Metrics details According to a growing body of neurobiological evidence the core symptoms of borderline personality disorder (BPD) may be linked to an opioidergic imbalance between the hedonic and stimulatory activity of mu opioid receptors (MOR) and the reward system inhibiting effects of kappa opioid receptors (KOR) is also likely to lead to epigenetic and neurobiological adaptations by extensive activation of the stress and endogenous opioid systems we investigated the methylation differences in the promoter of the KOR gene (OPRK1) in subjects with BPD (N = 47) and healthy controls (N = 48) Comparing the average methylation rates of regulatorily relevant subregions (specified regions CGI-1 we found no differences between BPD and HC Analyzing individual CG nucleotides (N = 175) we found eight differentially methylated CG sites with five showing highly interrelated methylation rates This differentially methylated region (DMR) was found on the falling slope (5’) of the promoter methylation gap whose effect is enhanced by the DMR hypomethylation in BPD A dimensional assessment of the correlation between disease severity and DMR methylation rate revealed DMR hypomethylation to be negatively associated with BPD symptom severity (measured by BSL-23) analyzing the influence of CT on DMR methylation we found DMR hypomethylation to correlate with physical and emotional neglect in childhood (quantified by CTQ) the newly identified DMR may be a biomarker of the risks caused by CT which likely epigenetically contribute to the development of BPD The rigidity of these experience-driven difficulties indicates the involvement of acquired biological factors which may ensue from epigenetic modification Inspired by the current state of research compiled here our study sought to analyze methylation changes of the KOR gene OPRK1 as a potential candidate with respect to the impact of CT on BPD We hypothesized that the stress system in BPD subjects was overly activated by CT leading to an epigenetic response in the EOS In view of the transcriptional and regulatory relevance we investigated the methylation differences of specific functional segments of the promoter region of OPRK1 between BPD and HC subjects to test for aberrant methylation in BPD of functionally important which could result in different gene activity; further within a 10 kb analysis window in the promoter to identify differentially methylated CG sites between BPD and HC; second to exploratorily analyze correlations between CG methylation rates and BPD severity; and third to investigate the effects of CT on methylation rates we studied 47 well-characterized females with BPD and compared them with 48 healthy age-matched female controls The relevant studies were approved by the appropriate Ethics Committee All participants provided written informed consent including DNA-based analyses We selected 47 medication-free, non-smoking female patients with a current diagnosis of BPD and 48 female, non-smoking healthy subjects, matched for age and BMI (Table 1) The general exclusion criteria comprised neurological disorders current alcohol or drug abuse or dependence in the last 12 months Analyses were performed on isolated genomic DNA from whole blood samples of each subject DNA concentration was measured and adapted by using a QubitTM 4 fluorometer (InvitrogenTM DNA was sheared into fragments of 150–200 bp by focused-ultrasonic fragmentation using the Covaris S220 sonication system (Covaris Ltd DNA methylation was analyzed via multiplex bisulfide PCR sequencing in accordance with the manufacturer’s instructions without modification (SureSelectXT Methyl-Seq Target Enrichment System for Illumina Multiplexed Sequencing Sequencing was done on an Illumina MiSeqTM System (2 × 150-bp paired-end) (Illumina Inc. USA) followed by demultiplexing and quality control for the generated reads (Q30 > 90%) a Gene structure of OPRK1 and covered 10 kb-target section b Localization of a priori specified functional gene regions EH1 c Localization of in BPD differentially methylated CG sites and DMR (CG34-CG38) For individual analyses of functionally relevant sites, three regions of interest were defined. CG-island 1 (CGI-1): chr8:53251468-53251883 (including 52 CG, 416 bp), CG-island 2 (CGI-2): chr8:53250744-53251285 (including 62 CG, 542 bp), and enhancer (EH1): chr8:53253201-53254400 (including 4 CG, 1.19 kb). These defined sections are all located in the promoter chr8:53250800–53252001 (GH08J053251, https://genome.ucsc.edu) EH1 is located 1.2 kb upstream from the promoter and 1.564 kb upstream of exon 1, respectively, while CGI-1 and CGI-2 are located closely together within the promoter region. Thus, CGI-1 starts 246 bp upstream of exon 1 and extends 169 bp inside. CGI-2 begins 162 bp downstream from the start of intron 1 and extends 341 bp into intron 2 (Fig. 1b) Statistical analyses were programed and carried out with R (version 4.3.0; R Core Team All cytosines with a coverage less than 25 reads were excluded from calculations we focused on methylation differences between patients and healthy individuals we assessed the a priori defined target regions (CGI-1 the mean methylation rate per target region by averaging the methylation rates of all CG sites within that target region we sought to determine whether these average methylation rates differed between patients and healthy individuals by modeling the mean methylation rate as a function of group (BPD vs HC) and testing the significance by the t-value associated with the difference between BPD and HC We assessed the prerequisites (notably normality and homoscedasticity of residuals) of all parametric regressions using Shapiro-Wilk and Breusch-Pagan tests Only for one analysis (the defined target region CGI-2) the normality assumption was violated while heteroscedasticity was never detected To assess the robustness of our parametric results we recalculated this regression using a bootstrapping algorithm (with 10,000 replicates) which gave similar results to the parametric version (including a non-significant difference in methylation rates between the two groups) we report in the remaining manuscript only the parametric results we report the estimated mean methylation rates at the mean age and mean BMI we defined a novel differentially methylated region (DMR) located on chromosome 8 (chr8:53252014-53252198) To assess the consistency in methylation rates of these CG sites we calculated correlations across participants analogous to the a priori defined target regions we calculated the DMR’s mean methylation rate by averaging the methylation rate of each CG site we assessed group differences using a regression Note that this approach does not account for the potentially different coverage across the individual CGs and across subjects the methylation rate may not be significantly different between BPD and HC even though each CG site (with the statistically more powerful analysis above) is we aimed to quantify the relationship between the methylation rate and symptom severity of this novel DMR we estimated a series of regression models in which we modeled the mean methylation rate of the DMR as a function of the linear symptom scales BSL-23 The resulting regression slope between symptom scale and methylation rate is similar to a correlation also with age and BMI having been controlled for we assessed the influence of childhood trauma we modeled the methylation rate as a function of CTQ severity We found no significant differences in the average methylation of the a priori defined target regions CGI-1 (mC = 4.2% The methylation rates of CG34 to CG38 were highly correlated with each other (Supplementary Table S1, Supplementary Fig. S1) and due to the adjacent localization of CG34-CG38 we defined this cluster for further analyses as a differently methylated region ((DMR) When mirroring the analysis for the a priori defined regions we found that the mean methylation rate of this DMR was significantly smaller for the BPD group (57.9%) than for the HC group (59.9%; difference: 2.04 ± −0.98 The analyses didn’t reach significance for the groups (HC and BPD) separately Both the new DMR localizing within the falling slope (5’) and CG163 within the rising slope (3’) of the methylation gap strengthen the effect of the promoter hypomethylation in BPD Detail: enlarged view of the clustering CG34-38 Functionally relevant gene regions such as CGI-1 did not differ significantly in their average methylation between BPD and HC our findings of hypomethylation within the OPRK1 promoter are both in line with and add to the current understanding of the interplay of opioid receptors They support the notion of a chronic KOR overactivity being related to BPD either overstimulation of KOR or under-stimulation of MOR can drive a KOR-MOR imbalance both being seminal in their individual effects in relation to BPD symptomatology while at the same time interacting inextricably It can be speculated that this MOR hypersensitization with hyperexcitation may in turn provoke a strong KOR counter-activation The decreased DMR methylation seen in our BPD sample which may be an adaptation to early environmental conditions As we did not study the MOR encoding of the OPRM1 gene we cannot comment on the KOR/MOR expression ratio high KOR expression can also be led by repeated high stress levels which trigger increased MOR or CRF activity with consequent KOR activation to dampen the overall stress system our overall results suggest that experiences of childhood neglect are particularly related to OPRK1 methylation changes and correlate with BPD symptomatology along with our findings regarding promoter hypomethylation in BPD suggests that heroin addiction and BPD lie at different poles of the opioid system imbalance no general functional conclusions with respect to psychopathology and patho-etiology can be drawn based on gene transcription we demonstrated that aberrant methylation of a DMR within the promoter of OPRK1 in peripheral blood cells may be an indicator of BPD and its symptom severity and that it is associated with CT at the sublevel of emotional and physical neglect we did not statistically control for any influence of these genetic variants on our methylation analyses of OPRK1 given that there is no known interaction between these genes and the OPRK1 methylation rate forcing us to control for all genes theoretically associated with BPD (without any relevant evidence thereof) and thus overstraining the statistical power of our cohort without a valid rationale As for the dimensional aspect of our investigations the study cohort was not optimally suited given that it consisted of a group of healthy individuals without psychopathology and patients with full-blown BPD thus lacking an intermediate group with lower BPD levels the study was conducted with samples from only 95 subjects The results must be replicated in a larger cohort to strengthen the evidence This suggests that such receptor-selective agents ought to be tested for the treatment of BPD highlighting the KOR receptor as a promising pharmacological target For a fundamental understanding of the EOS in the pathogenesis and maintenance of BPD the receptors involved in the EOS should be conceptualized as a functional ensemble in further research The data that support the findings of the current manuscript have been made available within the paper and its supplementary figures/tables as far as possible under data protection law Additional deidentified data is available from the corresponding author on reasonable request Bohus M, Stoffers-Winterling J, Sharp C, Krause-Utz A, Schmahl C, Lieb K. 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Neurosci Biobehav Rev. 2014;40:6–19. https://doi.org/10.1016/j.neubiorev.2014.01.003 Monoamine oxidase- a genetic variants and childhood abuse predict impulsiveness in borderline personality disorder childhood maltreatment and borderline personality disorder features among male inmates in China Download references This study was part of the central project of the Clinical Research Unit on Mechanisms of Disturbed Emotion Processing in Borderline Personality Disorder (KFO 256) which was supported by the German Research Foundation The research project was supported by the Short Term Program of the Faculty of Medicine RWTH Aachen University Open access funding provided by the Open Access Publishing Fund of RWTH Aachen University We would like to thank Felix Krueger for his valuable biotechnical support Open Access funding enabled and organized by Projekt DEAL These authors contributed equally: Thomas Frodl Department of Psychiatry and Psychotherapy Philip Wolff & Geraldine Zimmer-Bensch Center for Intervention and Research on adaptive and maladaptive brain Circuits underlying mental health (C-I-R-C) Department of Psychosomatic Medicine and Psychotherapy Implementation of the molecular biology: DS All authors approved the final version of 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 Download citation DOI: https://doi.org/10.1038/s41380-024-02628-z Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Volume 2 - 2014 | https://doi.org/10.3389/fbioe.2014.00016 Mixed-acid fermentation end products have numerous applications in biotechnology This is probably the main driving force for the development of multiple strains that are supposed to produce individual end products with high yields The process of engineering Escherichia coli strains for applied production of ethanol or acetate was initiated several decades ago and is still ongoing This review follows the path of strain development from the general characteristics of aerobic versus anaerobic metabolism over the regulatory machinery that enables the different metabolic routes major improvements for broadening the substrate spectrum of E coli toward cheap carbon sources like molasses or lignocellulose are highlighted before major routes of strain development for the production of ethanol Over decades, Escherichia coli has been studied regarding the multitude of factors that determine its physiology and the different phenotypes it can adopt. The molecular toolbox, enabling genetic engineering and studying of regulation and gene expression, is presumably the largest that exists for one particular organism (Richter and Gescher, 2012) coli has been widely used by applied microbiologists to try to steer the metabolism of this organism toward the production of molecules with biotechnological value This topic gains increasing interest due to the development of our society toward a bioeconomy Chemical production routines using fossil fuels as substrates are more and more being replaced by sustainable processes that work with biological catalysis and renewable substrates strains developed several years ago are still the starting points for the development of new strains that either broaden the spectrum of producible substances or increase the efficiency