University College London (UCL) said 5,000 to 6,000 people are diagnosed with the cancer every year - but estimated that true figure could be even higher up to 70 per cent of patients with the disease will die within just 12 months This is partially due to the fact bile duct cancer is notoriously tricky to spot Liver Cancer UK pinpointed two key symptoms of bile duct cancer that may appear when you're on the loo The first of these is unusually dark wee. When you're properly hydrated, your pee should be a pale straw colour. causing the bile to flow back into the blood and body tissues and tingeing urine a dark yellow Another symptom people with bile duct cancer might spot on the loo is pale, putty coloured poo Both these symptoms are potential signs of jaundice which can also cause additional symptoms such as: All these symptoms can be caused by other conditions, but it’s still important to see your GP to find out what’s causing them The number of people getting bile duct cancer has certainly increased in the last few years Researchers don't know for sure what's causing the number to increase Some studies suggest it might be related to factors such as smoking and drinking alcohol Liver Cancer UK stated: "It’s a myth that liver cancers are always related to alcohol it’s unclear whether alcohol is linked to bile duct cancer "Most people who get bile duct cancer are older "Some other liver or gallbladder conditions increase risk because they irritate the bile ducts "People with these may get bile duct cancer when they’re younger than that." such as cirrhosis or a long-term hepatitis infection may also increase the risk of bile duct cancer Game of Thrones actor Ian Gelder passed away at the age of 74 in 2024 after a five-month battle with the disease from the UK's only Cholangiocarcinoma Charity previously said that more awareness of this cancer and the diversity of people – and ages – at risk of developing it is urgently needed “Cholangiocarcinoma is no longer rare in many parts of the UK and cases in adults under the age of 65 are much more common than previously though," she said “People often don’t act on symptoms immediately or are misdiagnosed with other conditions when they do because they don’t fit the profile of what many assume a ‘liver cancer patient’ should look like "But unlike the other more well-known type of primary liver cancer – Hepatocellular carcinoma - there isn’t an established link to liver cirrhosis "The cause of rising cases in otherwise healthy adults is not known." In January this year, patients with bile duct cancer were offered new hope for treating the aggressive disease A study - which showed promising early results - matched patients' tumours to one or more of seven key medicines to drive the disease back Some people on the drugs saw their cancer go into remission while others with previously inoperable cancer became suitable for surgery Doctors said the study will lead to a set of new standards for treating the disease which currently kills the majority of patients in around a year does not mean that a person will get the disease And many people who get the disease have few or no known risk factors Our journalists strive for accuracy but on occasion we make mistakes. For further details of our complaints policy and to make a complaint please click this link: thesun.co.uk/editorial-complaints/ Metrics details Percutaneous transhepatic bile duct stent insertion is a useful alternative to the endoscopic approach for malignant biliary strictures This study retrospectively reviewed the cases of percutaneous metallic stent insertion at our institution to evaluate its safety and usefulness The study included cases of percutaneous bile duct stent insertion performed between April 2016 and August 2024 All patients included those with malignant biliary obstruction and those in whom an endoscopic approach was first attempted but could not reach or cannulate the papilla of Vater Two procedures were used: a two-stage procedure in which a drain was inserted to create an external or internal fistula in which the stent was inserted at the same time as the approach to the bile duct The causes of biliary strictures and complications were examined The study included 14 cases: seven patients had pancreatic head cancer including biliary tract cancer (n = 4) and postoperative gastric cancer (n = 3); three patients who underwent a one-stage insertion The number of inserted stents tended to increase in patients with postoperative cholangiocarcinoma recurrence and four had mild cholangitis; two patients who underwent one-stage procedures had moderate cholangitis and one had mild cholangitis In cases of two-stage expandable metal stent (EMS) insertion the average time from initial drainage to EMS insertion was 10.5 days (4–25) The stent can be safely inserted in a one-stage procedure without compromising the patient’s quality of life one-stage insertion of EMS for malignant biliary stricture may be performed aggressively unless the patient has severe cholangitis This study evaluated the use of percutaneous metal stents for malignant biliary strictures that cannot be drained using an endoscopic approach This study included 14 cases of percutaneous bile duct metal stent insertion performed at Tokai University Hachioji Hospital from April 2016 to August 2024 All 14 patients included those with malignant biliary obstruction and those in whom endoscopic approach was first attempted but could not reach or cannulate the papilla of Vater This study conformed to the ethical principles of the Declaration of Helsinki and was approved by the Institutional Review Board of Tokai University in December 2022 (Approval Number 22R-197) Informed consent was obtained from all the participants or their guardians Written informed consent was obtained from all patients before data release A RADIFOCUS guide wire M 0.035 is inserted from the percutaneous transhepatic gallbladder drainage into the duodenum through the gallbladder duct (black arrow) The tip of the guidewire is placed in the duodenum (white arrow) An 8.5-Fr bile drainage catheter with a side hole makes an internal/external fistula. The white triangle is the location of the stenosis. The black arrow indicates the gallbladder An Amplatz Super Stiff 0.035 wire and percutaneous stent are inserted. The white triangle is the location of the stenosis. The black arrow is the gallbladder The stent is successfully inserted with access through the gallbladder The procedure is terminated by removal of the guidewire The white triangle is the location of the stenosis The complications were assessed up to the time of patient discharge For percutaneous expandable metal stent (EMS) insertion a two-stage procedure was chosen for cases of severe cholangitis selection was done on the spot by the clinician All patients (six male and eight female) were Japanese, with a mean age of 66.5 (39–79) years (Table 1) and three had postoperative gastric cancer Three patients underwent a one-phase insertion One patient was approached through percutaneous transhepatic gallbladder drainage (PTGBD) including perihepatic bile leakage or pancreatitis All cholangiocarcinoma cases were postoperative with bile duct resection and choledochal anastomosis The causes of obstructive jaundice included lymph node recurrence (n = 3) and local recurrence (n = 1) The papilla of Vater was reachable in one case Although it was possible to guide the guidewire to the intestinal tract via a percutaneous approach percutaneous EMS insertion was possible without modification an endoscopic approach using the Rendez-vous technique was not performed A stent was inserted in a one-stage procedure in one case Two patients underwent total gastrectomy followed by Roux-en-Y reconstruction One patient underwent Billroth-2 reconstruction The papilla of Vater was unreachable in all cases guidewire insertion into the intestinal tract was possible the endoscopic approach using the Rendez-vous technique was not chosen because a percutaneous stent could be inserted directly One patient underwent a one-stage procedure There were no cases of stent occlusion during the course of the procedure that required reinsertion cholangiocarcinoma often requires multiple stent insertions This study included two cases of postoperative recurrence of cholangiocarcinoma in which three stents were inserted Side-by-side techniques are necessary in such cases pancreatitis does not need to be considered in pancreatic cancer EMS insertion for non-pancreatic duct dilatation should be performed with caution because of the high risk of pancreatitis We have been using bare metal stents for insertion when the main pancreatic duct might be obstructed We believe the main pancreatic duct is less likely to be obstructed by bare metal stents than by covered stents no pancreatitis occurred among the 10 patients who underwent bare EMS insertion through the papilla of Vater suggesting that the pancreatitis incidence due to stent type should not be a concern if a patient does not have severe cholangitis according to the Tokyo Guidelines 2018 (TG18) one-stage stent insertion is feasible on the day of drainage We performed one-stage insertions in three cases without any complications one-stage insertion should be performed to free the patient from the need of drainage tubing Making a simple comparison was difficult because percutaneous insertion was attempted only in cases where endoscopic insertion was impossible only a few studies from a single institution were available No significant complications were observed in this study possibly because of the small number of cases The number of cases needs to be increased to re-evaluate the usefulness of percutaneous metal stent insertion to increase the generalizability of our findings Percutaneous metal stent insertion is a useful alternative for malignant bile duct stenosis that is difficult to approach endoscopically The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request Percutaneous transhepatic gallbladder drainage Randomised trial of endoscopic stenting versus surgical bypass in malignant low bileduct obstruction Endoscopic management of benign biliary strictures Percutaneous treatment of benign bile duct strictures Endoscopic ultrasound-guided bilioduodenal anastomosis: a new technique for biliary drainage Percutaneous transhepatic balloon dilatation of benign bilioenteric strictures: long-term results in 110 patients Gianturco expandable metallic biliary stents: results of a European clinical trial Endoscopic placement of expandable metal stents for biliary strictures–a preliminary report on experience with 33 patients Obstructive jaundice: use of expandable metal endoprosthesis for biliary drainage Randomised trial of self-expanding metal stents versus polyethylene stents for distal malignant biliary obstruction controlled trial of metal stents for malignant obstruction of the common bile duct Common bile duct obstruction due to malignancy: treatment with plastic versus metal stents A randomized trial of endoscopic drainage methods for inoperable malignant strictures of the common bile duct Long-term experience in Wallstent therapy for malignant choledochal stenosis International multicenter comparative trial of transluminal EUS-guided biliary drainage via hepatogastrostomy vs Initial experience with EUS-guided cholangiopancreatography for biliary and pancreatic duct drainage: a Spanish National survey Endoscopic or percutaneous biliary drainage for gallbladder cancer: a randomized trial and quality of life assessment Percutaneous drainage and stenting for palliation of malignant bile duct obstruction Endoscopic and percutaneous preoperative biliary drainage in patients with suspected hilar cholangiocarcinoma Prediction of drainage effectiveness during endoscopic stenting of malignant hilar strictures: the role of liver volume assessment Risk factors of ineffective drainage in uncovered self-expandable metal stenting for unresectable malignant hilar biliary strictures Current status of percutaneous transhepatic biliary drainage in palliation of malignant obstructive jaundice: a review and risk factors for acute pancreatitis after percutaneous transhepatic biliary stent placement across the papilla of vater Acute pancreatitis after percutaneous insertion of metallic biliary stents in patients with unresectable pancreatic cancer Incidence and risk factors of pancreatitis in obstructive jaundice patients after percutaneous placement of self-expandable metallic stents Biliary stent placement is associated with post-ERCP pancreatitis Percutaneous transhepatic biliary stenting with uncovered self-expandable metallic stents in patients with malignant biliary obstruction– efficacy and survival analysis The number of wire placement in the pancreatic duct and metal biliary stent as risk factors for post-endoscopic retrograde cholangiopancreatography pancreatitis Risk factors for pancreatitis and cholecystitis after endoscopic biliary stenting in patients with malignant extrahepatic bile duct obstruction Lethal post-endoscopic retrograde cholangiopancreatography pancreatitis following fully covered metal stent placement in distal biliary obstruction due to unresectable cholangiocarcinoma Evaluation of post-ERCP pancreatitis after biliary stenting with self-expandable metal stents vs plastic stents in benign and malignant obstructions No benefit of covered vs uncovered self-expandable metal stents in patients with malignant distal biliary obstruction: a meta-analysis Covered versus uncovered double bare self-expandable metal stent for palliation of unresectable extrahepatic malignant biliary obstruction: a randomized controlled multicenter trial A prospective randomized study for efficacy of an uncovered double bare metal stent compared to a single bare metal stent in malignant biliary obstruction TG13 flowchart for the management of acute cholangitis and cholecystitis Single-stage endoscopic treatment for mild to moderate acute cholangitis associated with choledocholithiasis: a multicenter Download references The authors would like to thank Editage for English language proofreading All authors read and approved the final manuscript The protocol for this research project was approved by a suitably constituted Ethics Committee of the institution and conformed to the provisions of the Declaration of Helsinki (Committee of Tokai University in December 2022 A copy of the written consent form is available for review by the journal’s Associate Editor Download citation DOI: https://doi.org/10.1186/s12876-025-03767-5 Metrics details Bile acids (BAs) play important roles in the context of lipid homeostasis and inflammation Based on extensive preclinical mouse studies BA signaling pathways have been implicated as therapeutic targets for cardiovascular diseases differences in BA metabolism between mice and humans hamper translation of preclinical outcomes we generated Cyp2c70−/− mice with a human-like BA composition lacking mouse/rat specific muricholic acids We employed this model to assess the consequences of a human-like BA pool on atherosclerosis and heart function in hypercholesterolaemic mice We overexpressed a PCSK9 gain-of-function (GOF) mutation in the liver of male Cyp2c70−/− and Cyp2c70+/− control mice and fed these mice a Western-type diet (WD) for 12 weeks Cyp2c70−/− mice displayed a hydrophobic BA pool rich in chenodeoxycholic acid Cyp2c70−/− mice showed reduced hepatic total cholesterol and triglycerides (p < 0.05) combined with lower plasma total cholesterol (p < 0.05) and triglycerides (p = 0.05) due to lower VLDL levels Circulating white blood cells remained largely unaffected in Cyp2c70−/− mice we found a trend (p = 0.08) towards smaller atherosclerotic lesions in the aortic root of Cyp2c70−/− mice but no effect on cardiac morphology or function was observed a human-like BA composition ameliorated PCSK9-GOF-induced hypercholesterolaemia in WD-fed mice which translated into a tendency towards smaller atherosclerotic lesions this may differentially affect cardiovascular function we aimed to investigate the effect of a human-like BA pool on atherosclerosis development and heart function in male Cyp2c70−/− mice fed a Western-type diet (WD) Successful depletion of hepatic LDLR upon AAV-mediated transduction of PCSK9-GOF in Cyp2c70−/− mice possessing a hydrophobic BA pool with substantial amounts of CDCA (A) mRNA expression of PCSK9 in in livers of WT (n = 15) and PCSK9-GOF virus injected and WD-fed Cyp2c70+/− (Het) and Cyp2c70−/− (KO) mice (n = 11–13 per group) (B) LDLR and β-actin protein levels in livers of WT (n = 2) and PCSK9-GOF virus injected and WD-fed Cyp2c70+/− (n = 4) and Cyp2c70−/− (n = 5) mice (C) Abundance of BA species in plasma and bile of PCSK9-GOF Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD Plasma concentration of (D) total BAs and (E) (T)MCAs + (T)UDCA and (T)CA in PCSK9-GOF Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD Biliary concentration of (F) total BAs and (G) TMCAs and TCA in PCSK9-GOF Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD (I) ratio of 12α/non-12α-hydroxylated BAs in bile and (K) hepatic mRNA expression of Cyp7a1 and Cyp8b1 in PCSK9-GOF Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD Gene expression is normalized to cyclophilin A and depicted as fold change compared to the control group and ****p < 0.0001 as determined by Kruskal-Wallis followed by Dunn’s post hoc test (A) K) or Mann-Whitney U test (G) (n = 11–13 per group) these data confirmed a more human-like BA pool in Cyp2c70−/− mice and efficient PCSK9-GOF-induced knockdown of LDLR in livers of both Cyp2c70−/− and Cyp2c70+/− mice these findings indicate that WD-induced fatty liver is mitigated despite elevated liver damage in Cyp2c70−/− mice Diminished lipid accumulation with mild liver damage in Cyp2c70−/− mice (A) Body weight and (B) liver to body weight ratio of PCSK9-GOF Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD feeding (C) Plasma alanine transaminase (ALT) and aspartate aminotransferase (AST) levels (D) Representative images of liver sections stained with haematoxylin and eosin (E) Hepatic triglyceride (TG) levels in Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD feeding (F) Hepatic total cholesterol levels in Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD feeding H) Hepatic cholesteryl ester levels in Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD feeding Decreased plasma VLDL-cholesterol levels and limited effects on circulating white blood cells in Cyp2c70−/− mice (A) Total plasma cholesterol levels and triglyceride (TG) levels (B) Cholesterol distribution after lipoprotein fractionation of plasma sample by fast protein liquid chromatography (E) White blood cells subsets count and (F) percentage of blood leukocyte levels of Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD *p < 0.05 as determined by Mann-Whitney U test (A Atherosclerotic plaque size and heart function were not affected in Cyp2c70−/− mice (A) Representative of haematoxylin and eosin-stained sections and MAC2+ stained sections of the aortic root of Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD feeding (B) Quantification of atherosclerotic root lesions area (C) Quantification of MAC2+ area in atherosclerotic lesions left ventricular end-diastolic and end-systolic internal diameters (E) and fractional shortening (F) in Cyp2c70−/− and Cyp2c70+/− mice after 12 weeks of WD feeding Data represent mean ± SEM (n = 10–13 per group) Although hepatic inflammation was not measured in our study liver damage markers were significantly elevated in plasma of Cyp2c70-/- mice compared to controls our data indicates that the BA pool of Cyp2c70-/- mice exhibits an increased hepatic cytotoxic potential compared to the BA pool of Cyp2c70+/- mice implying that inflammation may have played a role in downregulating Cyp7a1 and Cyp8b1 in Cyp2c70-/- mice Since hepatic lipid levels were significantly reduced in our study the reduced ratio of 12α/non-12α-hydroxylated BAs in bile of Cyp2c70−/− mice may thus be indicative of impaired fat absorption thereby reducing plasma cholesterol levels in this current study strong BA receptor activators in the plasma BA pool are associated with risk factors of CVD in humans The effect of BAs on heart function and its physiological relevance remains therefore unclear Future studies should investigate the effect of human FXR on atherosclerosis development and heart function in Ldlr−/− or ApoE−/− mice Here we used the PCSK9-AAV model to downregulate the LDLR and induce atherosclerosis to investigate the impact of a humanized BA profile on early plaque development in Cyp2c70−/− mice fed a WD for 12 weeks We observed a marginal trend towards decreased atherosclerosis which may be explained by the small lesion sizes in our mice future studies should include a longer WD intervention when applying the PCSK9-GOF-AAV model to study atherosclerosis Studying the impact of CYP2C70 knockdown or overexpression in Ldlr−/− or ApoE−/− mice would be an alternative approach to provide more definitive data to clarify the role of hydrophobic BAs on atherosclerosis disease risk male WD-fed Cyp2c70−/− mice with a PCSK9-GOF showed a tendency towards smaller atherosclerotic lesions in the initiation phase of the disease Reduced total hepatic and plasma cholesterol (a tendency of) triglycerides and VLDL-cholesterol levels in Cyp2c70−/− mice could potentially explain these results All methods were performed in accordance with the relevant guidelines and regulations Mice were individually housed in a temperature-controlled room (21 °C) with a 12-hourslight/12-hours dark cycle and ad libitum access to food and water Directly after virus injection mice were fed a Western diet (D11111701-1.5; 45% kcal% Fat and 0.21% Cholesterol; Research Diets Inc body composition was measured in conscious mice using a Minispec Body Composition Analyzer (LF90; Bruker Bio-Spin GmbH four hours fasted mice were anesthetized with Hypnorm (fentanyl/fluanisone; 1 ml/kg) and diazepam (10 mg/kg) after which the bile duct was ligated and the gallbladder cannulated Mice were then placed into a humidified incubator to maintain body temperature and bile was collected continuously for 30 min Bile production was determined gravimetrically Blood was collected and centrifuged in EDTA-containing tubes and plasma was stored at −80 C for further analysis and fixed in formalin or snap-frozen in liquid nitrogen and stored at −80 °C The study is reported in accordance with the ARRIVE guidelines and conforms to its principles total cell lysates and liver homogenates were obtained using NP40 buffer (0.1% Nonidet P-40 (NP-40) freshly supplemented with protease and phosphatase inhibitors (Roche Protein concentration was determined using the Bradford assay (Bio-Rad 25 mg of protein was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Amersham™ HybondTM-P PVDF Transfer Membrane (GE Healthcare Membranes were blocked in 5% milk in tris-buffered saline with 0.01% Tween20 and incubated with rabbit anti-LDLR (1:1000 Germany) and mouse anti-beta-actin (1:5000 A5441 Proteins were visualized using a ChemiDoc™ XRS + System (Bio-Rad) using Image Lab software version 5.2.1 (Bio-Rad) Plasma transaminases were analysed using a Cobas 6000 analyser with standard reagents (Roche Diagnostics Total and free cholesterol was determined using a colorimetric assay and cholesterol standard FS was used as a reference (DiaSys Diagnostic Systems Cholesteryl ester was determined by subtracting the value of free cholesterol from the total cholesterol Triglyceride levels were measured using a Trig/GB kit and Precimat Glycerol standard was used as a reference (Roche For MAC2 staining paraffin sections were incubated in a preheated citric acid-based buffer (H-3300 Sections were then blocked in 10% goat serum for 30 min at RT and incubated overnight at 4 °C with rat anti-mouse/human MAC2 primary antibody (1/5000 dilution Sections were subsequently stained with biotinylated goat anti-rat secondary antibody for 30 min at RT (1:125 dilution) and Vectastain ABC-peroxidase kit according to the manufacturer’s instructions followed by staining with diaminobenzidene (DAB) for 10 min at RT and counterstaining with haematoxylin Echocardiographic measurements were performed using a Vevo 3100 preclinical imaging system equipped with a 40-MHz MX550D linear array transducer (FUJIFILM VisualSonics Left-ventricular (LV) short axis M-mode recordings were obtained at the mid-papillary level Vevo LAB software (version 5.7.1; FUJIFILM VisualSonics) was used to determine heart rate LV end-diastolic internal diameter (LVIDd) LV end-systolic internal diameter (LVIDs) and fractional shortening (FS) by outlining the epicardial and endocardial borders using the LV Trace tool Neutrophils were identified as CD45+CD115−Ly6G+ and monocytes as CD45+CD115+ Monocytes were further identified as Ly6Clow and Ly6Chigh subsets Flow cytometry measurements were performed on NovoCyte Quanteon (Agilent US) and analysed using NovoExpress (version 1.6.2) software All data are presented as mean ± standard errors of the mean (SEM) Statistical analysis was performed using GraphPad Prism 9 Software (Graphpad Software All data were tested for normality by d’Agastino and Pearson normality test non-parametric Mann-Whitney U test was used Data was considered significant if p < 0.05 The dataset that support the findings of this study have been deposited in Harvard Dataverse: https://doi.org/10.7910/DVN/TAEUOA Cardiovascular Diseases (CVDs). (2021). https://www.who.int/news-room/fact-sheets/detail/cardiovascular-diseases-(cvds) Targeting inflammation in atherosclerosis — from experimental insights to the clinic Emerging roles of gut microbial modulation of bile acid composition in the etiology of Cardiovascular diseases Diminished bile acids excretion is a risk factor for coronary artery disease: 20-year follow up and long-term outcome Simultaneous inhibition of FXR and TGR5 exacerbates atherosclerotic formation TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading Cholangiopathy and biliary fibrosis in Cyp2c70-Deficient mice are fully reversed by Ursodeoxycholic Acid Emerging roles of bile acids in control of intestinal functions Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions Relationship between Heart Disease and Liver Disease: a two-Way Street Low production of 12α-hydroxylated bile acids prevents hepatic steatosis in Cyp2c70-/- mice by reducing fat absorption Gut microbiome and bile acids in obesity-related diseases Atherogenic postprandial remnant lipoproteins; VLDL remnants as a causal factor in atherosclerosis VLDL best predicts aortic root atherosclerosis in LDL receptor deficient mice Of mice and men: murine bile acids explain species differences in the regulation of bile acid and cholesterol metabolism Genetic and Microbial associations to plasma and fecal bile acids in obesity relate to plasma lipids and liver Fat Content Circulating bile acids concentration is predictive of coronary artery disease in human Fasting serum total bile acid