« Back Abby brings us a story nothing short of a movie Abby Boretto is a resilient advocate for community Raised in a historic Connecticut town with deep family roots she developed a lifelong dedication to service and making a positive impact Abby dedicated herself to raising her three children in California becoming a pillar of her community while balancing parenting proving that reinvention and purpose have no limits As someone who’s seen what happens when the truth is distorted I know how unfair it feels when those who’ve sacrificed the most lose their voice you’re not just leveling the playing field—you’re standing with those who’ve already given so much ensuring they continue to serve by delivering stories that matter Every subscription means we can hire more veterans and keep their hard-earned knowledge in the fight You must become a subscriber or login to view or post comments on this article Anduril Is the Netflix of War—and the Old Defense Giants Are About to Get Blockbuster’d How Department of Defense Educational Activity Staff Co-Opted Diversity Equity and Inclusion Protests SOFREP Evening Brief: Israel Approves Gaza Seizure Plan Japanese Soryu-class Submarines Could Be Stealthiest in the World SOFREP Evening Brief: Trump Annoys 1.4 Billon Catholics by Posting Pic of Himself As Pope, Putin Says He Hopes He Won’t Have to Use Nukes in Ukraine, Mexico Rejects US Offer to Send Troops Across the Border · 13 hours ago The World is Gearing Up for War: $2.7 Trillion in Global Military Spending Signals a New Era of Conflict · 14 hours ago The Great Goat Heist: Inside the Navy’s Mascot Snatch · 15 hours ago SOFREP Evening Brief: Israel Approves Gaza Seizure Plan, Pakistan Conducts Second Missile Test · 16 hours ago Anduril Is the Netflix of War—and the Old Defense Giants Are About to Get Blockbuster’d · 20 hours ago Your Subscription Supports our Veteran Staff and more than 75 State Department-approved countries around the world Our ultra-portable washing machine makes your journey easier pocket-sized travel companion allows you to travel lighter while helping you save money Our roots in shooting sports started off back in 1996 with our founder and CEO His love of airguns took hold of our company from day one and we became the first e-commerce retailer dedicated to airguns customers turned to us for our unmatched product selection and continued support of the sport and airgun industry RecPak is a meal replacement for the outdoors that saves you weight space and time in the most challenging environments Metrics details Endometrial disorders represent a major gynaecological burden Current research models fail to recapitulate the nature and heterogeneity of these diseases thereby hampering scientific and clinical progress Here we developed long-term expandable organoids from a broad spectrum of endometrial pathologies Organoids from endometriosis show disease-associated traits and cancer-linked mutations Endometrial cancer-derived organoids accurately capture cancer subtypes replicate the mutational landscape of the tumours and display patient-specific drug responses Organoids were also established from precancerous pathologies encompassing endometrial hyperplasia and Lynch syndrome and inherited gene mutations were maintained Endometrial disease organoids reproduced the original lesion when transplanted in vivo we developed multiple organoid models that capture endometrial disease diversity and will provide powerful research models and drug screening and discovery tools Prices may be subject to local taxes which are calculated during checkout All other data supporting the findings of this study are available from the corresponding authors on reasonable request Unique biological materials can be made available to third parties depending on their research goals (that is mutual ethical permissions and a Material Transfer Agreement Reproductive tract function and dysfunction in women Classification of endometrial carcinoma: more than two types Endometrial cancer: experimental models useful for studies on molecular aspects of endometrial cancer and carcinogenesis Characterization of patient-derived tumor xenograft models of endometrial cancer for preclinical evaluation of targeted therapies Lkb1 inactivation is sufficient to drive endometrial cancers that are aggressive yet highly responsive to mTOR inhibitor monotherapy Models of endometriosis and their utility in studying progression to ovarian clear cell carcinoma Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability hormone-responsive organoid cultures of human endometrium in a chemically defined medium American Society for Reproductive Medicine Revised American Society for Reproductive Medicine classification of endometriosis: 1996 The role of matrix metalloproteinases in the pathogenesis of endometriosis DNA microarray analysis of gene expression markers of endometriosis Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis DNA microarray analysis of gene expression profiles in deep endometriosis using laser capture microdissection MMP-9 and PCNA in endometriosis and endometrial carcinoma Transcriptional characterizations of differences between eutopic and ectopic endometrium Reconstructing lineage hierarchies of mouse uterus epithelial development using single-cell analysis SSEA-1 isolates human endometrial basal glandular epithelial cells: phenotypic and functional characterization and implications in the pathogenesis of endometriosis p27kip1 overexpression regulates IL-1β in the microenvironment of stem cells and eutopic endometriosis co-cultures Cancer-associated mutations in endometriosis without cancer Clonal expansion and diversification of cancer-associated mutations in endometriosis and normal endometrium beta-catenin mediates glandular formation and dysregulation of beta-catenin induces hyperplasia formation in the murine uterus Human primary liver cancer-derived organoid cultures for disease modeling and drug screening Organoid cultures derived from patients with advanced prostate cancer A living biobank of breast cancer organoids captures disease heterogeneity Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition Proliferative and signaling activities of insulin analogues in endometrial cancer cells Induction of hepatocyte growth factor in stromal cells by tumor-derived basic fibroblast growth factor enhances growth and invasion of endometrial cancer PTEN mutation in endometrial cancers is associated with favorable clinical and pathologic characteristics Integrated genomic characterization of endometrial carcinoma Use of mutation profiles to refine the classification of endometrial carcinomas Endometrial carcinoma: molecular alterations involved in tumor development and progression Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation Prospective derivation of a living organoid biobank of colorectal cancer patients Functional expression of the mechanosensitive PIEZO1 channel in primary endometrial epithelial cells and endometrial organoids Functional expression of TRP ion channels in endometrial stromal cells of endometriosis patients Human pancreatic tumor organoids reveal loss of stem cell niche factor dependence during disease progression CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer Personalized in vitro and in vivo cancer models to guide precision medicine The use of endometrial cancer patient-derived organoid culture for drug sensitivity testing is feasible Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer Fast and accurate short read alignment with Burrows-Wheeler transform From FastQ files to high confidence variant calls: the genome analysis toolkit best practices pipeline Dindel: accurate indel calls from short-read data ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks TopHat2: accurate alignment of transcriptomes in the presence of insertions The functional expression of transient receptor potential channels in the mouse endometrium Download references Peetermans) for their valuable input and technical help Laureys (Department of Clinical and Experimental Medicine Eggermont for their expert help in mouse transplantation experiments staff and nurses at UZ Leuven for providing the clinical samples We thank the VIB Nucleomics Core and KU Leuven Genomics Core (particularly Á Calabuig) for their expert assistance in RNA-seq and aCGH analysis We are also grateful to InfraMouse (VIB−KU Leuven Hercules type 3 project ZW09-03) for the use of histological instruments and microscopes we acknowledge the use of the Electron Microscopy Platform of the Centre for Human Genetics (VIB−KU Leuven) This work was supported by grants from the KU Leuven Research Fund and from the FWO−Flanders (Belgium) are supported by a PhD Fellowship from the FWO is a PhD Fellow supported by a GOA grant from the Research Fund of the KU Leuven is a Senior Clinical Investigator of the FWO Laboratory of Tissue Plasticity in Health and Disease Stem Cell and Developmental Biology Cluster Department of Development and Regeneration Department of Anatomy and Structural Science Laboratory of Molecular Imaging and Photonics Unit of Translational Research in Gastrointestinal Disorders performed the experiments and the data analysis and interpreted the results gene expression analyses and routine organoid culturing provided essential help in the bioinformatic analysis of RNA-seq data performed and cointerpreted the functional ion channel analysis helped to perform gene expression analyses collected patient information and samples and helped to perform the molecular analyses guided and supervised the targeted sequencing analysis were driving forces in setting up the clinical collaboration to obtain human samples was a collaborating surgeon who provided many of the clinical samples are collaborating gynaecologists with joint grant applications in endometrial research supervised and cointerpreted the functional ion channel analysis and is a collaborating scientist with joint grant applications in endometrial research codesigned the experiments and coanalysed and cointerpreted the data All coauthors critically read and approved the manuscript The authors declare no competing interests Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations (a) Flowchart depicting organoid establishment from the indicated spectrum of endometrial diseases (b) Brightfield pictures of development of EM-O and matched ECT-O and EUT-O after seeding (P0) Representative pictures of 3 independent experiments (that is 3 independent donors per condition) are shown (c) Representative brightfield pictures showing clonal ECT-O formation Arrow indicates a single cell monitored for 20 days (n=3 biologically independent experiments) (d) Passaging time of EM-O (3 independent donors) and of matched EUT-O/ECT-O (3 other independent donors) minimum and maximum per individual organoid line (e) Brightfield and H&E images of organoids derived from rASRM stage I to IV endometriosis Representative pictures of 3 independent donors per rASRM stage are shown 200 µm for brightfield and 50 µm for H&E (f) aCGH plots of short-term (1–2 months; low passage) and long-term (4–6 months; high passage) cultured EM-O and EUT-O Representative plots of 3 independent experiments are shown (g) Gene expression analysis of endometrial markers in ECT-O and EM-O after short- and long-term culture presented as ΔCt (Ct of gene – Ct of GAPDH) (mean ± s.e.m of n=3 biologically independent experiments) (h) Rescue of IWP2-induced organoid growth inhibition represented as percentage of organoids formed after 10 days with the indicated treatment as compared to SOM (left graphs) and gene expression analysis of canonical (middle) and non-canonical (right) WNT target genes in ECT-O (top) (mean ± s.e.m of n=5 biologically independent experiments) and EM-O (bottom) (mean ± s.e.m of n=4 biologically independent experiments) P***<0.001; non-parametric Kruskal-Wallis test for multiple comparison with Dunn’s post-test (95% confidence intervals) (i) WNT ligand gene expression in ECT-O as determined by RT-qPCR and represented as ∆Ct (Ct of gene – Ct of GAPDH) (mean ± s.e.m of 8 independent donors) (left) and as extracted from the RNA-seq dataset and presented as heatmap of transcript per million values (right) in which colors range from white (low) to red (high) (a) H&E analysis of matched EUT-O and ECT-O Representative pictures of 5 independent experiments (that is 5 independent donors per condition) are shown (b) Immunohistochemical analysis of MMP2 and MMP7 in primary peritoneal endometriotic lesions and ECT-O Representative pictures of 4 independent experiments (that is 4 independent donors per condition) are shown as normalized to GAPDH and expressed as fold change relative to EM-O (mean ± s.e.m of n=4 biologically independent experiments) *P=0.0286 as compared to EM-O; two-tailed non-parametric T-test (95% confidence intervals) (c) E-cadherin immunofluorescence staining of ECT-O showing the epithelial nature and correct positioning of tight junctions which supports the cells’ polarization Representative pictures of 5 independent experiments (that is 5 independent donors) are shown (d) TEM pictures of matched EUT-O and ECT-O from an individual patient The EUT-O are composed of a single-cell layer bordering a lumen containing microvilli (magnified box) and ciliated cells (as revealed by acetylated (Ac) α-tubulin immunofluorescence) double-cell layer (*) is present in the ECT-O with extensive microvilli (magnified box) and ciliated cells subrenally transplanted according to the schedule give rise to lesions with endometriotic features H&E images and immunohistochemical analysis for ERα and PR of the kidney capsule and ECT-O grafts are presented Lower panels display H&E staining of the non-grafted kidney Representative pictures of 6 biologically independent experiments with 4 independent donors are shown (c) Expression of WNT ligand and Hippo pathway target genes in stage I to IV ECT-O as normalized to GAPDH and expressed as fold change relative to EM-O (mean ± s.e.m of n=3 biologically independent experiments for stage I and IV ECT-O n=5 for stage II ECT-O and n=4 for stage III ECT-O) *P<0.05; non-parametric Kruskal-Wallis test for multiple comparison with Dunn’s post-test (95% confidence intervals) (d) Expression of invasion and inflammatory marker genes in stage I to IV ECT-O as normalized to GAPDH and expressed as fold change relative to EM-O (mean ± s.e.m (e) PCA plot showing the distribution of matched EUT-O and ECT-O based on the RNA-seq data (n=4 independent donors for the EUT-O and matched ECT-O) (f) Heatmap of 75 differentially expressed genes (log2 normalized counts) as identified by RNA-seq analysis of 4 matched ECT-O and EUT-O organoid lines as indicated Colors range from blue (low expression) to red (high expression) LGR6 is among the top upregulated genes in the ECT-O (a) Organoid development from hyperplastic endometrium (HYP-O) after seeding (P0) Representative brightfield pictures of 6 independent experiments (that is 6 independent donors) are shown 200 µm) and magnified organoid pictures (right; scale bar and mucin detection (PAS) in primary biopsies and corresponding HYP-O of different types of endometrial hyperplasia as indicated H&E staining reveals glandular-like morphology with a well-defined lumen in the organoids of simple benign and complex atypical hyperplasia and a poorly-defined lumen in hyperplastic polyp being present in simple benign hyperplasia and endometrial polyp but absent in complex atypical hyperplasia Mucus production is only detected in the lumen of the endometrial polyp and derived organoids (*) Representative brightfield pictures of 2 independent donors for hyperplastic polyp and 3 independent donors for the other hyperplasia types are shown (c) TEM analysis reveals some stratified epithelium (*) Microvilli are present while cilia are not observed (magnified box) Representative pictures of 3 independent experiments (that is 3 independent donors) are shown (d) aCGH plots indicate the absence of SCNA in both primary hyperplastic tissue and corresponding HYP-O Representative plots of 3 independent experiments (that is 3 independent donors) are shown (q) PCA plot based on gene expression analysis (n=4 independent donors for EM-O and HYP-O (e) Gene expression of ion channels in EUT-O and ECT-O subdivided into different rASRM stages