of an applied process we review studies from several decades to show the strain development for production of mixed-acid fermentation substances After an overview of metabolic capabilities and regulatory mechanisms developments regarding the expansion of usable substrate spectrum will be described acetyl-CoA is further processed within the citric acid cycle This gives rise to the production of more ATP and reducing equivalents Anaerobic fermentative metabolism in Escherichia coli Chemical structures are shown for all mixed-acid fermentation products and pyruvic acid Bold gray arrows: glucose transport systems; thin black arrows: glycolysis; bold black arrows: fermentative reactions; dashed ptsG (fused glucose-specific PTS enzyme: IIB and IIC component) gap (glyceraldehyde 3-phosphate dehydrogenase) ArcA and FNR greatly influence the expression of genes involved in central metabolism (Salmon et al., 2003, 2005; Constantinidou et al., 2006). In fact, they are the most important regulators involved in aerobic and anaerobic growth (Gunsalus and Park, 1994; Unden et al., 1995; Unden and Bongaerts, 1997) Oxygen inactivates the active dimeric form of FNR that contains one 4Fe-4S-cluster per monomer (4Fe-4S FNR) Continuous production of new FNR molecules and reactivation of the inactive 2Fe-2S-form (2Fe-2S FNR) or the apoenzyme (apo FNR) leads to constant cycling of the three FNR-forms The absence of oxygen triggers a rapid accumulation of the 4Fe-4S-form which dimerizes and thereby becomes an active transcription factor The availability of cost-efficient and abundant carbon sources is a requirement for biotechnological production of fermentation products by E. coli. These carbon compounds include lignocellulose, molasses, and glycerol (da Silva et al., 2009) Regarding the utilization of waste streams the focus of research so far has been on ethanologenic strains of E most probably due to the growing demand for biofuels and inserted the genes fucO (encoding an NADH-dependent furfural oxidoreductase) and ucpA (a cryptic gene encoded next to a sulfur assimilation operon) into the genome of an ethanologenic strain and consequently furfural tolerance increased They inserted the csc operon into the genome of an E coli W3110 and determined lactate production in a sugar mixture and diluted molasses coli could use diluted molasses as a sole carbon source sucrose metabolism could be successfully transferred between different strains Levoglucosan is the most abundant sugar in bio-oil and therefore an attractive fermentation substrate. It can be converted to glucose-6-phosphate by levoglucosan kinase (Kitamura et al., 1991). Hence, Layton et al. (2011) introduced a codon-optimized version of this kinase (LGK) from Lipomyces starkeyi YZ-215 into the genome of E. coli KO11 (Table 1) The resulting strain could ferment levoglucosan as a sole carbon source to ethanol a complete fermentation of the substrate was not achieved The authors hypothesized that this was due to the high Km value (71.2 mM) of LGK or to transport limitations coli was adapted to the utilization of various carbon sources These are very important improvements of the use of metabolic capacity and mark essential steps in the development of biotechnological processes with renewable substrates or waste streams as substrates a maximum ethanol production yield with glucose and xylose was reached more than 20 years ago high alcohol concentrations in the fermentation broth were not reached at that point due to ethanol toxicity The authors set pyruvate dehydrogenase under control of the native pyruvate formate lyase promoter which converts glucose as well as xylose to ethanol with a yield of 90% under anaerobic conditions using only genes and promoters from E The authors used strain TCS083 with plasmid pLOI297 and further deleted malate dehydrogenase (mdh) The evolved strain produced ethanol under low-oxygen conditions with a yield of 0.37 g ethanol/g glycerol consumed which is close to the theoretical maximum of 0.5 g/g They were able to use the advantages of anoxic fermentative processes (low cell mass high catabolic rates) and combine them with a respiration-based formation of a product that is more oxidized than the growth substrate This could be an enabling technology for a variety of other biotechnological production processes that are so far impossible due to insufficient redox balance In fact, Causey et al. (2004) used this technology to develop the strain further for pyruvate production. Additional deletion of acetate kinase (ackA) and pyruvate oxidase (poxB) led to strain TC44. This strain produces 0.75 g pyruvate/g glucose in a mineral salts medium under microoxic conditions. This is the highest yield of pyruvate production in a mineral salt medium reported for E. coli so far (Causey et al., 2004) The production of lactic acid is of biotechnological interest mostly due to the production of polylactic acid- (PLA-) based plastic materials. Since lactic acid bacteria have high nutritional requirements (van Niel and Hahn-Hagerdal, 1999) coli seems to be better suited for the production of this carboxylic acid the authors could reach lactate concentrations of up to 138 g L−1 with a strain that was modified by deletion of aceEF the production of two PEP from one molecule of glucose leads to the production of only two instead of four NADH molecules The reason why a ptsG deletion could rescue the phenotype of the ldhA pfl deletion mutant is not totally clear yet One hypothesis is that the rather unspecific import via the other systems results in decreased uptake rates These would consequently lead to slower formation of glycolysis intermediates and thereby prevent an accumulation of high NADH concentrations before oxaloacetate is reduced to succinate and ΔptsG strain with a dual-phase fermentation This enzyme activity resulted in a decreased production of formate and increased the available pool of NADH fewer side products were detected in the fermentation broth and succinate was produced from glucose with a yield of 1.1 g/g They used an anode in a microbial fuel cell as an acceptor for a surplus of electrons Electrodes are exhaustless electron acceptors anodic electron transfer could be a sustainable process to broaden the spectrum of producible substances microbial fuel cells demand specific electron transport chains to the cell surface could also couple the metabolism of other microorganisms to anode reduction 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 We are grateful for financial support from the German Ministry of Education and Research (BMBF) under the program “BioEnergie 2021” (Grant No Requirement of ArcA for redox regulation in Escherichia coli under microaerobic but not anaerobic or aerobic conditions Pubmed Abstract | Pubmed Full Text | CrossRef Full Text and xylose by recombinant Escherichia coli Pubmed Abstract | Pubmed Full Text Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD(+)-dependent formate dehydrogenase Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The ArcBA two-component system of Escherichia coli is regulated by the redox state of both the ubiquinone and the menaquinone pool Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Engineering Escherichia coli for efficient conversion of glucose to pyruvate Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Engineering the metabolism of Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: homoacetate production Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1 Pubmed Abstract | Pubmed Full Text The fermentation pathways of Escherichia coli CrossRef Full Text A reassessment of the FNR regulon and transcriptomic analysis of the effects of nitrate and NarQP as Escherichia coli K12 adapts from aerobic to anaerobic growth Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase Pubmed Abstract | Pubmed Full Text Distinct metal cofactor-induced conformational states in the NAD-specific malic enzyme of Escherichia coli as revealed by proteolysis studies Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Glycerol: a promising and abundant carbon source for industrial microbiology Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli Pubmed Abstract | Pubmed Full Text Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars Pubmed Abstract | Pubmed Full Text | CrossRef Full Text A novel fermentation pathway in an Escherichia coli mutant producing succinic acid Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Current knowledge of the Escherichia coli phosphoenolpyruvate-carbohydrate phosphotransferase system: peculiarities of regulation and impact on growth and product formation Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Enabling unbalanced fermentations by using engineered electrode-interfaced bacteria Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160 Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Amplification of signaling activity of the arc two-component system of Escherichia coli by anaerobic metabolites An in vitro study with different protein modules Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Quinones as the redox signal for the arc two-component system of bacteria Pubmed Abstract | Pubmed Full Text | CrossRef Full Text In vitro phosphorylation study of the arc two-component signal transduction system of Escherichia coli Pubmed Abstract | Pubmed Full Text A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Methylglyoxal bypass identified as source of chiral contamination in l(+) and d(-)-lactate fermentations by recombinant Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Aerobic-anaerobic gene regulation in Escherichia coli: control by the ArcAB and Fnr regulons Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The universal stress protein paralogues of Escherichia coli are co-ordinately regulated and co-operate in the defence against DNA damage Pubmed Abstract | Pubmed Full Text | CrossRef Full Text D- and L-lactic acid oxidases of Escherichia coli CrossRef Full Text The universal stress protein UspC scaffolds the KdpD/KdpE signaling cascade of Escherichia coli under salt stress Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Discovery and characterization of a new family of lytic polysaccharide monooxygenases Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Engineering Escherichia coli for improved ethanol production from gluconate Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Metabolic flux analysis for succinic acid production by recombinant Escherichia coli with amplified malic enzyme activity Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Glucose-transport of Escherichia coli growing in glucose-limited continuous culture Pubmed Abstract | Pubmed Full Text The respiratory chains of Escherichia coli Genetic engineering of ethanol production in Escherichia coli Pubmed Abstract | Pubmed Full Text a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The arcB gene of Escherichia coli encodes a sensor-regulator protein for anaerobic repression of the arc modulon Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132 Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion Pubmed Abstract | Pubmed Full Text | CrossRef Full Text from discovery to biotechnical and biomedical applications Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Ethanol production from marine algal hydrolysates using Escherichia coli KO11 Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Construction of an Escherichia coli K-12 mutant for homoethanologenic fermentation of glucose or xylose without foreign genes Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Metabolism of levoglucosan (1,6-anhydro-beta-D-glucopyranose) in microorganisms CrossRef Full Text CrossRef Full Text Pyruvate formate-lyase of Escherichia coli: the acetyl-enzyme intermediate CrossRef Full Text Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The ArcB sensor kinase of Escherichia coli: genetic exploration of the transmembrane region Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Engineering ethanologenic Escherichia coli for levoglucosan utilization Pubmed Abstract | Pubmed Full Text | CrossRef Full Text DNA binding and dimerization of the Fe-S-containing FNR protein from Escherichia coli are regulated by oxygen Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Evidence against the physiological role of acetyl phosphate in the phosphorylation of the ArcA response regulator in Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Role of the ArcAB two-component system in the resistance of Escherichia coli to reactive oxygen stress Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Microbial cellulose utilization: fundamentals and biotechnology Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Signaling by the arc two-component system provides a link between the redox state of the quinone pool and gene expression Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Efficient synthesis of L-lactic acid from glycerol by metabolically engineered Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli Pubmed Abstract | Pubmed Full Text Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Munoz-Gutierrez Cell surface display of a beta-glucosidase employing the type V secretion system on ethanologenic Escherichia coli for the fermentation of cellobiose to ethanol Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II Pubmed Abstract | Pubmed Full Text Fueling industrial biotechnology growth with bioethanol Pubmed Abstract | Pubmed Full Text | CrossRef Full Text ArcA redox mutants as a source of reduced bioproducts Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Functional citric acid cycle in an arcA mutant of Escherichia coli during growth with nitrate under anoxic conditions Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Enhanced trehalose production improves growth of Escherichia coli under osmotic stress Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The effect of temperature on L-lactic acid production and metabolite distribution of Lactobacillus casei Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The pdhR-aceEF-lpd operon of Escherichia coli expresses the pyruvate dehydrogenase complex Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Biotechnological applications of acetic acid bacteria Pubmed Abstract | Pubmed Full Text | CrossRef Full Text The molecular toolbox for chromosomal heterologous multiprotein expression in Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Accelerated glycerol fermentation in Escherichia coli using methanogenic formate consumption Pubmed Abstract | Pubmed Full Text | CrossRef Full Text A whole cell biocatalyst for cellulosic ethanol production from dilute acid-pretreated corn stover hydrolyzates Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Continuous ethanol production from wheat straw hydrolysate by recombinant ethanologenic Escherichia coli strain FBR5 Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Ethanol