level is associated with coronary artery disease myocardial infarction and severity of coronary lesions Bile acid metabolites in serum: intraindividual variation and associations with coronary heart disease The amino acid residues asparagine 354 and isoleucine 372 of human farnesoid X receptor confer the receptor with high sensitivity to chenodeoxycholate Adeno-Associated virus-mediated gain-of-function mPCSK9 expression in the mouse induces hypercholesterolemia AAV9 transduction mediated by systemic delivery of vector via retro-orbital injection in newborn Advancing age increases the size and severity of spontaneous atheromas in mouse models of atherosclerosis Chronic infusion of taurolithocholate into the brain increases fat oxidation in mice Generation of analytic plasma lipoprotein profiles using two prepacked superose 6B columns A rapid method of total lipid extraction and purification Cholesterol Accumulation in dendritic cells links the Inflammasome to Acquired Immunity Download references We thank the personnel from the Central Animal Facility of the University Medical Center Groningen for their support with the animal experiments This work was funded by the Netherlands Heart Foundation to F Koonen and Folkert Kuipers contributed equally European Research Institute for the Biology of Ageing (ERIBA) FK and DK conceived and designed the study All others critically reviewed the manuscript and approved the final version Download citation DOI: https://doi.org/10.1038/s41598-025-86183-9 February marks Gallbladder Cancer and Bile Duct Cancer Awareness Month a time to shine a light on two often-overlooked cancers that can have a significant impact on those diagnosed Gallbladder cancer and bile duct cancer are rare but they can be aggressive and challenging to detect in the early stages and encourage a better understanding of these cancers to improve outcomes for those affected Gallbladder cancer is a rare cancer that begins in the gallbladder a digestive fluid that helps break down fats in food Most gallbladder cancers are adenocarcinomas which are cancers that start in the glandular cells lining the organ Because gallbladder cancer is often asymptomatic in its early stages it is frequently diagnosed at a later stage making it more difficult to treat effectively Common risk factors for gallbladder cancer include: which are the tubes that carry bile from the liver to the gallbladder and small intestine There are two main types of bile duct cancer: intrahepatic (inside the liver) and extrahepatic (outside the liver) Bile duct cancer is also rare but can be quite aggressive Symptoms often appear only when the disease is in advanced stages The symptoms of both gallbladder cancer and bile duct cancer can be vague and often resemble other Because these symptoms can be attributed to a range of conditions many people with gallbladder or bile duct cancer may not seek medical attention until the disease has advanced Early detection is key to improving outcomes which is why it is important to discuss any persistent or unusual symptoms with a healthcare provider While gallbladder and bile duct cancers are not always preventable understanding and managing risk factors can help reduce the likelihood of developing these cancers Treatment for gallbladder and bile duct cancers typically depends on the stage at diagnosis and the overall health of the patient A multidisciplinary approach involving surgery and supportive care is often necessary for managing these cancers Because both gallbladder and bile duct cancers are often diagnosed in later stages improving early detection is crucial to increasing survival rates Regular medical checkups and awareness of the risk factors and symptoms can help catch these cancers in their earlier we encourage everyone to take the time to learn more about gallbladder and bile duct cancer we can all play a role in improving outcomes for those diagnosed with these cancers If you or someone you know is experiencing symptoms such as unexplained abdominal pain it is important to seek medical attention right away Early diagnosis and treatment can improve the chances of a successful outcome we are committed to supporting our community with comprehensive care and information If you are experiencing symptoms or are at risk don’t wait—schedule an appointment with one of our healthcare providers today by calling 307-688-3636 and support those affected by gallbladder and bile duct cancer If the first thing you do when something is awry with your health is head to “Dr Sign Up for Our Newsletter Metrics details Bile acid overload critically drives the pathogenesis of cholestatic liver injury (CLI) While ceramide metabolism has garnered increasing interest in liver research the role of ceramides in CLI remains unclear This study investigates the function of alkaline ceramidase 3 (ACER3)-catalyzed hydrolysis of unsaturated ceramides in CLI this work finds that ACER3 expression is upregulated in the cholestatic liver and positively correlated with the severity of CLI in patients Acer3 ablation increases ceramide(d18:1/18:1) and attenuates bile duct ligation-induced CLI in female mice with reduced hepatic necrosis it does not significantly impact CLI in male mice ceramide(d18:1/18:1) treatment attenuates CLI in wild-type female mice ACER3 knockdown and ceramide(d18:1/18:1) treatment prevent lithocholic-acid-induced cell death in human-liver-derived HepG2 cells ceramide(d18:1/18:1) binds the ligand binding domain of the liver X receptor β acting as an agonist to activate its transcriptional functions This activation upregulates sulfotransferase 2A1-catalyzed bile acid sulfation thereby reducing bile acid overload in hepatocytes to attenuate CLI Our findings uncover the role of ceramide(d18:1/18:1)-liver X receptor β signaling in mitigating bile acid overload in the cholestatic liver offering mechanistic insights and suggesting therapeutic potential for targeting ACER3 and ceramide(d18:1/18:1) for CLI suggesting a plausible NR-mediated signaling interplay between CER and BA metabolism the specific roles of individual CERs in regulating CLI remain poorly understood the function of ACER3 and ULCCs in regulating liver pathogenesis remains unclear we show that ACER3 expression is upregulated in cholestatic livers and positively correlates with the severity of CLI in patients Targeting ACER3 increases CER(d18:1/18:1) in the cholestatic liver which binds to LXRβ and activates its signaling transduction to enhance BA sulfation and restore lipogenesis These mechanisms mitigate BA overload in hepatocytes and attenuate CLI Our findings highlight the therapeutic potential of targeting ACER3 and CER(d18:1/18:1) to attenuate CLI a–f Clinical relevance of ceramide (CER) dysregulation in cholestatic liver injury (CLI) Hepatic CERs in patients with CLI (n = 30) and non-CLI (n = 30) (a) Correlation of dysregulated CERs and their metabolic enzymes with serum CLI severity markers (SCSMs) in CLI patients (c) Hepatic alkaline ceramidase 3 (ACER3) staining (d) and quantification of ACER3-positive cells (e) the black arrows and black arrowheads indicate ACER3-positive cells and cholestasis Correlation between ACER3-positive cells and SCSMs in CLI patients (f) g–p CLI in Acer3fl/fl and Acer3ΔHep female mice (n = 8) Hematoxylin and eosin (H&E) staining with circle areas and red arrows indicating necrotic foci (g) and quantification of necrotic areas (h) Higher-magnification images highlighting necrotic areas in H&E staining of Fig with black arrows indicating regions of inflammatory infiltration (j) Lymphocyte antigen 6 complex locus G6D (Ly6G) staining (l) and quantification of Ly6G-positive cells (m) Sirius Red (left panel) and alpha-smooth muscle actin (αSMA) staining (right panel) (n) Statistical significances were tested by the unpaired two-sided Student’s t-test (a and one-way ANOVA with Tukey’s multiple comparisons (h These findings demonstrate that Acer3 ablation attenuates CLI in female mice indicating that ACER3 upregulation plays a pathogenic role in CLI a Differentially expressed genes (DEGs) of hepatic transcriptomes in Acer3+/+ and Acer3-/- female mice (n = 4) b–g Sulfotransferase 2A1 (Sult2a1)-catalyzed bile acid (BA) sulfation in Acer3fl/fl and Acer3ΔHep female mice (n = 8) mRNA (b) and protein levels of Sult2a1 (c and d) h–s Sult2a1-catalyzed BA sulfation and CLI in the liver of Acer3fl/fl and Acer3ΔHep BDL female mice with or without Sult2a1 knockdown (n = 6) H&E staining with the red arrows indicating necrotic foci (l) and quantification of necrotic areas (m) Ly6G staining (p) and quantification of Ly6G-positive cells (q) f) and one-way ANOVA with Tukey’s multiple comparisons (b These findings demonstrate that loss of Sult2a1 abrogates the protective function of Acer3 ablation against CLI in female mice suggesting that Acer3 ablation alleviates CLI through upregulating Sult2a1-catalyzed BA sulfation and normalizing BA metabolism in female mice a–f Hepatic liver X receptor β (Lxrβ) expression in Acer3fl/fl and Acer3ΔHep female mice (n = 8) Immunoblot of Lxrβ in female mice with sham operation (b) Immunoblot of Lxrβ in the liver of female mice under sham and bile duct ligation (BDL) conditions (c) Immunofluorescent co-staining with Lxrβ and albumin (Alb) (d) and quantification of nuclear-Lxrβ-positive hepatocytes (e) Immunoblot of cytoplasmic and nuclear Lxrβ (f) g–v Examination of Lxrβ-Sult2a1 pathway and CLI in the liver of Acer3ΔHep female BDL mice with or without Lxrβ knockdown (n = 6) Immunoblot of cytoplasmic and nuclear Lxrβ (h) H&E staining with the circle areas and red arrows indicating necrotic foci (n) and quantification of necrotic areas (o) Ly6G staining (r) and quantification of Ly6G-positive cells (s) Sirius Red staining (left panel) and αSMA staining (right panel) (u) Statistical significances were tested by the unpaired two-sided Student’s t-test (g t) and one-way ANOVA with Tukey’s multiple comparisons (a a–j Hepatic CERs and lipid content in Acer3fl/fl and Acer3ΔHep female mice (n = 8) mRNA levels of Lxrβ-driven lipogenic genes in the liver (b) Principal component analysis (PCA) analysis of discrimination in hepatic lipid content with scoring plot (c) and loading plot (d) in Acer3fl/fl and Acer3ΔHep female mice under both sham and BDL conditions (n = 4) the arrow labels indicate the enriched lipid content in the indicated mouse groups lysodimethylphosphatidylethanolamine (LMPE) TG and PLs were dramatically affected by BDL Volcano plot exhibiting the difference of individual lipid species between Acer3fl/fl female mice with BDL and sham operation (e) Acer3ΔHep and Acer3fl/fl female mice with sham operation (f) Acer3ΔHep female mice with BDL and sham operation (g) Acer3ΔHep and Acer3fl/fl female mice with BDL operation (h) Oil Red O (ORO) staining with black arrows illustrating ORO-positive areas (i) and quantification of ORO-positive areas (j) (k) ORO staining with black arrows illustrating ORO-positive areas and quantification of ORO-positive areas in the liver of Acer3ΔHep BDL female mice with or without Sult2a1 knockdown (n = 6) l ORO staining with black arrows illustrating ORO-positive areas and quantification of ORO-positive areas in the liver of Acer3ΔHep BDL female mice with or without Lxrβ knockdown (n = 6) Statistical significances were tested by the one-way ANOVA with Tukey’s multiple comparisons (a j) and unpaired two-sided Student’s t-test (e-h CER(d18:1/18:1)-treated C57BL/6 J wild-type (WT) female mice were subjected to BDL (n = 8) a Hepatic CER levels after CER(d18:1/18:1) treatment H&E staining with the circle areas and red arrows indicating necrotic foci (b) and quantification of necrotic areas (c) Serum transaminase levels (d) Inflammatory gene mRNA levels (e) with black arrows indicating regions of inflammatory infiltration and quantification of Ly6G-positive cells (g) Sirius Red staining (left panel) and αSMA staining (right panel) (i) Immunofluorescent co-staining with Lxrβ and Alb (l) and quantification of nuclear-Lxrβ-positive hepatocytes (m) s and t ORO staining with black arrows indicating ORO-positive areas (s) and quantification of ORO-positive areas (t) u The mRNA levels of Lxrβ-driven lipogenic genes a Virtual model of Lxrβ LBD-CER(d18:1/18:1) complex (b) Nuclear CER(d18:1/18:1) in the liver of Acer3fl/fl and Acer3ΔHep female mice with or without BDL (n = 6) c–g Sult2a1-catalyzed BA sulfation in the liver of CER(d18:1/18:1)-treated C57BL/6 J WT female BDL mice with or without Lxrβ knockdown (n = 6) and quantification of Sult2a1 proteins (f) in the liver h–j Lipid content and mRNA expression of lipogenic genes in the liver of CER(d18:1/18:1)-treated C57BL/6 J WT female BDL mice with or without Lxrβ knockdown (n = 6) ORO staining with black arrows indicating ORO-positive areas (h) and quantification of ORO-positive areas (i) k–s Examination of CLI in CER(d18:1/18:1)-treated C57BL/6 J WT female BDL mice with or without Lxrβ knockdown (n = 6) H&E staining with the circle areas and red arrows indicating necrotic foci (k) and quantification of necrotic areas (l) Ly6G staining (o) and quantification of Ly6G-positive cells (p) Sirius Red staining (left panel) and αSMA staining (right panel) (r) Statistical significances were tested by the one-way ANOVA with Tukey’s multiple comparisons (b) and unpaired two-sided Student’s t-test (c a SULT2A1 mRNA levels in the healthy liver tissues of male (n = 70) and female (n = 33) humans from the Genotype-tissue expression (GTEx) database b–g Expression levels of SULT2A1 in the collected liver tissues of male and female patients with or without cholestasis Immunoblot of SULT2A1 in the collected liver tissues of male (n = 12) and female (n = 12) patients with non-CLI (c) or CLI (d) and quantification of SULT2A1 expressions (e) Immunoblot of SULT2A1 (f) in the collected liver tissues of patients with non-CLI (n = 12) or CLI (n = 12) and quantification of SULT2A1 expressions (g) h ACER3 mRNA levels in the collected liver tissues of male and female patients i Heat map of the mRNA levels of CER-metabolizing enzymes in the healthy liver tissues of male (n = 70) and female (n = 33) humans from the GTEx database j Heat maps of the mRNA levels of CER-metabolizing enzymes in the collected liver tissues of male and female patients with non-CLI (left panel) and CLI (right panel) k–m CER(d18:1) levels in the collected liver tissues of male and female patients with non-CLI (k) and CLI (l) l) and one-way ANOVA with Tukey’s multiple comparisons (b and cleaved-poly ADP-ribose polymerase (C-PARP) in HepG2 cells transfected by shCON and shACER3 lentivirus following treatment of vehicle (transfection medium) b and c LCA-sulfate in HepG2 cells (b) and the conditional medium (c) with or without ACER3 knockdown and LCA treatment and C-PARP in ACER3- and SULT2A1-knockdown HepG2 cells with or without 200 μM LCA e and f LXRβ immunofluorescence (e) and luciferase activity on SULT2A1 promoter (f) in shCON and shACER3 HepG2 cells with or without LCA g and (h) Luciferase activity on SULT2A1 promoter (g) and immunoblots of SULT2A1 and C-PARP (h) in ACER3-knockdown HepG2 cells with or without LXRβ knockdown (i) Unsaturated CER(d18:1) in shCON and shACER3 HepG2 cells with or without LCA treatment j Surface plasmon resonance (SPR) titration curves reflect the interaction between recombinant human LXRβ and CER(d18:1/18:1) l CER(d18:1/18:1) in the immunoprecipitated LXRβ-FLAG proteins m Virtual structure of human LXRβ ligand-binding domain (LBD)-CER(d18:1/18:1) complex n Interaction between CER(d18:1/18:1) and the agonism-related residues within LXRβ LBD Images and results represent the results of three independent experiments Statistical significances were tested by the one-way ANOVA with Tukey’s multiple comparisons (b l) and unpaired two-sided Student’s t-test (f) underscoring the pivotal role of LXRβ in mediating the effects of ACER3 knockdown relative to LXRα These findings unraveled CER(d18:1/18:1) as an endogenous agonist of LXRβ indicating that CER(d18:1/18:1)-LXRβ signaling transduction crucially mediates the protective effects of ACER3 ablation against CLI This study presents the insights regarding the role of ACER3 and its endogenous substrate in regulating the metabolic resilience of BAs and lipids in the cholestatic liver We found that cholestasis upregulates ACER3 while ACER3 ablation promotes the binding of CER(d18:1/18:1) to LXRβ to activate LXRβ signaling thereby improving BA detoxification and lipogenesis to attenuate CLI These findings unravel the function of CER(d18:1/18:1)-LXRβ signaling in maintaining BA and lipid metabolic resilience to counter BA overload in hepatocytes serving as a promising therapeutic target of CLI These suggest that cholestasis may drive ACER3 upregulation through multiple regulatory pathways involving these transcription factors These findings provide a plausible explanation for the upregulation of ACER3 in cholestasis and reinforce the role of ACER3 in CER metabolism under cholestatic conditions These findings confirm that ACER3 ablation improves BA detoxification by upregulating SULT2A1 to attenuate CLI uncovering a function of CER metabolism in regulating BA detoxification indicating that LXRα does not play a primary role in mediating the upregulation of SULT2A1 expression induced by ACER3 knockdown These findings indicate that the regulation of SULT2A1 expression by ACER3 is primarily mediated through LXRβ indicating that ACER3 upregulation impedes the buildup of CER(d18:1/18:1) in the cholestatic liver preserving lipogenesis is also critical for Acer3 ablation to attenuate CLI suggesting that targeting the ACER3-CER(d18:1/18:1) metabolic axis may facilitate the formation of the LXRβ/RXRα heterodimer which in turn upregulates mRNA expression of LXRβ via a positive feedback mechanism our findings indicate that CER(d18:1/18:1) functions as an endogenous agonist of LXRβ mediating the protective effects of ACER3 ablation against CLI This provides a molecular insight into the therapeutic potential of CER(d18:1/18:1) for CLI our results indicate that the sex-specific expression patterns and functions for Sult2a1 and Acer3-mediated CER metabolism observed in mice are unlikely expected in humans suggesting that targeting ACER3 could be an effective therapeutic strategy for CLI in both male and female patients our study reveals the role of CER in regulating BA metabolism underscoring the importance of exploring the regulatory interactions between CER and other metabolic pathways to uncover broader pathophysiological roles of metabolic cross-regulation in liver diseases our study revealed that ACER3 plays a pathological role in CLI by impeding the buildup of CER(d18:1/18:1) while CER(d18:1/18:1) acts as an endogenous agonist of LXRβ to improve BA detoxification and lipogenesis in the liver with CLI Our work lays the groundwork for future therapeutic interventions targeting ACER3 or supplementing CER(d18:1/18:1) to treat cholestatic liver diseases The experiments using human samples were approved by the Medical Ethics Committee of Nanfang Hospital of Southern Medical University under ethical ID NFEC-2021-356 All research was conducted in accordance with relevant guidelines and regulations and written informed consent was obtained from all patients The animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Southern Medical University and 0.5% CMC-Na was used as vehicle control CER(d18:1/18:1) treatment was performed once every day for 10 days Mice were sacrificed 7 days after the BDL operation The tissues and serum were collected and stored at -80 °C Liver tissues were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich USA) or Tissue-Tek OCT compound (Sakura Finetek RNA sequencing data including 70 male and 33 female liver tissues were obtained from the GTEx database (https://www.gtexportal.org/) and used for analysis in this study R version 4.3.2 was used to conduct a gene expression analysis of CER-metabolizing genes between female and male livers and corresponding visualization ACER3-related transcription factor prediction was accomplished by the online predicted tool (https://jingle.shinyapps.io/TF_Target_Finder/) all of the ACER3-related potential transcription factors from different databases were selected and visualized in the form of a Venn chart The promoter of SULT2A1 (-2000 to -1 bp) was subcloned and inserted into a pGL3-basic vector (Promega) (Figure S11j) The ACER3-knockdown HepG2 cells and ACER3-knockdown HepG2 cells transfected with LXRβ siRNA were further transfected with pGL3-SULT2A1-luc or pGL3-basic vector plasmid using Lipofectamine 3000 (Invitrogen pRL-TK was transfected to normalize the efficiency of transfection Luciferase receptor assays were performed using a Dual-luciferase assay kit (Promega The luciferase activity was determined by the Gen5 (Biotek All reporter assays were repeated three times Formalin-fixed and paraffin-embedded (FFPE) slides of mouse liver tissues were subjected to ISH using RNA scope ISH kits and probes (Advanced Cell Diagnostics, CA, USA) as described by Wang F67 Liver sections were pretreated by repair reagents and then hybridized with the specific oligonucleotide probe targeting the region (93-1195 bp) of the ACER3/Acer3 gene After amplification of the staining signal sections were hybridized with a probe labeled with horseradish peroxidase (HRP) Positive staining was detected with a red color Each RNA transcript exhibited a distinct dot or cluster of signals FFPE slides of mouse liver tissues were subjected to hematoxylin and eosin (H&E) staining for histopathological examination The assessment of collagen formation was assessed by staining using commercial Sirius red dye (Solarbio Liver injury was evaluated by detecting the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse serum using ALT and AST Colorimetric Activity Assay Kits (Sigma-Aldrich Oil Red O staining was performed to assess the lipid content in the liver Liver frozen sections or cells were fixed and then incubated with Oil Red O solution (0.375% The samples were immersed in ddH2O for 1 min mounted using a water-soluble mounting medium and examined under the Intelligently Designed Microscope (Olympus IHC staining was performed using a VECTASTAIN® Elite® ABC Kit (Rabbit IgG) (VECTOR USA) and DAB Peroxidase Substrate Kit (VECTOR USA) following the manufacturer’s instructions Liver sections were subjected to IHC staining with antibodies against ACER3 (Sigma-Aldrich Alpha-smooth muscle actin (αSMA) (Cell Signaling Technology lymphocyte antigen 6 complex locus G6D (LY6G) (Abcam Necrotic and Oil Red O positive areas were quantified by analyzing 5 randomly selected fields (20 ×) per section using Image Pro Plus software (Media Cybernetics The assessment of Ly6G and ACER3 staining was performed by counting positively stained cells in 5 randomly selected fields per section within a 20 × field of view using a blind approach Representative pictures were taken using the Intelligently Designed Microscope (Olympus IF co-staining was performed on the liver frozen sections and HepG2 cells using LXRβ antibody (Abcam Alexa Fluor® 488-conjugated rabbit antibody (Abcam USA) and Alexa Fluor® 594-conjugated mouse antibody (Abcam The cell nucleus was counterstained with DAPI (Abcam The stained liver sections were analyzed using the Intelligently Designed Microscope (Olympus The confocal dishes were observed and analyzed using a Laser Scanning Confocal Microscope LSM 980 (ZEISS Nuclear LXRβ/Lxrβ-positive cells were quantified within 5 randomly selected fields (20 ×) per section using Image Pro Plus software The Flag-tagged human LXRβ (F-LXRβ) coding sequence was constructed from the respective cDNA clones using 3 × FLAG-tag-encoding oligonucleotides followed by insertion into the pcDNATM3.1 vector (Thermo Fisher Scientific HepG2 cells were transfected with plasmids containing F-LXRβ or empty vectors the cells were treated with CER(d18:1/18:1) for another 24 hours The proteins were extracted using a detergent-free MinuteTM Total Protein Extraction Kit (Invent Biotechnologies The expression efficiency of FLAG and LXRβ protein was verified by Western blot EZview Red ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich USA) was washed with Tris Buffered Saline solution (50 mM Tris HCl Diluted protein lysates (100 ul) were incubated with EZview Red ANTI-FLAG M2 Affinity Gel (20 ul) and shaken slowly at 4 °C overnight the samples were centrifuged at 8200 g for 30 s at 4 °C and the supernatant was removed The precipitates were gently mixed and incubated with Tris-buffered saline solution for 5 min The above precipitations were incubated with 150 μl FLAG peptide (Sigma-Aldrich The supernatants were verified for the efficiency of IP and then subjected to CER extraction for CER measurement Total RNA was extracted from the liver tissues of Acer3+/+ and Acer3-/- mice Illumina RNA-seq libraries were prepared by Shanghai Majorbio Bio-Pharm Technology Co. The libraries were sequenced on an Illumina Novaseq 6000 platform The mouse genomic and genetic information was obtained from the National Center for Biotechnology Information database Expression levels of mRNA were evaluated using StringTie software (v1.3.44 d) |log2FC | ≧1 and P-value ≤ 0.05 were considered as the threshold criteria to screen differentially expressed genes (DEGs) The obtained data were used to generate fold changes and transform them to draw volcano plots BA content was normalized to the protein levels of tissues and cells or the volume of serum and cell culture medium Equal amounts of liver tissues were used for lipid extraction The samples were analyzed using Thermo Fisher Scientific Vanquish Flex ultra-high-performance liquid chromatography (UHPLC) equipped with Thermo Fisher Scientific Orbitrap Fusion Tribrid High-Resolution Mass Spectrometer (Thermo Fisher Scientific The identification of lipid molecular species was conducted by Lipid Search software (Thermo Fisher Scientific Missing values that were not detected in all samples were excluded Log10-transformed data were scaled for principal component analysis (PCA) The differences of individual lipid species were statistically analyzed by Student’s t-test The alteration of individual lipid species by BDL or hepatocyte-specific Acer3 ablation was illustrated by volcano plots CER standards were purchased from Avanti Polar Lipids (Birmingham Amounts of sphingolipids were quantified using standard curves and normalized to protein contents Mobile phase A: a gradient system consisting of acetonitrile:methanol:water (45:45:40 Mobile phase B: acetonitrile:methanol:water (45:45:10 The gradient program was as follows: 0–2 min Amounts of OS were normalized to protein contents Discovery Studio software was used to analyze the hydrophobicity of the ligand-protein interaction An online protein structure prediction server was used to construct the three-dimensional structure of the LBD of mouse Lxrβ protein (UniProt ID: Q60644) resolution: 2.40 Å) was used as a template through BLAST (identity: 99.58%) Chain A of the protein with the known LXRβ ligand GW3965 was used