as normalized to the geometric mean of housekeeping genes HPRT1 and PGK1 and expressed as fold change relative to EM-O (mean (± s.e.m n=5 ECT-O stage I-II and n=5 ECT-O stage III-IV independent donors) **P<0.01 compared to EM-O; two-way ANOVA and Dunn’s multiple comparison test (f) Percentage of responding cells in EM-O EUT-O and ECT-O to specific ion channel activators The data represent the following n independent experiments encompassing the indicated total number of cells: GSK in EM-O: n=6 (g) Gene expression of ion channels in HYP-O and EC-O the latter subdivided into non-invasive (N-IN) and invasive (IN) phenotypes as normalized to HPRT1 and PGK1 and expressed as fold change relative to EM-O (mean ± s.e.m n=3 EC-O N-IN and n=4 EC-O IN independent donors) ***P<0.001 compared to EM-O; two-way ANOVA and Dunn’s multiple comparison test (h) EC-O biobanking with representative organoid line ID card Supplementary table titles/legends and Supplementary video titles/legends List of genes differentially expressed between EM-O and ECT-O List of genes differentially expressed between EUT-O and ECT-O List of genes differentially expressed between EM-O and EUT-O Top divergent pathways between ECT-O and EM-O as identified by KEGG PATHWAY analysis using the 277 differentially expressed genes Top divergent biological terms between ECT-O and EM-O as identified by DAVID Gene Ontology enrichment using the 277 differentially expressed genes Optimized culture medium for EC-O formation and expansion RT−qPCR gene expression analysis of organoid lines derived from multiple endometrial conditions z-stacks (up to 350 µm) from the bottom to the top of nuclear Hoechst-stained EM-O acquired using two-photon microscopy displaying a single-cell layer surrounding a central lumen z-stacks (up to 350 µm) from the bottom to the top of nuclear Hoechst-stained ECT-O acquired using two-photon microscopy displaying a stratified cell layer surrounding a central lumen z-stacks (up to 350 µm) from the bottom to the top of nuclear Hoechst- and membrane DiI-stained low-grade EC-O acquired using two-photon microscopy displaying a stratified cell layer and the presence of a lumen z-stacks (up to 350 µm) from the bottom to the top of nuclear Hoechst-stained high-grade EC-O combined with brightfield displaying a stratified cell layer and an absence of a lumen Download citation DOI: https://doi.org/10.1038/s41556-019-0360-z 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: Cancer newsletter — what matters in cancer research (ANS - Boretto) - The first presentation of the book "Artemide Zatti salesiano coadiutore - In bicicletta verso il cielo" (Elledici editions) written by Fr Postulator General for the Causes of Saints of the Salesian Family at the "Bottega del tempo libero" in front of about 100 people with several speakers bringing their contributions and recounting their experiences related to Zatti which is very attached to the figure of the saint and which participated joyfully and in large numbers in the canonization that took place in Rome last October 9 that the pastoral unit that includes Brescello and Lentigione in addition to Boretto is named During the evening - moderated by the journalist of the Gazzetta di Reggio Cameroni retraced the salient stages of the saint's life the spirit of service of the "relative of the poor": "Don Bosco," he explained "had said to his Salesians who left for America to take care of the sick Zatti as a Good Samaritan took them in the inn of his heart and in the 'San José' hospital in Viedma: in each of them His canonization tells us the beauty of the consecrated life and the value of a life wholly dedicated to God in service to the poor with the apostolic heart of Don Bosco It is a strong impulse to promote the vocation of the Salesian coadjutor who brings to all educational and pastoral fields the proper value of his laity which makes him in a specific way a witness to the Kingdom of God in the world." and the parish priest of Boretto and Brescello announcing that next year the Rector Major of the Salesians will be conferred honorary citizenship of Boretto Other speakers included: Boretto residents Anna Montagna Touching was the testimony of little Benedetta a sign of how even the little ones feel involved in Artemide Zatti's adventure "I came to tell my experience of my pilgrimage to Rome: I really enjoyed being with my family But the most beautiful thing was attending the canonization of Saint Artemide Zatti: I thought it was a dream to see the Pope in person I made many new friends and strengthened the friendships I already had The dinner together was very exciting; I thought I was a disciple of Jesus I also enjoyed the 'Color Zatti' experience where I was an extra; I felt like a close relative of Artemide because we looked like migrants from Boretto who wanted to go to Argentina." ANS - “Agenzia iNfo Salesiana” is a on-line almost daily publication the communication agency of the Salesian Congregation enrolled in the Press Register of the Tibunal of Rome as n 153/2007 This site also uses third-party cookies to improve user experience and for statistical purposes By scrolling through this page or by clicking on any of its elements The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission All content on this site: Copyright © 2025 Elsevier B.V. SAN DIEGO (KGTV) – A California woman set out on a journey to meet the man who found her father's long-lost Naval Academy ring to say the least," said Abby Pilger Boretto Pilger Boretto’s father, a Navy helicopter pilot, died in a crash during a NATO training exercise on the Island of Grytoya in Northern Norway She was just 15 months old at the time and never had the chance to know her father a piece of him found its way back to her when Dr Hans Krogstad came across the ring at the crash site "And then I look down and between two small boulders I saw this ring,” he said Although he did his best to ensure it made it back to the Pilgers He never knew the outcome and Pilger Boretto never had the chance to thank him they found one another and started making plans to meet during a very important time 23 and so we wanted to celebrate that and memorialize that “I think it’s this quest to visit this site that has been in me my whole life,” she added I think it's a very healing story for many out there.” Pilger Boretto is now in the middle of filming a documentary about her journey the crash and the others who lost their lives while serving their country And although she's managed to track some down she's still hoping to find more relatives of those other four men “So that I can make those connections and they know their family member has forever been memorialized,” she said This story was originally reported on 10news.com. Linda Merklinger was 22 in 1972 when her husband of three years died in a helicopter crash about 4,600 miles away in Norway was a 25-year-old first lieutenant in the U.S Linda said she was told at the time the crash came during a “special mission.”   A Department of the Navy document she provided stated he died Sept grew up; Linda remarried (she’s now Linda Patterson) and had a son But the circumstances of what happened to Gerald Merklinger remained a mystery “You’re thinking was it pilot error … You don't know.”  after texts and phone calls to family members from a woman in California Gerald Merklinger and three others died in the crash “That's how we found out they were on mission 'Strong Express,' and it was a NATO mission,” Patterson said NATO −The North Atlantic Treaty Organization is an intergovernmental military alliance created in 1949 by the United States and several Western European nations to provide collective security against the Soviet Union Its members agree to defend each other against attacks by third parties According to a New York Times article on the day of the crash the massive NATO “Strong Express” exercise involved in excess of 65,000 men about 700 aircraft and 350 ships from 12 NATO nations It was to demonstrate in part how quickly NATO members could come to the defense of Norway in the event northern Norway was captured by an enemy, according to Associated Press archival video.  Patterson and her family flew in a helicopter to the rocky crash site which she said is on the side of a mountain and devoid of vegetation “You finally now have a visual of this is where it happened While they couldn’t land because of the wind they hovered over the spot and Patterson got a good sense of the area who also attended Martin County High School said she wore one of her father’s flight jackets during the visit to Norway so I had no idea it existed,” Melissa Merklinger said Gerald Merklinger is buried at Fernhill Memorial Gardens & Mausoleum on South Kanner Highway in Stuart The cemetery is across the street from First United Methodist Church of Stuart where Linda and Gerald were married Patterson said over the years she tried to get information from the military Patterson said Boretto’s efforts to connect with Patterson’s family proved critical to learning more but Boretto’s journey to find out about her dad ‘We just carried on’Boretto was 15 months old when her father “I just didn't know all that much about him,” Boretto and it was just a sign of the times back in the 70s.”  Her mother remarried when Boretto was 8 years old and much of what she knew about Pilger came when people remarked she looked like him or shared a mannerism said her father had given her mother a sweetheart ring gave that ring to Boretto when she was a teen and she wore it daily because it was her only connection to her father she was swimming in Hawaii and the ring came off “I said out loud to my friends who were on the beach helping me .. ‘He knew to start with an embassy’A different ring The package arrived several months after a Norwegian man out hunting found it in 1993 — more than 20 years after the helicopter crash — on Grytoya Island “He knew to get this ring back to the family,” Boretto said It took maybe four or five months to get to me.” and I'm turning 50 in June 2021,” she said She turned to the ring and the package and the letter included from the hunter in Norway “I said I can't believe we never thanked this person,” she said Not too long after Boretto’s 50th birthday a Facebook message arrived from a journalist in Norway asking her specific questions about whether her father died in Norway and about a ring She learned that Krogstad wondered whether the ring made it back to the family and began his own investigation Krogstad’s son is friends with the Norwegian journalist Boretto decided she and her husband wanted to go to the crash site meet Krogstad and film a documentary on the 50th anniversary of the crash Boretto and her husband began a “deep dive” into the crash after getting a copy of the crash report from the Norwegian journalist Efforts to research the others who died in the crash brought Boretto to Gerald Merklinger’s sister Boretto said she told Melissa Merklinger they planned to return to Norway in September 2023 to premier the documentary and they would love to have her along Melissa Merklinger’s wife and her mother also went on the trip “I think Melissa has been one of my greatest treasures in this finding the next-of-kin has been a tremendous honor for me,” Boretto said 'The mission is everything': Stuart Marine remembers buddies lost, earlier generations on Veterans Day Vet recalls war experiences: Memorial Day for Vietnam vet is reminder of war 'I don't think we ever really understood' Will Greenlee is a breaking news reporter for TCPalm. Follow Will on X @OffTheBeatTweet or reach him by phone at 772-267-7926. E-mail him at will.greenlee@tcpalm.com (ANS - Boretto) - "I am truly proud to have been born in Boretto I’m very happy to meet a fellow citizen who is a saint!" Thus participated in the event organized on July 7 given the upcoming canonization of the beloved son of that land Our Brother," produced by the Salesian Bulletin and the Provinces of Argentina scripted and directed by Fr Ricardo Campoli which narrates the human and spiritual story of Zatti at a dramatic moment in his life when he was informed that the San Jose Hospital in Viedma to which he had dedicated all his energies It was at this moment that his faith and fortitude would be tested The city of Boretto had the privilege of seeing the film dubbed in Italian for the first time The emotion of those present was immediate and intense highlighting aspects and provocations that make Artemide Zatti's testimony relevant today The day was also marked by a meeting with the group of animators of the Brescello oratory and Fr Campoli gave a family presentation on the story of Artemide Zatti and his life dedicated to the service of the poor and the sick which was also attended by the mayor of Boretto they took stock of the initiatives in the pipeline to enliven the event of Artemide Zatti's canonization Of particular interest is the creation of a photographic exhibition Vicar of the Province of Italia Lombardo-Emiliana (ILE) which traces the story of Artemide Zatti around three key verbs that marked his entire life: "Credetti the setting up of an itinerary on the territory through the meaningful sites where the future Salesian saint lived his childhood and adolescence which offers some messages regarding family Metrics details Carcinogenesis results from the sequential acquisition of oncogenic mutations that convert normal cells into invasive Colorectal cancer exemplifies this process through its well-described adenoma–carcinoma sequence modeled previously using clustered regularly interspaced short palindromic repeats (CRISPR) to induce four consecutive mutations in wild-type human gut organoids we demonstrate that long-term culture of mismatch-repair-deficient organoids allows the selection of spontaneous oncogenic mutations through the sequential withdrawal of Wnt agonists epidermal growth factor (EGF) agonists and the bone morphogenetic protein (BMP) antagonist Noggin while TP53 mutations were selected through the addition of Nutlin-3 organoids sequentially acquired mutations in AXIN1 and AXIN2 (Wnt pathway) ACVR2A and BMPR2 (BMP pathway) and NRAS (EGF pathway) gaining complete independence from stem cell niche factors p53 and BMP) mutant organoids formed solid tumors upon xenotransplantation This demonstrates that carcinogenesis can be recapitulated in a DNA repair-mutant background through in vitro selection that targets four consecutive cancer pathways chose to exploit the MLH1-mutant background for our study we build on this experimental approach by selecting spontaneous oncogenic mutations of MLH1-mutant organoids by removing the niche factor components Wnt EGF and Noggin from the organoid culture medium or by adding the small-molecule p53 inhibitor Nut3 this study asks whether simple growth factor-based selection of MMR-deficient (yet otherwise WT) organoids would allow the derivation of full-blown colon cancer cells in vitro potentially mimicking features of MSI-related CRCs Strategy to select the survived organoids with deprivation of Wnt pathway factors Organoids were dissociated into single cells and then 200,000 single cells were cultured in niche-factor-deprived medium to select mutated stem cells (red) out from WT stem cells (blue) and differentiated cells (white) Surviving clone organoids were expanded briefly for WGS and WES analysis Accumulated mutations were determined by subtracting the mutations in the original clone (blue) from the mutations in the selected subclone (red) WT organoids and MLH1KO-1 organoids grew in complete medium (WRNE) but some of organoids grew out only from MLH1KO-1 organoids in −WR medium (representative pictures from n = 3 clonal organoid lines) (c) Quantification of survived organoids in −WR medium (error bars) of n = 4 clonal organoid lines P values (two-sided Welch’s t-test): MLH1KO-1 versus MLH1KO-2 Single survived organoids (MLH1KO-1−WR-1 and MLH1KO-1−WR-2) were expanded and passaged in −WR medium (representative pictures from n = 3 independent replicates) The Venn diagrams illustrate the number of base substitutions and indels found in WGS data of survived clones (MLH1KO-1−WR-1 and MLH1KO-1−WR-2) AXIN1 and AXIN2 candidate gene mutations are listed in the overlapping section The numbers indicate nonsynonymous mutations and the numbers in brackets indicate all mutations