production from wheat straw by recombinant Escherichia coli strain FBR5 at high solid loading Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Global gene expression profiling in Escherichia coli K12 The effects of oxygen availability and FNR CrossRef Full Text Global gene expression profiling in Escherichia coli K12: effects of oxygen availability and ArcA Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins Pubmed Abstract | Pubmed Full Text Effect of ArcA and FNR on the expression of genes related to the oxygen regulation and the glycolysis pathway in Escherichia coli under microaerobic growth conditions CrossRef Full Text Production of D(-)-lactate from sucrose and molasses Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Manipulating redox and ATP balancing for improved production of succinate in E Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Genes restoring redox balance in fermentation-deficient E Pubmed Abstract | Pubmed Full Text | CrossRef Full Text FNR and its role in oxygen-regulated gene expression in Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Glucose transport in Escherichia coli mutant strains with defects in sugar transport systems Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Nitrate regulation of anaerobic respiratory gene expression in Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant Pubmed Abstract | Pubmed Full Text Engineering a homo-ethanol pathway in Escherichia coli: increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Chemical characterization of D-lactate dehydrogenase from Escherichia coli B Regulation of aerobic-to-anaerobic transitions by the FNR cycle in Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Metabolic regulation analysis of wild-type and arcA mutant Escherichia coli under nitrate conditions using different levels of omics data Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Characterization of Escherichia coli [NiFe]-hydrogenase distribution during fermentative growth at different pHs Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Escherichia coli multiple [Ni-Fe]-hydrogenases are sensitive to osmotic stress during glycerol fermentation but at different pHs Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Metabolic engineering of Escherichia coli for efficient conversion of glycerol to ethanol Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Reprogramming of Escherichia coli K-12 metabolism during the initial phase of transition from an anaerobic to a micro-aerobic environment Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes Pubmed Abstract | Pubmed Full Text | CrossRef Full Text O2-sensing and O2-dependent gene regulation in facultatively anaerobic bacteria Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors Pubmed Abstract | Pubmed Full Text | CrossRef Full Text On the role of cyclic AMP and the Fnr protein in Escherichia coli growing anaerobically Pubmed Abstract | Pubmed Full Text | CrossRef Full Text An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Nutrient requirements of lactococci in defined growth media CrossRef Full Text Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Homofermentative production of D-lactic acid from sucrose by a metabolically engineered Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Biotechnological production of lactic acid and its recent applications Engineering improved ethanol production in Escherichia coli with a genome-wide approach Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Deletion of methylglyoxal synthase gene (mgsA) increased sugar co-metabolism in ethanol-producing Escherichia coli Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Re-engineering Escherichia coli for ethanol production Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Homofermentative production of optically pure L-lactic acid from xylose by genetically engineered Escherichia coli B Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Engineering a native homoethanol pathway in Escherichia coli B for ethanol production Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Functional replacement of the Escherichia coli D-(-)-lactate dehydrogenase gene (ldhA) with the L-(+)-lactate dehydrogenase gene (ldhL) from Pediococcus acidilactici Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Fermentation of 12% (w/v) glucose to 1.2 M lactate by Escherichia coli strain SZ194 using mineral salts medium Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Fermentation of 10% (w/v) sugar to D: (-)-lactate by engineered Escherichia coli B Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Homolactate fermentation by metabolically engineered Escherichia coli strains Pubmed Abstract | Pubmed Full Text | CrossRef Full Text Disclaimer: All claims expressed in this article are solely 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WindWind turbine snaps into pieces as boss says 'it was only checked last year'Ageing machine torn off at height of about 20 metres amid stormy weather at German wind farm Objective: Epigenetic mechanisms have been described in several mental disorders, such as mood disorders, anxiety disorders and schizophrenia. However, less is known about the influence of epigenetic mechanisms with regard to personality disorders (PD). Therefore, we conducted a literature review on existing original data with regards to epigenetic peculiarities in connection with personality disorders. Methods: Systematic literature review using PRISMA guidelines. Search was performed via NCBI PubMed by keywords and their combinations. Used search terms included “epigenetic,” “methylation,” “acetylation” plus designations of specified personality traits and disorders according to DSM-IV. Volume 9 - 2018 | https://doi.org/10.3389/fpsyt.2018.00579 This article is part of the Research TopicBack to the Future: On the Road Towards Precision PsychiatryView all 21 articles Objective: Epigenetic mechanisms have been described in several mental disorders less is known about the influence of epigenetic mechanisms with regard to personality disorders (PD) we conducted a literature review on existing original data with regards to epigenetic peculiarities in connection with personality disorders Methods: Systematic literature review using PRISMA guidelines Search was performed via NCBI PubMed by keywords and their combinations Used search terms included “epigenetic,” “methylation,” “acetylation” plus designations of specified personality traits and disorders according to DSM-IV Results: Search yielded in total 345 publications 23 original publications fulfilled the intended search criteria and were included Those are 13 studies on gene methylation pattern with aggressive 9 with borderline personality disorder (BPD) and 2 with antisocial personality disorder (ASPD) The results of these studies showed significant associations of PD with methylation aberrances in system-wide genes and suggest evidence for epigenetic processes in the development of personality traits and personality disorders of which childhood trauma showed a high impact interfered with many neurofunctional genes Methylation alterations in ASPD and BPD repeatedly affected HTR2A Summary: Epigenetic studies in PD seem to be a useful approach to elucidate the interaction of co-working risk factors in the pathogenesis of personality traits and disorders the complexity of pathogenesis leads to divergent results and impedes an explicit interpretation Differing methylation patterns within the selected PD could indicate subgroups which would benefit from patient-oriented therapeutic adjustments They might play a major role in the future design and observation of early therapeutic intervention and thus could help to prevent severe dysfunctional conduct or full-blown personality disorder in risk subjects The epigenetic view on genes presumably associated with psychiatric disorders is gaining increasing academic interest and enables auxiliary insights in the pathogenesis of a particular disease Severe psychiatric axis-I disorders like major depressive disorder (MDD) are currently widely investigated at the epigenetic level and results account for a substantial pathogenetic impact of gene epigenetic modifications although many details of molecular mechanisms remain unknown the present insights substantiate and refine the idea how environmental signals might be translated into intracellular information and molecular memory a considerable number of studies explore epigenetic changes in association with behavior or affect difficulties like aggression or fear in human and animal subjects particularly in connection with disturbances of the serotonergic system that meanwhile is well-known to be crucial in early brain development The objective of this work was to review the current original publications on epigenetic modifications associated with personality disorders (PD) in humans Literature search was performed as a systematic review according the Preferred Recording Items for Systematic Reviews and Meta-Analyses (PRISMA-P) guidelines (17) We based our search on the PubMed Central database of the U.S National Institutes of Health's National Library of Medicine (NIH/NLM) using terms oriented on the Medical Subject Headings (MeSH) of the NCBI Library For the search keywords were inserted in a double or triple combination to yield comprehensive hits The following keywords were utilized: “personality,” “personality disorder,” “personality trait,” each of them combined with “epigenetic,” “methylation,” “acetylation,” “phosphorylation,” “ubiquitation,” “sumoylation,” “microRNA,” “chromatin” and “chromatin remodeling,” respectively The search included publications until May 15th 2018 Flow diagram of study search and sequenced selection Concomitantly, we used the PubMed search function of f1000prime (Faculty of 1000 Limited, London, UK). Herewith we found one further expedient study that met all of the described inclusion criteria (18) 24 original studies were included in this review 13 explored the epigenetic influence on PT (two on impulsiveness seven on aggression) and 11 studied epigenetic differences in personality disorders (two in antisocial personality disorder (ASPD) 9 in borderline personality disorder (BPD)) and the number of investigated genes varied between the studies and ranged from single gene assays to genome-wide methylation analyses (GWA) with implications for the statistical power only methylation of the monoamine oxidase A gene (MAOA) has been examined The two studies differed decisively with respect to study design and methylation results Philibert et al. considered the known variable nucleotide repeat (VTNR) region of MAOA and introduced a new VTNR region upstream of the transcriptional start site (TSS) of the gene (MAOA VTNR P2). They found a genotype-dependent methylation level and gene activity, but only in females (19) Within a total of well characterized 571 subjects (312 female) of the Iowa Adoption Study (IAS) they measured ASPD lifetime symptom counts in a linear mode according to DSM-IV criteria Methylation patterns were analyzed at two promoter-associated CpG islands of MAOA in DNA extracted from EBV transformed lymphoblast cell lines from peripheral blood Sequence analyses of the VTNR P2 revealed five genotypes with each seven to 11 eleven repeats (7R of which the 9R genotype showed the lowest methylation in homozygous females and the greatest gene activity in the functional analysis via luciferase essay Presence of the low activity allele 10R was associated with higher vulnerability to the harming effects of childhood sexual and physical abuse and it accounted significantly for variances in symptom severity of ASPD in women In male subjects no significant effect of the P2 genotype on MAOA methylation status was found Studies on epigenetics in personality disorders Regarding studies on NR3C1 methylation and PD, two studies focused on exon 1F promoter, which is functionally crucial. In a cohort of BPD outpatients (n = 281) Martin-Blanco et al. (22) found a significant positive correlation between overall NR3C1 exon 1F methylation level of peripheral blood leucocytes and clinical severity Exon 1F methylation was further significantly associated with childhood physical abuse Individual CpG sites were associated with particular subscores of CTQ Perroud et al. (23) considered that a current severe mental illness might have epigenetic implications per se and could confound analyses that aim to isolating methylation characteristics specific for BPD in a comparison of subjects with BPD (n = 101) to those with MDD (n = 99) their results showed higher overall NR3C1 exon 1F methylation levels in BPD than in MDD subjects in peripheral blood leucocytes methylation was associated with CT scaled by the CTQ NR3C1 exon 1F hypermethylation in subjects with BPD was still significant when corrected for childhood maltreatment Regarding monoamine receptor genes, within a large-scaled study on subjects with bulimia spectrum disorders (BSD) with and without comorbid BPD Groleau et al. (24) found significant but marginally increased methylation of the dopamine D2 receptor gene (DRD2) exon 1 promoter region in whole peripheral blood cell (PBC) DNA of subjects with BSD and BPD compared with that of subjects with BSD only Methylation of the serotonin receptor 3A gene (5HTR3A) was found by Perroud et al. (25) to be correlated to clinical severity of BPD and other psychiatric disorders The authors compared PBC DNA methylation levels of eight CpG sites within the 5HT3A gene in subjects with BPD (n = 116) attention deficit hyperactivity disorder (ADHD) (n = 111) and bipolar disorder (BD) (n = 122) They also considered single nucleotide variants (SNP) of the gene and CT history as additional factors and explored associations with methylation levels of the particular CpG sites Methylation levels between the patient groups differed significantly in most of the CpG sites and showed a distinct pattern of hyper- and hypomethylation in the several disorders in selected CpG sites subjects showed the highest scores in the CTQ and the highest methylation level between the patient groups CT was associated with mean methylation status and CTQ total score and physical abuse each with different selected CpG sites CT was further associated with higher severity of disease Carrying the CC-allele was significantly associated with methylation at one specific CpG site independent of CT (in all disorders) Dammann et al. (26) analyzed five neuropsychiatric genes assumed to be of significance for psychopathological phenotype in particular genes coding for soluble catechol-O-methyltransferase (s-COMT) Methylation levels of each gene promoter were quantified in PBC DNA of individuals with BPD (n = 26 24 female) and in healthy controls (n = 11 quantitative DNA methylation analysis showed significant elevated overall methylation levels in BPD subjects within HTR2A Gene methylation