for model pocket construction the molecular docking between CER(d18:1/18:1) and mouse Lxrβ was accomplished as described above SPR analysis was performed using the PlexArray HT A100 (Plexera; Seattle USA) was loaded on the 3D photo-crosslinking chip the chip was immersed in Dimethylformamide (DMF) (Sigma-Aldrich the chip was dried under a continuous stream of nitrogen The recombinant human LXRβ (AntibodySystem France) (dissolved in PBS) with increasing concentrations (0 The data was collected with Plexera Data Explorer and analyzed with BIA evaluation software (version 4.1) Statistical analyses were performed with Statistical Product and Service Solutions software 20.0 (IBM; Armonk USA) and R version 4.3.2 with ggplot2 and psych packages Correlation analysis was performed by the Spearman correlation test analyzed by unpaired two-sided Student’s t-test or one-way ANOVA with Tukey’s multiple comparisons and a P value < 0.05 (bilateral) was considered significant Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Cholestatic Liver Disease: Current Treatment Strategies and New Therapeutic Agents Biliary bile acids in hepatobiliary injury - What is the link International Union of Basic and Clinical Pharmacology CXIII: Nuclear Receptor Superfamily—Update 2023 Enzymatic reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice Novel insights into bile acid detoxification via CYP Bile Acid sulfation: a pathway of bile acid elimination and detoxification Mouse organic solute transporter α deficiency enhances renal excretion of bile acids and attenuates cholestasis Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR New molecular insights into the mechanisms of cholestasis Sphingolipids and their metabolism in physiology and disease A systemic combined nontargeted and targeted LC-MS based metabolomic strategy of plasma and liver on pathology exploration of alpha-naphthylisothiocyanate induced cholestatic liver injury in mice Lithocholic acid disrupts phospholipid and sphingolipid homeostasis leading to cholestasis in mice Role of sphingolipids in the pathogenesis of intrahepatic cholestasis Sphingosine 1-Phosphate Receptor 2 and 3 Mediate Bone Marrow-Derived Monocyte/Macrophage Motility in Cholestatic Liver Injury in Mice Suppressing the intestinal farnesoid X receptor/sphingomyelin phosphodiesterase 3 axis decreases atherosclerosis Alkaline ceramidase family: The first two decades Alkaline Ceramidase 3 (ACER3) Hydrolyzes Unsaturated Long-chain Ceramides and Its Down-regulation Inhibits Both Cell Proliferation and Apoptosis Alkaline Ceramidase 3 Deficiency Results in Purkinje Cell Degeneration and Cerebellar Ataxia Due to Dyshomeostasis of Sphingolipids in the Brain Targeting alkaline ceramidase 3 alleviates the severity of nonalcoholic steatohepatitis by reducing oxidative stress Generation of sphingosine-1-phosphate by sphingosine kinase 1 protects nonalcoholic fatty liver from ischemia/reperfusion injury through alleviating reactive oxygen species production in hepatocytes Ceramides and sphingosine-1-phosphate mediate the distinct effects of M1/M2-macrophage infusion on liver recovery after hepatectomy Nonalcoholic steatohepatitis critically rewires the ischemia/reperfusion-induced dysregulation of cardiolipins and sphingolipids in mice Solute Carrier Organic Anion Transporter Family Member 3A1 Is a Bile Acid Efflux Transporter in Cholestasis Limb expression 1-like (LIX1L) protein promotes cholestatic liver injury by regulating bile acid metabolism Dysregulated miR-124 and miR-200 expression contribute to cholangiocyte proliferation in the cholestatic liver by targeting IL-6/STAT3 signalling Function and organization of the human cytosolic sulfotransferase (SULT) family Regulation of the cytosolic sulfotransferases by nuclear receptors Liver X receptors in lipid signalling and membrane homeostasis Natural products as modulators of the nuclear receptors and metabolic sensors LXR Structure of the retinoid X receptor α–liver X receptor β (RXRα–LXRβ) heterodimer on DNA Activation of LXRs prevents bile acid toxicity and cholestasis in female mice Mechanisms of MAFG Dysregulation in Cholestatic Liver Injury and Development of Liver Cancer Hepatic cell lines for drug hepatotoxicity testing: limitations and strategies to upgrade their metabolic competence by gene engineering Validation of gene expression profiles from cholestatic hepatotoxicants in vitro against human in vivo cholestasis Ligands of Therapeutic Utility for the Liver X Receptors X-ray Crystal Structure of the Liver X Receptor β Ligand Binding Domain Orally Efficacious Liver X Receptor (LXR) β Agonist Ceramide reduction and transcriptional up-regulation of glucosylceramide synthase through doxorubicin-activated Sp1 in drug-resistant HL-60/ADR cells P53-dependent upregulation of neutral sphingomyelinase-2: role in doxorubicin-induced growth arrest In vitro profiling of toxic effects of environmental polycyclic aromatic hydrocarbons on nuclear receptor signaling disruption of endogenous metabolism and induction of cellular stress Induces Oncogenic Virus-Infected Cell Autophagic Death and Represses Tumor Growth Targeting Src reactivates pyroptosis to reverse chemoresistance in lung and pancreatic cancer models Alterations in Lipid Metabolism Mediate Inflammation and Proliferation in a Mouse Model of Chronic Cholestatic Liver Injury Cholestasis impairs hepatic lipid storage via AMPK and CREB signaling in hepatitis B virus surface protein transgenic mice Structural analysis identifies an escape route from the adverse lipogenic effects of liver X receptor ligands Liver X receptor ligands suppress ubiquitination and degradation of LXRalpha by displacing BARD1/BRCA1 Liver X receptor (LXR) regulation of the LXRalpha gene in human macrophages Tissue-specific autoregulation of the LXRalpha gene facilitates induction of apoE in mouse adipose tissue Tissue Distribution and Ontogeny of Sulfotransferase Enzymes in Mice Profiles and Gender-Specifics of UDP-Glucuronosyltransferases and Sulfotransferases Expressions in the Major Metabolic Organs of Wild-Type and Efflux Transporter Knockout FVB Mice Sex differences in the activation of tamoxifen to DNA binding species in rat liver in vivo and in rat hepatocytes in vitro: role of sulfotransferase induction Age- and sex-dependent expression of multiple murine hepatic hydroxysteroid sulfotransferase (SULT2A) genes Sulphotransferase-mediated activation of the carcinogen 5-hydroxymethyl-chrysene Species and sex differences in tissue distribution of the enzyme activity and a possible participation of hydroxysteroid sulphotransferases Cloning and expression of human liver dehydroepiandrosterone sulphotransferase Sulfation of melatonin: enzymatic characterization is involved in colorectal cancer progression through ceramide synthesis Hydrolysis of lactosylceramide by human galactosylceramidase and GM1-beta-galactosidase in a detergent-free system and its stimulation by sphingolipid activator proteins Activator proteins stimulate lactosylceramide hydrolysis Convenient and Sensitive Measurement of Lactosylceramide Synthase Activity Using Deuterated Glucosylceramide and Mass Spectrometry Regulation of astrocyte activation by glycolipids drives chronic CNS inflammation Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease Bile duct ligation in mice: induction of inflammatory liver injury and fibrosis by obstructive cholestasis Simultaneous characterization of bile acids and their sulfate metabolites in mouse liver Distinct Plasma Bile Acid Profiles of Biliary Atresia and Neonatal Hepatitis Syndrome Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics Improved sphingolipidomic approach based on ultra-high performance liquid chromatography and multiple mass spectrometries with application to cellular neurotoxicity A multi-omics investigation of the composition and function of extracellular vesicles along the temporal trajectory of COVID-19 Enhancing UCSF Chimera through web services DOCK 6: Impact of new features and current docking performance Download references We would like to acknowledge the technical support provided by LipidALL Technologies (Changzhou China) and Central Laboratory of Southern Medical University (Guangzhou China) for lipidomics and bile acid analyses This work was supported by the National Natural Science Foundation of China (82170647 to K.W the Basic and Applied Basic Research Foundation of Guangdong Province (2023A1515010088 to C.J.L and 2024A1515013204 to K.W) the Project of Guangzhou Science and Technology (202201020604 and 20231A011030 to L.L) and the Innovation and Entrepreneurship Training Program for College Students (S202212121102 and S202312121240 supervised by K.W) These authors contributed equally: Leyi Liao Guangzhou Women and Children’s Medical Center Guangdong Provincial Clinical Research Center for Child Health The State University of New York at Stony Brook Nature Communications thanks David Montefusco and the other Download citation DOI: https://doi.org/10.1038/s41467-025-57330-7 Metrics details Bioconjugation is one of the most promising strategies to improve drug delivery Biomolecules such as bile acids (BAs) have been intensively explored as carriers due to their peculiar physicochemical properties and biocompatibility BAs trafficking is regulated by intracellular lipid-binding proteins and their transport in the liver can be studied using chicken liver Bile Acid-Binding Proteins (cL-BABPs) as a reference model we conceived the idea of developing a BA-conjugate with Mirin an exonuclease inhibitor of Mre11 endowed with different anticancer activities Following computational analysis of various BAs in complex with cL-BABP we identified cholic acid (CA) as the most promising candidate as carrier leading to the synthesis of a novel bioconjugate named CA-M11 As predicted by computational data and confirmed by X-ray crystallographic studies CA-M11 was able to accommodate into the binding pocket of BABP it can enter BAs trafficking in the hepatic compartment and here release Mirin evaluated in combination with varying concentrations of Doxorubicin on HepG2 cell line demonstrated a significant increase in cell mortality compared to the use of the cytotoxic drug or Mirin alone thus highlighting chemo-sensitizing properties The promising results regarding plasma stability for CA-M11 validate its potential as a valuable agent or adjuvant for hepatic cancer therapy Workflow of the strategy to build a bioconjugate of Mirin with BAs for targeting the DNA repair protein Mre11 in the liver A common feature observed amongst these BAs was the ability of the carboxylate moiety to interact with Lys77. Additionally, all compounds formed an H-bond between the hydroxyl 3 (atom numbering is displayed in Fig. 1) and the side chain of Gln101, except for UDCA. The pink lines depict HB interactions followed by the related percentage of occurrence during the whole simulations This is essential in our design since it can allow the accumulation of our bioconjugate in the hepatic compartment while simultaneously enhancing its potential to serve as a substrate for FABPs Therefore CA emerged as the optimal choice for our purposes Crystal structure of recombinant cL-BABP (pale brown cartoon and carbons) in complex with BAs The inner cavity of the protein is populated by two molecules Ligand-binding cavity of cL-BABP in complex with (A) DCA (in sticks water molecules are shown as red spheres and H-bonds as red dashed lines; oxygen atoms are colored red 2D structure of iminothiazolidin-4-one tautomer 6a and aminothiazolidin-4-one tautomer 6b of CA-M11 Geometrical comparison between the lowest-energy conformer of 6a and 6b (magenta carbon) and their respective best docked poses (green carbon) Top and front view of the best docking pose of (A) keto-imino (green carbon sticks) and (B) keto-amino tautomers (orange carbon sticks) of CA-M11 in complex with cL-BABP with the residues involved in pivotal contacts shown as carbon sticks The H-bonds are interactions are indicated as yellow dashed lines Schematic representation of ligand-protein interactions of (C) keto-imino and (D) keto-amino tautomers of CA-M11 into cL-BABP Interactions occurring more than 30% of the MDs are reported Synthesis of Mirin (5) and Cholic acid-Mirin bioconjugate CA-M11 (6a) Reagents and conditions are specified in the scheme Ligand-binding cavity of cL-BABP in complex with CA-M11 (in sticks The fitting of CA-M11 in the omit map (light blue contoured at the 2.5 σ level) is shown in the inset To better elucidate the ADME properties of CA-M11 and Mirin, both compounds were incubated at a fixed concentration in the presence of human plasma for several time points to investigate the kinetics of hydrolysis. As shown in Table 2; Fig. 9 Mirin exhibited higher plasma stability over time with a half-life (t1/2) greater than 24 h (96 h precisely) the percentage of unmodified compound never dropped below 80% up to 8 h of incubation with only a slight decrease in stability observed after 24 h CA-M11 suffered more significant degradation even at the earliest time points Approximately 30% of the compound was hydrolyzed by plasmatic esterases after a few minutes The percentage of unmodified CA-M11 remained constant for up to 1 h of incubation (70.27%) A significant decrease in plasma stability was observed after 8 h (54.99%) and 24 h (31.75%) Based on the percentage of plasma stability the calculated half-life (t1/2) for CA-M11 was about 18 h the stability of both compounds in the presence of Dulbecco’s Modified Eagle Medium (DMEM) added with 10% FBS with experiments conducted at the same time points for better comparison with the results on plasma stability the lack of significant passive permeability for CA-M11 may suggest that it exploits active transport mechanisms to reach the intracellular target Percentage of viable HepG2 cells after 24 h of contact with different concentrations of Doxorubicin Data are mean ± SD of three experiments run in six replicates *Values are statistically different versus negative control (complete medium Percentage of viable HepG2 after 24 h of contact with different concentrations of Mirin and CA-M11, as determined by the Neutral Red Uptake. Data are mean ± SD of three experiments run in six replicates. *Values are statistically different versus Mirin 5 µM, p < 0,05. Percentage of viable HepG2 after 24 h of contact with different concentrations of Doxorubicin (DOX) and Doxorubicin + Mirin 5 µM and Doxorubicin + CA-M11 5 µM *Values are statistically different versus Doxorubicin p < 0.05; # Values are significantly different from DOX-Mirin 5 µM in silico studies guided the selection of CA as the preferred BA for the interaction with cL-BABP emerging as the most promising carrier for a selected Mre11 inhibitor This latter is renowned for its anticancer activity albeit hindered by inadequate pharmacokinetic properties and significant off-target toxicity it has been demonstrated the successful accommodation of a CA-based bioconjugate with Mirin (CA-M11) within the ligand-binding cavity of cL-BABP as marked by the analysis of the crystal structure of the complex which provides strong evidence that our bioconjugate can serve as a substrate for the protein the similarity between cL-BABP and FABPs allows to speculate about the interaction of our conjugate with the mammal proteins and its trafficking in the districts of interest These preliminary yet encouraging findings demonstrate the potential of our approach in developing novel conjugates as adjuvant treatments for liver cancer delivered by CA-M11 in the hepatic compartment where BAs accumulates leading to the possibility of reducing the dose of Doxorubicin while achieving an equivalent antitumor effect considering that the cytotoxic agent is an Mre11 inhibitor our findings also indicate this strategy as a promising opportunity for a renewed application of DDR inhibitors in cancer therapy Our strategic design paves the way for the exploitation of iLBPs in the transport of bioactive substances to the liver we can reach out for the creation of different analogues of CA-M11 possibly bearing different anticancer agents or other drugs which has to be directed to the liver thanks to the sensor-activity of CA or other BAs as carriers investigation will be performed moving from the in vitro to the in vivo evaluation to further elucidate the activity and metabolic fate of BAs-bearing bioconjugates The calculation relies on the following equation: The initial step involves identifying the proton donor and acceptor atoms within the molecule followed by redistributing protons among these atoms to generate a range of tautomers the tautomers are assessed based on their semiempirical PM3 heat of formation with high-energy tautomers being eliminated The surviving tautomers then undergo conformational analysis using MacroModel to generate a set of conformers High-energy conformations are filtered out based on their semiempirical PM3 heat of formation the lowest-energy tautomers undergo DFT geometry optimizations using the B3LYP-D3/LACVP** level of theory and the obtained geometries were ranked computing single–point energies at the M06-2X/cc-pVTZ(-f) level of theory docking simulations were performed on the top conformers for the lowest-energy tautomeric forms of CA-M11 the best docked pose was subjected to 500 ns of MDs using the same aforementioned procedure All reagents and solvents were purchased from Merck and were used as received Merck silica gel 60 (230–400 mesh) was used for column chromatography Merck aluminum sheets coated with silica gel 60 F254 were used for TLC 1H spectra were recorded with a Bruker DRX 600 AVANCE spectrometer in the indicated solvent (the residual solvent peaks were used as the internal standard) The values of the chemical shifts (δ) are reported in ppm and the H–H coupling constants (J) are expressed in Hz An Agilent 1100 LC-MS running with an electrospray source (ESI) was used in mass spectrometry measurements The purity of compounds 5 and 10 was assessed by LC-MS analysis and was found to be higher than 95% An Infinity Lab Poroshell 120 EC-C18 column 2.1 × 50 mm 2.7 μm was used as the stationary phase while a 5–95% gradient of MeCN (+ 0.1% HCOOH) in H2O (+ 0.1% HCOOH) in 5 min represented the mobile phase A Bruker Tims-TOF instrument with an ESI source was used to measure the high-resolution mass (HRMS) values (Z)-5-(4-Hydroxybenzylidene)-2-iminothiazolidin-4-one (5) 8.2 mmol) and 2-iminothiazolidin-4-one (12 9.0 mmol) were heated at reflux in a mixture of NaOAc (2.0 g The resulting orange solid was filtered and washed several times with cold water The pure compound was obtained as an orange solid (1.4 g HRMS (ESI) m/z: [M + H]+ Calcd for C10H9N2O2S+ 221.03792 Found 221.03773; [M + Na]+ Calcd for C10H8N2NaO2S+ 243.01987 (4R)-4-((Z)-(2-Imino-4-oxothiazolidin-5-ylidene)methyl)phenyl 4-((3R,7R,10 S,12 S,13R,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanoate (10 To a stirred solution of cholic acid (100 mg The mixture was stirred at room temperature for 16 h Volatiles were removed under reduced pressure and the crude was purified by means of chromatography on silica gel (EtOAc in PE gradient 10:90 to 90:10) to afford pure compound CA-M11 (45 mg HRMS (ESI) m/z: [M + Na]+ Calcd for C34H46N2NaO6S+ 633.29688 samples for the crystallization experiment were prepared by adding 5 mM compounds (dissolved in dimethyl sulfoxide DMSO) to the protein solution (25 mg mL−1 in 100 mM sodium chloride and 20 mM TRIS consisting of equal volumes of these samples and precipitant solution were equilibrated over a 200 µL reservoir at 8 °C the precipitant formulation was 200 mM sodium chloride for cL-BABP-UDCA complex was 12% w/v PEG-3500 and 100 mM sodium formate while for cL-BABP-DCA and cL-BABP-LCA complexes was 25% w/v PEG-3500 The crystals of cL-BABP-CA-M11 complex grew in 2.4 M sodium malonate Prior to flash freezing in liquid nitrogen crystals were singularly transferred to the cryoprotectant solution prepared by adding to each precipitant 20% v/v of PEG-400 Final coordinates and structure factors were deposited in the Protein Data Bank (PDB) under the codes 9ETE (cL-BABP-DCA) and all the solvents used for cell culture were purchased from Lonza (Switzerland) Human hepatoma HepG2 cells were purchased from American Type Culture Collection (USA) The mutagenicity assay was supplied by Biologik s.r.l This test is suitable for sample with various shapes Among the recommended cells lines reported in the document ISO 10995-5:2009 to test Mirin Cells were maintained in DMEM at 37 °C in a humidified atmosphere containing 5% CO2 The culture medium was supplemented with 10% fetal calf serum (FCS) 1% L-glutamine-penicillin-streptomycin solution and 1% MEM Non-Essential Amino Acid Solution taken up with trypsin-EDTA solution and then centrifuged at 1000 rpm for 5 min The pellet was re-suspended in medium solution (dilution 1:15) Cells (1.5 × 104) suspended in 1 mL of complete medium were seeded in each well of a 24 well round multidish and incubated at 37 °C in an atmosphere of 5% CO2 the culture medium was discharged and the test compounds The following concentrations of Mirin and CA-M11 were tested: 5; 10; 20; 30; 40; 50; 60; 70; 80; 90 and 100 µM The tested concentrations of Doxorubicin were 0.3; 0.6; 1.2; 1.8; 2.4; 3.0; 6.0 and 12.0 µM the experiments were repeated adding to Doxorubicin concentrations ranging from 0.3 to 6.0 µM These values were chosen because of cytotoxic effect towards HepG2 cells and all samples were set up in six replicates A complete medium was used as negative control cell viability was evaluated by Neutral Red uptake the following solutions were prepared in order to determine the percentage of viable cells: (1) Neutral Red (NR) Stock Solution: 0.33 g NR Dye powder in 100 mL sterile H2O; (2) NR Medium: 1.0 mL NR Stock solution + 99.0 Routine Culture Medium pre-warmed to 37 °C); (3) NR Desorb solution: 1% glacial acetic acid solution + 50% ethanol + 49% H2O the routine culture medium was removed from each well and cells were carefully rinsed with 1 mL of pre-warmed D-PBS Multiwells were then gently blotted with paper towels 1.0 mL of NR Medium was added to each well and further incubated at 37 °C The cells were checked during the NR incubation for NR crystal formation cells were carefully rinsed with 1 mL of pre-warmed D-PBS the PBS was decanted and blotted from the wells and exactly 1 mL of NR Desorb solution was added to each sample Multiwells were then put on a shaker for 20–45 min to extract NR from the cells and form a homogeneous solution During this step the samples were covered in order to protect them from light After 5 min from the plate shaker removal the absorbance was read at 540 nm by a UV/visible spectrophotometer (UV5 The TA100 and TA98 strains of Salmonella Typhimurium were utilized for mutagenicity assay Approximately 107 bacteria were exposed to 6 concentrations of each test compound as well as a positive and a negative control for 90 min in a medium containing sufficient histidine to support approximately two cell divisions the exposure cultures were diluted in pH indicator medium lacking histidine and aliquoted into 48 wells of a 384-well plate cells which had undergone the reversion to His grew into colonies Metabolism by the bacterial colonies reduced the pH of the medium This color change can be detected visually or by a microplate reader The number of wells containing revertant colonies were counted for each dose and compared to a zero-dose control The following concentrations of Mirin and CA-M11 were tested: 5; 10; 30; 50; 80 and 100 µM The concentration values tested of Doxorubicin were: 0.3; 1.2; 1.8; 3.0; 6.0 and 30 µM The experiments were then repeated adding to Doxorubicin concentrations ranging from 0.3 to 30 µM Multiple comparison was performed by one-way ANOVA and individual differences tested by Fisher’s test after the demonstration of significant intergroup differences by ANOVA Differences with p < 0.05 were considered significant Chromatographic separations were accomplished at room temperature (rt) using a gradient elution made up of solvents A (H2O) and B (ACN) both acidified with 0.1% v/v formic acid (FA) The analyses began with 100% A (t = 0–1 min) and then returned in 1.0 min to the original conditions Spectra were acquired in both positive and negative modes within the scan range of m/z 100–2000 with UV detection being monitored at 254 nm Plasma Stability and stability in the cellular medium A DMSO stock solution of tested compounds was incubated in the presence of human plasma (55.7 µg protein/mL) and HEPES buffer (25 mM samples were treated with cold ACN to stop reactions through protein denaturation The supernatant was analyzed by UV/LC-MS to monitor the amount of unmodified compound Data were calculated with Excel and plotted using GraphPad Prism 8.0 (GraphPad Software Inc. The half-life value (t1/2) was calculated with the following formula: \(\:{t}_{1/2}=0.693/b\) Where b is the slope found in the linear fit of the natural logarithm of the fraction remaining of the parent compound vs Processed data that support the findings of this study are provided within the manuscript or the supplementary information file Bioconjugate therapeutics: current progress and future perspective Bile acids and their derivatives as potential modifiers of drug release and pharmacokinetic profiles Exploitation of bile acid Transport systems in Prodrug Design Liver-specific drug targeting by coupling to bile acids Synthesis and characterization of a new bile acid and platinum(II) complex with cytostatic activity Research progress in the application of bile acid-drug conjugates: a trojan horse strategy & Review The liver bile acid-binding proteins Structural and Biochemical Characterization of Toad Liver Fatty Acid-Binding Protein , Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid INTRACELLULAR LIPID-BINDING PROTEINS AND THEIR GENES Evolution of the family of intracellular lipid binding proteins in vertebrates Banaszak, L. et al. Lipid-binding proteins: a family of fatty acid and Retinoid Transport Proteins. 89–151 (1994). https://doi.org/10.1016/S0065-3233(08)60639-7 A binding protein for fatty acids in Cytosol of Intestinal Mucosa Evolutionary coupling of structural and functional sequence information in the intracellular lipid-binding protein family DNA repair mechanisms in cancer development and therapy Improving targeted small molecule drugs to overcome chemotherapy resistance The DNA-damage response in human biology and disease Requirements for MRN endonuclease processing of topoisomerase II-mediated DNA damage in mammalian cells MRE11-RAD50-NBS1 complex alterations and DNA damage response: implications for cancer treatment Targeting Mre11 overcomes platinum resistance and induces synthetic lethality in XRCC1 deficient epithelial ovarian cancers MRE11 as a molecular signature and therapeutic target for cancer treatment with radiotherapy DNA double-strand break repair pathway choice is Directed by distinct MRE11 nuclease activities Moiani, D. et al. Targeting Allostery with avatars to design inhibitors assessed by cell activity: dissecting MRE11 Endo- and exonuclease activities. 