found in WGS AXIN2 transcripts were compared between each clone cultured in the medium with or without Wnt3a and R-spondin 1 (error bars) of n = 3 biologically independent experiments P values (two-sided Welch’s t-test): MLH1KO-1 versus MLH1KO-1−WR-1 in −WR medium P = 0.0048; MLH1KO-1 versus MLH1KO-1−WR-2 in −WR medium Representative images of Sanger DNA sequencing of AXIN1 and AXIN2 mutations in Wnt-independent clones (MLH1KO-1−WR-1 and MLH1KO-1−WR-2) Source data MLH1KO organoids allowed the faithful selection of some of the specific mutations seen in MMR-deficient and MSI-H tumors in in vitro culture Source data These data clearly suggest that the mutational signature of d-MMR induced by MLH1 mutation remained the dominant driver of mutagenesis regardless of the accumulated genetic mutations Source data The acquisition of identical mutations was independently observed in different clones The available data indicated that these recurrent mutations arose independently yet were because of a shared mutational mechanism simple selection in an MLH1KO mutant organoid background allowed the sequential isolation of spontaneous quadruple-pathway mutant organoids Source data Each mean relative risk was calculated from mutational signatures of normal human colonic organoid (C in vitro normal human small intestinal organoid (SI in vitro normal human small intestinal tissue (SI in vivo n = 14) and the average mutant organoids (samples SBS mutations found in the mutant organoids (AXIN1Q351* and TP53R158L) are shown together Heat map diagram of expression levels for each representative genes in CRC driver pathways Columns and rows in the heat map represent mutant clones and genes Color scale indicates fold changes in gene expression (n = 3 clonal organoid lines) Triple-pathway mutant organoids (NRASWT) and quadruple-pathway mutant organoids (NRASQ61K) were injected subcutaneously in NSG immunodeficient mice Both triple-pathway mutant (NRASWT) and quadruple-pathway mutant (NRASQ61K) organoids developed visible nodules NRASQ61K organoids grew as tumors with vasculature in the subcutaneous lesion Representative pictures from n = 6–12 tumors Representative images of extracted tumors of NRASWT organoids (top) and NRASQ61K organoids (bottom) Tumor size measurement of xenografts at 8 weeks after xenotransplantation P values (two-sided Welch t-test): N-1 NRASWT versus N-1 KRASG12D P = 0.0023; N-2 NRASWT versus N-2 NRASQ61K 1 P = 0.0473; N-2 NRASWT versus N-2 NRASQ61K 2 P = 0.0334; N-2 NRASQ61K 2 versus N-1 KRASG12D E-cadherin and Ki67 immunostaining on nodules isolated from triple-pathway mutant (NRASWT) and quadruple-pathway mutant (NRASQ61K) injected mice Triple-pathway mutant organoids did engraft but remained thin with a cystic structure of organoids Quadruple-pathway mutant organoid-derived tumors showed a thick and irregular multilayered epithelium The analysis was performed on n = 3 tumors Source data The AKPS organoid line was engineered to carry the strongest mutations in each of the four pathways while the quadruple-pathway mutant MLH1KO carries much weaker versions We do not know how each of these mutations contributes to the observed in vivo growth difference yet they apparently add up to a notably more malignant phenotype of the AKPS organoids upon transplantation quadruple-pathway mutant clones isolated in this study were tumorigenic in vivo upon subcutaneous transplantation but not to the sequence changes observed in the current study Our approach is not easily generalizable to other tumor types CRC appears relatively unique in that the four most commonly mutated pathways can be easily and specifically selected for: Three of these pertain to growth factor dependencies (Wnt while Nut3 allows for selection of mutations in the fourth pathway (TP53) this study provides the example of the derivation of full-blown cancers from WT cells simply by providing selective pressure for four well-defined oncogenic pathways We chose to perform our study in an MLH1-mutant background which has an increased rate of accumulation of genetic mutations and is well known to confer CRC predisposition Similar approaches with WT cells or with organoids carrying other cancer predisposing mutations will likely yield similar results The study was approved by the UMC Utrecht and Diakonessenhuis ethical committee (TC-Bio 12-093) and was in accordance with the Declaration of Helsinki and according to Dutch law This study is compliant with all relevant ethical regulations regarding research involving human participants All mouse experiments were conducted under a project license (AVD 8010020209924) granted by the Central Committee Animal Experimentation of the Dutch government and approved by the KNAW–Hubrecht Institute Animal Welfare Body (HI 21.39.04) NOD scid gamma (NSG; NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; 8 weeks old) mice were used in the study The mice were kept in a constant-temperature environment of 21 °C (40–60% humidity) with a natural day–night light cycle in a conventional animal colony with free access to food and water All mice were housed in a pathogen-free vivarium in sterile disposable microisolator cages and fed a sterile irradiated diet with free access to sterile All the mice were monitored and weighed once a week Mice with symptoms of severe discomfort (for example >20% weight loss compared to beginning or >15% weight loss in 2 days) with tumor burden or with measured tumors larger than 1,000 mm3 were humanely killed three mice in the subcutaneous transplantation experiment were killed with the estimation of exceeded maximal tumor burden at an earlier time point As tumor size was weekly checked by manual palpation it was only possible to estimate the size of the tumors ahead of killing we noted that n = 1 tumor of the killed animals exceeded the size limit basal culture medium (AdDMF+++) contained advanced DMEM/F12 (Invitrogen) supplemented with 10 mM HEPES 100 U per ml penicillin and 100 μg ml−1 streptomycin The culture medium (WRNE) contained AdDMF+++ including B27 (Invitrogen) produced using R-spondin 1 cells (Trevigen)) produced using stably transfected L cells) TGFβ type I receptor inhibitor A83-01 (Tocris) and p38 inhibitor SB202190 (Sigma-Aldrich) Organoids were embedded in domes of Cultrex β-mercaptoethanol (BME; Trevigen) and covered by culture medium (WRNE) Established MLH1KO colon organoids were cultured in two wells of a six-well plate (480 μl of BME approximately 160,000–200,000 cells before passaging) Organoids were passaged weekly at a 1:2 split ratio keeping multiclonal cell diversity without selective pressure Organoids were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) in the presence of 10 µM Y-27632 (Tocris Bioscience) for 20 min and mechanically dissociated into single cells A total of 120,000 single cells were counted resuspended in 240 μl of Cultrex BME and seeded at low density on a prewarmed six-well culture plate (Corning) Survived organoids derived from single cells were manually picked up resuspended in 20 μl of Cultrex BME and seeded on a 48-well culture plate (Corning) Clonally selected organoids were cultured in selection medium to confirm as they grow in that culture condition For selection of Wnt pathway or BMP pathway mutants organoids were grown in culture medium lacking Wnt3a-conditioned medium and R-spondin 1-conditioned medium or Noggin organoids were cultured in the presence of 10 mM Nut3 (Cayman Chemical) and maintained in the presence of 1 mM Nut3 For selection of Ras and Raf pathway mutants organoids were grown in culture medium lacking EGF and containing 1 µM Gef (Selleck Chemicals) or 1 µM Afa (Selleck Chemicals) SBSs or indels were considered as driver mutations if they had an MQ of 30 had a minimum of 10× base coverage or were found in the driver genes COSMIC cancer gene consensus (version from 9 May 2019) these driver mutations were annotated as missense had either a high or moderate effect as annotated by SnpEff and were not in the SNP Database version 146 or a panel of unmatched normal human mesenchymal stem cells and fetal genomes (BED file available upon request) we extract the 96-trinucleotide mutational count matrix from the somatic mutation catalog with the mut_mat() function and the relative contribution plot was extracted with plot_96_profile() The COSMIC version 3 signatures were used and identified on the basis of a cosine similarity > 0.85 Gene expression and protein analyses were prepared as follows Organoids were passaged to single cells following an enzymatic dissociation with TrypLE for 10 min at 37 °C Cells were then resuspended in Matrigel and cultured in full human colon organoid expansion medium for 5 days to allow organoid formation Fully formed organoids were then washed three times in PBS and cultured for 24 h in either full expansion medium or selection medium (−WRNE + 1 mM Nut3 the organoids were collected and subjected to either RNA or protein extraction and used for RT–qPCR and western blot analyses Cells were lysed in radioimmunoprecipitation assay buffer containing cOmplete EDTA-free protease inhibitor cocktail tablets (4693132001 Protein quantification was performed using a standard Bradford assay (Bio-Rad) and equal amounts of protein were run on SDS–PAGE gels and transferred to PVDF membranes (Millipore) washed and incubated with antibodies against p53 (1:1,000; DO-1 glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000; ab9485 phospho-p44/42 MAPK (ERK1 and ERK2) (Thr202 Cell Signaling Technology) and β-actin (1:5,000; 13E5 The signals were detected using enhanced chemiluminescence (SuperSignal West Femto maximum sensitivity substrate Drug screenings were performed as described elsewhere66 outgrowing organoids were recovered from BME and enzymatically dissociated in TrypLE for 5 min at 37 °C dissociated organoids were resuspended in BME and allowed to recover for 2 days in a medium containing a low concentration of EGF (0.5 ng ml−1 before being cultured in a medium lacking EGF for two additional days prewarmed dispase was added and the organoids were incubated for 1 h at 37 °C after which the organoids were collected and washed with ice-cold AdDMF+++ The organoids were counted and dispensed at a density of 1,000 organoids per well in 40 µl of culture medium lacking EGF and containing 5% (v/v) BME using a multidrop dispenser (Thermo Fisher Scientific) in a 384-well plate The inhibitors and the vehicle for the normalization were added with an HP D300e digital dispenser (Hewlett-Packard) 30 µl of Cell Titer Glo (Promega) was added to each well using a multidrop dispenser and the plate was shaken for 5 min and incubated for an additional 15 min at room temperature before the luminescence was read using a SPARK luminescence detector (Tecan) with Tecan SparkControl software (version 2.1) The experiments were performed on two different clones each with three technical replicates; organoids before obtaining NRAS mutations were used as a control the tumor specimens were fixed overnight at 4 °C in 4% PFA paraffin-embedded and processed for histological analyses Tissues and organoids were fixed in 4% PFA dehydrated in ethanol and embedded in paraffin Sections were subjected to hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining Slides were cut at 5-μm thickness and used for histology Organoids were stained with H&E or primary antibodies for IHC the organoids were dehydrated in an ethanol gradient and then rinsed in PBS Heat-induced antigen retrieval was performed and the slides were sequentially incubated with the primary and secondary antibodies which were then counterstained with hematoxylin dehydrated and mounted with permanent mounting medium The imaging was performed on a LEICA microscope (DM6000) and images were processed using Leica LAS-X (version 1.1) software The following primary antibodies were used for IHC staining: mouse anti-E-cadherin clone 36 (1:1,000; 610182 mouse anti-Ki67 clone MM1 (1:1,000; 550609 rabbit anti-β-catenin H-102 (1:500; sc-7199 Santa Cruz Biotechnology) and rabbit anti-EGFR clone EP38Y (1:1,000; ab52894 No statistical method was used to predetermine sample size but our sample sizes are similar to those reported in a previous publication13 The investigators were not blinded to allocation during experiments and outcome assessment Data collection and analysis were not performed blind to the conditions of the experiments Data distribution was assumed to be normal but this was not formally tested Statistical analysis was performed with Prism 9.0 (GraphPad For comparisons between two independent groups a two-tailed unpaired Welch’s t-test was used P values < 0.05 were defined as statistically significant Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Mutation calling and filtering pipeline NF-IAP is available from GitHub (https://github.com/ToolsVanBox/NF-IAP). Somatic variants were extracted with SMuRF version 3.0.0 (available from GitHub: https://github.com/ToolsVanBox/SMuRF) and MutationalPatterns (available from GitHub: https://github.com/ToolsVanBox/MutationalPatterns) In-house R scripts (version 4.2.3) were used to generate VAF plots and the comparison of MANTIS and MSMutect2 with library ggplo2 Global burden of colorectal cancer: emerging trends Gene expression differences between the microsatellite instability (MIN) and chromosomal instability (CIN) phenotypes in colorectal cancer revealed by high-density cDNA array hybridization Comprehensive molecular characterization of human colon and rectal cancer A genetic model for colorectal tumorigenesis BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1 but not in hereditary nonpolyposis colorectal cancer Role of the serrated pathway in colorectal cancer pathogenesis Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating β-catenin/TCF signalling Frequent alterations in the Wnt signaling pathway in colorectal cancer with microsatellite instability Detection of point mutations of the AXIN1 gene in colorectal cancers Landscape of somatic single nucleotide variants and indels in colorectal cancer and impact on survival A molecular portrait of microsatellite instability across multiple cancers Loss of activin receptor type 2 protein expression in microsatellite unstable colon cancers Sequential cancer mutations in cultured human intestinal stem cells Modeling colorectal cancer using CRISPR–Cas9-mediated engineering of human intestinal organoids Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids RNF43 is frequently mutated in colorectal and endometrial cancers Microsatellite Instability in Colorectal Cancer Cancer risks associated with germline mutations in MLH1 Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines Biallelic inactivation of hMLH1 by epigenetic gene silencing a novel mechanism causing human MSI cancers CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients Genome-scale CRISPR screening in human intestinal organoids identifies drivers of TGF-β resistance Mutations and mechanisms of Wnt pathway tumour suppressors in cancer An AXIN2 mutant allele associated with predisposition to colorectal neoplasia has context-dependent effects on AXIN2 protein function Differential Roles of AXIN1 and AXIN2 in tankyrase inhibitor-induced formation of degradasomes and β-catenin degradation AXIN1 protects colon carcinogenesis by an immune-mediated effect The repertoire of mutational signatures in human cancer Genetic and epigenetic changes of components affecting the Wnt pathway in colorectal carcinomas stratified by microsatellite instability Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer Whole-exome sequencing analyses of inflammatory bowel disease-associated colorectal cancers Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA The bone morphogenetic protein pathway is inactivated in the majority of sporadic colorectal cancers Somatic frameshift mutations of bone morphogenic protein receptor 2 gene in gastric and colorectal cancers with microsatellite instability CRISPR–Cas9-mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes Transforming growth factor β superfamily signaling in development of colorectal cancer Signatures of mutational processes in human cancer Post, J. B. et al. Cancer modeling in colorectal organoids reveals intrinsic differences between oncogenic RAS and BRAF variants. Preprint at bioRxiv https://doi.org/10.1101/860122 (2019) Quantifying single-cell ERK dynamics in colorectal cancer organoids reveals EGFR as an amplifier of oncogenic MAPK pathway signalling Comprehensive characterization of RAS mutations in colon and rectal cancers in old and young patients NRAS status determines sensitivity to SHP2 inhibitor combination therapies targeting the Ras–MAPK pathway in neuroblastoma The Q61H mutation decouples KRAS from upstream regulation and renders cancer cells resistant to SHP2 inhibitors Performance evaluation for rapid detection of pan-cancer microsatellite instability with MANTIS Analysis of somatic microsatellite indels identifies driver events in human tumors Evaluating multiple next-generation sequencing-derived tumor features to accurately predict DNA mismatch repair status Classification and characterization of microsatellite instability across 18 cancer types Tissue-specific mutation accumulation in human adult stem cells during life Activating ERBB2/HER2 mutations indicate susceptibility to pan-HER inhibitors in Lynch and Lynch-like colorectal cancer The effects of mutational processes and selection on driver mutations across cancer types BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing Algorithmic methods to infer the evolutionary trajectories in cancer progression Long-term expansion of epithelial organoids from human colon Intra-tumour diversification in colorectal cancer at the single-cell level A colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis The different immune profiles of normal colonic mucosa in cancer-free Lynch syndrome carriers and Lynch syndrome colorectal cancer patients Immune profiling of premalignant lesions in patients with Lynch syndrome Deterministic evolution and stringent selection during preneoplasia Fast and accurate long-read alignment with Burrows–Wheeler transform Elevated mutational age in blood of children treated for cancer contributes to therapy-related myeloid neoplasms Measuring mutation accumulation in single human adult stem cells by whole-genome sequencing of organoid cultures A framework for variation discovery and genotyping using next-generation DNA sequencing data MutationalPatterns: comprehensive genome-wide analysis of mutational processes Establishment of patient-derived cancer organoids for drug-screening applications Genome engineering using the CRISPR–Cas9 system Efficient genetic engineering of human intestinal organoids using electroporation Determination of subcutaneous tumor size in athymic (nude) mice Download references Kranenburg of the Utrecht Platform for Organoid Technology (UMC Utrecht) for patient inclusion and tissue acquisition van den Brink for Wnt3a-conditioned medium and R-spondin 1-conditioned medium production and S van der Elst for help with fluorescence-activated cell sorting We also thank the members of the contributing labs for helpful suggestions and discussions This work was supported by an award from the Cancer Research UK Grand Challenge (C6307/A29058) the Mark Foundation for Cancer Research to the SPECIFICANCER team the ‘Sta op tegen Kanker (Stand Up To Cancer)’ International Translational Cancer Research grant to T.M a Dutch National Growth Fund project under grant number NGFOP22201 (Organoid Group) is a postdoctoral fellow supported by a long-term EMBO fellowship (ALTF 765-2019) is a New York Stem Cell Foundation Robertson Investigator and is supported by The New York Stem Cell Foundation Present address: Department of Gastroenterology and Hepatology Present address: Roche Pharmaceutical Research and Early Development These authors contributed equally: Tomohiro Mizutani Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht The Princess Máxima Center for Pediatric Oncology performed major experiments and analyzed the data performed the organoid culture and western blotting experiments All authors discussed the results and commented on the manuscript H.C. is the inventor of several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/ The other authors declare no competing interests Eduardo Vilar and Omer Yilmaz 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 (a) MLH1KO-1 organoids were selected by inhibition of other niche factor pathways Triple independent MLH1KO-1 organoid lines grew in complete medium (WRNE) but all of small organoids formed in all conditions died within a week (representative pictures from n = 3 clonal organoid lines) (b) Quantification of formed organoids in each condition Mean and SD (error bars) of n = 4 clonal organoid lines (c) MLH1KO-1 organoid clones established from another individual were selected with the deprivation of Wnt factor Some of organoids grew out only from MLH1KO-1 clone in -WR medium (d) Quantification of survived organoids in each condition Source data Graphs show Variant Allele Frequency (VAF) distribution of somatic mutations per organoid clone (MLH1KO-1-WR-1 and MLH1KO-1-WR-2) in WGS A VAF-filtering threshold of 0.3 (dotted line) is applied to discard mutations that are introduced after the single-cell sort Source data Lollipop diagram corresponding colorectal cancer studies selected in cBioPortal (https://www.cbioportal.org) A black dot indicated a truncating mutation; a green dot indicated a missense mutation; and a purple dot indicated other types of mutation The length of each lollipop represents the number of patients with the specific mutation Specific mutations found in MLH1KO-1 organoid are represented on top of each lollipop The medium selection experiments with the MLH1KO AXIN1/2-mutant organoids (MLH1KO-1) and each pathway driver CRISPR-mutant organoids (CRISPR-APCKO Representative pictures from n = 3 clonal organoid lines Graphs show Variant Allele Frequency (VAF) distribution of somatic mutations per organoid clone (MLH1KO-1-WR-1-N Source data Graphs show Variant Allele Frequency (VAF) distribution of somatic mutations per organoid clone (MLH1KO-1-WR-1+Nu-N-1 Source data These transcripts were compared between NRAS wildtype parental clone (WT) and NRAS-Q61K clone cultured in the medium with or without afatinib (Afa+/-) (b) Dose-response curves showing the sensitivity of NRAS-WT and NRAS-Q61K clones to inhibitors of the EGFR (Gefitinib) mTOR/PI3Kα (Everolimus/Alpelisib) and CDKs (Dinaciclib) Y-axis represent the viability of the samples while X-axis represent the drug concentration (power of 10 µM) Mean and SD (error bars) of n = 3 technical replicates for n = 2 independent organoid clones for each genetic background (c) Representative bar graphs showing the sensitivity of NRAS-WT and NRAS-Q61K organoids to different inhibitors (d) Heatmaps reporting the viability of the drug screening performed on NRAS-WT and NRAS-Q61K organoids as normalized to vehicle treated organoids Color mapping range from low viability (red) to high viability (green) (a) Triple-pathway mutant organoids were transfected with Cas9 sgRNA and the oligonucleotide previously designed for KRASG12D mutation KRASG12D mutants were selected in the medium lacking EGF with the EGFR inhibitor gefitinib (-WRNE+Nut3+Gef) Small single organoids survived and grew (Inset) (b) Picked up surviving KRASG12D mutant organoid (representative pictures from n = 3 clonal organoid lines) (c) Single survived organoids were expanded and passaged in the medium lacking EGF Representative pictures from n = 3 independent replicates (d) Sequence analysis of the targeted KRAS exon Oncogenic GGT > GAT mutation is indicated in green; silent mutations are in blue; protospacer adjacent motif (PAM) is underlined in red (a) the quadruple-pathway mutant organoids (NRASQ61K) and AKPS mutant organoids grew in complete medium (WRNE) (b) Quantification in cell numbers of the quadruple-pathway mutant organoids (NRASQ61K) and AKPS mutant organoids Mean and SD (error bars) of n = 3 clonal organoid lines Source data Download citation DOI: https://doi.org/10.1038/s43018-024-00841-x the city of Boretto experienced a historic day celebrating thanksgiving for the canonization of fellow citizen Artemide Zatti In the morning in the Basilica dedicated to St Vicar of the Lombardo-Emilian Province (ILE) and several Salesians experienced the Eucharist in honor of the new saint Postulator General for the Causes of the Saints of the Salesian Family who grew up in a family marked by deep bonds of affection and faith A relic of the new saint was solemnly carried in procession as a sign of his living presence as an intercessor and on the road companion expressed the joy of the entire civic community by sharing Pope Francis' greeting to him last Oct "You have a very important fellow citizen." was when the Postulator General noted in the book of Baptisms - where Artemide Zatti was registered on October 12 1880 - that that child was canonized by Pope Francis on 9 October 2022 emphasizing how holiness is indeed the ripe fruit of baptismal grace The celebration continued in the afternoon with the first edition of the "Color Zatti" event: a walk animated by the children of the Boretto music school that captured everyone’s attention; there was an interpretation of the figure of Artemide and the story of his life; moments of games to see who most resembles St Artemide in caring for others and who is most colorful; with the beauty of the songs of the Salesian school of the Daughters of Mary Help of Christians of Bibbiano All this accompanied by the "Hurrahs" for St Artemide and Jesus along the route that took the pilgrims to some of the most significant places in the life of St attracting the curiosity of many who stopped along the way with their mouths open as they watched the river of people pass by culminating in prayer in the basilica and ending with games always under the reassuring gaze of the Auser volunteers so beautiful that it has already given rise to the desire to organize the second "Saint Artemide gave a sign of his presence precisely by making different realities and generations walk together Holiness unites people of goodwill and eager for happiness," commented Fr Metrics details Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes We envision that our approach can be readily adapted to create in vitro models for tumorigenesis of solid human tumors for a vast number of tissues a Protein encoded by the gene CTNNB1 harbors hot-spot mutations in the N-terminus which correlate with principles of CTNNB1 degradation by kinases CK1 and GSK3 followed by proteasomal destruction by E3-ligase β-TrCP b Design of 3 sgRNA’s for introduction of hot-spot mutations in exon 3 of CTNNB1 sgRNA-1 can be used with SpCas9-ABE to introduce priming kinase mutation S45P sgRNA-2 can be used with SpCas9-ABE to introduce D32G or with SpCas9-CBE to introduce S33F and sgRNA-3 in combination with SpCas9-NG-ABE introduces T41A c Principles of hepatocyte organoid electroporation for CTNNB1 mutagenesis d–g Sanger trace and quantification of editing efficiency by Sanger sequencing of hygromycin-resistant organoids harboring CTNNB1S45P (d) Correct mutations are highlighted in green and synonymous but unintended mutations are highlighted in red h–k CBE and ABE mutated hepatocyte organoids on residue S45 (h) and D32 (k) show increased intracellular localization of CTNNB1 Source data are provided as a Source data file these results indicate that the hygromycin resistance system can be utilized to increase the percentage of mutated organoids in our cultures and enable quantification of the editing efficiency associated with different CRISPR technologies suggesting that beta catenin was translocated to the nucleus for activation of downstream target genes These data successfully demonstrate the application of ABEs and CBEs for oncogene activation in hepatocyte organoids a Amount of hot-spot mutations as quantified by the COSMIC database for somatic mutations in cancer The y-axis shows number of mutations across all tumor types in the database b CTNNB1 is localized to the plasma membrane in wild-type organoids c Illustration of the line scans through the plasma membrane and nucleus to quantify CTNNB1 immunofluorescence intensity d–f Quantification of mean relative intensity of CTNNB1 through the plasma membrane and cytoplasm/nucleus across wildtype (d) and D32G (f) genotypes (inset: representative cells for each genotype) Solid line represents the mean fluorescence intensity Error band represents standard error of the mean (s.e.m.) g The CTNNB1 intensity ratio of intracellular to plasma membrane is significantly different across different mutants when compared to the wild-type control (n = 15 cells analyzed over two independent organoid clones) Error bars represent standard error of the mean (s.e.m.) h Three-way venn diagram showing overlap of significantly enriched genes between wild type i Volcano plot showing genes that were differentially expressed between the S45P and D32G genotypes Genes that have an absolute fold change of greater than 3 have been labeled j–l Heatmaps showing transcription factors (j) and the 20-most enriched membrane-bound genes (l) selected from all significantly enriched genes extracted from the volcano plot these results confirm that hotspot mutations in the third exon of CTNNB1 lead to significant Wnt pathway activation and that both conventional and evolved forms of Cas9 can be used to introduce point mutations in human ASC-derived organoids by ABEs and CBEs a Bar graph displaying the percentage of PTEN mutations in various cancer types ordered according to frequency The endometrium harbors the highest percentage of PTEN mutations b Structure of the PTEN protein showing the different domains Mutational hotspots are highlighted in red c Sequence alignment of the PTENQ245 locus showing successful C > T transition The asterisk highlights the edited residue d Pie chart reporting the efficiency of the PTENQ245* sgRNA The number of clones per genotype is indicated e Pie chart reporting the efficiency of the PTENR130* sgRNA f Panel showing representative pictures of PTEN WT and mutant organoids PTEN nonsense mutations result in lower immunoexpression of PTEN protein and increased phosphorylated mTOR expression Scale bar 500 µm for brightfield and 50 µm for histology g Dose–response curve reporting the sensitivity to Everolimus (mTOR inhibitor) of WT and PTEN The viability is indicated on the Y-axis while the inhibitor concentration is indicated on the X-axis in logarithmic scale Data is derived from three technical replicates h Bar graph representing the IC50 of Everolimus in different organoid lines Statistics derived from three technical replicates a Principles of intestinal organoid electroporation and subsequent functional selection by growth factor manipulation b Brightfield images of organoids selected for TP53 c Sanger sequencing of APC Q1406* mutant clones that survive Wnt-pathway selection Asterisk highlights mutation of interest and PAM is shown in blue d Sanger sequencing of TP53W53* mutant clones that survive Nutlin-3 selection e Sanger sequencing of heterozygous PIK3CAE545K mutant clones that survive Meki selection f Sanger trace of homozygous PIK3CAE545K mutant clones that survive Meki selection g Quantification of mutations found in clones that are selected on Wnt-pathway withdrawal h Quantification of editing efficiency of the PIK3CAE545K guide by Sanger sequencing of 16 clones surviving only APC and TP53 selections we have established conventional and evolved variants of Cas9-CBEs and -ABEs as tools to generate organoid lines that harbor either mutations that activate oncogenes or inactivate tumor suppressors to demonstrate their combinatorial efficacy we turned to colorectal cancer tumorigenesis modeling Nutlin-3 resistant clones transfected with the W53* sgRNA revealed correct mutation on the W53 residue without off-target mutations we selected this sgRNA for subsequent multiplexing experiments a Schematic overview of the problem arising when CBE and ABE is applied using the same cas9-homolog This can be circumvented by using Sa-CBE together with Sp-ABE b Brightfield images showing hepatocyte organoid outgrowth upon Nutlin-3 selection on 3 SaKKH-sgRNA’s compared to a scrambled control sgRNA and our optimal TP53W53* sgRNA in combination with SpCas9-CBE c Sanger sequencing of hepatocyte organoids surviving Nutlin-3 selection upon transfection of SaKKH-CBE and a TP53W146* sgRNA Edited residue is highlighted with an asterisk and SaKKH PAM is shown in yellow d Quantification of editing