of MAOA and MAOB could only be analyzed in female subjects and methylation of MAOA was significantly higher in BPD (of MAOB only by trend) across the five genes that were investigated average methylation level across all quantified regions was significantly higher in BPD patients compared to controls Implications of attendant data as trauma history of the subjects weren't presented as part of the study but it was noted that aberrant methylation had not been associated with traumatic experience in appropriate statistical tests Meanwhile epigenetic assays include genome-wide association studies (GWA) that indicate specific CpG sites of aberrant methylation levels across the whole genomic DNA and facilitate comprehensive aspects within epigenetic evaluations In extension to the aforementioned study, Teschler et al. (27) performed a GWA in PBC DNA between female BPD (n = 24) and HC (n = 11) subjects Results showed a total of 256 significantly hypermethylated CpG sites in BPD but significance of any of each didn't persist post Bonferroni correction The research group selected seven hypermethylated genes for validation analyses and could endorse increased methylation in five of the genes with specified CpG sites related to the amyloid beta (A4) precursor protein-binding family A member 2 (APBA2) and member 3 (APBA3) genes potassium voltage-gated channel KQT-like subfamily member 1 gene (KCNQ1) MCF2 cell line derived transforming sequence gene (MCF2) and the ninjurin 2 gene (NINJ2) GATA binding protein 4 (GATA4) and holocarboxylase synthetase (HLCS) genes showed increased methylation in BPD in the GWA Methylation studies of the ribosomal RNA gene (rDNA) promoter, 5′ external transcribed spacer gene (5′ETS) and of the proline rich membrane anchor 1 gene (PRIMA1) promoter in PBC DNA of female subjects with BPD (n = 24) and HC (n = 11) revealed significantly less methylation of the rDNA promoter region in BPD compared with HC subjects, and hypomethylation of the 5′ETS in BPD by trend. PRIMA1 showed a higher methylation in BPD subjects (28) Another GWA with PBC DNA was performed by Prados et al. (29) on BPD subjects affected with high levels of childhood adversity (n = 96) and subjects with MDD and a history of low levels of childhood adversity (n = 93) Uni- and multivariate analyses revealed significant methylation differences in a significant number of particular CpG sites associated with BPD compared with MDD subjects or related to childhood maltreatment most significant results of multivariate analyses resulted in significantly different methylated CpG sites located e.g. within the gene coding for a ligand of Eph-related receptor tyrosine kinases (EFBN1) closely to the gene coding for a suppressor of cytokine signaling (SOCS) family member (SPSB2) and near the gene coding for a protein similar to mouse cystatin 9 (CST9L) Univariate analyses detected hypomethylated CpG sites in BPD i.e. near the encoding region of microRNA 124 (miR124-3) which targets several genes that have been described to be correlated with BPD (including NR3C1) near the gene coding for the WD repeat domain 60 (WD60) and a CpG site within the gene of the family with sequence similarity 163 member A (FAM163A) Many of the significantly hypermethylated sites were found on chromosome X results of multivariate analyses with the CTQ revealed strong associations with loci within or near the genes of human homolog of the mouse p (pink-eyes dilution) (OCA2) microfibrillar-associated protein 2 (MFAP2) 5′-nucleotide domain containing 2 (NT5DC2) and rabphilin 3A-like (RPH3AL) Univariate analyses yielded most significant alterations located within the gene coding for the interleukin 17 receptor A (IL17RA) in an intergenic region on chromosome 6p22.1 Methylation of miR124-3 was associated with both severity of childhood adversity (higher methylation) and with BPD (lower methylation) Most original publications on methylation and personality traits were found with antisocial or aggressive features in connection with epigenetic alterations associated with the functionality of the serotonergic system or the hypothalamic–pituitary–adrenal (HPA-) axis functionality which is the main neuroendocrine system for regulation of stress reaction and adaptation theory-driven studies examined genes associated with these systems such as the serotonin transporter gene (SLC6A4) dopamine and serotonin receptor genes (DRD1 With respect to its role in socio-affective perception and processing some studies exist that pertain to antisocial behavior and oxytocin and oxytocin receptor genes (OXT the role of cytokines and other factors were considered Consistent with a large body of literature that implicates the serotonergic system in the regulation of anxiety, aggression, and stress response, epigenetic alterations in the serotonin transporter gene (SLC6A4) were found to be related to a history of child sexual abuse as well as to antisocial behavior in adulthood by Beach et al. (30) The authors performed methylation analyses on the participant's peripheral blood lymphocyte DNA that was EBV transformed into lymphoblast cell lines all female) were gradually diagnosed by means of symptom score of ASPD Child sexual abuse was highly associated with mean methylation level and methylation was highly significantly associated with symptoms of ASPD thereby playing a modulating role to develop antisocial traits after childhood sexual abuse influence of SLC6A4 methylation on ASPD severity was impacted by genotype since association of methylation with the ss and the sl genotype of SLC6A4 was significant These results suggest an aggravating effect of methylation in SLC6A4 risk (s) alleles for ASPD methylation was increasingly associated with ASPD in carriers with greater number of s alleles Further, in a prospective, longitudinal study, Cecil et al. (18) studied 84 youth with early-onset and persistent conduct problems with regard to early life risks and to methylation changes of OXTR in PBC DNA and collected an environmental risk score (kind and time of risk factor) methylation analyses were performed longitudinally with sampling at birth (cord blood) and at age 7 and 9 years (peripheral blood) for evaluation subjects were divided by severity of internalizing problems collected by maternal reports at each study time Peripheral morning cortisol plasma levels were associated with hypermethylation of CpG9 in PBC DNA only the represented studies in antisocial and BPD-associated personality traits yet cannot provide a base for general conclusions Within a 21 year longitudinal study on early onset aggression the research group Wang et al specified children by severity and persistence of aggressive behavior during their age of 6–15 years and established four states of childhood aggression trajectories: the first consisted in subjects with no physical aggression at any time points (no physical aggression a second in subjects with low to moderate aggression that declined between age 10–15 (low physical aggression the third in subjects with high rates of aggression that subsequently declined (childhood-limited high physical aggression CLHPA) and finally in subjects with consistently high physical aggression until age 15 (chronic high physical aggression They analyzed DNA from peripheral T cells and found early onset CPA being associated with clustered and genome-wide methylation differences at 900 sites within 448 distinct gene promoters The detected genes comprised several that have been associated with aggression earlier: arginine vasopressin receptor 1A (AVPR1A) serotonin receptor 1D (HTR1D) and glutamate metabotropic receptor 5 (GRM5) genes with less methylation in the CPA group and the dopamine receptor D1 gene (DRD1) and SLC6A3 with higher methylation in the CPA group most functional categories of genes with different methylation in CPA included behavior (hyperactivity) inflammatory response (chemotaxis and phagocytes) and gene expression (transcription factors Concerned specific canonical pathways included cytokine signaling and G-protein coupled receptor signaling In a further GWA Guillemin et al. studied methylation aberrancies in DNA from isolated peripheral T-cells in female subjects with CPA trajectories since childhood (n = 5) compared to women without any aggression history (n = 16) (40) A total of 917 probes corresponding to 430 distinct gene promoters were differentially methylated of which many are involved in immune and inflammatory responses such as FOS some had previously been shown to be associated with aggressive behavior and Corticotrophin-releasing hormone-binding protein (CRHBP) all of them were significantly less methylated in the CPA subjects Van Dongen et al. (41) studied methylation differences with respect to aggressive behavior in 40 monozygotic twins (20 pairs) highly discordant for aggressive traits They performed a GWA with PBC DNA and received about 24 methylation sites with high methylation difference yet differences were generally twin pair-specific; thus statistically no genome-wide significant methylation differences were identified in this sample The mentioned studies were performed in a small sample size of 8 (men) and 5 (women) subjects None of the studies considered the experience of childhood trauma the depicted studies confirm the need of genome-wide methylation assays or at least a focus on a comprehensive set of functional associated genes Future studies should consider childhood trauma Studies on personality traits are summarized in Table 2 Studies on epigenetics in personality traits with the insight in a genome-wide involvement of epigenetic modified gene loci the validity of the studies on particular genes today seems to be restricted the sample size is very small and might impact the results High requirements in the technical methods and in an expedient study design make the disentangling of personality-specific methylation patterns a sensitive challenge especially since there might be more than one way to (epi-)genetically forward the development of a PD This also might be a reason for the diversity of the final clinical epiphenotypes of these disorders the variety of pathogenesis and of the individually developed clinical shapes impedes the attribution of possibly identified genetic or epigenetic aberrances to a specific personality trait or disorder If the functional attribution of epigenetic aberrations to personality disorders is so highly impeded As in other mental disorders, epigenetic patterns can help to predict medical response. Thus, in depression, selected methylation aberrations could predict different therapy response to escitalopram and between escitalopram and nortriptyline (51, 52). In BPD, methylation levels of APBA3 and MCF2 were predictive for psychotherapy outcome (53) These merely exemplary findings suggest a high dynamic in epigenetic processes in mental disorders and their course The interaction of psychotropic drugs and other therapeutic interventions with epigenetic remodeling seems still understudied the fact of this interaction then could be utilized as an intraindividual control of lasting therapy response The pathophysiological principle of gene-environment-interaction not only explains the obvious differences in the severity or combination of a person's personality traits but further implicates the existence of genetic and epigenetic risk and protective factors during the formation of the ultimate personality structure Hitherto epigenetic studies underline the impact of early life adversity on the multifactorial pathway to PD They reveal several relevant gene loci that are epigenetically affected in PD but differ in between the studies This can partially be explained by the multifactorial and multi-step genesis of each PD leading to different pathogenetic subtypes Epigenetic analyses in connection with PD represent a complex but suitable amendment in the elucidation of personality development and pose as valuable diagnostic step in the specification of an individual's premorbid risks and for the development of individually tailored therapeutic strategies They might play a valuable role in the future design and observation of early and personalized therapeutic intervention and thus could help to prevent the unfolding in severe dysfunctional conduct or personality disorder in risk subjects DG conducted the full literature search process KK critically overworked focus and manuscript TH and HF provided expert advice in epigenetics JK and TF reviewed the manuscript and collaborated in the interpretation of the results and for the discussion DNA methylation: a mechanism for embedding early life experiences in the genome Epigenetic programming by maternal behavior Lasting epigenetic influence of early-life adversity on the BDNF gene Dynamic DNA methylation programs persistent adverse effects of early-life stress Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse Broad epigenetic signature of maternal care in the brain of adult rats Conserved epigenetic sensitivity to early life experience in the rat and human hippocampus The signature of maternal rearing in the methylome in rhesus macaque prefrontal cortex and T cells Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions DNA-methylation gene network dysregulation in peripheral blood lymphocytes of schizophrenia patients Histone acetylation by p300 is involved in CREB-mediated transcription on chromatin Biochim Biophys Acta (2001) 1541:161–9 The methylated-DNA binding protein MBD2 enhances NGFI-A (egr-1)-mediated transcriptional activation of the glucocorticoid receptor The transcription factor nerve growth factor-inducible protein a mediates epigenetic programming: altering epigenetic marks by immediate-early genes Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2 Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation oxytocin receptor gene (OXTR) methylation and youth callous-unemotional traits: a 13-year longitudinal study Gene environment interactions with a novel variable monoamine oxidase A transcriptional enhancer are associated with antisocial personality disorder Monoamine oxidase A gene promoter methylation and transcriptional downregulation in an offender population with antisocial personality disorder Response to psychotherapy in borderline personality disorder and methylation status of the BDNF gene Association between methylation of the glucocorticoid receptor gene and clinical severity in borderline personality disorder Increased methylation of glucocorticoid receptor gene (NR3C1) in adults with a history of childhood maltreatment: a link with the severity and type of trauma Methylation of the dopamine D2 receptor (DRD2) gene promoter in women with a bulimia-spectrum disorder: associations with borderline personality disorder and exposure to childhood abuse Methylation of serotonin receptor 3A in ADHD and bipolar disorders: link with severity of the disorders and childhood maltreatment Increased DNA methylation of neuropsychiatric genes occurs in borderline personality disorder Aberrant methylation of gene associated CpG sites occurs in borderline personality disorder Aberrant DNA Methylation of rDNA and PRIMA1 in borderline personality Disorder Borderline personality disorder and childhood maltreatment: a genome-wide methylation analysis Methylation at 5HTT mediates the impact of child sex abuse on women's antisocial behavior: an examination of the Iowa adoptee sample Serotonin 1B receptor gene (HTR1B) methylation as a risk factor for callous-unemotional traits in antisocial boys Methylation of the oxytocin receptor gene and oxytocin blood levels in the development of psychopathy MAOA methylation is associated with nicotine and alcohol dependence in women Individual differences in childhood behavior disorders associated with epigenetic modulation of the cortisol receptor gene Normative data for the strengths and difficulties questionnaire in Australia CrossRef Full Text Epigenetic modifications of the glucocorticoid receptor gene are associated with the vulnerability to psychopathology in childhood maltreatment Peripheral SLC6A4 DNA methylation is associated with in vivo measures of human brain serotonin synthesis and childhood physical aggression Differential DNA methylation regions in cytokine and transcription factor genomic loci associate with childhood physical aggression Association of childhood chronic 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Text | Google Scholar Epigenetic biomarkers as predictors and correlates of symptom improvement following psychotherapy in combat veterans with PTSD Kuhn J and Frodl T (2018) Epigenetics in Personality Disorders: Today's Insights Received: 05 July 2018; Accepted: 23 October 2018; Published: 19 November 2018 Copyright © 2018 Gescher, Kahl, Hillemacher, Frieling, Kuhn and Frodl. 