205–241 (2018). https://doi.org/10.1016/bs.mie.2017.11.030 The DNA damage Sensor MRE11 regulates efficient replication of the Autonomous Parvovirus Minute Virus of mice Viral modulation of the DNA damage response and innate immunity: two sides of the same Coin Relocalization of the Mre11-Rad50-Nbs1 complex by the Adenovirus E4 ORF3 protein is required for viral replication A forward chemical genetic screen reveals an inhibitor of the Mre11–Rad50–Nbs1 complex a small-molecule inhibitor of the Mre11–Rad50–Nbs1 complex MRE11 inhibition highlights a replication stress-dependent vulnerability of MYCN-driven tumors AKT overactivation can suppress DNA repair via p70S6 kinase-dependent downregulation of MRE11 Characterization of crystalline rat liver fatty acid binding protein produced in Escherichia coli The Crystal structure of the liver fatty acid-binding protein Crystal Structure of Chicken Liver Basic Fatty Acid-Binding Protein Complexed with Cholic Acid, Validation of recombinant chicken liver bile acid binding protein as a Tool for Cholic Acid Hosting Present Scenario of bioconjugates in Cancer Therapy: a review Synthesis and biological evaluation of cholic acid-conjugated oxaliplatin as a new prodrug for liver cancer Bile acids and bile acid derivatives: use in drug delivery systems and as therapeutic agents Structural and Dynamic Determinants of Molecular Recognition in bile acid-binding proteins further evidence of the usefulness of bile acids as molecules for shuttling cytostatic drugs toward liver tumors Recent advances in Biomedical Applications of Cholic Acid-based macromolecules Updates in bile acid-bioactive molecule conjugates and their applications and mechanistic investigations of bile acid–tamoxifen conjugates for breast Cancer therapy Effect of bile acids on the expression of MRP3 and MRP4: an in vitro study in HepG2 cell line Current trends in the treatment of hepatocellular carcinoma with transarterial embolization: a cross-sectional survey of techniques Oral lipid nanocomplex of BRD4 PROteolysis TArgeting chimera and vemurafenib for drug-resistant malignant melanoma Delineating effect of Headgroup and Preparation method on transfection Versus Toxicity of DNA-Loaded lipid nanocarriers Galactose-decorated liver tumor-specific nanoliposomes incorporating selective BRD4-targeted PROTAC for hepatocellular carcinoma therapy Schrödinger Release 2018-1: Protein Preparation Wizard; Schrödinger LLC: New York Development and testing of the OPLS All-Atom Force Field on Conformational Energetics and properties of Organic liquids Schrödinger Release 2021 – 4: LigPrep (LLC Schrödinger Release 2021-4: Glide (Schrödinger LLC Schrödinger Release 2021-4: Desmond Molecular Dynamics System (D.E Comparison of simple potential functions for simulating liquid water Assessing the performance of the MM/PBSA and MM/GBSA methods The accuracy of binding Free Energy calculations based on Molecular Dynamics simulations The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities Influence of the solvent representation on vibrational entropy calculations: generalized born versus distance‐dependent dielectric model End-point binding Free Energy calculation with MM/PBSA and MM/GBSA: strategies and applications in Drug Design Jaguar: a high-performance quantum chemistry software program with strengths in life and materials sciences Crystallization of soluble proteins in vapor diffusion for x-ray crystallography An introduction to data reduction: space-group determination Overview of the CCP 4 suite and current developments REFMAC 5 for the refinement of macromolecular crystal structures PROCHECK: a program to check the stereochemical quality of protein structures Biological Evaluation of Medical devices—Part 5: Tests for in vitro Cytotoxicity (International Organization for Standardization Biological evaluation and in Vitro characterization of ADME Profile of In-House Pyrazolo[3,4-d]pyrimidines as dual tyrosine kinase inhibitors active against Glioblastoma Multiforme [1,2,4]Triazolo[3,4-b]benzothiazole Scaffold as Versatile Nicotinamide Mimic allowing Nanomolar inhibition of different PARP enzymes an effective way to overcome HIV-1 resistance targeting host proteins Privileged Scaffold decoration for the identification of the First Trisubstituted Triazine with Anti-SARS-CoV-2 activity Download references Authors would like to thank the University of Siena for the support to the open access publication through the F-OPEN ACCESS program Consorzio Interuniversitario Risonanze Magnetiche di Metallo Proteine (CIRMMP) laid down the rationale behind the project was responsible for the synthesis and characterization of Mirin and CA-M11 were responsible for the crystallographic section produced and purified cL-BABP and performed all the crystallographic analysis of BAs and CA-M11 in complex with cL-BABP performed crystallographic data collection and structure solution and refinement; E.D are responsible for the early ADME evaluation is responsible for the biological evaluation I.R and S.A designed and performed the computational studies The manuscript was written through contributions of all authors All authors have given approval to the final version of the manuscript Download citation DOI: https://doi.org/10.1038/s41598-024-73636-w Research on a new protocol developed by internationally respected specialists at the Orlando Health Digestive Health Institute was recently published in Digestive Endoscopy entitled Orlando Protocol for single session ductal clearance of common bile duct stones at endoscopic retrograde cholangiopancreatography concluded that a predetermined protocol optimized outcomes by enabling single-session ductal clearance of CBDS with high technical success and low adverse events Endoscopic retrograde cholangiopancreatography (ERCP) with biliary sphincterotomy and stone extraction is accepted procedural treatment for common bile duct stones (CBDS) extraction by standard methodology does not work for more difficult cases “There has been no standardized approach to managing common bile duct stones by endoscopic retrograde cholangiopancreatography despite not-infrequent technical challenges,” says Shyam S president of Orlando Health Digestive Health Institute “Our proposed ‘Orlando’ protocol provides a framework whereby the underlying challenge is clearly defined and then objectively managed.” The study analyzed 409 patients treated from February 2022 to May 2023 A management protocol taking into consideration stone size and bile duct diameter was applied prospectively on consecutive patients with CBDS aged ≥18 years who underwent ERCP All procedures were performed by four therapeutic endoscopists with Orlando Health Digestive Health Institute By adopting a protocol-based treatment approach single-session ductal clearance was achieved in more than 99 percent of patients with severe to mild adverse events occurring in less than 5 percent “Our research concluded the ‘Orlando’ protocol enables efficient single session ductal clearance of CBDS with high rate of technical success and low adverse events,” says Dr we believe this protocol could offer improved clinical outcomes and yield cost savings.” Principal investigators of the study published in Digestive Endoscopy include physicians Ji Young Bang Orlando Health Digestive Health Institute provides comprehensive and coordinated care to evaluate diagnose and treat a multitude of digestive diseases The institute’s internationally respected specialists are frequent contributors to leading publications on gastroenterology and endoscopy and spearhead numerous research and educational initiatives globally ER Wait Times are approximate and provided for informational purposes only Metrics details Recent studies suggest the role of gut microbes in bile acid metabolism in the development and progression of colorectal cancer the surveys of the association between fecal bile acid concentrations and colorectal cancer (CRC) have been inconsistent We searched online to identify relevant cross-sectional and case-control studies published online in the major English language databases (Medline We selected studies according to inclusion and exclusion criteria and extracted data from them RevMan 5.3 was used to perform the meta-analyses and UDCA (ursodeoxycholic acid) were significantly higher (CA: standardized mean difference [SMD] = 0.41 P = 0.009; DCA: SMD = 0.33,95% CI: 0.03–0.64 and the combined effect size was significantly higher in the high-risk than the low-risk CRC group (SMD = 0.36 the effect sizes of CA and CDCA were significantly higher (CA: SMD = 0.42 and their combined effect size was also significantly higher in the high-risk compared to low-risk CRC group (SMD = 0.39 Only one cross-sectional study suggested a higher concentration of CDCA and UDCA in the stool of the CRC high-risk group than the low-risk group These findings indicate that higher fecal concentrations of bile acid may be associated with a higher risk/incidence of CRC Several cross-sectional and case-control studies have attempted to find a relationship between fecal BAs and CRC; however their conclusions have not been consistent More conclusive assessment of fecal BA content between CRC patients and healthy people is needed and systematic comparisons between countries and regions are lacking due to differences in dietary habits and fecal composition there is a strong need to perform an updated systematic analysis of fecal BA concentrations and CRC risk/incidence to understand their relationship all the studies included in this study were observational and the central issue was the relationship between fecal BAs and the risk/incidence of CRC the data were derived from cross-sectional studies among specific populations and case-control studies on the presence or absence of risk factors (adenomatous polyps) Studies on the incidence of CRC are based on comparisons between CRC patients and healthy people (or non-CRC patients) The chemical compositions studied included a variety of joint BAs (cholic acid and total BA data are directly obtained from various studies rather than calculated by addition This systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis reporting guidelines The study is registered in the PROSPERO database (registration code: CRD42024533773) An online search identified relevant cross-sectional and case-control studies published online in the major English language databases (Medline The papers search for this study included only English peer-reviewed articles published before January 1st Steps for searching: (I) Search for articles and (II) Analyze and summarize subject words and keywords (IV) Screening of articles according to the inclusion and exclusion criteria Cross-sectional and case-control studies of fecal BA levels in patients with CRC or those at high risk for CRC have been officially published as of 1 January 2024 The studies used similar objectives and methods to compare fecal BA levels in patients with CRC or those at high risk for CRC with healthy people The case-control study consisted of patients with CRC confirmed by endoscopic pathology as a case group and patients without CRC as a control group Information on their fecal BA content was collected and analyzed patients at high risk for CRC identified through epidemiological investigation served as the case group a comparable group of non-high-risk CRC patients within the same time and scope was risk colorectal cancer patients within the same time and scope were selected as the control group The primary outcome of the studies was to investigate the contents and types of various BAs in the feces of the case or control groups The local ethics committee approved all studies All studies were required to have original paper and extractable data available smaller datasets (the total number of subjects is less than ten) studies with incomplete or contradictory information and significant errors in statistical methods were excluded and studies with non-primary data were also excluded We imported all search records (including author names and abstracts) into EndNote X7 and deleted duplicate records Search results were screened according to “inclusion criteria” and “exclusion criteria,” and then basic experiments and animal experiments were excluded Two researchers (Shaohui Yang and Yu Wang) did the study screening independently and a third researcher (Wei Cui) would decide if there was disagreement researchers (Lijuan Sheng and Chenyang Ma) read the study They extracted the primary study information (e.g. NOS scores range from 0 to 9; studies with 0–3 Data from studies with a high risk of bias were unreliable The meta-analysis was performed using the RevMan 5.3 statistical software provided by www.cochrane.org Various BA components were analyzed by subgroup analysis and then the results of each subgroup analysis were combined for analysis A fixed-effect model was used to analyze data with no significant heterogeneity (I2 < 50%) The data in each study were recorded differently and converted to “mean ± standard deviation” form before the analysis began The expression of concentrations varied across studies; therefore A funnel plot was drawn to detect publication bias the difference was statistically significant Some articles had no specific data or statistical graphs so they are considered qualitative analyses These studies were conducted in multiple regions and countries (including China and Zimbabwe) and included 1,265 patients and healthy people All subjects had explicit inclusion and exclusion criteria Endoscopy and pathology identified and diagnosed CRC/adenomatous polyp cases and healthy controls the BA conditions of the case and control groups were described to ensure they were comparable two similar populations were compared at a specific time and within a particular area to find the cause (A) Forest plots of a meta-analysis of the fecal concentrations of CA, CDCA, DCA, LCA, and UDCA in the CRC risk category. (B) Forest plots of a meta-analysis of the fecal concentrations of primary and secondary BAs in the CRC risk category. (C) Forest plots of a meta-analysis of the fecal concentrations of total BAs in the CRC risk category. (A) Forest plots of a meta-analysis of the fecal concentrations of CA (B) Forest plots of a meta-analysis of the fecal concentrations of primary and secondary BAs in the CRC incidence category (C) Forest plots of a meta-analysis of the fecal concentrations of total BAs in the CRC incidence category suggested that the level of UDCA in the stool of HC was higher the results of the other three cross-sectional and case-control studies did not show differences in fecal BA levels between CRC high-risk vs Some of the qualitative analysis results were consistent with our meta-analysis We used a funnel plot to detect publication bias for several primary BAs in stool, and the results are shown in Fig. 4. The asymmetric distribution of each point in the funnel plot suggested the presence of publication bias in this study. This systematic review and meta-analysis included several clinical studies investigating the relationship between the concentration of BAs in stool and the risk and incidence of CRC We found that individuals with a high risk of CRC had a higher concentration of fecal BAs than those with a low risk in the CRC risk category suggesting a potential association between fecal BAs and CRC development and this finding was confirmed in the CRC incidence category Some types of BAs can be classified as primary or secondary BAs the primary or secondary BA data was directly obtained from the original study rather than by adding the data of BAs This finding could have occurred because different studies adopted different detection methods; some BA detection methods needed more accuracy in earlier studies and superimposed calculations may have caused inaccurate data it is necessary to study the relationship between them we designed two different NOS to evaluate the quality of the research and provide a more comprehensive understanding of the results of the meta-analysis The detection methods used in each study are different and the accuracy of the detection results is also different which will inevitably affect the final analysis results the number of studies included in this study must be more significant in order to perform subgroup analysis these studies come from different countries and regions we must admit that is unavoidable heterogeneity is this study’s most significant limitation the fecal composition of CRC patients at different stages can be analyzed and compared The updated meta-analysis differs from previous results because it includes recent clinical studies Our study showed that CRC and high-risk patients had higher concentrations of some BAs in their stool than healthy controls and low-risk patients Because of the possible correlation between fecal BA concentration and colorectal risk/morbidity more attention is likely to be paid to predicting and treating CRC Some or all data are available from the corresponding author by request Global Cancer statistics 2020: GLOBOCAN estimates of incidence and Mortality Worldwide for 36 cancers in 185 countries Association of Colonoscopy Adenoma findings with Long-Term Colorectal Cancer incidence Urea cycle activation triggered by host-microbiota maladaptation driving colorectal tumorigenesis Association between fecal bile acids and colorectal cancer: a meta-analysis of observational studies Interplay between bile acids and the gut microbiota promotes intestinal carcinogenesis Metagenomic and metabolomic analyses reveal distinct stage-specific phenotypes of the gut microbiota in colorectal cancer Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice Grape seed Proanthocyanidin alleviates intestinal inflammation through gut microbiota-bile acid crosstalk in mice Short-chain fatty acid concentrations in the incidence and risk-stratification of colorectal cancer: a systematic review and meta-analysis Panethnic differences in blood pressure in Europe: a systematic review and Meta-analysis and microbial metabolites in colon cancer risk in rural africans and African americans Faecal bile-acids and clostridia in patients with cancer of the large bowel Fecal bile acids and neutral sterols in colon cancer patients and patients with adenomatous polyps Faecal bile acids and clostridia in the aetiology of colorectal cancer Fecal bile acids and neutral sterols in Japanese with large bowel carcinoma Faecal bile acid profiles in patients with large bowel cancer in Japan Comparison of faecal bile acid profiles between patients with adenomatous polyps of the large bowel and healthy subjects in Japan Fecal bile acid excretion pattern in colonic cancer patients Fecal bile acids in patients with adenomatous polyps of the colon Steroids and cancer: faecal bile acid screening for early detection of cancer risk Fecal free and conjugated bile acids and neutral sterols in vegetarians Faecal steroids and colorectal cancer: steroid profiles in subjects with adenomatous polyps of the large bowel and pH of fecal water from patients with colorectal adenomas Fecal primary bile acids and serum cholesterol are associated with colorectal adenomas Effects of a 3-mo consumption of short-chain fructo-oligosaccharides on parameters of colorectal carcinogenesis in patients with or without small or large colorectal adenomas Stool microbiome and metabolome differences between colorectal cancer patients and healthy adults Simultaneous assay of fecal short-chain fatty and bile acids and ratio of total bile acids to butyrate in Colon cancer butyric acid and t10,c12-CLA changes in colorectal adenomatous polyp patients Association between low colonic short-chain fatty acids and high bile acids in high colon cancer risk populations short-chain fatty acids and bile acids Link Colorectal Cancer Risk to Dietary Changes Associated with Urbanization among zimbabweans A prospective cohort analysis of gut microbial co-metabolism in Alaska native and rural African people at high and low risk of colorectal cancer Comparison of Acetaminophen (paracetamol) with Ibuprofen for Treatment of Fever or Pain in children younger than 2 years: a systematic review and Meta-analysis Gut microbiota-derived bile acids in intestinal immunity Does cholecystectomy predispose to colo-rectal cancer Cholecystectomy Risk Colorectal Cancer 100 Bacterial alterations in Post-cholecystectomy patients are Associated with Colorectal Cancer Gallbladder-derived surfactant protein D regulates gut commensal bacteria for maintaining intestinal homeostasis Transport and biological activities of bile acids The Effect of Lithocholic Acid on the gut-liver Axis Meta-analysis of fecal metagenomes reveals global microbial signatures that are specific for colorectal cancer Prediagnostic plasma bile acid levels and Colon cancer risk: a prospective study Bile salt hydrolase in non-enterotoxigenic Bacteroides potentiates colorectal cancer A gut microbiota-bile acid axis promotes intestinal homeostasis upon aspirin-mediated damage The gut microbiota-bile acid axis links the positive association between chronic insomnia and cardiometabolic diseases Dietary fiber-based regulation of bile salt hydrolase activity in the gut microbiota and its relevance to human disease Vitro Antibacterial Activity of Unconjugated and conjugated bile salts on Staphylococcus aureus Lithocholic acid and derivatives: antibacterial activity Gut microbiota-bile acid-interleukin-22 axis orchestrates polycystic ovary syndrome Bias control in the analysis of case-control studies with incidence density sampling Download references This study was supported by Medicine and Health Science and Technology Plan Projects in Zhejiang Province (No Ningbo Medical and Health Brand Discipline (No and the Science Foundation of Lihuili Hospital (No.2022FZ002) Gulou Street Community Health Service Center writing - original draft; Yu Wang: formal analysis methodology; Lijuan Sheng: data curation; Wei Cui: formal analysis methodology; Chenyang Ma: conceptualization Download citation DOI: https://doi.org/10.1038/s41598-024-84801-6 Lewczyk | Date published: February 18 Siva Karthik Varanasi Investigates the Role of Bile Acids in Liver Diseases  has proven to be much less effective for certain cancers assistant professor of medicine in the Division of Innate Immunity and a faculty member in the Program in Innate Immunity explored the interplay between the immune system and liver microenvironment to understand why immune cells are less sensitive to immunotherapy during liver cancer and offer new molecular targets for improving liver cancer treatment and immunotherapy.  The study team found that certain bile acids in liver cancer could affect the activity of cancer-fighting immune cells They identified several liver bile acids associated with impaired T cell function and tumor growth By blocking the production of these bile acids they successfully halted tumor growth and significantly sensitized tumors to immunotherapy the team found that one specific bile acid had a positive effect on T cell activity as well as reducing existing tumors in the liver Given that UDCA is safe in humans and is FDA approved to treat primary biliary cholangitis Varanasi advocates that UDCA can be easily repurposed to treat liver cancer especially in combination with immunotherapy.   “Considering how T cell performance varies across different organs and tumors puts us at a great vantage point for looking at ways to optimize cancer treatment,” said Dr we’re able to see that bile acids in the liver are hugely influencing T cells’ ability to do their job and may be a useful therapeutic target.”  