observed in Nutlin-3 resistant clones highlights increased indel induction by SaKKH-CBE Clones with homozygous C > T mutations are shown in green and clones with indels on one of their alleles are shown in orange e Quantification of editing observed in Nutlin-3 resistant clones highlights increased amount of unintended edits upon SaKKH-CBE editing Pie-chart shows clones with homozygous C > T (green) mutations and clones harboring a C > A (red) or indel (orange) on at least one of their alleles f Brightfield images of organoids upon Cas9-homolog multiplexing using SpCas9-ABE to target CTNNB1 and SaKKHcas9-CBE to target TP53 Organoids are selected for TP53 mutations by addition of Nutlin-3 to the culture media and selected for CTNNB1 mutations by addition removal of CHIR and Rspondin-1 g Brightfield images of Cas9-homolog multiplexing Organoids are transfected with SaKKH-CBE targeting TP53 and APC and SpCas9-NG targeting PIK3CAH1047R Organoids are selected on medium lacking wnt and Rspondin-1 (rpso) and with the addition of Nutlin-3(Nut-3)Scale bars are 2000 µm h Sanger sequencing of the PIK3CAH1047R locus showing heterozygous mutation induction PAM is shown in blue and mutation is highlighted with an asterisk i Quantification of editing efficiency of the SpCas9-NG PIK3CAH1047R sgRNA by Sanger sequencing These results validated our approach for combinatorial gene targeting using Cas9 base editors derived from different species These results indicate that SaKKH-CBE can be effectively used to introduce stop codons in human ASC-derived organoids while editing outcomes other than C > T can be observed when using SaKKH-CBE high editing efficiencies can ensure that organoids harboring the desired and targeted mutations can be easily selected using Sanger sequencing mutations in APC render cells Wnt-pathway independent This is followed by mutational activation of growth pathways while TP53 mutations occur broadly in many cancer types a Brightfield images of intestinal organoids transfected with only SpCas9-CBE and a sgRNA targeting APCQ1406* and organoids transfected with our cocktail of 4 sgRNA’s targeting APC b Sanger sequencing of 96 clones surviving Wnt-pathway selection by removal of Rspondin-1 and Wnt-surrogate from culture medium Starting with a cystic structure (top) to a more dense phenotype (bottom) c Quantification of editing efficiency of our 4 sgRNA by Sanger sequencing and quadruple mutants acquired from a single transfection experiment in human intestinal organoids e Brightfield images highlighting increasingly dense morphology upon introduction of additional mutations on top of APC f Sanger sequencing traces showing induction of KRASG12S and KRASG12N mutations upon using SpCas9-NG in multiplexing five mutations common in colorectal cancer tumorigenesis g Quantification of editing efficiency of our 5 sgRNA CBE multiplexing experiment utilizing evolved Cas9 variant SpCas9-NG and quintuple mutants acquired from a single transfection experiment in human intestinal organoids using SpCas9-NG CBE This data shows that evolved Cas9 variants can be used to increase the target scope of tumor modeling in organoids By using these Cas9 variants that recognize alternative PAM motifs organoid models can be created that genotypically reflect the distinct stages of oncogenesis To study potential off-target effects of our multiplexing approach, we performed whole-genome sequencing (WGS) analysis on three colon organoid clones (M1-3) that were co-transfected with SpCas9-CBE and the cocktail of APC/TP53/SMAD/PIK3CA sgRNA’s (Supplementary dataset 2) We directly compared these data to sequentially 2 distinct electroporation events with two different sgRNA’s) a Total number of mutations accumulated during either multiplexed or sequential base editor tumor modeling in organoids Colors in the bars represent mutational signature analysis fit b Average number of mutations in either multiplexed or sequentially edited organoids n = 3 independent biological samples in case of the multiplexed experiment and n = 4 independent biological samples in case of the sequential experiment “Data are presented as mean values +/− SD” c Number of mutations in gene bodies (light) compared to expected (dark) based on their size and total amount of mutations in both SBS2-like (blue) and other (orange) observed mutations d log2 normalized depletion scores for both SBS2-like and observed mutations that do not correlate with SBS2 Target scope of adenine and cytosine base editors showing the total amount of pathogenic variants in oncogenes (e) and tumor suppressors (f) Pie-charts showing the percentage of oncogenes (g) and tumor suppressors (h) in which at least one pathogenic mutation can be modeled by adenine and cytosine base editors Asterisks show significance based on binominal testing just outside of the targeted spacer region No sgRNA-dependent off-target effects were observed in the sequentially edited cells CBE and ABE multiplexing may have a great impact on functional characterization of individual SNVs and the impact of oncogene and tumor suppressor mutation during tumorigenesis these data indicate that while CBEs sometimes result in off-target effects they remain a safe strategy to create tumor models in organoids fewer mutations are introduced in the clonal organoid lines compared to when the mutations are introduced sequentially we significantly extend their application by demonstrating the opportunities that reside in multiplexing CBE’s and ABE’s as well as Cas9 homologs and evolved Cas9 variants By performing these experiments in hepatocyte we show the versatility of this genome engineering strategy for generation of 3D models of human tumorigenesis Since base editor multiplexing can be done in a single reaction it outperforms the use of conventional CRISPR/Cas9-mediated genome engineering for tumor modeling Editing outcomes of these C > G base editors are still relatively heterogeneous but they represent a potentially valuable addition to the base editor multiplexing toolbox most mutations that we observed were actually caused by processes that are intrinsic to in vitro cell culturing We show that base editor multiplexing results in less off-target mutations by decreasing the time these organoid clones are kept in culture and the clonal steps required to achieve the desired genotype base editor multiplexing in ASC-derived organoids is a versatile tool that allows for generation of tumor models from a wide variety of tissues As these engineered organoids can be clonally expanded they could lead to a better understanding of tumorigenesis and potential strategies to develop therapeutic regimens The study was approved by the UMC Utrecht (Utrecht and the Diakonessenhuis ethical committee (Utrecht The Netherlands) and was in accordance with the Declaration of Helsinki and according to Dutch law 1% Noggin conditioned medium (U-Protein express) 0.5 μM A83-01 100 ng/ml FGF10 (Peprotech) and was supplemented with 100 μg/ml Primocin (Invivogen) expansion medium was refreshed every 2 days Outgrowing organoids were mechanically passaged with a glass Pasteur pipet every week Domes of cultrex Pathclear Reduced Growth Factor Basement Membrane Extract (BME) were covered with the following expansion medium: Advanced DMEM/F12 (Gibco) 100 μU/ml penicillin–streptomycin and 1× B27 minus vitamin A (all supplied by Thermo Fisher Scientific) plus 15% RSPO1 conditioned medium (home-made) and 20 ng/ml TGFa and was supplemented with 100 μg/ml Primocin (Invivogen) Expansion medium was refreshed every 2 days and organoids were split 1:4 every week cells were resuspended in BME and seeded in a pre-warmed 48-wells plate at 20 µl drop per well pre-warmed culture medium (supplemented with Y-27632) was added and the plate was imaged at 24 h to monitor GFP expression selection based on transfection (Hygromycin B gold InvivoGen) or by phenotype was started APC mutations were selected for by removal of Wnt-Surrogate and Rspo1-conditioned medium TP53 mutations were selected for by addition of Nutlin-3 and PIK3CA mutations were selected for by addition of Meki to the culture medium Organoid RNA was reverse-transcribed and subjected to SYBR Greenbased quantitative real-time PCR (qPCR) using the forward and reverse primers described in Supplementary Table 5 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (Hprt) were used as housekeeping genes Relative gene expression levels were calculated as ΔCt values (Ct ‘target’ minus Ct ‘housekeeping gene’) and in most analyses compared between ‘sample’ and ‘reference’ to express fold change Mutant organoids were cultured in medium without Wnt/Rspondin for 24 h following which RNA was extracted using commercially available kit (Qiagen RNeasy) The quality of RNA was checked on an RNA pico chip and only high-quality RNA was used for library preparation and sequencing The raw data was aligned to the human genome assembly using STAR aligner (ref) following read trimming using Cutadapt (ref) RNA-seq analysis was performed using DESeq2 (ref) in R programming language Genes that were upregulated more than 2-fold were considered differentially expressed All heatmaps and plots were also made in R the organoids were recovered from BME and enzymatically dissociated in TrypLE for 3 min at 37 °C dissociated organoids were resuspended in BME and allowed to recover for 2 days in a medium containing a low concentration of Egf (0.5 ng/ml) and Fgf10 (1 ng/ml) 40 µl of pre-warmed dispase were added per ml of culture medium and the organoids were incubated for 1 h at 37 °C the organoids were collected and washed with ice-cold Advanced DMEM+++ counted and resuspended at a density of 1000 organoids per well in culture medium containing 5% (v/v) BME and without Egf and Fgf10 We finally dispensed 40 µl per well of the organoids containing medium using a ThermoFisher multidrop dispenser in a 384-wells plate and the drugs were added with an HP D300e digital dispenser 30 µl of Cell Titer Glo (Promega) were added to each well and the plate was incubated for 20 min at RT before the luminescence was read with a Tecan SPARK luminescence detector Hepatocyte organoids were harvested using Cell Recovery solution (Corning The organoids were allowed to settle to the bottom of the tube after which the supernatant was removed and replaced with Formalin washed twice with PBS containing 0.5% Triton X-100 and blocked in buffer supplemented with 0.5% BSA The samples were incubated with the primary antibody solution (Rabbit anti-CTNNB1 Cat#Sc-7199; RRID: AB_634603) overnight at 4 °C and washed three times the next day in PBS-0.5% Triton They were then incubated with the secondary antibody solution (Goat anti-rabbit Alexa Fluor 647 the organoids were mounted in Vectorshield (Vector labs) and imaged using a Leica SP8 confocal microscope (63x water objective) Images were saved as LIF files and imported in FIJI (v 1.53) for downstream processing Endometrium organoids were fixed overnight in 4% paraformaldehyde at 4 °C followed by dehydration and paraffin embedding To prepare organoids for histological stainings intact BME drops containing organoids were collected from the culture plates and incubated in Cell Recovery Solution (Corning Organoids were then allowed to settle to the bottom of the tube by free gravitation supernatant removed and the material fixed in 4% paraformaldehyde at room temperature for 1 h Sections were cut and hydrated before staining Sections were subjected to H&E staining or immunohistochemistry with antibodies Rabbit anti-human PTEN (Cell Signaling Technology 9552 1:200) for PTEN and Mouse anti-human p-mTOR (Santa Cruz sc-293133 The images were acquired on Leica DM4000 microscope and processed using Leica LAS X software we calculated the intensity profile along lines drawn through individual cells of different organoids using FIJI (v1.53) (Analyze>Plot Profile) We wrote a custom script in R programming language to identify the membrane boundaries for each cell in an unbiased manner using Phalloidin signal as a reference we calculated the mean intensity within the membrane (“plasma membrane”) and between membranes (“intracellular”) and normalized them to the maximum intensity observed for each genotype to plot intensities for cells of different widths on the same axis we separately normalized the length of all membranes and intracellular regions All plots were made using the package ggplot in R Statistical significance was calculated using ANOVA followed by Tukey’s HSD variants were multisample called with the GATK HaplotypeCaller v4.1.3.0 and GATK-Queue v.4.1.3.0 based on default settings and the additional option “EMIT_ALL_CONFIDENT_SITES.” Subsequently GATK VariantFiltration v4.1.3.0 was used to evaluate the quality of the variant positions To control for removal of true variants in clusters all variants that failed the SnpCluster filter were retrieved as well the same potential off-target genomic regions were intersected with all start and end coordinates of the structural variations called by GRIDSS-purple-linx To exclude the formation of driver mutations by off-target base editor activity all clone-specific mutations were filtered for predicted effect levels ‘HIGH’ or ‘MODERATE’ based on the variant annotation as described above variants were filtered for presence in the Cancer Gene Census (COSMIC No statistical methods were used to predetermine sample size The experiments were not randomized and the investigators were not blinded to the sample allocation during experiments and outcome assessment Value of n is always displayed in the figure as individual data points Sanger sequencing validation for editing efficiency was always performed in clones derived from at least two individual transfection experiments to ensure reproducibility Statistical analysis was performed with the GraphPad Prism 9 software All schematics in this manuscript are created using Adobe Illustrator 2023. Part of Fig. 1c was created with the use of Biorender.com Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article All software tools used for sequencing data analysis can be found online at: https://github.com/ToolsVanBox. The code that we have used to calculate the targeting scope of base editors in tumor modeling can be found in Supplementary code 1 Modeling development and disease with organoids Patient-derived organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer An organoid biobank of neuroendocrine neoplasms enables genotype-phenotype mapping Cancer modeling meets human organoid technology Oral mucosal organoids as a potential platform for personalized cancer therapy A rectal cancer organoid platform to study individual responses to chemoradiation Patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients Organoid profiling identifies common responders to chemotherapy in pancreatic cancer Patient-derived organoids model treatment response of metastatic gastrointestinal cancers Patient-derived organoids predict chemoradiation responses of locally advanced rectal cancer Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids CRISPR-Cas9-mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes CRISPR engineering in organoids for gene repair and disease modelling Evolved Cas9 variants with broad PAM compatibility and high DNA specificity Engineered CRISPR-Cas9 nucleases with altered PAM specificities Engineered CRISPR-Cas9 nuclease with expanded targeting space In vivo genome editing using Staphylococcus aureus Cas9 Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity RNA-guided human genome engineering via Cas9 Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9 CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage Programmable base editing of A T to G C in genomic DNA without DNA cleavage Base editing: precision chemistry on the genome and transcriptome of living cells Long-term expansion of functional mouse and human hepatocytes as 3D organoids Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche B-Catenin Signaling and Cancer 1021–1030 (1999) Optimized base editors enable efficient editing in cells One-step generation of conditional and reversible gene knockouts assembly and deconvolution of sanger chromatogram trace files Identification of novel human Wnt target genes using adult endodermal tissue-derived organoids NOTUM from Apc-mutant cells biases clonal competition to initiate cancer Up-regulation of REG3A in colorectal cancer cells confers proliferation and correlates with colorectal cancer risk Role of regenerating islet-derived protein 3A in gastrointestinal cancer invasion and angiogenesis by targeting the PKA pathway in hepatocellular carcinoma RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-β2 CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations Altered PTEN Expression as a diagnostic marker for the earliest endometrial precancers Predicting base editing outcomes with an attention-based deep learning algorithm trained on high-throughput target library screens PIK3CA and PTEN mutations in uterine endometrioid carcinoma and complex atypical hyperplasia The mutational landscape of normal human endometrial epithelium Spatiotemporal dynamics of clonal selection and diversification in normal endometrial epithelium Clinical actionability of molecular targets in endometrial cancer base editors induce genome-wide off-target mutations in rice ClinVar: Improving access to variant interpretations and supporting evidence COSMIC: the catalogue of somatic mutations in cancer Probing the tumor suppressor function of BAP1 in CRISPR-engineered human liver organoids Mutational landscape and significance across 12 major cancer types CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells Search-and-replace genome editing without double-strand breaks or donor DNA Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids Enhanced prime editing systems by manipulating cellular determinants of editing outcomes Engineered pegRNAs improve prime editing efficiency Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos Next-generation surrogate wnts support organoid growth and deconvolute frizzled pleiotropy in vivo Driehuis, E., Kretzschmar, K. & Clevers, H. Establishment of patient-derived cancer organoids for drug-screening applications. Nat. Protoc. 