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: Dorothee Maria Gescher, ZG9yb3RoZWUuZ2VzY2hlckBtZWQub3ZndS5kZQ== †These authors share first authorship Metrics details Biofilms are the natural form of life of the majority of microorganisms These multispecies consortia are intensively studied not only for their effects on health and environment but also because they have an enormous potential as tools for biotechnological processes Further exploration and exploitation of these complex systems will benefit from technical solutions that enable integrated where readily available microfluidic chips are connected by automated liquid handling with analysis instrumentation chromatography and optical coherence tomography The system is operable under oxic and anoxic conditions allowing for different gases and nutrients as feeding sources and it offers high spatiotemporal resolution in the analysis of metabolites and biofilm composition We demonstrate the platform’s performance by monitoring the productivity of biofilms as well as the spatial organization of two bacterial species in a co-culture which is driven by chemical gradients along the microfluidic channel we here report on the development of an integrated platform for machine-assisted biofilm research The focus was on the development of a generic automatable approach that makes it possible to investigate native multi-species biofilms that are not accessible with genetic engineering methods a number of modules have been developed combining powerful standard methods such as flow cell technology and optical imaging with new robotic approaches for spatiotemporal characterisation to enable investigation of variations in the composition of the biofilm community and the culture broth In order to minimize the effort and possible errors caused by manual handling we aimed at a high degree of automation for the entire biofilm platform Overview of the integrated platform for machine-assisted cultivation and analysis of biofilms It is very easy to process into any shape by replica casting which allows to puncture the material for sampling purposes without causing irreparable damage in the form of holes and leaks Of particular importance for cell culture applications is that PDMS has a very high gas permeability which allows the fluidic system to operate under well defined environmental conditions in terms of gas composition (see below) Optical analyses of flowcell biofilms labeled by automated FISH Overview image and high-resolution (inset) micrographs of a pure B Biofilms were grown in triplicates in linear flow chambers for 12 h (e) subjected to automated FISH and analyzed with the integrated fluorescence reader at the 13 measurement points to obtain mean fluorescence values for the nine parallelly cultivated biofilms (f) coli were labeled with probes LGC354B-A488 (colored in green) and Ent-A546 (red) Error bars represent the standard deviation between the mean fluorescence values of triplicate experimental samples carried out in individual flow chips Note that DAPI staining indicates approximately equal growth density and FISH data allow for reliable identification of pure E (g) Epifluorescence micrographs of mixed species B coli biofilms (same color code as in a–d) confirm the topographical biofilm features visualized by means of OCT (h) As a result of microbial activity and/or the diffusion of gases out of or into the medium via the PDMS materials the microfluidic channels of our platform allow for the formation of gradients along the medium flow This feature can be exploited for the cultivation of interdependent communities that are fluidically connected but spatially separated The self-organized separation of mixed cultures along autonomously created gradients can be controlled and steered by the applied growth conditions and it may also allow to determine specific characteristics of novel environmental isolates such as growth auxotrophies that are difficult if not impossible to study by conventional approaches It is an aerobic organism whose chromate tolerance stems from its capability to efficiently reduce toxic Cr(VI) to Cr(III) species the latter of which are highly insoluble and therefore less toxic our hypothesis was that flow cultivation of mixed E chromiiresistens cultures with Cr(VI)-containing medium should lead to a spatial separation of the two species The front part of the channel should be inhabited mainly by L coli should accumulate in the lower parts of the chip To achieve an optimal characterization of the biofilm composition we took advantage of both the automated FISH procedure and the robotic sampler The latter was used to draw 10 μl samples from the biofilms which were subjected directly to 16S rRNA gene amplification and subsequent Illumina sequencing Interdependent biofilm consortia self-organize along an autonomously created gradient of chromate A series of four identical inocula was used and cultivated for seven days in the absence (a,b) 2 mM (e,f) or 3 mM (g,h) chromate containing medium The analysis was performed by robotic cell sampling and subsequent staining by the auto FISH procedure Epifluorescent images and phylogenetic composition in (a,c,e,g) represent the front section of the microfluidic chip while (b,d,f,h) show representative data from the rear end The error bars represent the standard deviation between the ratio of reads from the 16S rDNA amplicon sequencing of two independently cultivated flowcells Sampling points are indicated in the scheme of the setup Unspecific labeling with DAPI is indicated in blue The bar charts on the left side of each sample represent the community composition derived from 16S rDNA amplicon sequencing Culturing and analysis of a productive E (a) Reaction scheme of the stereoselective reduction of the prochiral nitro-diketone substrate (NDK) into hydroxyketone (HK) and diol products by the R-selective ketoreductase LbADH (b) Representative chiral HPLC chromatograms of samples drawn from selected points of the meandric cultivation channel (c) The decrease of NDK educt and HK/diol products is clearly evident from the analysis of the reaction samples retrieved along the flowpath at a constant flowrate of 2 µL/min (d) The error bars represent the standard deviation of two independent samples which where withdrawn sequentially from the indicated sampling points dynamic studies can also be performed to analyse temporal fluctuations on relevant time scales of biofilm growth 16S rRNA gene amplicon sequencing was used to study a synthetic two species biofilm of Leucobacter chromiiresistens and E Changing compositions of the biofilm along the flowpath as well as with varying feed medium could be observed It was also confirmed by subsequent FISH analysis that L coli cells in the rear part of the flowcell by the reduction of toxic chromate it could be shown how the online sampling in combination with HPLC can be used to determine concentration levels of metabolic compounds and biotechnologically-relevant products this approach gives access to the composition and dynamics of the microbial community cultivated within the flowcells and it demonstrates the possibility to evaluate online process parameters for biocatalytic transformation reactions The latter is considered one of the basic prerequisites for the breakthrough of the use of productive biofilms in biotechnological applications microfluidic biofilm cultivation devices and tailored robotic instrumentation were combined with powerful standard methods such as (CARD-) FISH HPLC and next generation sequencing to create a unique set of tools for multispecies biofilm research All here described techniques for biofilms analysis do not rely on the genetic labelling of cells and enable the investigation of native communities studies on productive biofilms or the exploration of complex native consortia will largely benefit from the developed platform It is therefore anticipated that this work represents an important step towards the realization of machine-assisted processes for the next generation of biotechnology Further technical details of the robotic setup and the control software will be published elsewhere templates consisted of 1 µl of the robotically withdrawn samples Primers F_NXT_Bakt_341F (5′-CCT ACG GGN GGC WGC AG-3′) and R_NXT_Bakt_805R (5′-GAC TAC HVG GGT ATC TAA TCC-3′) were used for amplification Sequencing was conducted by IMGM Laboratories GmbH (Martinsried trimming of adapter sequences was conducted by IMGM Laboratories with the MiSeq Reporter 2.5.1.3 software Further bioinformatic analysis of the 16S rRNA gene amplicon sequencing (primer cutting and phylogenetic analysis) was carried out with the CLC Genomic Workbench software 10.0.1 equipped with the additional microbial genomic module 2.0 The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request Biofilms: an emergent form of bacterial life Bacterial biofilms: from the natural environment to infectious diseases Synthesis of many different types of organic small molecules using one automated process Computer-Assisted Synthetic Planning: The End of the Beginning Continuous-flow technology-a tool for the safe manufacturing of active pharmaceutical ingredients On-demand continuous-flow production of pharmaceuticals in a compact Single cells in confined volumes: microchambers and microdroplets Microfluidics for single-cell genetic analysis Commercialization of microfluidic point-of-care diagnostic devices Revisiting 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Start Fabrication of microfluidic systems in poly(dimethylsiloxane) Let there be chip—towards rapid prototyping of microfluidic devices: one-step manufacturing processes Poly(dimethylsiloxane) as a Material for Fabricating Microfluidic Devices Biofilm streamers cause catastrophic disruption of flow with consequences for environmental and medical systems Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques An integrated microfluidic chip for chromosome enumeration using fluorescence in situ hybridization A novel integrated microfluidic platform to perform fluorescence in situ hybridization for chromosomal analysis FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool Microfluidics on liquid handling stations (muF-on-LHS): an industry compatible chip interface between microfluidics and automated liquid handling stations Globally optimal stitching of tiled 3D microscopic image acquisitions Draft genome sequence of Leucobacter chromiiresistens Chromate Resistance Mechanisms in Leucobacter chromiiresistens Effect of Chromate Stress on Escherichia coli K-12 Enantiogroup-Differentiating Biocatalytic Reductions of Prochiral C -Symmetrical Dicarbonyl Compounds to meso Compounds Orthogonal Surface Tags for Whole-Cell Biocatalysis Specific oligonucleotide probes for in situ detection of a major group of gram-positive bacteria with low DNA G + C content Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures In situ probing of gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides Download references We acknowledge financial support of this work by the Helmholtz program BioInterfaces in Technology and Medicine Institute for Biological Interfaces (IBG-1) Section IV: Biomolecular Separation Engineering developed the robotic sampler and established the microfluidic cultivation setup together with S.K conceived conceptual and experimental work on the automated FISH method conceived the biofilm cultivation experiments All authors discussed the results and commented on the manuscript Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Reprints and permissions Download citation DOI: https://doi.org/10.1038/s41598-019-45414-6 a shareable link is not currently available for this article Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research Biotechnology AdvancesCitation Excerpt :Various strategies have been used for enhancing H2 solubilization in the liquid and making it readily available to hydrogenotrophic methanogens (Aryal et al. H2 and CO2 addition promote microbial growth Several studies have shown a considerable difference in the microbial community between the liquid and biofilm phases (Jensen et al. International Journal of Hydrogen EnergyCitation Excerpt :Over the last few years researchers have examined the potential of achieving biogas upgrading in various types of membrane bioreactors (MBR) which employ pressurized membranes for supplying a gaseous substrate to a bio-film attached on the membrane surface [100] which combine anaerobic digestion with membrane filtration and present the advantages of bioenergy recovery separation of sludge retention time (SRT) and hydraulic retention time (HRT) [102] investigated the suitability of a pseudo-dead-end MBfR for ex-situ biogas upgrading and showed that the concept of MBfR is especially advantageous for ex-situ hydrogenotrophic methanation of biogas CO2 yielding high product gas qualities of up to 99% methane [103] developed a novel MBfR for simultaneous biogas upgrading and liquid chemicals production Journal of Environmental Chemical EngineeringCitation Excerpt :Biomethanation efficiency highly relies on H2 transfer from gas to liquid phase special focus has been given on improving reactor designs (e.g membrane based reactors) and characteristics (i.e diffusers) to increase the availability of gases to the microbes [7–9] Despite regularly achieving a product gas for direct feed-in to the gas grid (>95% CH4) studies are still witnessing loses of H2 not converted through the methanogenic pathway [10,11] FuelCitation Excerpt :This is due to the biogas sector that attracts increasing interests considering the advantages of using biomethane as fuel for heat and transportation [13] The four technologies which are the most commonly applied in biogas upgrading include physical/chemical absorption [14] A comprehensive comparison between these technologies has been presented in earlier study [12] All content on this site: Copyright © 2025 Elsevier B.V., its licensors, and contributors. 