Read the full publication. Privacy Statement to study how two of the most common ICC mutations drive cancer and contribute to each subtype’s unique treatment vulnerabilities “Standard first-line treatment for ICC hasn’t changed since the last decade Patients need more targeted therapies,” Boila said His two-year, $100,000 DOD Rare Cancers Program Concept Award will enable Boila to study a subtype of ICC that currently has no targeted treatment The DOD Rare Cancers Research Program was developed to address the burden of rare cancers in military personnel and veterans About 16% of the cancers affecting military vets are rare cancers A deeper understanding of this subtype’s genetic underpinnings and potential drug susceptibilities will lay the groundwork Boila’s one-year, $50,000 Linda Blum Memorial Research Fellowship from the CCF will allow him to discover what underlies a different ICC subtype’s drug sensitivities These findings could help scientists develop strategies to stave off drug resistance or more effective combination therapies The CCF provides seed funding to creative early-career investigators whose promising projects are likely to have significant clinical impact which predominantly occurs in older people who have a median age of 71 years and are the U.S.’ largest veteran cohort Because ICC often produces no symptoms, it is usually diagnosed at a late stage when surgery is no longer an option. According to the American Cancer Society, only 9% as many patients with intra-liver bile duct cancer will survive five years compared to the percentage of people without the same disease who will survive five years This relative survival rate is 23% for patients whose ICC is diagnosed early but can be as low as 3% if the tumor is diagnosed after it has spread through the body Only about half of patients diagnosed with ICC will live a year past treatment More effective and targeted treatments are desperately needed Liver cancer incidence, including ICC, is rising it’s difficult to gather enough tissue samples to make confident inferences about what’s driving the cancer and where it may be vulnerable “It’s true of most rare cancers: small cohorts make it difficult to get statistically significant observations from patient data It’s why we depend more on experimental models,” Boila said He has several novel preclinical models he can use to better understand ICC. The late Supriya “Shoop” Saha, MD, PhD developed genetically engineered mouse models (or GEMMs) that grow ICC tumors in their bile ducts These include GEMMs that model the top two mutated genes seen in ICC One GEMM has a liver-specific cancer-associated mutation in the gene encoding the enzyme isocitrate dehydrogenase The other is a completely novel GEMM strain with a liver-specific knockout of a gene called ARID1A She now leads a combined lab focused on both liver and pancreatic cancer About 20% of human ICC cases have a mutation in the ARID1A gene ARID1A is part of a protein complex that restructures DNA packaging to influence expression of certain genes Its loss may shift which genes are turned on or off in a way that promotes tumor formation Boila’s DOD funding will allow him to begin untangling this mechanism we’ll be able to gather data showing the driving factors promoting ICC in ARID1A-mutated bile duct cells,” Boila said “We’re trying to find the mechanisms that regulate tumor formation in this ICC subtype.” he’ll use cell lines and organoids — which recapture in a lab dish some degree of a tumor's 3D nature — to reveal how ARID1A mutations promote ICC development Boila’s suite of preclinical models will also aid his efforts to discover potential new therapies for patients with ARID1A-mutated ICC Currently the only targeted treatments for patients with ICC are aimed at tumors with mutations in IDH or a growth factor gene Boila will use his DOD funding to support the hunt for potential new drugs He will screen a compendium of FDA-approved drugs for efficacy against ICC with mutated ARID1A testing them first in his cell line and organoid models Those that seem most promising will be tested in “preclinical” trials run in patient-derived xenograft (PDX) models in which human tumor tissue is implanted into mice PDX models can give scientists a better idea of how human tumors may respond to a potential treatment than a genetically manipulated mouse tumor would Boila’s CCF award will allow him to better understand another ICC subtype, driven by mutations in a gene involved in metabolism. This gene encodes the enzyme isocitrate dehydrogenase which creates an important metabolite in our cells’ energy-generating cycle Unlike cancer-promoting mutations in ARID1A cancer-promoting mutations in the IDH1 gene give the IDH enzyme a new function IDH breaks down a molecule called isocitrate Alpha-ketoglutarate is an important co-factor in a wide array of enzymatic reactions within our cells Mutant IDH gains the ability to create a new High concentrations of D-2HG and low levels of α-ketoglutarate alter the activity of many enzymes that regulate gene expression shifting the pattern of genes that are turned on and off Tumor cells tend to be less “differentiated” than normal cells which means they can’t perform the specialized functions that are unique to various cell types The gene expression shifts in IDH1-mutant cells help promote cancer by blocking the liver stem cells’ ability to differentiate into functional liver cells Boila's CCF funding will support his studies of an enzyme called PP2A which regulates sensitivity to chemotherapies called nucleoside analogs These anti-cancer drugs damage DNA by mimicking its components Standard treatments for many types of cancer nucleoside analogs include drugs like gemcitabine Whether IDH mutation status influences PP2A’s ability to promote sensitivity to gemcitabine is unknown Kugel’s team revealed that a new signaling complex removes activating modifications from growth-promoting proteins Boila will work to better understand the critical role that PP2A plays in IDH1-mutant ICC biology and how modifying PP2A function may influence tumor cell growth Boila recently attended and presented their latest findings at the CCF annual conference It was an opportunity to connect with pioneers in the CCA research community as well as patients and patient advocates He was able to interact with a global community of scientists who presented clinical and research data from around the world including regions where CCA is more prevalent than in the U.S He also learned about how regulatory bodies work to help make drugs more accessible to patients and spoke with interested patients and caregivers “It was a wonderful experience,” Boila said “It reminds me how my work impacts patients’ lives Boila also commended Kugel and his other Fred Hutch mentors for their guidance and support “Sita has been an incredibly motivating and supportive mentor,” he said Having trained in leukemia research during his PhD Boila wanted to utilize his expertise in cancer biology and passion for understanding how cells regulate modifications of DNA and DNA packaging in the study of rare cancers Kugel’s expertise in gastrointestinal cancers and careful mentorship allowed Boila to dive into the intricacies of ICC research And her guidance doesn’t end at the lab bench: Kugel has designed a dynamic training plan to ensure Boila achieves his long-term goal of leading his own research group Kugel has also modeled the perseverance and resilience needed to weather — and grow from — scientific setbacks He pointed to her recent McDougall Mentoring Award as further testament to her outstanding support for trainees Fred Hutch prostate and bladder cancer physician-scientist Andrew Hsieh, MD, co-mentored Boila for his DOD award. Fred Hutch’s Jonathan Cooper, PhD an expert in the critical enzyme inhibited by dasatanib “Debraj is a fantastic scientist — intellectually curious and rigorous,” Kugel said “I am so happy and proud to support him for this project and into the future as he grows into an independent scientist and establishes his own lab.” Read more about Fred Hutch achievements and accolades Sabrina Richards, a senior editor and writer at Fred Hutch Cancer Center, has written about scientific research and the environment for The Scientist and OnEarth Magazine. She has a PhD in immunology from the University of Washington, an MA in journalism and an advanced certificate from the Science, Health and Environmental Reporting Program at New York University. Reach her at srichar2@fredhutch.org Fred Hutchinson Cancer Center is an independent organization that serves as UW Medicine's cancer program © 2025 Fred Hutchinson Cancer Center, a 501(c)(3) nonprofit organization 1100 Fairview Ave. N., P.O. Box 19024, Seattle, WA 98109-1024 206.667.5000Contact Us Volume 11 - 2024 | https://doi.org/10.3389/fnut.2024.1447878 high-fat diets and unhealthy lifestyles have led to an epidemic of obesity and obesity-related metabolic diseases seriously placing a huge burden on socio-economic development A deeper understanding and elucidation of the specific molecular biological mechanisms underlying the onset and development of obesity has become a key to the treatment of metabolic diseases Recent studies have shown that the changes of bile acid composition are closely linked to the development of metabolic diseases Bile acids can not only emulsify lipids in the intestine and promote lipid absorption but also act as signaling molecules that play an indispensable role in regulating bile acid homeostasis Disorders of bile acid metabolism are therefore important risk factors for metabolic diseases is abundantly expressed in liver and intestinal tissues Bile acids act as endogenous ligands for the farnesol X receptor and erroneous FXR signaling triggered by bile acid dysregulation contributes to metabolic diseases non-alcoholic fatty liver disease and diabetes Activation of FXR signaling can reduce lipogenesis and inhibit gluconeogenesis to alleviate metabolic diseases It has been found that intestinal FXR can regulate hepatic FXR in an organ-wide manner The crosstalk between intestinal FXR and hepatic FXR provides a new idea for the treatment of metabolic diseases This review focuses on the relationship between bile acids and metabolic diseases and the current research progress to provide a theoretical basis for further research and clinical applications a well-studied receptor belonging to the nuclear receptor (NR) superfamily. serves as a transcription factor activated by BAs and tightly regulates the synthesis of BAs and their enterohepatic circulation Disruption of bile acid metabolism can lead to aberrant FXR signaling which is a significant predisposing factor in the pathogenesis of metabolic diseases Current investigations are focused on targeting BAs-FXR for the treatment of metabolic diseases by regulating bile acid levels to balance and restore the FXR signaling mechanism It is important to note that FXR exhibits tissue specificity in regulating metabolic diseases and its physiological functions being complex or even contradictory in different tissues This article will first discuss the biological properties of bile acids and their relationship with metabolic diseases we will focus on the latest research on the bile acid receptor FXR as a therapeutic target These studies suggest that activation of the bile acid replacement pathway may offer potential improvements for metabolic disorders glyco(tauro)- chenodeoxycholic acid; α(β)MCA α(β)-muricholic acid; T(α/β)-MCA tauro-α/β-muricholic acid; DCA Na + −taurocholate co-transporting polypeptide; ASBT apical sodium-dependent bile acid transporter; OSTα/β organic solute transporter α/β FXR plays a crucial role in regulating bile acid synthesis and enterohepatic circulation to maintain balanced distribution throughout the body and prevent potential health hazards The contradictory effects of FXR in intestinal L cells and pancreatic β-cells suggest that the FXR signaling pathway is tissue-specific and different tissues may have opposing roles sterol regulatory element-binding protein-1; FAS peroxisome proliferators-activated receptors; CPT1 This results in an elevation of bound bile acids concentration which counteracts the intestinal FXR pathway intestinal FXR is an important drug target for metabolic disease intervention and acts as a mediator of intestinal flora which plays a crucial role in the body’s metabolism FXR agonists/antagonists and metabolic diseases This can be attributed to the pharmacokinetic understanding that oral administration may stimulate intestinal FXR through absorption while intraperitoneal injection directly activates liver FXR via the circulatory system Determining the role of FXR in the body’s metabolism is a complex task especially in regards to whether activating intestinal FXR could improve metabolic diseases Previous studies have produced conflicting results indicating that the previously proposed mechanisms are insufficient to explain the diverse physiological functions of FXR in various tissues It remains uncertain whether the adverse impacts of agonists are confined to specific tissues or if the overall effect of various mechanisms is influenced by governing factors Further investigation is necessary to determine the specific molecular mechanisms involved derived from hepatic cholesterol metabolism play a pivotal role in maintaining the body’s homeostasis They serve not only as intestinal surfactants to facilitate the absorption of dietary lipids but also as signaling molecules that modulate pathways involved in insulin resistance There is mounting evidence suggesting that individuals with diabetes and obesity display perturbed bile acid profiles and elevated levels of secondary bile acids This leads to aberrant modulation of the intestinal FXR signaling pathway resulting in lipid accumulation and insulin resistance the treatment options for metabolic diseases are limited Activation of liver FXR has shown potential in improving metabolic diseases by inhibiting lipogenesis and glycogenesis making FXR agonists a promising new target for the treatment of conditions such as obesity and diabetes as potential therapeutic drugs for NAFLD/NASH is ongoing it has been observed that the use of GW4064 agonist exacerbates metabolic symptoms possibly because the agonist is not tissue-specific and activates FXR signaling at various sites throughout the body it is important to note that the intestinal FXR signaling pathway operates through different mechanisms activating intestinal FXR reduces lipid absorption and promotes hepatic gluconeogenesis across organs inhibiting intestinal FXR increases GLP-I secretion from intestinal L cells improving insulin sensitivity and increasing hepatic cholesterol metabolism the mechanism of the intestinal flora-BAs-FXR axis has been further elucidated the intestinal FXR pathway can be inhibited by decreasing the level of BSH enzyme-containing bacteria This reveals the connection between intestinal flora constituting a well-established intestinal flora-BAs-FXR metabolism network This presents a novel perspective for enhancing and managing the molecular mechanisms underlying the contradictory impact between this approach and the treatment of metabolic diseases with FXR agonists remain ambiguous and the correlation between hepatic and intestinal FXR has not been firmly established Given the diversity in bile acid pool composition in both mice and humans further comprehensive investigation is imperative prior to translating findings from mouse experiments into clinical therapy The above indicates that FXR may have opposing effects in various tissues and cells highlighting the importance of considering tissue specificity when studying the paradoxical effects of FXR It is anticipated that ligand drugs targeting FXR will prove efficacious in addressing metabolic diseases LW: Writing – review & editing QY: Writing – review & editing LL: Writing – review & editing YX: Writing – review & editing The author(s) declare that financial support was received for the research This work was supported by the Project of the National Natural Science Foundation of China (Nos The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 1. Lobstein, T, Jackson-Leach, R, Powis, J, Brinsden, H, and Gray, M. World obesity atlas 2023. (2023). Available at: https://data.worldobesity.org/publications/?cat=19 Google Scholar and inflammation: An update for anaesthetists caring for patients with obesity The global burden of metabolic disease: data from 2000 to 2019 Jiang-Tang-san-Huang pill alleviates type 2 diabetes mellitus through modulating the gut microbiota and bile acids metabolism Bisphenol A induced hepatic steatosis by disturbing bile acid metabolism and FXR/TGR5 signaling pathways via remodeling the gut microbiota in CD-1 mice Effect of lotus seed resistant starch on small intestinal flora and bile acids in hyperlipidemic rats New insights in the multiple roles of bile acids and their signaling pathways in metabolic control The liver under the spotlight: bile acids and oxysterols as pivotal actors controlling metabolism Bile acids and GPBAR-1: dynamic interaction involving genes Crossref Full Text | Google Scholar Bile acid receptors and the gut-liver Axis in nonalcoholic fatty liver disease Bile acid and receptors: biology and drug discovery for nonalcoholic fatty liver disease Crossref Full Text | Google Scholar Review article: therapeutic aspects of bile acid signalling in the gut-liver axis Bile acid metabolism in liver pathobiology PubMed Abstract | Crossref Full Text | Google Scholar The protective mechanism of Lactobacillus plantarum FZU3013 against non-alcoholic fatty liver associated with hyperlipidemia in mice fed a high-fat diet Gut microbial metabolites as multi-kingdom intermediates Parabacteroides distasonis alleviates obesity and metabolic dysfunctions via production of succinate and secondary bile acids Review on bile acids: effects of the gut microbiome and alterations in the bioaccessibility of bioactive compounds Intestinal crosstalk between bile acids and microbiota in irritable bowel syndrome Crossref Full Text | Google Scholar Apple polyphenol extract improves high-fat diet-induced hepatic steatosis by regulating bile acid synthesis and gut microbiota in C57BL/6 male mice Bile acid receptors FXR and TGR5 signaling in fatty liver diseases and therapy Crossref Full Text | Google Scholar PubMed Abstract | Crossref Full Text | Google Scholar Discovery of farnesoid X receptor and its role in bile acid metabolism Bile acids in glucose metabolism and insulin signalling – mechanisms and research needs Crossref Full Text | Google Scholar Bile acids as metabolic regulators and nutrient sensors PubMed Abstract | Crossref Full Text | Google Scholar Low production of 12α-hydroxylated bile acids prevents hepatic steatosis in Cyp2c70(−/−) mice by reducing fat absorption Association between 12α-hydroxylated bile acids and hepatic steatosis in rats fed a high-fat diet Alteration of bile acid metabolism by a high-fat diet is associated with plasma transaminase activities and glucose intolerance in rats A dysregulated bile acid-gut microbiota axis contributes to obesity susceptibility Organic solute transporter α-β protects Ileal enterocytes from bile acid-induced injury Intestinal Farnesoid X receptor activation by pharmacologic inhibition of the organic solute transporter α-β Determination of individual bile acids in acute respiratory distress syndrome reveals a specific pattern of primary and secondary bile acids and a shift to the acidic pathway as an adaptive response to the critical condition Bile acid metabolism and signaling: emerging pharmacological targets of dietary polyphenols The molecular insights of bile acid homeostasis in host diseases PubMed Abstract | Crossref Full Text | Google Scholar Bile acid modifications at the microbe-host Interface: potential for nutraceutical and pharmaceutical interventions in host health PubMed Abstract | Crossref Full Text | Google Scholar Crossref Full Text | Google Scholar The influence of probiotics on bile acids in diseases and aging Contribution of the microbiome for better phenotyping of people living with obesity Role of the gut microbiota and its metabolites in tumorigenesis or development of colorectal Cancer Dietary influences on the microbiota-gut-brain Axis Gut microbiome and health: mechanistic insights Gut-microbiota derived bioactive metabolites and their functions in host physiology Gut microbial pathways for bile acid metabolism PubMed Abstract | Crossref Full Text | Google Scholar PubMed Abstract | Crossref Full Text | Google Scholar Bile salt hydrolases: gatekeepers of bile acid metabolism and host-microbiome crosstalk in the gastrointestinal tract Targeting gut microbial bile salt hydrolase (BSH) by diet supplements: new insights into dietary modulation of human health Crossref Full Text | Google Scholar Bile acids and microbes in metabolic disease A gut-restricted Lithocholic acid analog as an inhibitor of gut bacterial bile salt hydrolases Diversification of host bile acids by members of the gut microbiota PubMed Abstract | Crossref Full Text | Google Scholar Bile acid signaling in the regulation of whole body metabolic and immunological homeostasis Characterization of gut microbial structural variations as determinants of human bile acid metabolism Crossref Full Text | Google Scholar Western diet induces Paneth cell defects through microbiome alterations and farnesoid X receptor and type I interferon activation Gut microbial bile acid metabolite skews macrophage polarization and contributes to high-fat diet-induced colonic inflammation Role of bile acids in the regulation of food intake and their dysregulation in metabolic disease Crosstalk between bile acids and gut microbiota and its impact on Farnesoid X receptor Signalling Alterations of bile acids and gut microbiota in obesity induced by high fat diet in rat model Gut microbiota regulates postprandial GLP-1 response via ileal bile acid-TGR5 signaling Chenodeoxycholate in females with irritable bowel syndrome-constipation: a pharmacodynamic and pharmacogenetic analysis Crossref Full Text | Google Scholar FXR in liver physiology: multiple faces to regulate liver metabolism FXR isoforms control different metabolic functions in liver cells via binding to specific DNA motifs and drug development for NASH and fibrosis diseases Progress and challenges of selective Farnesoid X receptor modulation Bile acid-induced tissue factor activity in hepatocytes correlates with activation of farnesoid X receptor The ameliorative effect of probiotics on diet-induced lipid metabolism disorders: A review Obeticholic acid and INT-767 modulate collagen deposition in a NASH in vitro model Crossref Full Text | Google Scholar Intestinal bile acid receptors are key regulators of glucose homeostasis Crossref Full Text | Google Scholar Probiotic Lactobacillus rhamnosus GG prevents liver fibrosis through inhibiting hepatic bile acid synthesis and enhancing bile acid excretion in mice Dihydromyricetin prevents obesity via regulating bile acid metabolism associated with the farnesoid X receptor in Ob/Ob mice Postbiotics prepared using Lactobacillus reuteri ameliorates ethanol-induced liver injury by regulating the FXR/SHP/SREBP-1c Axis Dan-shen Yin promotes bile acid metabolism and excretion to prevent atherosclerosis via activating FXR/BSEP signaling pathway The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases Emerging drugs for the treatment of non-alcoholic steatohepatitis: a focused review of farnesoid X receptor agonists Inonotus obliquus and its bioactive compounds alleviate non-alcoholic fatty liver disease via regulating FXR/SHP/SREBP-1c axis Transcriptome analysis of dual FXR and GPBAR1 Agonism in rodent model of NASH reveals modulation of lipid droplets formation Bile acids mediated potential functional interaction between FXR and FATP5 in the regulation of lipid metabolism Crossref Full Text | Google Scholar SREBP-1c and lipogenesis in the liver: an update1 PubMed Abstract | Crossref Full Text | Google Scholar Flavonoids from whole-grain oat alleviated high-fat diet-induced hyperlipidemia via regulating bile acid metabolism and gut microbiota in mice Arabinoxylan combined with different glucans improve lipid metabolism disorder by regulating bile acid and gut microbiota in mice fed with high-fat diet Extract of Schisandra chinensis fruit protects against metabolic dysfunction in high-fat diet induced obese mice via FXR activation Treatment with the natural FXR agonist chenodeoxycholic acid reduces clearance of plasma LDL whilst decreasing circulating PCSK9 The role of FXR and TGR5 in reversing and preventing progression of Western diet-induced hepatic steatosis Bile acids regulate gluconeogenic gene expression via small heterodimer partner-mediated repression of hepatocyte nuclear factor 4 and Foxo1 Beneficial effect of farnesoid X receptor activation on metabolism in a diabetic rat model Knockout of farnesoid X receptor aggravates process of diabetic cardiomyopathy The hypoglycemic effect of Berberine and Berberrubine involves modulation of intestinal Farnesoid X receptor signaling pathway and inhibition of hepatic gluconeogenesis Hepatic thyroid hormone signalling modulates glucose homeostasis through the regulation of GLP-1 production via bile acid-mediated FXR antagonism Curcumin compensates GLP-1 deficiency via the microbiota-bile acids Axis and modulation in functional crosstalk between TGR5 and FXR in Ob/Ob mice The glucose-lowering effects of α-glucosidase inhibitor require a bile acid signal in mice Hepatic protective effects of Shenling Baizhu powder against inflammatory damage via TLR4/NLRP3 signalling pathway in rats with nonalcoholic fatty liver disease Protective effect of Danshen Zexie decoction against non-alcoholic fatty liver disease through inhibition of ROS/NLRP3/IL-1β pathway by Nrf2 signaling activation Eriobotrya japonica leaf triterpenoid acids ameliorate metabolic syndrome in C57BL/6J mice fed with high-fat diet Black chokeberry (Aronia melanocarpa L.) polyphenols attenuate obesity-induced colonic inflammation by regulating gut microbiota and the TLR4/NF-κB signaling pathway in high fat diet-fed rats Dityrosine aggravates hepatic insulin resistance in obese mice by altering gut microbiota and the LPS/TLR4/NF-κB inflammatory pathway Microbial bile acid metabolites modulate gut RORγ(+) regulatory T cell homeostasis Bile acid metabolites control T(H)17 and T(reg) cell differentiation FXR inhibits endoplasmic reticulum stress-induced NLRP3 Inflammasome in hepatocytes and ameliorates liver injury FXR mediates T cell-intrinsic responses to reduced feeding during infection Bacteroides uniformis-induced perturbations in colonic microbiota and bile acid levels inhibit TH17 differentiation and ameliorate colitis developments Gut-microbiota-derived metabolites maintain gut and systemic immune homeostasis Signaling from intestine to the host: how bile acids regulate intestinal and liver immunity Crossref Full Text | Google Scholar Melatonin mitigates aflatoxin B1-induced liver injury via modulation of gut microbiota/intestinal FXR/liver TLR4 signaling axis in mice Intestinal barrier and permeability in health Fucoidan ameliorated dextran sulfate sodium-induced ulcerative colitis by modulating gut microbiota and bile acid metabolism Bile acid-gut microbiota Axis in inflammatory bowel disease: from bench to bedside The direct and gut microbiota-mediated effects of dietary bile acids on the improvement of gut barriers in largemouth bass (Micropterus salmoides) Microbiota-induced obesity requires farnesoid X receptor The role of the gut microbiome and its metabolites in metabolic diseases Metabolic-associated fatty liver disease and the gut microbiota Crossref Full Text | Google Scholar Lactobacillus reuteri J1 prevents obesity by altering the gut microbiota and regulating bile acid metabolism in obese mice Association between body mass index and Firmicutes/Bacteroidetes ratio in an adult Ukrainian population Changes seen in gut bacteria content and distribution with obesity: causation or association Crossref Full Text | Google Scholar Theabrownin from Pu-erh tea attenuates hypercholesterolemia via modulation of gut microbiota and bile acid metabolism Diammonium Glycyrrhizinate ameliorates obesity through modulation of gut microbiota-conjugated BAs-FXR signaling The triterpenoid sapogenin (2α-OH-Protopanoxadiol) ameliorates metabolic syndrome via the intestinal FXR/GLP-1 axis through gut microbiota remodelling Emodin palliates high-fat diet-induced nonalcoholic fatty liver disease in mice via activating the farnesoid X receptor pathway Chlorothalonil induces obesity in mice by regulating host gut microbiota and bile acids metabolism via FXR pathways FXR activation protects against NAFLD via bile-acid-dependent reductions in lipid absorption Effects of flaxseed powder in improving non-alcoholic fatty liver by regulating gut microbiota-bile acids metabolic pathway through FXR/TGR5 mediating Intestinal FXR agonism promotes adipose tissue browning and reduces obesity and insulin resistance Intestine farnesoid X receptor agonist and the gut microbiota activate G-protein bile acid receptor-1 signaling to improve metabolism Intestinal FGF15/19 physiologically repress hepatic lipogenesis in the late fed-state by activating SHP and DNMT3A Farnesoid X receptor activation induces the degradation of hepatotoxic 1-deoxysphingolipids in non-alcoholic fatty liver disease Crossref Full Text | Google Scholar Hepatic cholesterol accumulation ascribed to the activation of ileum Fxr-Fgf15 pathway inhibiting hepatic Cyp7a1 in high-fat diet-induced obesity rats Dietary acetic acid suppress high-fat diet-induced obesity in mice by altering taurine conjugated bile acids metabolism Chlorogenic acid inhibits lipid deposition by regulating the enterohepatic FXR-FGF15 pathway Prevention of high-fat-diet-induced dyslipidemia by Lactobacillus plantarum LP104 through mediating bile acid enterohepatic Axis circulation and intestinal Flora Caffeic acid phenethyl ester suppresses intestinal FXR signaling and ameliorates nonalcoholic fatty liver disease by inhibiting bacterial bile salt hydrolase activity Highland barley β-glucan alleviated western diet-induced non-alcoholic fatty liver disease via increasing energy