1–30. https://doi.org/10.1038/s41596-020-0379-4 (2020) Unscrambling cancer genomes via integrated analysis of structural variation and copy number Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases BEDTools: the Swiss-army tool for genome feature analysis Download references Present address: Miller Institute for Basic Research in Science Present address: Princess Maxima Center for Pediatric Oncology Present address: Pharma Research Early Development These authors contributed equally: Maarten H Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center Utrecht Princess Maxima Center for Pediatric Oncology performed sanger-based genotyping reactions and organoid cell culturing were responsible for WGS-based off-target analysis under the supervision of R.v.B aided in experimental design and data analysis regarding hepatocyte and endometrium experiments aided in securing material for hepatocyte organoids This study was performed under the supervision of J.H.v.E H.C. is an inventor on several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/ is currently head of pharma Research Early Development (pRED) at Roche holds several patents on organoid technology followed by their publication numbers (if applicable) The remaining authors declare no competing interests Nature Communications thanks Nozomu Yachie reviewer(s) for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-023-40701-3 (ANS – Boretto) – As a sign of homage to the first Salesian Brother proclaimed a saint by the Church – Saint Artemides Zatti – the Rector Major of the Salesians of Don Bosco on the occasion of the first anniversary of his canonisation which took place on 9 October 2022 in St Peter's Square The Rector Major arrived at the city basilica of San Marco in the early afternoon welcomed by representatives from the city and diocesan community already prepared to reflect on the figure of Zatti from the presentation of the book Artemide giocava a campana (a text written with the contribution of children from the primary schools in Boretto and Brescello) which had been held in the morning of the same day at the municipal theatre starting with the Superior of the Lombard-Emilian Salesian Province (ILE) attended the presentation of the exhibition on Zatti curated by Fr Erino Leoni used all the parts of a bike to present the seventeen panels in an impassioned explanation involving theology and spiritual life that captured the attention of the community gathered in the basilica: from the drive chain to the handlebars The presentation was also attended by the Postulator General for the Causes of Saints of the Salesian Family who highlighted two aspects of his holiness: his complete attention to the individual and the fact that he was a great man of communion immediately after the conclusion of the first meeting on Saturday afternoon a provocation: the paintings do not follow a documentary structure but rather the three actions of the most famous motto that Saint Zatti experienced personally: “I believed edifying and informal moment of listening took place in the church with Fr Á.F who engaged in dialogue with the pastoral community displaying the qualities of a well-practised educator Answering the questions from some lay people – Giulia Marco – the Salesian cardinal addressed a varied range of topics: school teaching Fr Artime offered several pieces of advice from his pastoral experience and urged them to continue “practising face to face” and to cultivate  tenderness and loving-kindness The first day of his weekend in Boretto ended for the Rector Major with a time for informal greetings and the friendly response to requests for photos and selfies waiting for the completion of the next day's activities with the celebration of the Sunday Eucharist for the community and the reception of Boretto's honorary citizenship SAN DIEGO (KGTV) — This Season of Hope story is more of a journey. A journey that begins on the rocky island of Grytoya in northern Norway How the ring traveled all the way from that rugged Norwegian island is a bit of a miracle It has its own powers I think," says Abby Pilger Boretto with a smile Abby Boretto couldn't be happier to have that ring "Captain Easy" they called him at Annapolis, Md., a star athlete and student fromSyracuse he received his letter of recommendation to the Naval Academy by Robert F Henry Pilger would become a helicopter pilot and in September 1972 Henry Pilger was killed in a helicopter crash during a NATO training exercise on the island of Grytoya "I do like to look at it," Abby says trying to hold back tears It certainly had the power to return to her and to bring people together "This thing is like a beacon," Abby says admiring the ring "It was wedged between two rocks for 22 years." That little beacon of a ring lay dormant for over two decades until it was spotted by Hans Krogstad in 1993 Hans Krogstad during a recent Zoom video call Hans Krogstad lives in Harstad, Norway. He was hunting grouse on the island of Grytoya in '93 when... "And then I look down and between two small boulders, I saw this ring," adds Krogstad. "It was in mint condition because it was golden so I picked it up and it's a massive big ring." But it's what Hans noticed on the inside that struck him most. Engraved was the name Henry N. Pilger. "My first thought was this ring must be returned to the family," says Krogstad. So, Hans started his own journey to find the ring's rightful owner. With no internet in the early '90s, it was a slow one researching the crash and writing letters. He eventually placed the ring in a brown envelope to the U.S. Embassy in Oslo, Norway. That was Dec. 17, 1993. He was never told if the family ever received it. "I used to go hunting there every autumn in that mountain," adds Krogstad. "And every time I came near to the sight, I got kind of a flashback and would start thinking, 'I wonder what happened to that ring.'" It wasn't until this summer Hans would have his answer. Abby, about to turn 50, was going through memories of her life when she came across the brown envelope and the letter from Hans her mother had tucked away for years. "It just sort of struck me like, 'We never said thank you to this person.' It's just wrong," says Boretto. Abby began her own journey to find Hans, but little did she know he was still trying to find her. That's when she got a call, out of the blue, from the Naval Academy, asking if she had the ring. A man named Hans Krogstad wanted to know. "I remember thinking, what! Huh! How is this possible!" adds a stunned Boretto. The two have since connected but never spoken or seen each other in person. They're saving that moment for a special day. The ring is still working its magical powers. "It's pretty amazing," says Krogstad with a smile. "Yes, it is, and I'm so glad that she made that connection with her father." Abby's story began to get out. And since then, she has heard from her father's classmates at the Naval Academy and high school. It's as if she's being reintroduced to her father. "I'm meeting him for the first time," says Boretto with a satisfied smile. "This is the next best thing. I get to live vicariously through these letters from his friends." All because of the magical little ring. Connecting people from across the globe in this season of hope. "I want people to know that at the end of the day hope is all we have," adds Boretto. "People should not give up. There's always a positive side to everything. It might take some time, it took me 49 years, so I think it's pretty exciting for me." Abby Pilger Boretto and Hans Krogstad are making plans to meet later next year. They'll reconnect on the island where her father died, the two of them with family, and of course the ring. Volume 10 - 2020 | https://doi.org/10.3389/fcimb.2020.00416 The obligate intracellular bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections trachomatis undergoes a biphasic developmental cycle within a membrane-bound compartment Successful establishment of the intracellular niche relies on bacterial Type III effector proteins In vitro and in vivo systems have contributed to elucidating the intracellular lifestyle of C but additional models combining the archetypal environment of infection with the advantages of in vitro systems are needed Organoids are three-dimensional structures that recapitulate the microanatomy of an organ's epithelial layer bridging the gap between in vitro and in vivo systems Organoids are emerging as relevant model systems to study interactions between bacterial pathogens and their hosts we took advantage of recently developed murine endometrial organoids (EMOs) and present a C trachomatis-murine EMO infection model system Confocal microscopy of EMOs infected with fluorescent protein-expressing bacteria revealed that inclusions are formed within the cytosol of epithelial cells trachomatis strain that allows for the tracking of RB to EB transition indicated that the bacteria undergo a full developmental cycle which was confirmed by harvesting infectious bacteria from infected EMOs the inducible gene expression and cellular localization of a Chlamydia Inc protein within infected EMOs further demonstrated that this model is compatible with the study of Type III secreted effectors we describe a novel and relevant system for the study of Chlamydia-host interactions While each of the above-mentioned systems has its benefits additional model systems that better combine the advantages of in vitro systems and in vivo models would be valuable to gain a better understanding of the molecular mechanisms underlying the interaction of C Despite the great potential of EMOs to increase the relevance of the study of STIs they have not been used in the context of infections with pathogens we describe a novel murine EMO model system to study Chlamydia infection trachomatis successfully infects and completes its developmental cycle within murine EMOs we show that this system is compatible with inducible gene expression and detection of tagged Chlamydia Inc proteins by immunofluorescence microscopy trachomatis-EMO model system has the potential to preserve the tools of in vitro models while contributing aspects of the cellular environment of in vivo systems All genetic manipulations and containment work were approved by the UVA Biosafety Committee and are in compliance with the section III-D-1-a of the National Institutes of Health guidelines for research involving recombinant DNA molecules Mice were not bred for the sole purpose of harvesting endometrial tissues Endometrial tissues for EMO generation was sourced from otherwise discarded murine reproductive tracts The method for generating murine endometrial organoids was adapted from Boretto et al. as follows (Boretto et al., 2017) Uterine horns were isolated from otherwise discarded genital tract tissue from naïve 6–8 week-old female C57BL/6J mice Tissue fragments were incubated in 30 mM ethylenediaminetetraacetic acid (EDTA) in PBS for 1 h at 37°C followed by mechanical dissociation in cold PBS Dissociated tissue and supernatant were passed through a 70 μm cell strainer (Corning) to isolate endometrial glands and supplemented with 10% heat inactivated FBS Isolated glands in PBS/FBS were centrifuged at 230 g for 5 min The sample was centrifuged at 230 g for 5 min and the pellet was resuspended in mixture of 70% growth factor reduced Matrigel (Corning) in DMEM Ten to twenty microliters drops of the suspension were plated onto pre-warmed non-treated 24 well plates (1–2 drops per well) and incubated at 37°C for ~20–30 min to allow for Matrigel solidification Mouse IntestiCultTM organoid growth medium (STEMCELL Technologies) supplemented with 1X penicillin-streptomycin-glutamine (PSQ; Gibco) was added to cover the Matrigel drops Cultures were maintained at 37°C with 5% CO2 and culture medium was changed every 2 days EMOs were passaged every 7–14 days and TrypLETM Express Enzyme (1X) without phenol red (Gibco) was added for 15 min at 37°C TrypLETM was inactivated by addition of DMEM EMOs were collected and centrifuged at 230 g for 5 min at 4°C The pellet was resuspended in DMEM and subjected to mechanical disruption to fully dissociate EMOs into single cells Dissociated cells were centrifuged at 230 g for 5 min at 4°C and the pellet was resuspended in 70% Matrigel in DMEM and plated as described above Culture medium supplemented with 1X PSQ and 10 μM Y-27632 (ROCK inhibitor; Millipore Sigma) was added Culture medium containing ROCK inhibitor was used for the first subsequent medium change culture medium was replaced with medium free of PSQ and cold PBS was added to dissolve Matrigel followed by vigorous pipetting to break apart EMOs The fragmented EMOs were incubated in DMEM for 15 min at 37°C for 5% CO2 followed by centrifugation at 290 g for 5 min The pellet of ~50–100 fragmented EMOs was incubated with ~5 × 109 inclusion forming units (IFU) of the indicated strain of C trachomatis in 100 μl of DMEM for 1 h at 37°C to allow for infection Matrigel was added to generate a 70% Matrigel mixture which was plated as described above IntestiCultTM without PSQ or Rock inhibitor was used Uninfected or infected EMOs embedded in Matrigel were plated in eight well-chambered coverglass wells (Thermo Scientific Nunc) EMOs were fixed in 1X PBS containing 4% paraformaldehyde for 1 h at room temperature (RT) Antibodies and fluorescent dyes were diluted in PBS containing 0.5% Triton X-100 and 5% bovine serum albumin (BSA) Incubation with primary and secondary antibodies was conducted overnight at 4°C and 1–2 h at RT Primary and secondary staining of fixed samples was followed by three washes with 1X PBS HeLa cells seeded onto glass coverslips were fixed for 20 min in PBS containing 4% paraformaldehyde Coverslips were stained with Hoechst dye diluted in PBS containing 0.1% Triton X-100 for 1 h at RT Coverslips were mounted with DABCO antifade-containing mounting media Samples were imaged with a Nikon epifluorescence microscope or a Leica DMi8 confocal microscope equipped with the Andor iXon ULTRA 888BV EMCCD camera and driven by the IQ software live samples were imaged in a humidified live cell environmental chamber set at 37°C and 5% CO2 Images were processed using the Imaris software (Bitplane The following primary antibodies and dyes were used: goat polyclonal anti-Chlamydia major outer membrane protein (MOMP; 1:200 Virostat) and mouse monoclonal anti-FLAG (1:1,000 The following secondary antibodies were used: Alexa