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Volume 9 - 2018 | https://doi.org/10.3389/fmicb.2018.01313 Nitrogen based eutrophication of ecosystems is a global problem that gains momentum through a growing global population The water quality of nitrate or ammonium contaminated rivers and streams cannot always be amended in centralized waste water treatment plants Field denitrification plants were suggested as a solution for a decentralized reduction of nitrate to dinitrogen stable and cheap organic carbon sources serve as carbon and electron source for a microbial community our knowledge on the impact of these organic carbon sources on the development and diversity of these cultures is sparse the stability of these denitrification plants at different nitrate loading rates especially in the higher concentration regime were not tested so far we compare the fate of carbon and nitrogen as well as the microbial community of wood pellet (WP) (pressed sawdust) and wood chips (WC) based laboratory denitrification reactors Our study reveals that the diversity and composition of the community is strongly dependent on the carbon source The three reactor types were characterized by different nitrate reduction kinetics and were affected differently by high nitrate loading rates While the nitrate reduction kinetics were negatively influenced by higher nitrate doses in the wheat straw reactors WPs as carbon source sustained the opposite trend and WC lead to an overall slower but concentration independent nitrate reduction rate the concentration of soluble organic carbon was highest in the WP reactors but methane emission was not detectable This is corroborated by the microbial diversity data in which methanogenic species were highly underrepresented compared to the other two reactor types the methane emissions in the wheat straw and WC reactors were comparable to each other investigations regarding the exact microbial composition and the catalyzed processes within these denitrification beds are necessary to model and predict nitrate elimination and to analyze their overall sustainability One assumption is that the microbial composition in these beds is strongly dependent on the carbon material and influences the nitrate elimination process and the formation of side products the exact microbial composition of denitrification beds was recently investigated for the first time in WC filled systems we revealed that the microbial community changed depending on the nitrate loading rate and that high nitrate loading rates (low Corg/NO3-) could lead to a diversification of anaerobic nitrate reduction toward the production of ammonium through DNRA which contradicts to textbook knowledge claiming that DNRA should be favored under high Corg/NO3- ratios it was elucidated that higher nitrate loading rates can increase methane emissions in WC based denitrification beds The effect of nitrate loading rates on DNRA and methane emissions was unexpected and also highly unwanted regarding the desired application of these systems as sustainable and ecosystem-friendly nitrate elimination technology The aim of this study was to elucidate whether this effect of high nitrate loading rates on the fate of carbon and nitrogen is specific to denitrification beds based on WC or whether other sustainable stable organic carbon materials would lead to the development of different microbial communities that would consequently also respond differently to variations in the nitrate loading rate We expected the result of this study to be generic with regard to the suitability of a carbon source for the elimination of nitrate in a specific concentration window Our motivation is the planned construction of one of the first field denitrification beds in middle Europe which is supposed to eliminate nitrate from agricultural drainage waters that run directly into a fen The construction of such a field denitrification system at this but also at every other field site necessitates knowledge regarding the nitrate elimination rates and the resilience of the system toward different nitrate concentrations we show here the results from a 200-day laboratory triplicate experiments with wood pellets (WPs) and wheat straw as denitrification substrates and aim to correlate the results to the microbial community in the reactors and to our previous study with WC Our results reveal that all three materials lead to unique microbial community compositions and sustain also individual responses toward nitrate pulses Also the used artificial moor media and inoculum were the same as in the previous study The reactors were incubated at room temperature (approx The starting concentration of nitrate was 1.18 mmol L-1 which correlated to the highest measured nitrate concentration of an agricultural drainage from a field site – a fen in the Vulkaneifel (Germany) – that we observe regarding the eutrophication effects of agricultural drainage waters This fen is surrounded by agricultural fields and drainages lead directly into this sensible ecosystem In the experiment runtime of 200 days, samples were taken two to three times a week and analyzed for NO3-, NO2-, NH4+, and TOC according to Grießmeier et al. (2017) with a spectral photometer DR3900 and cuvette tests (Hach) as well as a TOC-analyzer (Multi N/C 2100 S Analytic Jena, Germany). Organic carbon compounds were analyzed via HPLC according to Kipf et al. (2013) Whenever the nitrate concentration decreased below 0.24 mmol L-1 nitrate was added to reach the initial NO3- concentration of 1.18 mmol L-1 The nitrate concentration was increased to the twofold (2.4 mM L-1) and 20-fold (24 mM L-1) of the initial concentration to test the response of the systems to variations in the nitrate loading rates On day 70 all reactors of the triplicates were opened and samples were taken to analyze the biodiversity in the planktonic phase as well as in the biofilms growing on the surface of the organic carbon sources (WPs and wheat straw) genomic DNA was extracted from drainage water from the study site (collected December 8 Genomic DNA was extracted from 800 μl of the planktonic and from 200 to 300 mg of the solid samples using the innuSPEED Soil DNA Kit (Analytic Jena) as per the manufacturer’s guidelines The maximum nitrate removal rate was determined using the maximum slope of the nitrate reduction kinetics for every reactor type A one-way analysis of variance (ANOVA) and a following Tukey’s honestly significant difference (HSD) post-hoc test with a significance level of 0.05 was performed to analyze the statistical significance of the nitrate elimination rates sustained by the different carbon sources All bioinformatic analysis as well as the rarefaction curves and PCoA of the 16S rRNA gene amplicon sequencing were conducted as described previously with the CLC Genomic Workbench software 11.0.1 and the additional microbial genomic module 3.0 First all reads of every triplicate were quality trimmed with a limit of 0.05 a primer sequences trim and merging of the paired reads was performed The OTU clustering was performed against the SILVA 16S v128 97% database with a similarity percentage specified by the OTU database and new creations were allowed with a taxonomy similarity of 80% OTUs with a minimum combined abundance less than 50 were removed OTUs belonging to Bacteria detected with the Archaea primer pair were excluded before the visualization of the relative abundance of the OTUs Out of the triplicate of every sample the mean value for every OTUs was calculated Alpha diversity was described with the phylogenetic diversity the total number of OTUs and the Shannon index by aligning the OTUs using MUSCLE 2.0 and building a maximum likelihood based phylogenetic tree with the tree algorithm Neighbor Joining and nucleotide substitution model Jukes Cantor A Principle Coordinate analysis (PCoA) was performed on the D_0.5 UniFrac distance For the analysis of the nirS gene amplicons the merged sequencing reads were analyzed using the blastN algorithm and a subset of sequences derived from the database of the European Nucleotide Archive (ENA) containing only nitrite reductases (E.C Reads were accounted as nirS sequences if the gained E-value of the alignment was below 10-10 The phylogenetic assignment was conducted by grouping the hits with more than 100 reads of the blast analysis on the level of the bacterial order S2 provides the results of the sequencing analysis parameters for each sample All raw reads of the amplicon sequencing that were retrieved for this study are publicly available through NCBI BioProject PRJNA445677 under SRA accession: SRP145155 The objective of this study was to analyze the effect of different organic carbon sources in laboratory surrogates of environmental denitrification beds on the fate of nitrogen and carbon at varying nitrate loading rates and to analyze how this response would be catalyzed by the developing microbial communities only the mean values are shown for all parameters A detailed analysis of each triplicate is depicted in the Supplementary Figures S1–S4 the mean maximum acetate concentration in these reactors was only 4.2 mM acetate (Supplementary Figure S5) FIGURE 1. Development of the nitrogen species and total organic carbon (TOC) in the laboratory reactors. Mean value concentration of nitrate, nitrite, ammonium, and TOC in the laboratory denitrification reactors filled with (A) wood pellets (WP), (B) wheat straw, or (C) wood chips (WC). Data from the wood chip filled reactors derived from the earlier published investigation of Grießmeier et al. (2017) Dashed vertical lines represent the different nitrate addition events: dotted lines represent 1× (1.18 mmol L-1) nitrate addition dotted and dashed lines represent 2× (2.36 mmol L-1) nitrate addition on Day 72 5× (5.9 mmol L-1) nitrate addition on Day 112 10× (11.8 mmol L-1) nitrate addition on Day 131 and 20× (23.6 mmol L-1) nitrate addition on Day 148 The measured concentration of organic acids (acetate and fumarate) was very low at this time point in the wheat straw reactors comparable low organic acid concentrations were also detected in the WC reactors and this did not affect the nitrate elimination rates to the same extent as in the wheat straw reactors (Supplementary Figures S5 Maximal and mean nitrate elimination rate in each reactor type for the different nitrate addition events with additional p-values for the analysis of variance (ANOVA) Maximum nitrate elimination rate for the 5× (A) Differences between the samples were determined by analysis of variance (ANOVA) followed by a Tukey post-hoc test (D) Depicts the maximum nitrate elimination of each triplicate for every nitrate addition event Significant differences (p < 0.05) between the samples are marked with an asterisks The rather robust nitrate reduction in the WP systems went along with short pulses of nitrite that could be detected after every nitrate addition, no matter if at the beginning of the experiment – to reach the initial nitrate concentration - or in the later phase with higher nitrate concentrations (Figure 1A and Supplementary Figure S2) This indicates that the nitrate elimination was probably conducted by a different biocenosis compared to the other two systems This value is significantly lower than the starting concentration of NO3-N used in this study (74 mg l-1 NO3-/16.7 mg l-1 NO3-N) CWs work rather well regarding the elimination of different pesticides and they might in fact be superior compared to denitrification beds regarding this application the removal of higher nitrate concentrations can most probably not be superior to denitrification beds as the latter provide a robust anoxic niche for denitrifying organisms and a constant supply of carbon and electrons the similarity of the material might be the reason why the conditions selected in both reactor types for Clostridiales FIGURE 3. Microbial diversity in the laboratory reactors. Relative abundance of the 16S rDNA amplicon sequencing results for (A) OTUs of Bacteria (>1% of the total number of amplicons) and (B) OTUs of Archaea. SILVA 16S v 128 97% served as reference database. Results of the WC reactors were first published in Grießmeier et al. (2017) Maximum values of different diversity indices – Shannon entropy total number of OTUs and phylogenetic diversity – for each triplicate of the different samples as well as their mean value and standard deviation (SD) These two known denitrifying orders were accompanied in the WC reactors by members of the order Xanthomonadales (mainly from the genus Pseudoxanthomonas) which is also known to contain denitrifying organisms (Lycus et al., 2017) and could possibly also play an important role in the denitrification process no order commonly known to contain denitrifiers was found in the solid phase of the wheat straw reactors One possibility could be that organisms that were underrepresented (less than 1% relative abundance) may be responsible for the denitrification process here or are so far not known to be able to accomplish this process division of labor could also be a reason why a small number of nirS encoding organisms could be sufficient to sustain the observed denitrification rates Total abundance of reads which could be assigned to nirS genes Only nitrite reductases which inhabited an E-value lower than 10-10 and minimum read amount of 100 were used for further analysis further elemental analysis of the WPs used in this study will show if there is an increased heavy metal concentration which could lead to the inhibition of methanogens Volume percent [% v/v] of produced CH4 and CO2 in the different denitrification reactors (A) wheat straw Dashed vertical lines represent the added nitrate concentrations: dotted lines represent 1× (1.18 mmol L-1) nitrate addition This study represents an enlargement of an earlier investigation (Grießmeier et al., 2017) gas production and microbial composition of denitrification