expenditure and regulating bile acid metabolism in mice Ferulic acid attenuates high-fat diet-induced hypercholesterolemia by activating classic bile acid synthesis pathway Astragaloside IV ameliorates diet-induced hepatic steatosis in obese mice by inhibiting intestinal FXR via intestinal flora remodeling Glycine-β-muricholic acid antagonizes the intestinal farnesoid X receptor-ceramide axis and ameliorates NASH in mice Sterol 12α-hydroxylase aggravates dyslipidemia by activating the ceramide/mTORC1/SREBP-1C pathway via FGF21 and FGF15 Effect of Gegen Qinlian decoction on hepatic gluconeogenesis in ZDF rats with type 2 diabetes mellitus based on the Farnesol X receptor/ceramide signaling pathway regulating mitochondrial metabolism and endoplasmic reticulum stress Luo L and Xiong Y (2024) Regulation of bile acids and their receptor FXR in metabolic diseases Received: 12 June 2024; Accepted: 13 November 2024; Published: 11 December 2024 Copyright © 2024 Li, Wang, Yi, Luo and Xiong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Yuxia Xiong, eHl4X2NlbGxAc3dtdS5lZHUuY24= Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Metrics details The levels of fasting-state serum bile acids (BAs) in individuals with polycystic ovary syndrome (PCOS) differ from those of control subjects there is a lack of research on the BAs profile in lean women with PCOS and whether these changes are linked to the host metabolism our objective was to investigate the synthesis and metabolism of serum BAs in lean women with PCOS and assess the correlation between BAs and clinical characteristics This study employed a cross-sectional design of lean women with PCOS (n = 240) in comparison to a control group (n = 80) consisting of healthy lean women The findings revealed significant increases in the levels of non-12-OH BAs and chenodeoxycholic acid (CDCA)% (both P < 0.05) in lean women with PCOS a positive correlation was observed between CDCA% and total testosterone (T) (r = 0.130 P = 0.044) and free androgen index (FAI) (r = 0.153 a decreased ratio of cholic acid/chenodeoxycholic acid (CA/CDCA) (P < 0.001) was observed in lean women with PCOS suggesting the depletion or downregulation of CYP8B1 Receiver operating characteristic curve analysis indicated that the combination of CDCA/CA and DHEAS could potentially be used as a characteristic factor for PCOS in lean women It is possible that enzymatic modifications in the liver could play a role in regulating hyperandrogenism in this specific subgroup of lean women with PCOS Despite extensive research efforts aimed at elucidating the mechanisms underlying the development of PCOS the specific mechanisms associated with lean women with PCOS remain incompletely understood the existing studies have primarily concentrated on PCOS patients who are classified as obese or overweight the specific patterns of BAs metabolites that contribute to the development of PCOS in lean women remain unknown Given that cholesterol serves as a metabolic substrate for both BAs and sex hormones it is reasonable to hypothesize that a significant correlation exists between BAs metabolism and hyperandrogenism in lean women with PCOS we conducted a comprehensive analysis of the serum BAs profiles and the synthesis pathway of BAs in lean women with polycystic ovary syndrome (PCOS) and BMI-matched controls using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach we investigated the association between BA metabolism and hyperandrogenism in Chinese women with PCOS The primary objective of our research was to enhance our understanding of the relationship between BA metabolism and hyperandrogenism in this specific population The criteria were as follows: (1) oligomenorrhoea and/or anovulation (OA); (2) biochemical and/or clinical manifestations of hyperandrogenemia (HA); (3) polycystic ovarian morphology (PCOM): ultrasound shows the diameter of one or both ovaries 2–9 mm follicles ≥ 12 and/or one side or both ovarian volume ≥  10 cm3; subjects who meet any 2 of the above 3 items and ruled out congenital adrenal hyperplasia Cushing’s syndrome would be confirmed diagnosis of PCOS Control group comprised lean women with normal menstruation no clinical or biochemical signs of hyperandrogenemia and normal ovaries morphology in ultrasound Exclusion criteria for all participants were as follow: (1) combined with severe liver function test abnormality (defined as alanine aminotransferase (ALT) 2.5 times or higher than the upper limit of normal range) or renal dysfunction (serum creatinine > 132 umol/L and/or eGFR < 60 ml/min.1.73m2); (2) combined with hepatic diseases (including autoimmune and previous gastrointestinal surgery; (3) antibiotics use for the past three months; (4) regularly use of medicine known to affect glucolipid metabolism or hormone in one month; (5) infectious diseases within two weeks; (6) take pharmacological doses of cholestyramine and ursodeoxycholic acid for any reason; (7) hyperthyroidism and hypertension; (8) Pregnancy was excluded through a urine pregnancy test The exclusion criteria were also applied to the control subjects All participants were enrolled in the study after the research protocols were explained and written informed consent were provided and waist circumference for all participants were obtained using established protocols and skilled medical professionals The weight and height of each individual were measured with precision using a digital scale and stadiometer (inbody Body Mass Index (BMI) was calculated by dividing the weight in kilograms by the square of the height in meters The waist circumference was determined at the narrowest point between the costal margin and iliac crest with measurements recorded to the nearest 0.1 cm Bile acids were separated using an ACQUITY BEH C18 column (1.7 μm 100 mm × 2.1 mm internal dimensions) (Waters The column elution solvents consist of water + 0.01% formic acid (A) and acetonitrile/methanol (87:13) + 0.01% formic acid (B) The flow rate was 450 µL/min as follows: 0–1 min (5% B) The raw data obtained from UPLC-MS were collected with multiple reaction monitor (MRM) and the cone and collision energy for each bile acid used the optimized settings from QuanOptimize application manager (Waters) Several internal standards were added to each experimental and process standard sample prior to injection into the mass spectrometer The measure of platform variability was determined by calculating the median relative standard deviation (RSD) for the internal standards Quantified bile acids contained six primary species taurochenodeoxycholic acid (TCDCA); and nine secondary species glycolithocholic acid (GLCA) and taurolithocholic acid (TLCA) The detection instruments are API3200MD triple quadrupole mass spectrometer (American ABSciex company) and Shimadzu 20AD liquid chromatograph (Japan Shimadzu company) Statistical analyses were performed using SPSS 22.0 software (SPSS Inc. USA) and GraphPad Prism 8.0 (GraphPad Software Distribution normality of each continuous variable was assessed using Kolmogorov-Smirnov test Normally distributed data were expressed as mean ± SD and assessed using the two independent samples t-test whereas for non-normal distributed data were presented as median (25th-75th interquartile range) and evaluated using the Mann-Whitney U test The difference levels of BAs between different two groups were compared by logistic regression models adjusted UA The cross-sectional association of BAs with sex hormonal and metabolic parameters was assessed using Spearman correlation analysis Receiver operator characteristic (ROC) curve analysis was used to evaluate the potential characteristic factor of BAs in lean women for PCOS A two-tailed P value < 0.05 was considered statistically significant for all analyses A summary of each subject’s clinical and biochemical characteristics can be found in Table 1 lean women with PCOS and controls were well matched The mean age (years) of our population was 27.66 in lean women with PCOS and 27.19 in controls LDL-c (P = 0.001) and AUCglu calculated by OGTT (P = 0.010) were higher in lean women with PCOS than those in controls The sex hormonal levels of LH (P < 0.001) and AMH (P < 0.001) were higher in lean women with PCOS compared with the levels in controls Ovarian volumes (P < 0.001) were significantly higher in the PCOS group compared to the control group There were no significant differences in waist circumference the characteristics of our study population in accordance with the Rotterdam criteria were presented 34.17% exhibited features consistent with HA and PCOM This was followed by 25% of women who displayed OA only 19.16% of the population lacked HA characteristics Serum BAs profile is significantly altered in lean women with PCOS Data are presented as the median with 95%CI and analyzed using Mann-Whitney U test Proportion of individual BAs in lean women with PCOS and controls (G) Correlation between BAs and the clinical-biological parameters of all subjects at baseline Spearman′s correlation coefficient (R) is presented for each pair of parameters Significant values are presented: *p<0.05 Ratios of bile acids reflective of liver and gut microbiome enzymatic activities Three types of ratios were calculated to inform about possible enzymatic activity changes in lean women with PCOS These ratios reflect one of the following: (1) shift in bile acid metabolism from primary to alternative pathway; (2) changes in gut microbiome correlated with production of secondary bile acids; (3) changes in glycine and taurine conjugation of secondary bile acids Associations of the BAs ratio with DHEAS Spearman’s correlation coefficient (R) is presented for parameters ROC curves for ratio CDCA/CA and DHEAS.ROC receiver operating characteristic we have elucidated a potential role for the activation of the alternative pathway of bile acids (BAs) in the liver in relation to hyperandrogenism in lean women with polycystic ovary syndrome (PCOS) Our findings indicate a significant increase in serum levels of non-12-OH BAs and a primary BA derived from the liver (CDCA) with a notably lower ratio of CA/CDCA in lean women with PCOS compared to the control group we have observed a positive correlation between CA/CDCA and DHEAS levels.We have identified the decreased level of CA/CDCA as a compensatory response to the elevated DHEAS in lean women with PCOS our data suggests that the combination of CDCA/CA and DHEAS may serve as potential characteristic marker it is imperative to allocate more attention to the issue of BAs synthesis and metabolism disorders in lean women with PCOS suggesting that elevated androgen levels may contribute to increased CDCA levels the underlying mechanisms linking bile acids (BAs) and hyperandrogenism in patients with PCOS remain unclear Our study’s findings indicate that the activation of alternative pathways in the synthesis of bile acids (BAs) may serve as a compensatory mechanism for hyperandrogenism in lean women with PCOS we investigated the presence of a similar association between BAs ratio and DHEAS there is no significant correlation between BAs and DHEAS in the control group the regression analysis revealed that the BAs ratio does not possess significant predictive value for DHEAS levels within the control population highlighting the specificity of the BAs profile in the PCOS population These findings suggested that the manipulation of BAs could potentially be employed as a potential therapeutic approach to mitigate hyperandrogenism in lean women with PCOS it is recommended that future research endeavors focus on assessing the profile of bile acids (BAs) across various PCOS phenotypes Our findings suggest that modulating BAs metabolism could serve as a potential therapeutic approach for addressing hyperandrogenism in PCOS patients it is important to note that the current body of evidence is limited additional investigations employing mechanistic studies are necessary to validate and substantiate our results This study possesses several notable strengths meticulous control for numerous confounding variables and rigorous standardization of the BAs test it is crucial to acknowledge several limitations inherent in this study the current cross-sectional design did not investigate the potential causal role of BAs alteration in the initiation and progression of PCOS To comprehensively understand the relationship between BAs synthesis and hyperandrogenism in PCOS further investigations employing long-term and multi-center studies with substantial sample sizes are warranted our study has established a correlation between the modified profile of bile acids and hyperandrogenism in lean women diagnosed with PCOS we have observed that the alterations in BAs synthesis and metabolism particularly the shift from the classical to the alternative metabolic pathway serve as a compensatory mechanism for hyperandrogenism in lean women with PCOS These significant findings regarding the role of bile acids in PCOS provide a foundation for future researchin this domain The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request Cholesterol associated with high density lipoproteins Cholesterol associated with low density lipoproteins Homeostasis model assessment of insulin resistance Liquid chromatography tandem mass spectrometry Homeostatic model assessment for insulin resistance Consensus on women’s health aspects of polycystic ovary syndrome (PCOS).Hum Meta-analysis of gene expression profiles of lean and obese PCOS to identify differentially regulated pathways and risk of comorbidities Lean polycystic ovary syndrome (PCOS): an evidence-based practical approach Combining a nontargeted and targeted metabolomics approach to identify metabolic pathways significantly altered in polycystic ovary syndrome Metabonomics reveals plasma metabolic changes and inflammatory marker in polycystic ovary syndrome patients Bile acids and metabolic regulation: mechanisms and clinical responses to bile acid sequestration A dysregulated bile acid-gut microbiota axis contributes to obesity susceptibility.EBioMedicine 55 Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism.Cell bile salt biotransformations by human intestinal bacteria The presence and severity of nonalcoholic steatohepatitis is associated with specific changes in circulating bile acids Bile acid alterations in nonalcoholic fatty liver disease insulin resistance and type 2 diabetes: what do the human studies tell Increased circulating conjugated primary bile acids are associated with hyperandrogenism in women with polycystic ovary syndrome Serum metabolomics analysis of patients with polycystic ovary syndrome by mass spectrometry The use and Interpretation of Anthropometry Report of a WHO Expert Committee 8541–452 (World Health Organ Tech Rep Ser ROTTERDAM E A-S P C W G Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome Recommendations from the 2023 International evidence-based guideline for the assessment and management of polycystic ovary syndrome CHRIST J P & CEDARS M I Current guidelines for diagnosing PCOS Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man Insulin secretion in response to glycemic stimulus: relation of delayed initial release to carbohydrate intolerance in mild diabetes mellitus Quantification of the relationship between insulin sensitivity and beta-cell function in human subjects insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp Assessment of insulin sensitivity and beta-cell function from measurements in the fasting state and during an oral glucose tolerance test Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry Polycystic ovary syndrome with hyperandrogenism is characterized by an increased risk of hepatic steatosis compared to nonhyperandrogenic PCOS phenotypes and healthy controls independent of obesity and insulin resistance Quantification of bile acids: a mass spectrometry platform for studying gut microbe connection to metabolic diseases Differences in the regulation of the classical and the alternative pathway for bile acid synthesis in human liver No coordinate regulation of CYP7A1 and CYP27A1 The influence of biological sex and sex hormones on bile acid synthesis and cholesterol homeostasis Bile acids in glucose metabolism in health and disease Testosterone-induced permanent changes of hepatic gene expression in female mice sustained during Plasmodium chabaudi malaria infection Targeting the alternative bile acid synthetic pathway for metabolic diseases Attenuated effects of bile acids on glucose metabolism and insulin sensitivity in a male mouse model of prenatal undernutrition Chenodeoxycholic Acid as a potential prognostic marker for Roux-en-Y gastric bypass in Chinese obese patients Increased prevalence of Elevated DHEAS in PCOS Women with non-classic (B or C) phenotypes: a retrospective analysis in patients aged 20 to 29 years and severity of androgen excess in 1205 consecutively recruited women Age-related changes of plasma bile acid concentrations in healthy adults–results from the cross-sectional KarMeN study Download references We thank the women who participated in the study and gratefully acknowledge the assistance of the nursing staff and of the technical assistants at Shanghai Renji Hospital This work was supported by the National Natural Science Foundation of China (82170807) the Medical Guidance Science and Technology Support Projects of Shanghai Municipal Science and Technology Commission (18411968700); and the Natural Science Foundation of Shanghai (12ZR1417800) Yuchen Zhu and Siyu Lin contributed equally to this work Department of Endocrinology and Metabolism Shanghai Jiao Tong University School of Medicine and was a major contributor in writing the manuscript SL followed up the patients and collected the patient data they contributed equally to this work and share first authorship This study was approved by the Ethical Committees of Shanghai Renji Hospital and written informed consent was obtained from all subjects Spearman’s correlation coefficient (R) is presented for each pair of parameters Significant values are presented: *p < 0.05 Download citation DOI: https://doi.org/10.1038/s41598-024-77645-7 Sorry, a shareable link is not currently available for this article. Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma. Cellular and Molecular Mechanisms of Brain-aging Volume 8 - 2016 | https://doi.org/10.3389/fnagi.2016.00263 a structurally related group of molecules derived from cholesterol have a long history as therapeutic agents in medicine from treatment for primarily ocular diseases in ancient Chinese medicine to modern day use as approved drugs for certain liver diseases Despite evidence supporting a neuroprotective role in a diverse spectrum of age-related neurodegenerative disorders including several small pilot clinical trials little is known about their molecular mechanisms or their physiological roles in the nervous system We review the data reported for their use as treatments for neurodegenerative diseases and their underlying molecular basis While data from cellular and animal models and clinical trials support potential efficacy to treat a variety of neurodegenerative disorders and the precise molecular mechanism(s) by which they confer neuroprotection are not known delaying translation to the clinical setting Model system and human data implicating bile acids in neurodegenerative disorders Genomic and metabolomics data implicating bile acids in neurodegenerative disorders Two aspects of bile acid metabolism are relevant to their role in neurodegenerative disorders, bile acids that circulate systemically and that are synthesized by neurons. Circulating bile acids are largely synthesized from cholesterol in the liver (Prawitt et al., 2011) Ingestion of food causes bile acid secretion from the gallbladder through the common bile duct to the duodenum in order to facilitate the absorption of lipids and lipid-soluble vitamins via formation of micelles bile acids are transported by specific transport proteins to the portal circulation for recycling back to the liver The process is highly efficient with over 95% of bile acids resorbed the remaining 5% proceeding to the colon and excreted through the stool Enterohepatic recycling of the bile acid pool occurs about 12 times per day thus the net flux of bile acids through primarily the portal Despite documented neuroprotective roles in models of neurodegenerative disorders the primary signaling pathways (TGR5 and FXR) and the potential role of endogenous bile acids have not yet been studied TUDCA appeared to interact with the ligand binding domain of the mineralocorticoid receptor to prevent its binding to heat shock protein 90 and subsequent trafficking for proteosomal processing allowing for translocation to the nucleus suggesting a possible involvement of bile acids In humans, a recent study of cognitively intact patients identified and validated a set of blood-based biomarkers that included glycoursodeoxycholic acid (GUDCA) that could predict the onset of either AD or amnestic mild cognitive impairment, considered an early precursor of AD, within 2–3 years with an accuracy of over 90% (Mapstone et al., 2014) This suggests a potential association of bile acids in the progression or preclinical neurodegenerative phase of AD providing further genetic evidence for a role of bile acids in PD Mitochondrial dysfunction has been associated with PD (Luo et al., 2015). To identify compounds that could restore mitochondrial function in skin fibroblasts obtained from patients with a PD parkin (PARK2) gene mutation, a 2000 compound library was screened for significant improvement in mitochondrial membrane potential (Mortiboys et al., 2013) Ursocholanic acid and the related compound dehydro(11,12)ursolic acid lactone were among the top 15 compounds that had dose response characteristics favorable for drug development and lacked many of the disadvantages of the other top hits The structurally related bile acid UDCA was also found to rescue mitochondrial function to a similar extent which was dependent upon activation of the glucocorticoid receptor and increased phosphorylation of Akt UDCA was also found to restore mitochondrial function in fibroblasts obtained from a PD patient with a LRRK2-G2019S mutation CYP27A1 is a key enzyme in the alternative bile acid synthesis pathway and mutations in this enzyme can cause cerebrotendinous xanthomatosis as described below These results suggest that the mechanisms of bile acid protection may be similar both in vitro and in vivo Despite the successful administration of TUDCA using a similar subcutaneous injection protocol as described above for the R6/2 transgenic HD mouse and documentation of increased levels in the brain no effects on cell survival or on the neurological phenotype were noted The disparate effects in two different genetic models of neurodegenerative disease suggest that bile acids target specific pathways TUDCA and UDCA also reduced neuronal loss in a prion organotypic slice culture model of intracerebral infection that assesses prion replication occurring ex vivo through infection of brain slices with prion infected brain homogenate UDCA treatment also reduced astrocytosis and prolonged survival in prion infected male C57BL/6 mice although whether bile acids interacted with the PrPC to PrPSc conversion or mediated protective effects through some other mechanism is not known Cerebrotendinous xanthomatosis (CTX) is a very rare autosomal recessive disorder caused by mutations in the CYP27A1 gene (Björkhem and Hansson, 2010; Bjorkhem, 2013) These mutations lead to deficiency of the sterol 27-hydroxylase inner mitochondrial membrane protein Sterol 27-hydroxylase oxidizes cholesterol to 27-hydroxycholesterol in the alternative bile acid synthesis pathway that leads to the generation of CDCA Sterol 27-hydroxylase deficiency leads to a reduction of CDCA and upregulation of cholesterol 7α-hydroxylase (CYP7A1) the rate-limiting enzyme in the classic bile acid synthesis pathway resulting in elevated levels of cholestanol and bile alcohols CTX patients have a mean age of diagnosis of 35 years and manifest multiple neurologic symptoms including dementia as well as a variety of non-neurological manifestations including premature atherosclerosis Long-term treatment with CDCA can result in amelioration of neurological symptoms and an improved prognosis The phosphorylation of MerTK was significantly increased by TUDCA in a concentration-dependent manner but did not affect expression of the ER stress marker glucose regulated protein-78 (GRP-78) TUDCA also decreased the amount of apoptosis-inducing factor (AIF) released from the mitochondria and its subsequent accumulation in the nucleus Production of protein carbonyls and ROS were also significantly decreased after TUDCA treatment administration of TUDCA prior to the intravitreal injection of NMDA was found to increase survival of retinal ganglion cells essentially no data is available on the primary signaling pathways through which bile acids act the cellular receptor TGR5 and the nuclear receptors FXR and RXRα despite the well-known function of retinoic acid as a potent neurotrophic molecule Determining the precise molecular mechanism(s) of neuroprotection by bile acids in neurodegenerative disorders will be important to realize their future therapeutic potential Molecular pathways implicated in the neuroprotective effects of bile acids in neurodegenerative disease models Despite the relatively large structurally related group of bile acids relatively few have been studied in neurodegenerative disorders A major focus has been on apoptotic pathways and the PI3 kinase and AKT signaling pathway the primary signaling pathways through which bile acids act and FXR/RXR have received essentially no attention (designated by “?”) despite that retinoic acid is known to be a potent neurotrophic molecule Both authors approved the submitted version of the manuscript This work was funded by the Lewis Katz School of Medicine at Temple University Department of Medical Genetics and Molecular Biochemistry and the Joseph & Rebecca Goodfriend Endowed Chair in Genetics Inactivation of liver X receptor beta leads to adult-onset motor neuron degeneration in male mice Björkhem Cerebrotendinous xanthomatosis: an inborn error in bile acid synthesis with defined mutations but still a challenge Tool from ancient pharmacopoeia prevents vision loss Current neurogenic and neuroprotective strategies to prevent and treat neurodegenerative and neuropsychiatric disorders The bile acid tauroursodeoxycholic acid modulates phosphorylation and translocation of bad via phosphatidylinositol 3-kinase in glutamate-induced apoptosis of rat cortical neurons Tauroursodeoxycholic acid prevents MPTP-induced dopaminergic cell death in a mouse model of Parkinson's disease Molecular genetics of 3beta-hydroxy-Delta5-C27-steroid oxidoreductase deficiency in 16 patients with loss of bile acid synthesis and liver disease Ursodeoxycholic acid suppresses mitochondria-dependent programmed cell death induced by sodium nitroprusside