FluorTM 488-conjugated donkey anti-goat IgG (1:500 Molecular Probes) and Alexa FluorTM 488- or 594-conjugated goat anti-mouse IgG (1:500 The following dyes were used: CellMaskTM Green (1:200 EMOs were infected with mCherry-expressing C the culture medium was removed and replaced with cold water to dissolve the Matrigel followed by vigorous pipetting to break apart and lyse epithelial cells of the EMOs and release the bacteria Bacteria and unlysed cells were collected and centrifuged at 8,000 rpm for 20 min at 4°C Pelleted sample was resuspended in cold water followed by additional vigorous pipetting and centrifugation with the same conditions to ensure that all infected cells were lysed Pelleted sample was resuspended in RT DMEM supplemented with 10% FBS and plated onto a confluent HeLa cell monolayer followed by centrifugation at 1,200 rpm for 20 min at RT HeLa cells were incubated with harvested EBs for 32 h at 37°C in the presence of 5% CO2 trachomatis expressing mCherry constitutively and IncV-3xFLAG under the control of the anhydrotetracycline inducible promoter the culture medium was replaced with culture medium supplemented with 20 ng/ml aTc Infected EMOs were incubated in the presence of aTc at 37°C with 5% CO2 for 6 h Images presented in this study are representative of multiple infected EMOs analyzed from at least three independent experiments performed with independent EMO preparations (A) Cartoon diagram depicting the method used to generate EMOs from female C57BL/6J mice (B) Brightfield image of mature murine EMOs embedded in Matrigel following passage and expansion (C–E) Confocal micrographs of a live EMO stained with CellMaskTM Green to mark plasma membranes A top view of a 3D reconstruction of an EMO (C) a side view of a 3D reconstruction of an EMO (D) and a single plane across the middle of an EMO (E) are shown which were indicated by mCherry fluorescence and SYTOXTM Green staining were detected within the cytosol of individual cells trachomatis successfully infects murine EMOs and forms inclusions within the cytosol of epithelial cells (A) Cartoon diagram depicting the method of infecting EMOs with C (B) Confocal micrographs of an EMO infected with mCherry-expressing C Infected EMOs were fixed at 32 hpi and immunostained with anti-C Higher magnification of the indicated boxed area is shown in the inset of each panel A single plane across the middle of the inclusion is shown (C) Confocal micrographs of an EMO infected with mCherry-expressing C mCherry) fixed at 32 hpi and stained with SYTOXTM Green (green A single plane across the middle of the EMO and inclusions is shown Bottom panels: higher magnification of indicated boxed area The presence of mCherry-positive inclusions within the HeLa cell monolayer indicated that infectious EBs were indeed produced within EMOs trachomatis undergoes a full developmental cycle and generates infectious progeny within murine EMOs trachomatis completes a full developmental cycle in murine EMOs (A) Live epifluorescence micrographs of two different EMOs infected with the RB-to-EB reporter strain of C trachomatis for 24 h (top panels) or 48 h (bottom panels) Bacteria at the RB stage expressing mCherry alone are shown in red (left panels and bacteria that have transitioned to EB stage expressing GFP are shown in green (middle panels (B) Immunofluorescence image of a HeLa cell monolayer infected with mCherry-expressing C trachomatis (red) collected from infected EMOs at 48 hpi the infected HeLa cells were fixed and stained with the Hoechst DNA dye (blue) The ability to induce and detect the expression of IncV-3xFLAG in infected EMOs demonstrates that the EMO model system is compatible with the study of the cellular localization of Chlamydia inclusion membrane proteins and Induction of gene expression and inclusion localization of an Inc protein within infected murine EMOs (A) 3D confocal micrographs of an EMO infected for 30 h with mCherry-expressing C trachomatis constitutively (red) and IncV-3xFLAG under the control of an aTc inducible promoter (green) A low magnification merge image corresponding to the whole EMO is shown on the left (Whole EMO) A high magnification of individual channels and the merge image of the indicated boxed area is shown in the three panels to the right Scale bar: 20 μm (B) Confocal micrographs of an EMO infected for 30 h with C trachomatis expressing mCherry constitutively (red) and IncV-3xFLAG under the control of an aTc inducible promoter (yellow) A single plane across the middle of the EMO and inclusion is shown Much of the current understanding of the molecular processes used by Chlamydia to interact with its host cell comes from in vitro systems (Dolat and Valdivia, 2019) Although these models are more easily maintained and manipulated than in vivo models they do not fully recapitulate the cell-cell interactions and 3D nature present in the natural environment of the infected tissues EMOs present an ex vivo model system that bridges the gap between less relevant in vitro studies and less tractable in vivo systems Our data demonstrate that murine EMOs can serve as a novel model system in which to study Chlamydia host-pathogen interactions Microinjection is especially appropriate for invasion or early infection studies and offers the advantage of minimally disrupting the integrity of the EMO at the time of infection It also allows for a tight control of the number of bacteria delivered in the EMO lumen is infection of mechanically fragmented organoids This approach leads to a temporary disruption of the 3D architecture of the EMOs and does not allow for a precise control of the MOI which would preclude it from invasion studies the cellular organization of each EMO fragment is preserved and post-invasion stages of the infection can be monitored in the 3D environment of the primary cell epithelium of the resealed EMO This method is also less technically challenging and does not require any special equipment trachomatis infected EMOs indicated that Chlamydia undergoes a productive developmental cycle in EMOs we observed ruptured inclusions (not shown) however this phenomenon was not accompanied by the formation of secondary inclusions 72 h post-infection Further studies will be required to determine if this phenomenon is due to technical limitations of keeping infected EMOs alive for a long period of time or reflective of the response of EMOs to infection and/or inclusion lysis and the methods developed here for infecting murine EMOs could be easily translatable our results indicate that murine EMOs can be used as a novel system with which to study C This model offers a simple system to study direct interactions between C trachomatis and the epithelial cells of the endometrium providing the relative ease of maintenance and microscopy methods of in vitro cell culture as well as the tissue architecture and relevant cell types of an in vivo model The datasets generated for this study are available upon request to the corresponding author and HV provided expertise and edited the manuscript All authors contributed to the article and approved the submitted version This work was supported by National Institute of Allergy and Infectious Diseases grants AI007046 to RB and AI146649 and AI101441 to ID 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 Laura Shankman and Samantha Fleury (University of Virginia) for assistance with organoid culturing techniques and Francesca Azar (University of Virginia) for assistance in providing otherwise discarded murine endometrium tissues and Rachel Ende (University of Virginia) for critical reading of the manuscript PubMed Abstract | CrossRef Full Text | Google Scholar Expression of the effector protein IncD in Chlamydia trachomatis mediates recruitment of the lipid transfer protein CERT and the endoplasmic reticulum-resident protein VAPB to the inclusion membrane Modeling infectious diseases and host-microbe interactions in gastrointestinal organoids PubMed Abstract | 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Chlamydial infection from outside to inside PubMed Abstract | CrossRef Full Text | Google Scholar Differences in Chlamydia trachomatis serovar E growth rate in polarized endometrial and endocervical epithelial cells grown in three-dimensional culture Characterization of estrogen-responsive epithelial cell lines and their infectivity by genital Chlamydia trachomatis Risk of sequelae after Chlamydia trachomatis genital infection in women A co-infection model system and the use of chimeric proteins to study Chlamydia inclusion proteins interaction Growth and effect of chlamydiae in human and bovine oviduct organ cultures Mechanisms of host cell exit by the intracellular bacterium Chlamydia An in vitro model for immune control of chlamydial growth in polarized epithelial cells Enterohemorrhagic Escherichia coli reduces mucus and intermicrovillar bridges in human stem cell-derived colonoids A human fallopian tube model for investigation of C The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids Chronic Chlamydia infection in human organoids increases stemness and promotes age-dependent CpG methylation Chlamydia trachomatis disturbs epithelial tissue homeostasis in fallopian tubes via paracrine Wnt signaling Expression of secretory leukocyte protease inhibitor and elafin in human fallopian tube and in an in-vitro model of Chlamydia trachomatis infection Persistence and toxin production by Clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function Evolution and conservation of predicted inclusion membrane proteins in chlamydiae Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells PubMed Abstract | Google Scholar Lentivirus-based stable gene delivery into intestinal organoids Global mapping of the inc-human interactome reveals that retromer restricts Chlamydia infection The chlamydial inclusion preferentially intercepts basolaterally directed sphingomyelin-containing exocytic vacuoles Reconceptualizing the chlamydial inclusion as a pathogen-specified parasitic organelle: an expanded role for Inc proteins Interaction of Chlamydiae and host cells in vitro PubMed Abstract | CrossRef Full Text | Google Scholar Chlamydia trachomatis inclusion membrane protein MrcA interacts with the inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) to regulate extrusion formation Characterization of the growth of Chlamydia trachomatis in in vitro-generated stratified epithelium Seventy years of Chlamydia vaccine research–limitations of the past and directions for the future Pleguezuelos-Manzano Mutational signature in colorectal cancer caused by genotoxic pks(+) E Gastric organoids: an emerging model system to study Helicobacter pylori pathogenesis Chlamydia trachomatis disrupts N-cadherin-dependent cell-cell junctions and sequesters β-catenin in human cervical epithelial cells Hypoxia abrogates antichlamydial properties of IFN-gamma in human fallopian tube cells in vitro and ex vivo Three-dimensional organotypic culture: experimental models of mammalian biology and disease Three temporal classes of gene expression during the Chlamydia trachomatis developmental cycle tethers the endoplasmic reticulum to the pathogen-containing vacuole Frizzled proteins are colonic epithelial receptors for C Expression and localization of predicted inclusion membrane proteins in Chlamydia trachomatis Organoid and enteroid modeling of Salmonella infection CrossRef Full Text | Google Scholar Integrated phosphoproteome and transcriptome analysis reveals Chlamydia-induced epithelial-to-mesenchymal transition in host cells Salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions Conservation of extrusion as an exit mechanism for Chlamydia Vankelecom H and Derré I (2020) Murine Endometrial Organoids to Model Chlamydia Infection Received: 04 May 2020; Accepted: 07 July 2020; Published: 14 August 2020 Copyright © 2020 Bishop, Boretto, Rutkowski, Vankelecom and Derré. 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: Isabelle Derré, aWQ4bUB2aXJnaW5pYS5lZHU= Royal Netherlands Academy of Arts and Sciences 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 Drought blighting country’s longest waterway continues as economic hub battles climate crisis When the amateur photographer Alessio Bonin had a few hours to spare one afternoon in late March he decided to venture to a nature reserve in Gualtieri an Italian town on the banks of the Po River in Emilia-Romagna The drought that had been blighting the country’s longest waterway since one of its driest winters was showing no sign of easing The 450-mile (650km) Po flows from the Alps in the north-west nourishing several Italian regions before entering the Adriatic But unusually low water levels had transformed it affecting everything from the production of tomatoes and watermelons to hydroelectric power of a 50-metre cargo boat sunk during the second world war emerging from its watery grave that brought home the gravity of the drought to those living in Gualtieri and nearby towns along the river View image in fullscreenA photo taken by Alessio Bonin using a camera attached to a drone of a sunken cargo boat that has re-emerged from the Po. Photograph: Alessio BoninView image in fullscreenAnother view of the cargo boat Photograph: Alessio Bonin“In recent years you could see the bow of the boat “I’ve never seen such a drought at this time of year – our main worry used to be our river flooding including a German tank found close to Mantua and the remains of an ancient hamlet in Piedmont have also re-emerged amid what Italy’s river observatory said last week was the worst drought to affect Po valley in 70 years The drought is caused by higher-than-usual temperatures scant rainfall and much less snow during the winter which in turn contributed to lower snowmelt flowing into the Po The situation is so acute that leaders of Lombardy, Piedmont, Veneto and Emilia-Romagna said on Thursday they would ask for a state of emergency to be declared in their regions. Some northern towns require water supplies to be delivered by trucks, and calls have been made for 125 towns to ration supplies of drinking water to restore reservoir levels The river’s depth currently measures up to 2.7 metres below the zero gauge while its flow rate into the sea has slowed to 300 cubic meters a second – a fifth of the average for this time of year The Po valley experienced droughts in 2007 and scientists say their growing prevalence is a further indication of the climate crisis “This drought is unique in history due to the combination of two anomalies – the lack of rain which is directly linked to climate change,” said Luca Mercalli the president of the Italian Meteorological Society View image in fullscreenThe drought affecting the Po is the worst in the region for decades Photograph: Piero Cruciatti/AFP/GettyMercalli added: “It’s as if it’s the end of July in the Alps water is running out as little snow remains and in a week’s time there will be no more reserves We recently measured the snow level at a 3,000-metre altitude – usually in June there are two metres but this year not only is there no snow but flowers are already blooming “The situation is only going to get worse as the next few months are forecast to be hot and dry.” The Po valley is a crucial economic area for Italy, enabling industrial hubs such as Turin, Milan and Brescia, along with a vast variety of sectors, to thrive, and is one of the most important agricultural zones in Europe Gualtieri is a pertinent example of how small communities flanking the river depend on it The nature reserve where the submerged cargo ship which was built in Venice and used to transport grain between the Atlantic and Cremona in Lombardy came to light was the same area where Italian soldiers who had survived German concentration camps created jobs in timber upon their return home hence it later became known as Isola Degli Internati (Island for Prisoners) View image in fullscreenParched land under a bridge on the Po riverbed in Boretto Photograph: Luca Bruno/AP“Our veterans needed work and this area was rich in willow trees,” said Renzo Bergamini “They were used to build infrastructure to protect the river’s embankment.” Bergamini was born in Gualtieri and said climatic events over the past decade had become more abrupt “If before we used to get consistent rainfall over 10 months in more recent