reactors filled with either wheat straw or WPs were compared to this former study where WC were used as carbon source The results reveal that the choice of the carbon material not only determines the fate of the nitrate reduction it also defines the microbial composition in the denitrification beds The reactors filled with WPs seem to be suitable for high nitrate concentrations we could not measure methane emissions even at high TOC concentrations and over a time period of 200 days the occurring nitrite peaks and the gel like character of the soaked WPs are disadvantages of this material The reactors with straw showed the best nitrate elimination rates for moderate nitrate concentrations Still this material does not seem to be suitable for high nitrate loading rates high methane production and ammonia concentrations of almost 4 mM make this carbon source also unsuitable for a field denitrification bed The clearest advantage of the WC over the other two materials seems to be the highly diverse microbial community that developed over time The more diverse an ecosystem is the more robust it will be regarding process perturbations the temperature and the water flow through the systems in field scale compared to lab scale is hard to predict we will be able to judge on this hypothesis by the analysis of field system that is supposed to rescue a fen in the Vulkaneifel in Germany The raw data supporting the conclusions of this manuscript will be made available by the authors VG performed the experimental work including reactor design and bioinformatic evaluation of the 16S rRNA amplicon sequencing data and prepared all figures of the manuscript The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018.01313/full#supplementary-material Denitrifying bioreactors for nitrate removal: a meta-analysis Nitrogen pollution removal from areas of intensive farming-comparison of various denitrification biotechnologies Phylogenetic distribution of potential cellulases in bacteria Nitrous oxide: emission from soils during nitrification of fertilizer nitrogen Nitrate removal and hydraulic performance of organic carbon for use in denitrification beds CrossRef Full Text | Google Scholar The evolution and future of earth’s nitrogen cycle Nitrate removal and greenhouse gas production in a stream-bed denitrifying bioreactor CrossRef Full Text | Google Scholar How a century of ammonia synthesis changed the world CrossRef Full Text | Google Scholar Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics Grießmeier Investigation of different nitrogen reduction routes and their key microbial players in wood chip-driven denitrification beds Inhibition of methanogenesis in pure cultures by ammonia and protection against heavy metal toxicity by sewage sludge Systematic screening of carbon-based anode materials for microbial fuel cells with Shewanella oneidensis MR-1 Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies Phenotypic and genotypic richness of denitrifiers revealed by a novel isolation strategy Water Quality and Agriculture: Meeting the Policy Challenge CrossRef Full Text | Google Scholar II) Wood pellets for home heating can be considered environmentally friendly fuels Heavy metals determination by inductively coupled plasma-optical emission spectrometry (ICP-OES) in their ashes and the health risk assessment for the operators Enhancement of nitrate removal in constructed wetlands utilizing a combined autotrophic and heterotrophic denitrification technology for treating hydroponic wastewater containing high nitrate and low organic carbon concentrations Simultaneous cellulose degradation and electricity production by Enterobacter cloacae in a microbial fuel cell A comparison of the contribution of various gases to the greenhouse effect Wood chips and wheat straw as alternative biofilter media for denitrification reactors treating aquaculture and other wastewaters with high nitrate concentrations Nitrate removal from three different effluents using large-scale denitrification beds CrossRef Full Text | Google Scholar “Development and application of nucleic acid probes,” in Nucleic Acid Techniques in Bacterial Systematics Google Scholar Throbäck nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE nitric oxide and ammonia by gut bacteria under physiological conditions Technical report: human alteration of the global nitrogen cycle: sources and consequences Removal of nutrients in various types of constructed wetlands A comparison of different approaches for measuring denitrification rates in a nitrate removing bioreactor controls and potential adverse effects of nitrate removal in a denitrification bed communities of denitrifiers and adverse effects in different carbon substrates for use in denitrification beds Citation: Grießmeier V and Gescher J (2018) Influence of the Potential Carbon Sources for Field Denitrification Beds on Their Microbial Diversity and the Fate of Carbon and Nitrate Copyright © 2018 Grießmeier and Gescher. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited *Correspondence: Johannes Gescher, am9oYW5uZXMuZ2VzY2hlckBraXQuZWR1 Water ResearchCitation Excerpt :The two sets of variables were separated by the metal (Fe) the bacterial communities reacted differently to various organic matters (Reitter et al. the organic matter had a negative effect on bacterial activity in both the MG (standardized coefficient = 0.520) and SG (standardized coefficient = -0.056) Science of the Total EnvironmentCitation Excerpt :Mycobacterium neoaurum and Rhodococcus erythropolis in the LJH reservoir play a key role in negative associations (Lipko and Belykh They were found to have widely negative associations with other actinobacteria because actinobacterial communities would compete for similar resources (Reitter et al. Mycobacterium neoaurum occurred frequently in the hypolimnion layer which had a higher concentration of Fe and Mn Metrics details Micrarchaeota is a distinctive lineage assigned to the DPANN archaea which includes poorly characterised microorganisms with reduced genomes that likely depend on interactions with hosts for growth and survival we report the enrichment of a stable co-culture of a member of the Micrarchaeota (Ca Micrarchaeum harzensis) together with its Thermoplasmatales host (Ca We show that symbiont-host interactions depend on biofilm formation as evidenced by growth experiments comparative transcriptomic analyses and electron microscopy extracellular polymeric substances and lipid content analyses indicate that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host Our study of the cell biology and physiology of a Micrarchaeon and its host adds to our limited knowledge of archaeal symbioses we report the isolation of a stable co-culture of this Micrarchaeon together with its host a previously unknown member of the Thermoplasmatales This allows us to conduct experiments aiming at understanding the interaction of the two organisms and the response of the Thermoplasmatales member to growth in co-culture with the Micrarchaeon we were able to isolate the host B_DKE by enriching at pH 2.5 for planktonic organisms The composition of cultures was verified via PCR with organism-specific primers and CARD-FISH Based on the isolation of the Thermoplasmatales host and the reconstruction of complete genome sequences of both organisms we propose the names”Candidatus Micrarchaeum harzensis” sp pertaining to the German region of the Harz Mountains where the organism was isolated) and “Candidatus Scheffleriplasma hospitalis” gen Horst Scheffler and in recognition of his work on mine geology and commitment to our work; Gr plasma something shaped or moulded; hos.pi.ta′lis referring to its ability to serve as a host for “Candidatus Micrarchaeum harzensis”) we used physiology informed strategies for deselecting against additional community members and show that selection for biofilm formation of the Thermoplasmatales host population was the critical factor for obtaining a stable co-culture Depicted are the mean cell numbers of Ca Micrarchaeum harzensis (empty squares) and Ca Scheffleriplasma hospitalis (full squares) calculated via qPCR and the ferrous iron concentration (light grey circles) over time Error bars show standard deviation of triplicates Source data are provided as a Source Data file To examine the impact of the co-cultivation with the Micrarchaeon on Ca. Scheffleriplasma hospitalis its growth characteristics were compared between pure and co-culture under otherwise identical conditions (growth curve of Ca. Scheffleriplasma hospitalis pure culture in Supplementary Fig. 2) Cells of the Thermoplasmatales member showed a similar growth and ferric iron reduction in pure culture in comparison with the co-culture Scheffleriplasma hospitalis in co-culture (7.49 ± 1.45 days n = 3) was not significantly different (unpaired significance level 0.05) to the doubling time of the organism cultivated without its interaction partner (7.19 ± 3.80 days growth of the host seems to be little affected by the Micrarchaeon under the tested conditions Scheffleriplasma hospitalis was sequenced using a combination of PacBio and Illumina sequencing Illumina sequencing was also performed on the Ca The two organisms have circular chromosomes of 1,959,588 base pairs (bp) (Ca Scheffleriplasma hospitalis) and 989,838 bp (Ca Scheffleriplasma hospitalis isolate and the strain within the co-culture were 100% identical which was important for the later comparative transcriptomic analysis An analysis of clusters of orthologous groups (COGs) revealed that Ca Micrarchaeum harzensis contains more proteins with unknown function (29%; 300 putative proteins without an arCOG-assignment) relative to the overall number of genes compared to Ca Scheffleriplasma hospitalis (20%; 419 putative proteins without an arCOG-assignment) Given are the enzyme commission (EC) numbers of the involved enzymes in bold and the Transcripts Per Million (TPM) values (mean value of four co-culture samples) of the corresponding Ca Missing genes and metabolites that the Micrarchaeon cannot produce itself are highlighted in dark red Question marks label the unknown anchoring of the S-layer protein and an unknown flippase Among the tested lectins, only those specific to galactose- and mannose-related conjugates bound the extracellular matrix of the co-culture and of Ca. Scheffleriplasma hospitalis cells in pure culture (see Fig. 3 and Table 1) and PTA seemed to discriminate between the extracellular matrix of Ca Scheffleriplasma hospitalis in the presence or absence of Ca Micrarchaeum harzensis on the matrix chemistry or composition Micrarchaeum harzensis cells was weak and only possible with some of the lectins that bound to Ca Micrarchaeum harzensis to build carbohydrate polymers or the production of less common polymers for which we did not have a lectin It suggests that the detected signals on Ca Micrarchaeum harzensis are likely due to growth within the biofilm matrix of Ca Shown are example images of the microscopic analysis of the pure culture (left) and co-culture (right) The images show the results for staining with lectin CA (co-culture) and HHA (pure culture) Scheffleriplasma hospitalis was stained with the general archaea probe Arch915 (blue) which does not stain Ca Micrarchaeum harzensis was stained using the Micrarchaeota-specific ARMAN980 probe (magenta) The experiment was repeated at least three times with equivalent results The different pathways are indicated with dark blue (MVA pathway I) violet (MVA pathway III) and magenta arrows (MVA pathway IV) Scheffleriplasma hospitalis are indicated with black and grey circles Abbreviations are AMPD anhydromevalonate phosphate decarboxylase DGGGPS 2,3-bis-O-geranylgeranyl glycerol-1-phosphate synthase GGGPS 3-O-geranylgeranyl glycerol-1-phosphate synthase IDI isopentenyl-diphosphate-delta-isomerase MBD mevalonate-3,5-bisphosphate-decarboxylase Membrane localisation was verified using the TMHMM algorithm which detected eight transmembrane helices in the protein Ca. Scheffleriplasma hospitalis and Ca. Micrarchaeum harzensis genes are highlighted in light blue and violet, respectively. The Maximum likelihood phylogenetic trees shown here included our archaeal backbone dataset (Supplementary Data 7) and were inferred for arCOG02937 (149 sequences with 304 amino acids) arCOG00570 (1877 sequences with 132 amino acids) and COG2074 (arCOG01968 and arCOG01967 121 sequences with 171 amino acids) with the LG+C10+F+R model with an ultrafast bootstrap approximation run with 1000 replicates that the inclusions of bacterial and eukaryotic homologues did not change the interpretation of our findings Note that arCOG02937 and arCOG02074 represent the key enzymes of the Type-III mevalonate pathway characteristic of Thermoplasmatales Only bootstrap support values above 90 are shown as indicated in the panel Both organisms possess the enzymes to convert gluconic acid to glycerate our results reveal that the pattern of metabolites do not seem to deviate between isolated Ca Scheffleriplasma hospitalis and co-culture and that the kinetics of consumption show minor differences towards faster consumption of some compounds in the co-culture either both organisms employ similar pathways and compounds or Micrarchaeum harzensis predominantly uses metabolites provided by Ca The latter assumption is in agreement with the sparsity of transporters encoded in the Ca which may indicate that it relies on direct cell-cell interaction for nutrient and metabolite exchange Normalisation was done using a z-score and significance was calculated by a two-tailed t-test were observed (a) in physical contact with Ca as well as (b) free living and (c) undergoing cell division The freeze-etching experiment was repeated once each time micrographs showing more than 100 cells were analysed The detailed characterisation of our co-culture in comparison with pure host cultures indicate a specific regulatory response of Ca Scheffleriplasma hospitalis as a consequence of growth with Ca The combination of genomic analyses with comparative metabolomics lipidomics and determination of EPS composition revealed that the growth of Ca Micrarchaeum harzensis is dependent on interaction with its host Ca whose growth seemed little impaired by the symbiont Cryo-ET micrographs and comparative transcriptomics indicate that Ca Scheffleriplasma hospitalis might initiate the interaction by biofilm formation change of EPS composition and development of a filamentous structure which was observed in contact with Micrarchaeota cells A physical interaction involving surface remodelling is established which is likely crucial for the uptake of various metabolites and building blocks for Due to the limited metabolic capabilities of Ca Micrarchaeum harzensis and its dependency on Ca it is very likely that the DPANN member initiates the interaction The close cell-cell interactions between acidophilic