in SH-SY5Y cells Tauroursodeoxycholic acid and secondary damage after spinal cord injury in rats and prolong male survival in models of prion disease Genes and mutations causing retinitis pigmentosa Neuroprotection in glaucoma: recent and future directions Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation Mapping of gene expression reveals CYP27A1 as a susceptibility gene for sporadic ALS TUDCA slows retinal degeneration in two different mouse models of retinitis pigmentosa and prevents obesity in Bardet-Biedl syndrome type 1 mice Tauroursodeoxycholic acid in the treatment of patients with amyotrophic lateral sclerosis Fernandez-Sanchez Tauroursodeoxycholic acid prevents retinal degeneration in transgenic P23H rats Pathophysilogical mechanism and treatment strategies for Leber congenital amaurosis Steroids and neuroprotection: new advances Tauroursodeoxycholic acid protects retinal neural cells from cell death induced by prolonged exposure to elevated glucose Genetic variants in microRNAs and their binding sites are associated with the risk of Parkinson disease Gómez-Vicente Neuroprotective effect of tauroursodeoxycholic acid on N-methyl-D-aspartate-induced retinal ganglion cell degeneration Therapeutic potential of Takeda-G-protein-receptor-5 (TGR5) agonists Human FXR regulates SHP expression through direct binding to an LRH-1 binding site Deletion of mouse FXR gene disturbs multiple neurotransmitter systems and alters neurobehavior JNK-mediated activation of ATF2 contributes to dopaminergic neurodegeneration in the MPTP mouse model of Parkinson's disease Potential role of ursodeoxycholic acid in suppression of nuclear factor kappa B in microglial cell line (BV-2) Creatine-supplemented diet extends Purkinje cell survival in spinocerebellar ataxia type 1 transgenic mice but does not prevent the ataxic phenotype is neuroprotective in a transgenic animal model of Huntington's disease A bile acid protects against motor and cognitive deficits and reduces striatal degeneration in the 3-nitropropionic acid model of Huntington's disease The bile acid receptor TGR5 (Gpbar-1) acts as a neurosteroid receptor in brain Rexinoids as therapeutics for Alzheimer disease: role of APOE Bile acid inhibition of N-type calcium channel currents from sympathetic ganglion neurons GPBA: a GPCR for bile acids and an emerging therapeutic target for disorders of digestion and sensation Tauroursodeoxycholic acid (TUDCA) supplementation prevents cognitive impairment and amyloid deposition in APP/PS1 mice Mitochondria: a therapeutic target for Parkinson's disease Differentiated NSC-34 motoneuron-like cells as experimental model for cholinergic neurodegeneration Nutrients as trophic factors in neurons and the central nervous system: role of retinoic acid Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice Tauroursodeoxycholic acid (TUDCA) protects photoreceptors from cell death after experimental retinal detachment Plasma phospholipids identify antecedent memory impairment in older adults Cellular and circuit mechanisms underlying spinocerebellar ataxias Oral solubilized ursodeoxycholic acid therapy in amyotrophic lateral sclerosis: a randomized cross-over trial Ursocholanic acid rescues mitochondrial function in common forms of familial Parkinson's disease TUDCA promotes phagocytosis by retinal pigment epithelium via MerTK activation attenuates amyloid precursor protein processing and amyloid-beta deposition in APP/PS1 mice Doxycycline plus tauroursodeoxycholic acid for transthyretin amyloidosis: a phase II study protect against oxidative stress-induced retinal degeneration and cerebrospinal fluid penetration of ursodeoxycholic Acid in patients with amyotrophic lateral sclerosis Tauroursodeoxycholic acid preservation of photoreceptor structure and function in the rd10 mouse through postnatal day 30 Bile acid metabolism and the pathogenesis of type 2 diabetes Tauroursodeoxycholic acid suppresses amyloid beta-induced synaptic toxicity in vitro and in APP/PS1 mice Retinoids and motor neuron disease: potential role in amyotrophic lateral sclerosis Tauroursodeoxycholic acid reduces apoptosis and protects against neurological injury after acute hemorrhagic stroke in rats Neuroprotection by a bile acid in an acute stroke model in the rat Tauroursodeoxycholic acid partially prevents apoptosis induced by 3-nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition Challenges and promises in the development of neurotrophic factor-based therapies for Parkinson's disease PubMed Abstract | CrossRef Full Text The bile steroid chenodeoxycholate is a potent antagonist at NMDA and GABAA receptors Neuritic growth impairment and cell death by unconjugated bilirubin is mediated by NO and glutamate and prevented by glycoursodeoxycholic acid and interleukin-10 Functional modulation of nuclear steroid receptors by tauroursodeoxycholic acid reduces amyloid beta-peptide-induced apoptosis Tauroursodeoxycholic acid prevents amyloid-beta peptide-induced neuronal death via a phosphatidylinositol 3-kinase-dependent signaling pathway Pathway analysis of genome-wide association studies for Parkinson's disease Theofilopoulos Brain endogenous liver X receptor ligands selectively promote midbrain neurogenesis Peptide therapeutics in neurodegenerative disorders Trophic factors as modulators of motor neuron physiology and survival: implications for ALS therapy Glycoursodeoxycholic acid reduces matrix metalloproteinase-9 and caspase-9 activation in a cellular model of superoxide dismutase-1 neurodegeneration Veyrat-Durebex Advances in cellular models to explore the pathophysiology of amyotrophic lateral sclerosis Effects of tauroursodeoxycholic acid and alpha-lipoic-acid on the visual response properties of cat retinal ganglion cells: an in vitro study All-trans retinoic acid regulates hepatic bile acid homeostasis Yanguas-Casás Tauroursodeoxycholic acid reduces glial cell activation in an animal model of acute neuroinflammation Waking action of ursodeoxycholic acid (UDCA) involves histamine and GABAA receptor block 25- and 24-hydroxycholesterol in rat glial cells and neurons Chemical chaperone TUDCA preserves cone photoreceptors in a mouse model of Leber congenital amaurosis Citation: Ackerman HD and Gerhard GS (2016) Bile Acids in Neurodegenerative Disorders Received: 11 August 2016; Accepted: 21 October 2016; Published: 22 November 2016 Copyright © 2016 Ackerman and Gerhard. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) or licensor are credited and that the original publication in this journal is cited *Correspondence: Glenn S. Gerhard, Z3NnZXJoYXJkQHRlbXBsZS5lZHU= Metrics details Clostridium septicum infections are highly predictive of certain malignancies in human patients septicum spores must first germinate and regain vegetative growth septicum germinates in response to specific bile salts Putative bile salt recognition genes are identified in C septicum based on their similarity in sequence and organization to bile salt-responsive csp genes in Clostridioides difficile Inactivating two of these csp orthologs (cspC-82 and cspC-1718) results in mutant spores that no longer germinate in the presence of their respective cognate bile salts inactivating the putative cspBA or sleC genes in C septicum abrogates the germination response to all bile salt germinants suggesting that both act at a convergent point downstream of cspC-82 and cspC-1718 Molecular dynamics simulations show that both CspC-82 and CspC-1718 bear a strong structural congruence with C The existence of functional bile salt germination sensors in C septicum may be relevant to the association between infection and malignancy In many cases (and especially for colorectal cancer) these cancers were unknown and only discovered after the infection septicum infections may be exceedingly rare but its presence in a patient is highly predictive of a co-existing malignancy septicum’s host tropism may differ from other pathogenic clostridia septicum infections are so different from the other pathogenic clostridia and especially why they are so closely linked with malignancy Since a clostridial spore can only colonize an environment that supports its germination we reasoned that studying the germination triggers of C septicum preferentially infects cancer patients an unbiased screen of single factor germinants for C septicum revealed that sodium deoxycholate sodium chenodeoxycholate and sodium glycocholate are potent germinants difficile’s cspC were first identified as candidate bile salt-sensors Mutational inactivation of two of these cspC orthologs abolished C septicum’s bile salt germination response to distinct sets of bile salts cspBA and sleC mutant spores were unresponsive to all three bile salt germinants which is consistent with both proteins acting downstream of the cspC orthologs structural modeling and molecular dynamics simulations showed high congruence between C difficile’s CspC and both CspC orthologs in C The striking similarity between the bile salt-germinant sensing mechanisms of C difficile raises the possibility that other gut bacteria may use the same pathway A significant increase in OD was observed at pH 6.0 but not at pH 6.5 or pH 7.0 This pH-dependent effect was specific to DCA and not observed with either CA or GCA To avoid this artifact in future experiments all absorbance assays from this point were performed at pH 7.0 a Germination measured by optical density (OD) is shown OD values were normalized to the starting OD Purified wild-type spores were incubated with 25 mM of each bile salt b Representative phase contrast images of the spores at the end of the OD assay in a are shown c Germination response curves were plotted for the three bile salt germinants Germination was measured at 900 min post-exposure to increasing bile salt concentrations d Maximal germination velocity curves were plotted for the three bile salt germinants d) graphed data represent the average at least 3 biologically independent experiments using at least two different spore preparations Error envelopes (dashed lines) represent the standard error of the mean statistical significance relative to water was determined by one-way ANOVA and Dunnett’s test statistical significance relative to CDCA was determined by unpaired t test between groups at each time point All three Vmax curves plateaued at different points in the concentration range with its Vmax maxing out at a low value after 6.25 mM the Vmax of GCA exceeded both DCA and CDCA after 12.5 mM which was the highest concentration tested DCA’s Vmax response straddled both GCA and CDCA and plateaued at around 12.5 to 50 mM What is clear is that all three germinants have distinguishable germination kinetics The effect of heat activation on bile salt-induced germination was also investigated (Supplementary Fig. S7) the kinetics of germination increased slightly when spores were heat activated at 60 °C or 68 °C as compared to no heat activation This was reflected most noticeably by the steeper slopes for DCA and CDCA and the increased ability of the spore to germinate to the weak germinant GDCA the extent of germination induced by GCA decreased as spores went from no heat activation to heat activation at 68 °C No germination was observed at ≥ 78 °C for all bile salts With the exception of the weak germinant GDCA heat activation below 78 °C did not enhance bile salt germination heat activation was not incorporated into downstream germination assays in this study A correlation of bile acid structure with germinant activity revealed an intriguing observation. Taking GCA as the reference, the loss of either the R2 hydroxyl (as in GDCA) or the R4 conjugated glycine (as in CA) destroyed germination activity, implying that the presence of both features were important for the recognition of GCA (Supplementary Fig. S8) DCA lacked both these features but was still a germinant This raised the possibility that there are at least two distinct proteins which separately respond to DCA and GCA We next set out to identify the relevant sensors for the three bile salt germinants a Genetic layout of the csp operons for C cspC-82 and sleC-83 is part of a pentacistronic operon as predicted by OperonMapper The other two upstream genes were not predicted to be part of the germination mechanism and were hence omitted to avoid clutter b Schematic of insertion sites of the TargeTron vector for generation of mutants in the csp/sleC operon and cspC-1718 These four mutants were sufficient to provide an insight into the bile salt germination mechanism of C cspC mutants (cspC-82::ermB and cspC-1718::ermB) and complemented cspC mutants (cspC-82::ermB pcspC-82 and cspC-1718::ermB pcspC-1718) were incubated with 10 mM bile salts in the presence of oxyrase a Heat map depicting %OD drop values at the end of the assay Colors indicate extent of germination (red: germination The cspC-82 mutant was able to germinate only in the presence of GCA while the cspC-1718 mutant was able to germinate in the presence of CDCA and DCA Complementation of the mutant with their respective gene rescued the mutant phenotype Data represent the average of n = 2 biologically independent experiments b Representative phase contrast images of WT cspC mutants and their respective complemented cspC mutants after 900 minutes of incubation with various bile-salts Purified spores of cspBA or sleC mutants and their complemented counterparts were incubated with 10 mM bile salts in the presence of oxyrase cspBA-81 and sleC-83 mutants were unable to germinate with any bile salts cspBA and sleC mutants and their respective complements after 900 minutes of incubation with various bile-salts Complementing cspC-1718 restored viability to WT levels suggesting that the cspC-1718 may somehow influence spore Ca-DPA content cspBA and sleC mutant spores were not viable further corroborating our observations in the previous germination assays complementing cspBA and sleC restored colony counts to WT levels Having established that CspC-82 and CspC-1718 were similar to C we wondered what structural similarities existed between the three proteins A Molecular Dynamics (MD) simulation approach was used to study this question These MD experiments were not intended to provide functional insight just to cross-compare the overarching physical attributes of all three proteins septicum’s (CS) CspC-82 and CspC-1718 were compared with C a The change in RMSD with respect to time for three independent protein simulations of C Only non-hydrogen atoms were considered for the calculation of RMSDs b The time evolution of RMSD from the initial energy minimized structure is shown for the three proteins d The time evolution of RMSD measured in (a) but resolved for the individual domains shown in b were calculated to understand the local structural fluctuations of the three studied proteins the similar RMSDs for domains three and four suggest that all three proteins exhibit similar rigidity in these regions the previous observation of higher RMSD values for CspC-82 and CspC-1718 were due to the higher flexibility of domains one and two difficile’s CspC showed the highest interSASA while the C septicum CspC orthologs had similar and smaller interSASA values This result supports the RMSD analysis showing the comparatively greater structural flexibility of C septicum’s CspC orthologs at both the protein and domain levels there was a high degree of general structural congruence between the 3 proteins septicum CspC orthologs play a similar bile salt-sensing role to C there were also microscopic differences in domain flexibility and interSASA which remain to be explored and could shed light on differences in bile salt specificity or signaling we investigated the germinant vocabulary of C The most noteworthy finding was the strong germination response to bile salts germinants and the identification of two cspC genes mediating this effect difficile has hitherto provided the only example of a cspC gene playing a bile salt germinant-sensing role septicum create more opportunities for understanding the nature of bile salt-sensing by CspC we speculate that bile salt dysregulation may plausibly link C If Ger proteins are the canonical sensors for amino acid germinants it is conceivable that Csp proteins may likewise form the molecular basis for bile acid germinant sensing The evidence in this study seems to suggest otherwise septicum’s bile salt-sensing orthologs (CspC-1718 and CspC-82) possess catalytic triads showing that a catalytic triad is not a barrier to gaining a new bile salt-sensing function this appears to be true regardless of whether the catalytic triad is DHC (as for CspC-1718) or DHS (as for CspC-82) It remains to be seen whether these triads in C it would imply that CspC’s bile salt sensing and proteolytic activity are independent of each other One major limitation of these simulations is that they are limited only to bile salt-responsive CspCs A full molecular dynamical comparison of bile salt responsive CspC’s from C against bile salt non-responsive CspC’s from other clostridia is warranted This may reveal structural features within CspC which are essential for bile salt sensing We have not included potential mediators of amino acid sensing in this model A dotted arrow joins CspBA-81 and SleC-83 because it is unclear if SleC is activated solely by CspB (like in C redundantly by multiple Csp proteins (like in C This suggests that cleavage is necessary for function perfringens because both CspB and CspA are already expressed as distinct functional proteins If the same bile acids which promote carcinogenesis also trigger C then bile acid dysbiosis could very well be the common cause linking C septicum strains were grown on BHIS (Brain Heart Infusion supplemented with 0.5 g/l L-cysteine) in an anaerobic chamber (Plas lab) at 37 °C (85% N2 Antibiotics were added as needed (10 μg/mL thiamphenicol polymyxin B 60 U/ml or 2.5 μg/mL erythromycin) XL1-Blue and S17-1 (Biomedal S.L) were cultured aerobically at 37 oC on either LB plates or 2xYT broth and supplemented with 10 μg/ml chloramphenicol XL1-Blue was used as the host for plasmid construction while S17-1 was used for conjugal transfer of plasmids to C Oligonucleotides and synthesized DNA fragments were listed in Supplementary Materials 1, 2 The Targetron plasmid pJIR750ai (Sigma) was used as the starting plasmid for generation of mutants and was modified in several ways pRJ1 was created by cloning the oriT site into pJIR750ai BtsMutI restriction sites were knocked out and re-assembled with NEBuilder HiFi DNA assembly (NEB) to yield the plasmid pRJ2 The original promoter for the transcription of the group II intron machinery was replaced with the promoter for the alpha toxin gene in C The ErmB retrotransposition activated selectable marker (RAM) which confers erythromycin resistance upon insertion of the intron into the genome was inserted into the MluI site of PFGv2-Pcsa to finally yield the base plasmid pRJ4 Our choices of insertion sites were chosen according to two criteria 1) high scores 2) position of insertion site relative to the N-terminus of the protein so that the protein is truncated early and abolishment of the function can be determined The identified retargeting regions were synthesized by Gene Universal and ligated into pRJ4 at the HindIII and BsrGI restriction site These plasmids were then sequenced and used for electroporation of the transfer strain S17-1 csp and sleC mutants were generated in C. septicum ATCC 11424 with the TargeTron mutagenesis system. Conjugal transfer of the Targetron plasmid (Supplementary Material 3) to the recipient C septicum strains was performed in the anaerobic chamber by dripping 200 μl of the conjugation mixture on BHIS plates for six hours The mix was then harvested with 1 ml of PBS and 100 μl of the slurry was spreaded on a BHIS plate supplemented with 10 μg/ml thiamphenicol and 60U/ml polymyxin B for 16–24 h Replica plating was then performed on erythromycin containing BHIS plates (2.5 μg/ml) and grown for a further 16–24 h before screening with PCR to confirm insertion of introns Mutants were then further streaked on BHIS plates to shed the TargeTron plasmid Conjugal transfer of the respective plasmids to the respective mutant recipient strains was performed as described above The recipient strains carrying their respective plasmids were verified by PCR Genomic DNA (gDNA) was isolated with a few modifications from the original protocol described in ref. 70 RNAseA (Sigma) and Proteinase K (Roche) treatment was only done following crude DNA extraction for 2 h each at 37 oC gDNA was purified by phenol/chloroform extraction and quantified with Picogreen (Life Technologies) using lambda DNA (NEB) as standards 0.5 μg of gDNA was digested with 10 U of AseI separated by gel electrophoresis in a 0.8% agarose gel and transferred to a nylon membrane (Amersham Hybond-N + ) with a vacuum blotting system (GE Hybridization of the blot with the intron probe was done following manufacturer instructions (Roche) The blot was hybridized with a 378 bp DIG-labeled intron probe (Roche) generated from the plasmid pRJ3 The assembled genome can be found on NCBI’s GenBank through accession number JARRAV000000000 under Bioproject ID PRJNA872817 septicum CDS sequences with the Protein Blast (Blastp) algorithm Protein Blast (Blastp) was performed over nucleotide blast (Blastn) to account for synonymous codons septicum were identified by querying the sequences of C Identification of putative cspC orthologs of C bifermentans (taxid: 1490) was performed with Blastp using CspC (accession number: WP_003433821) from C septicum were analyzed in MegAlign Pro v13 (DNASTAR) using the Clustal Omega algorithm C. septicum spores were purified as described previously78 septicum were diluted 50X into 100 ml of BHI-S and incubated until the OD600 is 1.5 to 3 before the entire culture was added into 900 ml of sporulation media (0.5% L-cysteine 5% dehydrated cooked meat medium and 10% fetal bovine serum) Thiamphenicol (5 μg/ml) or erythromycin (2.5 μg/ml) were added when necessary This sporulation media was then incubated for a further 5 days Spores were purified from vegetative cells on a 80% discontinuous Percoll gradient resuspended in water and stored at 4 oC until use Purified spores were observed to be >99% phase bright All germination assays were conducted at 37 oC in the presence of 0.2x Oxyrase for broth (Oxyrase®). Single factor screening experiments of WT spores (Supplementary Table T1) were performed (Oxyrase®) in a 384 well plate (Greiner) and covered with a plastic film (Excel Scientific) Determination of the optimum pH for WT spore germination was performed as above except WT spores were germinated in a germination assay buffer containing 10 mM sodium phosphate buffer with a range of pH from 6 to 8 20 mM DCA and bacterial spores adjusted to an initial OD600 of 1.0士0.2 Data from the above experiments were collected from the infinite 200 Microplate reader (Tecan) Subsequently all data was collected from the Spark Multimode Microplate reader (Tecan) To investigate whether Ca-DPA release from the spores was responsible for the high OD artifact observed with DCA induced spore germination we performed germination assays as described above except that Ca2+ and DPA was added instead of spores a germination solution consisting of 10 mM sodium phosphate buffer at the tested pH of 6 50 uM Ca2+ and DPA was incubated at 37 oC and the OD was measured every 5 mins over 15 hours Germination assays of WT spores in serial dilutions of bile salts was performed in a germination assay media containing 10 mM sodium phosphate buffer pH 7.0 serial dilutions of bile salts (final concentrations 50 mM–0.78 mM) and lastly bacterial spores adjusted to an initial OD600 of 1.0士0.2 in a 384 well UV transparentplate (Greiner UV Star plate) covered with an optically clear qPCR film (ABI technologies) to maintain anaerobicity All measurements were taken in intervals of five minutes for 15 hours Amino acid co-germination assays were performed as above with sub-optimal concentrations of CDCA (1.56 mM) except that a 20- amino acid mastermix was added to the germination buffer at a final concentration of 5 mM for each amino acid Individual amino acids (5 mM) were also tested with GCA (3.13 mM) as described above The final percentage of OD decrease relative to its initial OD (ODfinal/ODinitial) of each individual amino acid with GCA is then subtracted by the GCA only control to establish the effect of the amino acid WT spores were incubated in a thermocycler (AIT Biotech) in their respective temperatures for 15 minutes before being cooled on ice for five minutes prior to performing the germination assay as described above with a few modifications The respective bile salts and TbCl3 were added to a final concentration of 10 mM and 0.1 mM respectively Released DPA complexes with terbium to form a fluorescence complex and is measured at an excitation of 272 nm with emission of 545 nm at z = 18216 μm and a time lag of 20 μs before measurement All measurements were taken in kinetic mode in 5 minute steps for 15 hours sleC mutants and complemented mutants were conducted in the same way as heat activation assays except spores were not heat activated All values below zero were considered to be zero Due to low Ca-DPA content in cspC-1718 mutants and its complemented mutant spores spores were added to an initial OD600 of 1.3 士 0.2 and the acquisition parameters were optimized as follows: number of flashes (50) To assess the total DPA content in the spore preparation spores suspensions (OD 0.5 in 100 ul) were heated at 100 oC for an hour The spores were then cooled on ice for five minutes the spore suspension is spun at 17,000 g for 5 minutes and 20ul of the supernatant was assayed for DPA content in the same buffer as described above for the spore germination assay for the mutant septicum spores were incubated in 0.1% solutions of various bile acids for 10 min in the anaerobic chamber before they were plated on LB overnight The colonies were counted the next day and compared to GCA exposed spores mutant and complemented spores (2 × 107 spores/ml) were serially diluted and 5ul was plated on BHI-S plates and left to dry for 30 minutes The end residues for all proteins were properly capped (Acetyl group at the N-terminal and N-methyl group at the C-terminal) The side-chain protonation states of asparagine and histidine were considered and resolved through optimization of local hydrogen bonding The three proteins were solvated by cubic TIP3 water boxes and the overall charge of each system was neutralized by adding counter ions Each of the three systems were energy minimized by performing 50,000 steps of Steepest Descent to eliminate the close van der Waals contacts the temperature of the systems was gradually increased to room temperature (300 K) followed by 50 ps equilibration using the NPT algorithm where temperature was controlled by the Langevin algorithm The three systems were then run for 50 ns under a constant pressure of 1 atm and a constant temperature of 303 K (NPT ensemble) The time steps for each simulation was 2 fs and trajectories were stored every 2 ps for analysis The long-range electrostatic forces were calculated using Particle Mesh Ewald method Periodic boundary condition and a 10 Å cutoff were applied for non-bonded short range interactions.The interface Solvent-Accessible Surface Area (interSASA) for a specific domain i among the set of all domains P is defined as: interSASA{i} = SASA{i} + SASAP-{i} - SASAP where SASA{i} SASAP-{i} and SASAP are the SASA values for all domains domain i and all domains excluding i respectively Spores of the order of 109 CFU/ml were used for all germination assays All experiments were independently replicated at least twice with two embedded technical replicates All attempts at replicating our findings were successful spores from a different batch were also assayed For viability and recovery of spores on agar at least 2 × 107 spores/ml were used and this amount produced colonies which were serially diluted for accurate counting Figures were generated on Graphpad Prism 9 for Windows (Graphpad software) Error bars represent the standard error of the mean Statistical analysis of germination curves heat activation and total DPA content was done with one way ANOVA and Dunnett with the final endpoint data Comparison of OD increase of different bile acids in varying pH was performed with two way ANOVA and Dunnett with the final endpoint data OD drop over the moving average of 6 timepoints (25 min) was calculated and the maximum OD drop velocity value over the duration of the experiment was used 100% germination response was defined as the maximal germination response with DCA since it showed the highest OD drop while 0% germination response was defined as the germination response in water instead of bile salts The standard curve of each bile salt germinant was generated using a four-parameter logistic curve and EC50 values were interpolated from the generated curves Vmax was calculated after the assay started for 30 min over a moving window of three time points (10 min) and the maximum velocity of each concentration was used The curve of each bile salt was generated using the built-in allosteric sigmoidal curve fit An unpaired t-test was conducted to assess if there were differences in EC50 and Vmax values between the germinants Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article All data is available in the main text or the Supplementary Information. The assembled genome can be found on NCBI’s GenBank through accession number JARRAV000000000. The source data behind the graphs in the paper can be found in Supplementary Data 3 Clostridium septicum infection and associated malignancy Report of 2 cases and review of the literature Phenotyping Clostridium septicum infection: a surgeon’s infectious disease septicum gas gangrene: A literature review Pore-Forming Activity of Alpha-Toxin Is Essential for Clostridium septicum-Mediated Myonecrosis Clostridium septicum α-toxin activates the NLRP3 inflammasome by engaging GPI-anchored proteins and medicosurgical management of Clostridium septicum infection Taxonomic relationships among the Clostridia in Clostridial Diseases of Animals 1–5 (John Wiley & Sons Reclassification of Clostridium difficile as Clostridioides difficile (Hall and O’Toole 1935) Prévot 1938 Germinants and Their Receptors in Clostridia Sporulation and Germination in Clostridial Pathogens Bile salts and glycine as cogerminants for Clostridium difficile spores Clostridium perfringens spore germination: characterization of germinants and their receptors Functional characterisation of germinant receptors in Clostridium botulinum and Clostridium sporogenes presents novel insights into spore germination systems germinants and temperature on the germination of spores of Clostridium bifermentans The activation of spores of Clostridium bifermentans Germination of spores of Clostridium bifermentans by certain amino acids lactate and pyruvate in the presence of sodium or potassium ions Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores Inorganic phosphate and sodium ions are cogerminants for spores of Clostridium perfringens type A food poisoning-related isolates Variability in spore germination response by strains of proteolytic Clostridium botulinum types A Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B E and F to a wide range of germinants and conditions Germination of Spores of the Orders Bacillales and Clostridiales Clostridioides difficile Spore Formation and Germination: New Insights and Opportunities for Intervention The serine proteases CspA and CspC are essential for germination of spores of Clostridium perfringens SM101 through activating SleC and cortex hydrolysis The protease CspB is essential for initiation of cortex hydrolysis and dipicolinic acid (DPA) release during germination of spores of Clostridium perfringens type A food poisoning isolates Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens Bile acids as modulators of gut microbiota composition and function Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation Bile acid recognition by the Clostridium difficile germinant receptor The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals The requirement for co-germinants during Clostridium difficile spore germination is influenced by mutations in yabG and cspA Structural and functional analysis of the CspB protease required for Clostridium spore germination A Revised Understanding of Clostridioides difficile Spore Germination Rapid determination of spore germinability of Clostridium perfringens based on microscopic hyperspectral imaging technology and chemometrics First Comparative Analysis of Clostridium septicum Genomes Provides Insights Into the Taxonomy Operon-mapper: a web server for precise operon identification in bacterial and archaeal genomes ClosTron-mediated engineering of Clostridium A Clostridium difficile alanine racemase affects spore germination and accommodates serine as a substrate Identification of a new gene essential for germination of Bacillus subtilis spores with Ca2+-dipicolinate Regulation of Clostridium difficile spore germination by the CspA pseudoprotease domain SleC is essential for germination of Clostridium difficile spores in nutrient-rich medium supplemented with the bile salt taurocholate Functional analysis of SleC from Clostridium difficile: an essential lytic transglycosylase involved in spore germination Conservation of the ‘Outside-in’ Germination Pathway in Paraclostridium bifermentans Progesterone analogs influence germination of Clostridium sordellii and Clostridium difficile spores in vitro Comparison of sporulation and germination conditions for Clostridium perfringens type A and G strains Differential effects of ‘resurrecting’ Csp pseudoproteases during Clostridioides difficile spore germination Requirements for germination of Clostridium sordellii spores in vitro A design of experiments screen reveals that Clostridium novyi-NT spore germinant sensing is stereoflexible for valine and its analogs Effect of diet on human fecal flora: comparison of Japanese and American diets Towards the human colorectal cancer microbiome Low prevalence of Clostridium septicum fecal carriage in an adult population Trusting Your Gut: Diagnosis and Management of Clostridium septicum Infections Influence of Bile Acids on Colorectal Cancer Risk: Potential Mechanisms Mediated by Diet - Gut Microbiota Interactions The role of bile acids in cellular invasiveness of gastric cancer Diagnosis of hepatocellular carcinoma using a novel anti-glycocholic acid monoclonal antibody-based method Rethinking the bile acid/gut microbiome axis in cancer Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome Bile Acids and Cancer: Direct and Environmental-Dependent Effects Urinary metabolic profiling identifies a key role for glycocholic acid in human liver cancer by ultra-performance liquid-chromatography coupled with high-definition mass spectrometry Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes A modular system for Clostridium shuttle plasmids A versatile quick-prep of genomic DNA from gram-positive bacteria NCBI prokaryotic genome annotation pipeline faster version of the PHAST phage search tool ISEScan: automated identification of insertion sequence elements in prokaryotic genomes CheckM: assessing the quality of microbial genomes recovered from isolates QUAST: quality assessment tool for genome assemblies KBase: The United States Department of Energy Systems Biology Knowledgebase Combination bacteriolytic therapy for the treatment of experimental tumors Optimization of the Additive CHARMM All-Atom Protein Force Field Targeting Improved Sampling of the Backbone ϕ ψ and Side-Chain χ1 and χ2 Dihedral Angles I-TASSER: a unified platform for automated protein structure and function prediction Highly accurate protein structure prediction with AlphaFold Download references This work was supported by Temasek Life Sciences Laboratory Core Funding (http://www.tll.org.sg) (to I.C.) We thank colleagues in Temasek Life Sciences Laboratory Ding Yichen for guidance on the assembly of the C Cai Lin and Ji Liang Hui for equipment and technical advice This study was supported by Temasek Life Science Laboratories These authors contributed equally: Madhumitha Ayyappan NUS High School of Mathematics and Sciences Communications Biology thanks Ernesto Abel-Santos and the other Download citation DOI: https://doi.org/10.1038/s42003-024-06617-4 Metrics details The incidence of duodenal tumors (DTs) is increasing the mechanisms underlying its development remain unclear including the microbiome and bile acids (BAs) are believed to influence tumor development observational study to investigate the potential differences between patients with DTs and healthy controls (HCs) based on these factors the BAs in the duodenal fluid were measured using liquid chromatography-tandem mass spectrometry We recruited 41 patients and performed 16S rRNA-seq There was no difference in the observed ASVs or PCoA plot of Bray–Curtis dissimilarity between the DTs and HCs The lithocholic acid concentration was significantly lower in the DT group than in the control group The ratio of CDCA to LCA was significantly higher in patients with DTs No significant differences in microbiota were observed between DTs and HCs the lithocholic acid concentration in duodenal was significantly lower than in HCs which are synthesized in the liver and excreted into the bile are vital for lipid digestion and absorption Cholic acid (CA) and chenodeoxycholic acid (CDCA) are synthesized in the liver as primary BAs Primary BAs are converted to secondary BAs by the intestinal microbiome the microbiome and BAs are closely associated we considered that the gastrointestinal microbiome and metabolites may differ between patients with duodenal tumors and healthy donors The workflow for microbiome analysis between study samples and negative control using QIIME2 and the Venn diagram of observed ASVs in samples and negative control The workflow for microbiome analysis using QIIME2 First, we compared the composition of microbes in the duodenum by observing Amplicon Sequence Variants (ASVs) and Bray–Curtis dissimilarity. In the observed ASVs alpha rarefaction curve, 862 reads that reached a plateau were utilized for alpha and beta diversity analyses (Fig. 2c) The observed ASVs of DT groups and HC groups PCoA plot of Bray–Curtis dissimilarity between DT groups and HC groups microbiome The profiles of duodenal microbiome at the genus level The PCoA plot of Bray–Curtis dissimilarity between DT group and HC group in 44–66 s The PCoA plot of Bray–Curtis dissimilarity between Young and Old people microbiome We also did not observe a difference between the young and old age groups most of the healthy patients are in the young group and most of the DT patients are in the old group we did not observe unique components of the microbiome in the DT group compared to HCs In addition, we used ANCOM for differential abundance analysis. ANCOM failed to detect any significant differences in dominance across all the classification hierarchies (Fig. 4). Comparison at species level between DT and HC groups and the W value represents the number of times of the null-hypothesis (the average abundance of a given species in a group is equal to that in the other group) was rejected for a given species Comparison at genus level between DT and HC groups The log scale of concentration of total bile acids and each bile acid in duodenal fluid Proportion of each bile acid in total bile acid an increase in the number of DTs has emerged little is known about the environmental factors in the duodenal mucosa of patients with DTs and HCs we explored the mucosal microbiome and its metabolites Further studies are needed to determine whether the accurate positive ratio of the patients in whom Actinobacteriota was detected in the DT group was lower than that in the HC group on a large scale we need to show that the lower detection rate of Actinobacteriota in the DT group was the cause or effect of the DT progression we suspected that reducing the LCA in patients with DT was not a DT trigger BAs and the microbiome indirectly affect duodenal tumors; for example high amounts of gastric acids in the duodenum suppress microbiome growth resulting in the reduction of secondary BAs we could not exclude the possibility of gastric acid influx into the duodenum owing to morphological factors As it is quite challenging to show the connection between gastric acids and duodenal tumors the unique composition of BA might be a biomarker for duodenal tumors even after compiling evidence on a large scale in a prospective study We collected mucus-related microbiome by obtaining duodenal tissue biopsies without washing we did not reveal the microbiome profiles on the three samples because of low amplification Further studies are required to obtain sufficient mucus-related microbiome using a safe method such as an abrasion brush we analyzed the microbiome composition and BAs in patients with DT and HCs We did not observe a difference in the observed ASVs and PCoA plots of Bray–Curtis dissimilarity between DT and HCs The LCA concentration in the duodenum of patients with DTs was lower than that in HCs and the balance of CDCA/LCA was higher in patients with DTs Patients aged ≥ 20 years who underwent endoscopic treatment for duodenal tumor or esophagogastroduodenoscopy and willingly consented to participate in this study were included inability to manage anticoagulation or platelet medication following the guidelines for gastrointestinal endoscopy or other cases deemed inappropriate for enrollment by the physician in charge were excluded The study was conducted between April 2021 and December 2022 This study was conducted following the 2008 revised Declaration of Helsinki Written informed consent was obtained from all participants and the study protocol was approved by Keio University School of Medicine Ethical Review Board (20,200,185) and the procedure was performed with at least 12 h of abstinence from food and scopolamine butylbromide or glucagon was used as antispasmodic The endoscope was inserted into the descending part of the duodenum without suction the duodenal fluid was aspirated through the endoscope tube and collected two pieces of normal mucosa were collected from the descending part of the duodenum using sterile biopsy forceps for microbiome analysis specimens were soaked overnight in RNAlater (Thermo Fisher Scientific the tissue was collected from the normal mucosa at a distance that would not affect tumor resection We collected individual patient information Three samples were removed because the taxonomic string contained any of mitochondria An internal standard mixture consisting of [2H4]CA (41.6 ng) v/v) was added to 4–20 µL of duodenal fluid The samples were then diluted with 1 mL of 0.5 M potassium phosphate buffer (pH 7.4) BAs were extracted using Bond Elut C18 cartridges (200 mg and an aliquot was injected into the LC–MS/MS system The LC–MS/MS system using the electrospray ionization (ESI) mode consisted of a TSQ Vantage triple stage quadrupole mass spectrometer (Thermo Fisher Scientific USA) equipped with an HESI-II probe and a Prominence ultra-fast liquid chromatography (UFLC) system (Shimadzu Thermo Fisher Scientific) was used for each BA’s chromatographic separation at 40 °C The following gradient system was used at a flow rate of 200 µl/min: first the mobile phase composed of 20 mM ammonium acetate buffer (pH 7.5)–acetonitrile–methanol (70:15:15 v/v/v); then it was programmed in a linear manner to 20 mM ammonium acetate buffer (pH 7.5)–acetonitrile–methanol (30:35:35 The final mobile phase was kept constant for an additional 10 min The general MS/MS conditions for selected reaction monitoring (SRM) were as follows: spray voltage 15 arbitrary units; ion transfer capillary temperature The monitoring ions and optimal collision energies were m/z 407 → 407 (20 V) for CA A calibration plot was established for each BA Different amounts of authentic BAs were mixed with deuterated internal standards and quantified as described above relative to the corresponding deuterated internal standard and the peak area ratio of the authentic BA to the deuterated variant measured by SRM was plotted on the ordinate Because deuterium-labeled BAs for all authentic BAs were not available [2H4]CA was used as an internal standard for GCA and samples that deviated from the calibration curve were diluted and reassayed Total BA means the sum of all unconjugated and conjugated BAs; bile acids without a prefix were defined as the unconjugated BAs in this study the reference to a CA is an unconjugated CA We performed univariate analyses using the Fisher exact test for categorical data and the Wilcoxon rank-sum test for continuous data; statistical significance was set at P < 0.05 Statistical analyses were performed using the JMP Pro software (version 17.0.0; SAS Institute USA) and GraphPad Prism version 9 (GraphPad Software) The datasets generated and analyzed during the current study are available in the DNA Data Bank of Japan repository Schottenfeld, D., Beebe-Dimmer, J. L. & Vigneau, F. D. The epidemiology and pathogenesis of neoplasia in the small intestine. Ann. Epidemiol. 19, 58–69. https://doi.org/10.1016/j.annepidem.2008.10.004 (2009) Bojesen, R. D., Andersson, M., Riis, L. B., Nielsen, O. H. & Jess, T. Incidence of, phenotypes of and survival from small bowel cancer in Denmark, 1994–2010: A population-based study. J. Gastroenterol. 51, 891–899. https://doi.org/10.1007/s00535-016-1171-7 (2016) Legué, L. M. et al. Trends in incidence, treatment and survival of small bowel adenocarcinomas between 1999 and 2013: A population-based study in The Netherlands. Acta Oncol. 55, 1183–1189. https://doi.org/10.1080/0284186x.2016.1182211 (2016) Yoshida, M. et al. The incidence of non-ampullary duodenal cancer in Japan: The first analysis of a national cancer registry. J. Gastroenterol. Hepatol. 36, 1216–1221. https://doi.org/10.1111/jgh.15285 (2021) Nakagawa, K. et al. Clinical practice guidelines for duodenal cancer 2021. J. Gastroenterol. 57, 927–941. https://doi.org/10.1007/s00535-022-01919-y (2022) Cameron, J. L., Riall, T. S., Coleman, J. & Belcher, K. A. One thousand consecutive pancreaticoduodenectomies. Ann. Surg. 244, 10–15. https://doi.org/10.1097/01.sla.0000217673.04165.ea (2006) Kim, J., Choi, S. H., Choi, D. W., Heo, J. S. & Jang, K. T. Role of transduodenal ampullectomy for tumors of the ampulla of Vater. J. Korean Surg. Soc. 81, 250–256. https://doi.org/10.4174/jkss.2011.81.4.250 (2011) Asbun, H. J. Management of duodenal polyps in the era of maximal interventional endoscopy and minimally invasive surgery: A surgical perspective. Gastrointest. Endosc. 84, 697–699. https://doi.org/10.1016/j.gie.2016.07.054 (2016) Uemura, N. et al. Helicobacter pylori infection and the development of gastric cancer. N. Engl. J. Med. 345, 784–789. https://doi.org/10.1056/NEJMoa001999 (2001) Kostic, A. D. et al. Genomic analysis identifies association of Fusobacterium with colorectal carcinoma. Genome Res. 22, 292–298. https://doi.org/10.1101/gr.126573.111 (2012) Yachida, S. et al. Metagenomic and metabolomic analyses reveal distinct stage-specific phenotypes of the gut microbiota in colorectal cancer. Nat. Med. 25, 968–976. https://doi.org/10.1038/s41591-019-0458-7 (2019) Andoh, A. et al. Multicenter analysis of fecal microbiota profiles in Japanese patients with Crohn’s disease. J. Gastroenterol. 47, 1298–1307. https://doi.org/10.1007/s00535-012-0605-0 (2012) Carroll, I. M., Chang, Y. H., Park, J., Sartor, R. B. & Ringel, Y. Luminal and mucosal-associated intestinal microbiota in patients with diarrhea-predominant irritable bowel syndrome. Gut. Pathog. 2, 19. https://doi.org/10.1186/1757-4749-2-19 (2010) Ley, R. E., Turnbaugh, P. J., Klein, S. & Gordon, J. I. Microbial ecology: Human gut microbes associated with obesity. Nature 444, 1022–1023. https://doi.org/10.1038/4441022a (2006) Tian, Y. et al. The microbiome modulating activity of bile acids. Gut Microbes 11, 979–996. https://doi.org/10.1080/19490976.2020.1732268 (2020) Sannasiddappa, T. H., Lund, P. A. & Clarke, S. R. In vitro antibacterial activity of unconjugated and conjugated bile salts on staphylococcus aureus. Front. Microbiol. 8, 1581. https://doi.org/10.3389/fmicb.2017.01581 (2017) Sato, Y. et al. Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians. Nature 599, 458–464. https://doi.org/10.1038/s41586-021-03832-5 (2021) Salter, S. J. et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12, 87. https://doi.org/10.1186/s12915-014-0087-z (2014) Dyrhovden, R. et al. Managing contamination and diverse bacterial loads in 16S rRNA deep sequencing of clinical samples: Implications of the law of small numbers. mBio 12, e0059821. https://doi.org/10.1128/mBio.00598-21 (2021) relevance and response for Ralsfonia respiratory infections Jhung, M. A. et al. A national outbreak of Ralstonia mannitolilytica associated with use of a contaminated oxygen-delivery device among pediatric patients. Pediatrics 119, 1061–1068. https://doi.org/10.1542/peds.2006-3739 (2007) Ivnitsky, H. et al. Bacterial community composition and structure of biofilms developing on nanofiltration membranes applied to wastewater treatment. Water Res. 41, 3924–3935. https://doi.org/10.1016/j.watres.2007.05.021 (2007) Frank, D. N. et al. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc. Natl. Acad. Sci. U. S. A. 104, 13780–13785. https://doi.org/10.1073/pnas.0706625104 (2007) Sekirov, I., Russell, S. L., Antunes, L. C. & Finlay, B. B. Gut microbiota in health and disease. Physiol. Rev. 90, 859–904. https://doi.org/10.1152/physrev.00045.2009 (2010) Angelakis, E. et al. A metagenomic investigation of the duodenal microbiota reveals links with obesity. PLoS One 10, e0137784. https://doi.org/10.1371/journal.pone.0137784 (2015) Meng, Y., Li, X., Zhang, J., Wang, C. & Lu, F. Effects of different diets on microbiota in the small intestine mucus and weight regulation in rats. Sci. Rep. 9, 8500. https://doi.org/10.1038/s41598-019-44994-7 (2019) Kayashima, A. et al. Unique bile acid profiles in the bile ducts of patients with primary sclerosing cholangitis. Hepatol. Commun. https://doi.org/10.1097/hc9.0000000000000452 (2024) Wang, S. et al. Bile acid-microbiome interaction promotes gastric carcinogenesis. Adv. Sci. (Weinh) 9, e2200263. https://doi.org/10.1002/advs.202200263 (2022) Noto, J. M. et al. Iron deficiency linked to altered bile acid metabolism promotes Helicobacter pylori-induced inflammation-driven gastric carcinogenesis. J. Clin. Invest. https://doi.org/10.1172/jci147822 (2022) Kundu, S., Kumar, S. & Bajaj, A. Cross-talk between bile acids and gastrointestinal tract for progression and development of cancer and its therapeutic implications. IUBMB Life 67, 514–523. https://doi.org/10.1002/iub.1399 (2015) Nguyen, T. T. et al. Lithocholic acid stimulates il-8 expression in human colorectal cancer cells via activation of Erk1/2 MAPK and suppression of STAT3 activity. J. Cell Biochem. 118, 2958–2967. https://doi.org/10.1002/jcb.25955 (2017) Hernández, S. B., Cota, I., Ducret, A., Aussel, L. & Casadesús, J. Adaptation and preadaptation of Salmonella enterica to bile. PLoS Genet. 8, e1002459. https://doi.org/10.1371/journal.pgen.1002459 (2012) Leverrier, P. et al. Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: Physiological and proteomic analysis. Appl. Environ. Microbiol. 69, 3809–3818. https://doi.org/10.1128/aem.69.7.3809-3818.2003 (2003) Kazumori, H., Ishihara, S., Rumi, M. A., Kadowaki, Y. & Kinoshita, Y. Bile acids directly augment caudal related homeobox gene Cdx2 expression in oesophageal keratinocytes in Barrett’s epithelium. Gut 55, 16–25. https://doi.org/10.1136/gut.2005.066209 (2006) Effect of type and amount of dietary fat and 1,2-dimethylhydrazine on biliary bile acids Kechin, A., Boyarskikh, U., Kel, A. & Filipenko, M. cutPrimers: A new tool for accurate cutting of primers from reads of targeted next generation sequencing. J. Comput. Biol. 24, 1138–1143. https://doi.org/10.1089/cmb.2017.0096 (2017) Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583. https://doi.org/10.1038/nmeth.3869 (2016) Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37, 852–857. https://doi.org/10.1038/s41587-019-0209-9 (2019) Miyamoto, K. et al. The gut microbiota-induced kynurenic acid recruits GPR35-positive macrophages to promote experimental encephalitis. Cell Rep. 42, 113005. https://doi.org/10.1016/j.celrep.2023.113005 (2023) Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucl. Acids Res. 41, D590-596. https://doi.org/10.1093/nar/gks1219 (2013) Dahl, E. M., Neer, E., Bowie, K. R., Leung, E. T. & Karstens, L. microshades: An R package for improving color accessibility and organization of microbiome data. Microbiol. Resour. Announc. 11, e0079522. https://doi.org/10.1128/mra.00795-22 (2022) Mandal, S. et al. Analysis of composition of microbiomes: A novel method for studying microbial composition. Microb. Ecol. Health Dis. 26, 27663. https://doi.org/10.3402/mehd.v26.27663 (2015) Murakami, M. et al. Detection of gut dysbiosis due to reduced clostridium subcluster xiva using the fecal or serum bile acid profile. Inflamm. Bowel Dis. 24, 1035–1044. https://doi.org/10.1093/ibd/izy022 (2018) Download references This work was supported by Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS) (21K18272 and 23H02899 to T.S.; 23K07423 to M.K.;23H00425 to T.K.) the Japan Agency for Medical Research and Development (19ek0109214 to T.S.; JP21gm1510002 to T.K.) the Mochida Memorial Foundation 2021 (to T.S.) the Yakult Bioscience Research Foundation (to M.K.) Division of Research and Development for Minimally Invasive Treatment Cancer Center Center for Diagnostic and Therapeutic Endoscopy Tokyo Medical University Ibaraki Medical Center T.K.; Methodology: Y.K.; Investigation: Y.K. Kentaro Miyamoto is an employee of Miyarisan Pharm Takanori Kanai have no competing interests Download citation DOI: https://doi.org/10.1038/s41598-024-69820-7