years it has come in two or three heavy bursts over a shorter period of time turning the river into a torrent,” he said “Today we’re worrying about the lack of water which doesn’t only serve farmers for irrigation but energy production and human nutrition – in this area we extract drinking water from aquifers in the Alps but in some areas it is also extracted from the Po and purified.” a man who usually swims across the river every day was able to walk to the middle of it last week “He is known as the god of the Po,” said Jennifer Bacchi a company that organises boat tours and fishing trips “You used to see a lot more ships in this stretch either for the transport of goods or passengers we’re in a motorway of water that is completely empty.” She said there had been a high rate of holiday cancellations among anglers due to the low water level Boretto really depends on fishing tourism.” a manager for commercial shipping at the Boretto unit of Aipo an agency that provides engineering and environmental services for the Po said navigation of cargo vessels in the area had to be suspended because of the drought Please enable JS and disable any ad blocker (ANS - Boretto) - On Sunday 8 October 2023 in a day full of symbolic and meaningful gestures and after a heartwarming Eucharistic celebration participated in by the entire city community received the honorary citizenship of the municipality of Boretto and thus became a fellow citizen of the 'most famous Boretto resident in the world' the Salesian lay Brother Saint Artemide Zatti The events on Sunday opened with a procession in homage to Zatti through the streets of the municipality presided over by the Tenth Successor of Don Bosco Among the latter was the presence of the Provincial of Lombardia-Emilia (ILE) accompanied by the Rectors of his Province and the Provincial Superiors of the Daughters of Mary Help of Christians (FMA) of Lombardia and Emilia the parish priest and head of the local pastoral unit emphasized that saints are not wax statues in museums but still walk alongside the humanity of today He noted that the very knowledge of the fact there is also a nurse specializing in material and spiritual assistance to the very poor he invited the people to realize that they have these powerful intercessors present and always close at hand in the hope that even in Zatti's birthplace there will be at least a little of the popularity and devotion that he is accorded in what became his adopted homeland Then the Vicar General of the Archdiocese of Reggio Emilia-Guastalla praised the example offered by Zatti in living the Salesian "Da mihi animas" and expressed the Archdiocese's joy at having a "great joy and an invitation to conversion" after expressing the archdiocese's gratitude to the Salesian community for its educational work he concluded: 'thanks to our dear Saint Artemide also feel even closer to the Sons of Don Bosco scattered around the world' the Rector Major explained the meaning of sanctity clarifying that through beatification and canonization the Church offers its faithful models of Christian life: "people in the flesh who have been extraordinary in living the faith practicing charity"; he recounted his experience as the Provincial of South Argentina and his direct knowledge of Zatti's places and activities but carried out discreetly and in everyday life the secret of Zatti's fidelity and service - the experience of illness and of healing at the hands of Mary with the subsequent promise that committed him for the rest of his life also drew a comparison between Zatti's service and the page of the Gospel dedicated to the figure of the Good Samaritan He concluded by observing that if a figure of holiness as fascinating as Zatti has come out of that Emilian land then "there is so much beauty and goodness here that should not be wasted" The celebration ended with a final significant gesture with the Rector Major handing over to the community Boretto a copy of the "Lettera Decretale" (Decree) of Zatti's canonisation the official pontifical document attesting to his canonization The culmination of the day was finally reached with the presentation of honorary citizenship to the Rector Major by the Mayor of Boretto "Today is a day of celebration for our community not only of the Christian community but also of the civil community" listing the many reasons for the celebration he specified: "The conferring of honorary citizenship on Cardinal Fr Ángel Fernández Artime is meant to be a recognition of the Rector Major of the Salesians in continuity with his predecessors But it also wants to be a sign of participation and friendship towards the entire Salesian world: the citizenship of the Rector Major means that every Salesian who comes to Boretto will be considered a citizen the importance of celebrating a person who who welcomed his neighbour and respected him and who -while he bandaged the wounds of the body did not neglect those of the soul "How nice it would be if we all treated each other with the gentleness that Zatti had!" he finished before officially presenting the Rector Major with the plaque conferring honorary citizenship The festive day was completed in the afternoon with a new edition of the "Color Zatti" event led by the Salesians of Parma and the FMA of Bibbiano music and colours - on shirts and faces - along the places where Artemide Zatti was born and grew up where he became a Christian and where he experienced the misery that saw him as a farm labourer at an early age and which later led his family to emigrate to Bahía Blanca LAKE ALFRED | What would a championship be without a little controversy and then a mechanical failure in the final race captured the 125cc hydro title Saturday in the 2014 UIM World Boat Racing Championships at Lion's Park on Lake Alfred but still ended up with a higher score than fellow Boretto driver Manuel Zambelli after Zambelli was flagged for moving out of his line on the start in the third heat and the German/Italian Demmler Racing Team filed a protest Like baseball and football officials going to a "replay booth," a jury of officials meet and make a decision whether to uphold the decision The jury decided Zambelli was not at fault Zambelli's red and white boat stopped dead in the water after the first turn of the course I'm tremendously disappointed," Zambelli said "It cost me a world title because I was unable to finish the final heat." then take the total of the best three times "We think it might be the carburetor," said Demmler team manager Bjorn Emmerich "After we got our $100 back from filing the protest Zilioli had a best time of 5:04.71 in Heat 1 and a total time of 21:07.65 Massimo Rossi and Estonia's Marek Peeba finished three boats will finish the meet in the 500cc runabout Tim Small of Lighthouse Point and Derek Gessler of Belleplaine leads with 400 points and a four-lap best time of 2:53.46 The 250cc class has Zambelli leading with 400 points and a best time of 5:45.69 Despite the Demmler Team dominating the event Emmerich said it takes about $10,000 to get their three boats four drivers and a "mechanical" crew of 20 from Italy and their home base "We have to get the ship container ready a month ahead," he said The Demmler Team was in Florida for races in 2008 and 2010 and he added: "We know the area and the people Emmerich said they are trying out a "borrowed" capsule boat in this meet "This is really new to us," he said of the 1100cc boats that are capable of more than 125 mph with their 300 horsepower engines who currently is stationed at Hickam Field in Hawaii Move affecting Piedmont and Lombardy regions comes amid worst drought to affect Italy’s longest river in 70 years More than 100 towns in the Po valley have been asked to ration water amid the worst drought to affect Italy’s longest river in 70 years Northern Italy has been deprived of significant rainfall for months with the effects of drought along the 400-mile (650km) Po River which stretches from the Alps in the north-west and flows through the Po delta before spilling out into the Adriatic The issue has been exacerbated by higher-than-usual temperatures and much less snow in the winter which contributed to lower snowmelt flowing into the Po Italy’s river observatory said the Po was suffering its worst drought in seven decades adding that demand for water in the Po valley basin was high Italy is experiencing a protracted heatwave with temperatures in some areas of the Po valley forecast to hit 36C by the end of the week which is severely impacting agricultural activities as well as commercial shipping to ask for the nightly suspension of drinking water supplies in 125 towns 100 in the Piedmont region and 25 in Bergamo province in Lombardy The Po also flows through Emilia-Romagna and Veneto serving one of the most important agricultural zones in Europe “The snow in the Alps in Piedmont and Lombardy has totally run out,” Utilitalia said in a statement “If this helped to replenish the flows of the Po in May the large reservoir that usually acts as a buffer in the summer months is depleted with flow rates well below the average for the period.” are also registering historically low water levels for the time of year and member of a consortium that distributes water for the irrigation of farmland said: “The situation is dramatic but we have known it since January,” he added which serve the basins that replenish whole Po valley (ANS - Vatican City) - During the Audience granted on Saturday to His Most Reverend Eminence Cardinal Marcello Semeraro Prefect of the Congregation for the Causes of Saints the Holy Father decided to convene a Consistory which will concern the canonization of Blessed Artemide Zatti a Salesian coadjutor who was born on 12 October 1880 in Boretto but moved to Argentina at a very young age especially in the service of the sick and the poor and the miracle that will bring him to the glory of the altars was recognized by decree on April 9 Also to be canonized with Artemide Zatti will be Blessed Giovanni Battista Scalabrini Founder of the Congregation of the Missionaries of St  and of the Congregation of the Missionary Sisters of St the Holy Father Francis received in audience His Eminence Cardinal Marcello Semeraro Prefect of the Vatican Congregation for the Causes of Saints the Supreme Pontiff authorized him to promulgate the Decree concerning: - the miracle attributed to the intercession of Blessed Artemide Zatti Professed Layman of the Salesian Society of St John Bosco; born on 12 October 1880 in Boretto (Italy) and died on 15 March 1951 in Viedma (Argentina) This act of the Holy Father opens the way for the Canonization of Blessed Artemide Zatti The date of the Canonization will be decided by the Supreme Pontiff during an ordinary Consistory Artemide Zatti was born in Boretto (Reggio Emilia) on 12th October 1880 It did not take long for him to experience the hardship of sacrifice that at the age of nine he was already earning his living as a farmhand the Zatti family emigrated to Argentina at the beginning of 1897 and settled in Bahia Blanca The young Artemide immediately began to frequent the parish run by the Salesians finding his spiritual director in Fr Carlo Cavalli the parish priest It was he who directed him towards Salesian life He was 20 years old when he went to the aspirantate in Bernal While caring for a young priest suffering from tuberculosis The paternal concern of Fr Cavalli - who followed him from afar – facilitated Zatti to go to the Salesian House in Viedma where there was a more suitable climate and above all a missionary hospital with a good Salesian nurse who was like a “doctor”: Fr Evasio Garrone Evasio Garrone invited Artemide to pray to Mary Help of Christians to be healed suggesting that he make a promise: “If she heals you you will dedicate your whole life for these sick people” Artemide made this promise willingly and was mysteriously cured His path was now clearly marked out and he embarked on it with great enthusiasm He made his first profession as a lay brother on 11th January 1908 and his Perpetual Profession on 8th February 1911 In keeping with the promise he had made to Our Lady he immediately consecrated himself totally to the Hospital initially taking charge of the adjoining pharmacy the entire responsibility of the hospital fell on his shoulders esteemed by all the patients and by the doctors themselves who gave him greater and greater freedom of action His service was not limited to the hospital and even to the two towns on the banks of the Negro River: Viedma and Patagones he travelled at all hours of the day and night His fame as a saintly infirmarian spread throughout the South and he received sick people from all over Patagonia It was not uncommon for the sick to prefer the visit of the holy infirmarian to that of the doctors Artemide Zatti loved his sick in a very touching way so much so that when he asked the sisters for a dress for a new boy who had just arrived do you have a dress for a 12-year-old Jesus?” His care for the sick was done with great care that it had its own delicate nuances Some people remember seeing him carrying the body of a patient who had died during the night on his shoulders towards the mortuary to remove it from the sight of the other patients: and he did this while reciting the De profundis Faithful to the Salesian spirit and to the motto bequeathed by Don Bosco to his sons - "work and temperance" - he carried out a prodigious activity with habitual readiness of spirit with absolute detachment from any personal satisfaction It’s said that the only five days of the rest he had were spent.. because of the escape of a prisoner in hospital He was absolved and his return home was a triumph with a great energy of sympathy that he manifested and gladly spent time to talk with the simple people And another exclaimed: “I started believing in God ever since I met Br the tireless infirmarian from a ladder and it was then that the symptoms of a cancer appeared he continued to carry out his mission for another year he passed away on 15 March 1951 in full consciousness surrounded by the affection and gratitude of an entire population He was declared Venerable on 7 July 1997 and beatified by St Argentina – March 2025 – The 11th edition of the pilgrimage to Viedma for Saint Artemide Zatti took place over the weekend of March 15–16 commemorating his 74th death anniversary on March 15 This year also marks the 150th anniversary of the first Salesian missionary expedition sent to Argentina by Saint John Bosco in 1875 The pilgrimage included various events: on Saturday a cycling trip from the "Don Zatti" chapel to the Don Bosco parish—the church where Saint Artemide Zatti’s remains rest—was followed by a lively Salesian oratory celebration and a Eucharistic celebration presided over by the Bishop of Viedma who also administered the sacrament of the Anointing of the Sick The main celebrations took place on Sunday beginning in the morning with a pilgrimage from Viedma Cathedral Monsignor Laxagüe presided over the Mass in front of Don Bosco Parish artistic performances were held at the Saint Artemide Zatti School Viedma’s City Council hosted the first online conference with Boretto and Monsignor Laxagüe took part in the meeting to strengthen ties between the two towns Representing Boretto were Mayor Andrea Codelupi and Deputy Mayor Andrea Vaccari who emphasized the immense legacy of Saint Artemide Zatti Buenos Aires, Argentina - December 2022 - On the evening of Thursday, December 1, the first episode of a new documentary entitled "Artemide Zatti, the Nurse of the Poor," a production of the Salesian Bulletin of Argentina, with the support of the two Argentine Provinces and the entire Salesian Congregation, was released in Argentina. The documentary, which can be viewed on the Argentine Salesian Bulletin's YouTube channel includes numerous interviews and never-before-seen historical material the documentary allows us to learn more about the man who dedicated himself to caring for the poorest of Argentina's Patagonia directing one of the first hospitals in the region at a time when public health care did not yet exist This new work shows that the canonization on Oct who showed his way of living the faith and left a legacy of concrete actions so that everyone can follow a simple spiritual path focused on caring for the poorest