Micrarchaeota and Thermoplasmatales may also provide a route for horizontal gene transfer among these DPANN symbionts and their hosts The cultures typically reached the exponential growth phase after incubation for 2–8 weeks Imaging was conducted using a Zeiss Axiovert 200 M fluorescence microscope equipped with the software Axio Vision 4.7 Sequences with ≥90% gaps were removed using a custom script (faa_drop.py) and used for a phylogenetic analysis with FastTree (v2.1.10 Differential expression was assessed by using the Create Expression Browser 1.1 implementation of CLC Genomics Workbench 20.0.1 (QIAGEN Results were filtered for log2fold changes higher or lower than 2 or -2 and a false discovery rate of 0.01 (Bonferroni) For differential expression analyses transcriptomic data was compared to Ca The experiment was performed two times with equivalent results A response factor derived from an archaeol:GDGT-0 standard (1:1) was used to correct for the difference in ionisation between archaeol and GDGTs The ToFMS was operated in extended dynamic range mode (2 GHz) with a scan rate of 2 Hz We assessed archaeal lipid distributions by monitoring m/z 600–1400 Archaeal lipids were identified by searching within 10 ppm mass accuracy for relevant [M + H] + signals Scheffleriplasma hospitalis and a co-culture of Ca Scheffleriplasma hospitalis were inoculated as described above for 42 (pure culture) or 49 days (co-culture) until all available ferric iron was reduced and stationary phase was reached 1 mL culture was sampled and stored at −80 °C until further analyses along with samples for ferrous iron quantification to estimate growth and samples for DNA extraction and CARD-FISH for further detection of Ca 500 µL samples were amended by inducing sulphur precipitation through the addition of a spatula tip of CaCO3 to each sample mixing for 5 min at 2,000 rpm at room temperature This treatment also led to cell lysis so that the analysis included also intracellular metabolites After centrifugation for 5 min at 17,000 × g at room temperature 50 µL of the supernatant was transferred to glass vials and dried under vacuum at 4 °C Dried samples were stored at −80 °C until further analysis 100 µL of anaerobically grown culture was removed aseptically from the growth flask 2.5 µL of the culture was immediately applied to a freshly glow-discharged Quantifoil R2/2 Cu/Rh 200 mesh or R3.5/1 Au 200 mesh grid blotted for 4–5 s and plunge-frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific Inc. while the blotting chamber was maintained at 100% humidity at 10 °C For half of the prepared grids 10 nm protein-A gold (CMC Utrecht Netherlands) was pelleted by centrifugation (100,000 × g 4 °C) resuspended in 1:10 dilution of the growth medium and additionally added to the samples immediately prior to grid preparation Micrarchaeum harzensis cells was performed using medium magnification cryo-EM image data collected at a pixel size of 3.998 nm Dividing cells were classified into associated or un-associated whether they were in direct contact to the larger host cell Ca Further information on research design is available in the Nature Research 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interactive sequence similarity searching InterProScan 5: genome-scale protein function classification MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses Near-optimal probabilistic RNA-seq quantification MetaboMAPS: pathway sharing and multi-omics data visualization in metabolic context A rapid method of total lipid extraction and purification Intact polar and core glycerol dibiphytanyl glycerol tetraether lipids in the Arabian Sea oxygen minimum zone Part II: Selective preservation and degradation in sediments and consequences for the TEX86 The effect of improved chromatography on GDGT-based palaeoproxies Analytical methodology for TEX86 paleothermometry by high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry An improved method to determine the absolute abundance of glycerol dibiphytanyl glycerol tetraether lipids Metabolite detector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis Automated electron microscope tomography using robust prediction of specimen movements Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging Computer visualization of three-dimensional image data using IMOD CTFFIND4: fast and accurate defocus estimation from electron micrographs Tomo3D 2.0 – Exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction Cryo-tomography tilt-series alignment with consideration of the beam-induced sample motion Fiji: an open-source platform for biological-image analysis Download references The authors thank Carola Berg for excellent technical assistance is a recipient of a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (202231/Z/16/Z) would like to thank the Vallee Research Foundation the European Molecular Biology Organization the Leverhulme Trust and the Lister Institute for Preventative Medicine for support was supported by the European Research Council (ERC STG ASymbEL: 947317) the Swedish Research Council (VR starting grant 2016-03559 to A.S.) and the NWO-I foundation of the Netherlands Organisation for Scientific Research (WISE fellowship) was supported by the NWO-I foundation of the Netherlands Organisation for Scientific Research (WISE fellowship to A.S.) was supported by the Soehngen Institute of Anaerobic Microbiology (SIAM) Gravitation grant (024.002.002) of the Netherlands Ministry of Education Culture and Science (OCW) and the Netherlands Organization for Scientific Research (NWO) We thank Marianne Baas for technical support with the lipid analysis were partly funded by the German Research Foundation (DFG) (grant number: 252014092) These authors contributed equally: Susanne Krause Kerstin Schmidt-Hohagen & Karsten Hiller Braunschweig Integrated Centre for Systems Biology (BRICS) Department of Marine Microbiology and Biogeochemistry Royal Netherlands Institute for Sea Research interpreted metagenomic and metatranscriptomic data and conducted together with T.R.N contributed to the writing of the manuscript performed PacBio and Illumina sequencing and were involved in bioinformatic analysis of the data interpretation of the respective data and contributed to the writing of the manuscript interpreted the data and was involved in writing the manuscript performed and interpreted the metabolome analysis and contributed to the writing of the manuscript contributed to metabolome analysis and writing of the manuscript constructed phylogenetic trees and contributed to the writing of the manuscript performed and interpreted the lipid analysis and contributed to the writing of the manuscript Nature Communications thanks Mircea Podar and the other reviewer for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-022-29263-y Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly. Volume 10 - 2019 | https://doi.org/10.3389/fmicb.2019.00126 Shewanella oneidensis is one of the best-understood model organisms for extracellular electron transfer Endogenously produced and exported flavin molecules seem to play an important role in this process and mediate the connection between respiratory enzymes on the cell surface and the insoluble substrate by acting as electron shuttle and cytochrome-bound cofactor the addition of riboflavin to a bioelectrochemical system (BES) containing S oneidensis cells as biocatalyst leads to a strong current increase an external application of riboflavin to increase current production in continuously operating BESs does not seem to be applicable due to the constant washout of the soluble flavin compound we developed a recyclable electron shuttle to overcome the limitation of mediator addition to BES Riboflavin was coupled to magnetic beads that can easily be recycled from the medium The effect on current production and cell distribution in a BES as well as the recovery rate and the stability of the beads was investigated The addition of synthesized beads leads to a more than twofold higher current production which was likely caused by increased biofilm production 90% of the flavin-coupled beads could be recovered from the BESs using a magnetic separator although bioelectrochemical systems (BESs) are perfectly suited for continuous biofilm based biotechnological production the constant stream of fresh media over the biofilm would result in a continuous wash out of flavin compounds neither the exogenous addition of soluble flavins nor the endogenous overproduction seem to be key strategies to enhance biotechnological production or consumption rates The aim of this research was to study the application of a recyclable electron shuttle and its effect on current production in a BES in order to overcome disadvantages and limitations of flavin addition such as losses by washing out or high process costs riboflavin was coupled to magnetic beads and the effects on current production in MR-1 as well as their stability and recyclability were investigated All strains used in this study are listed in Table 1. S. oneidensis barcode is a strain that was developed by Dolch et al. (2016). It contains a 72 bp synthetic genomic integration that can be used for the quantification of cells via quantitative PCR (qPCR, see below). The gfp strain constitutively expresses a gfp gene and was used for fluorescence microcopy (Stöckl et al., 2016) All strains were pre-cultured under oxic conditions in LB medium at 30°C Cells were then transferred into anoxic minimal medium containing 12 mM HEPES buffer 70 mM lactate as electron donor and 100 mM fumarate as electron acceptor the medium contained (per liter): 0.221 g K2HPO4 and 8.77 g NaCl supplemented with 1 mM MgSO4 The pH was adjusted to 7.4 and all bottles were sealed with rubber stoppers oxygen was removed from the medium via the repeated application of a vacuum and following sparging of the headspace with N2 Before inoculation into bioelectrochemical reactors the cells were harvested by centrifugation (5 min 6000 g) and washed twice with medium containing neither electron donor nor electron acceptor the cells were resuspended to an OD600 of 0.07 in medium containing 70 mM lactate but no electron acceptor The gfp-strain was cultivated with the addition of kanamycin (50 mg L-1) and L-rhamnose (2.2 g L-1) for plasmid maintenance and continuous eGFP expression All bioelectrochemical experiments were conducted in triplicates using a single chamber MFC with a working volume of 270 ml (Bursac et al., 2017) Germany) and platinum mesh (projected area of 1.25 cm2 Germany) were used as anode and cathode material and an Ag/AgCl electrode (Sensortechnik Meinsberg the anode was first rinsed with isopropanol The complete bioelectrochemical setup was sterilized by autoclaving the working electrode was poised to 0 mV vs SHE using a potentiostat (Pine Instruments United States) and current was monitored for 46 h BES were constantly flushed with N2 gas in order to provide anoxic conditions and constant mixing of the liquid phase The magnetic beads (PureCube MagBeads; Cube Biotech Germany) used in this work are spherical magnetic agarose beads The beads were synthesized by the company as a solid phase-based material via EDC/NHS-mediated coupling for covalent modification A riboflavin-modified epoxide function was coupled to the magnetic agarose with three different alkane carbon chains with a respective chain length of 4 (C4) glycine-modified epoxide function was used as a control and is from here on referred to as glycine-coupled beads The beads had an average particle diameter of 25 μm and were stored in a 25% (w/v) suspension in 100% isopropanol the beads were rinsed three times with sterile medium without electron donor and acceptor as stated above The number of beads was quantified using a Neubauer chamber (Roth The recovering process from the anolyte of the BES was performed using a magnetic separator and the recovery efficiency was calculated based on the difference between particle number added before the bioelectrochemical experiments and after the recovering process at the end of the experiments Riboflavin was decoupled from the beads chemically by adding 100 mM NaOH for 30 min. The beads were then separated from the riboflavin solution using a magnetic separator. Riboflavin was quantified using a plate reader (Infinite Pro M200 Tecan, Switzerland) at 440 nm (Bartzatt and Wol, 2014) The riboflavin concentration was determined based on a calibration curve Several dilutions of a riboflavin solution in 100 mM NaOH with concentrations ranging from 0.5 to 10 μM were prepared as standards Genomic DNA isolation as well qPCR based cell quantification were conducted according to Dolch et al. (2016) Wild type and ΔOMC cells were pre-grown harvested and centrifuged for 5 min at 6000 g The optical density was adjusted in minimal medium with 50 mM lactate as electron donor and 10 mM fumarate as electron acceptor to OD600 of 1 1 ml of cells was mixed with 100 μl of beads (coupled to either riboflavin or glycine) and cells were incubated overnight under anoxic conditions the beads were washed twice in anoxic medium lacking electron donor and acceptor using a magnetic separator The binding of cells was assessed qualitatively under the microscope Significance of the data was determined via Welch Two Sample T-test and F-test Experiments in the impedance flow cell were performed according to Stöckl et al. (2016) in a previously described flow cell (40) The flow cell was modified by the addition of an Ag/AgCl reference electrode (RE-3VT Japan) and assembled under sterile conditions Sterile Na2SO4 [electrolyte counter electrode (CE)] and lactate containing medium [LM for working electrode (WE)] were filled into the respective half-cell chambers The flow cells were tempered under an incubation hood to 30°C and the solutions were pumped through the flow cell by a peristaltic pump The WE solution was flushed with N2 with a flow rate of 5 mL min-1 in order to ensure anoxic conditions in the WE chamber The polarization and EIS routines were subsequently started SHE) was applied to the indium tin oxide (ITO) WE and EIS was measured with a potentiostat (Reference 600 The frequency (f) range varied between 100 kHz and 50 mHz with an amplitude (U) of 10 mV rms and 10 points per decade Polarization of the WE was started prior to the addition of bacteria to an OD600 of 0.1 Beads were added simultaneously to the cells to the WE chamber Similar to the bacteria the beads were circulated between the flow cell and WE chamber reservoir EIS measurements were performed after 24 h of incubation Microscopic images of cells growing on the ITO WE were taken 24 h after inoculation Samples were viewed on a Leica DM 5500B and images were taken with a Leica DFC 360 FX camera and the corresponding Leica LAS AF Lite software Pumping was paused during image acquisition to prevent image disturbance by the peristaltic pump A 50-fold higher shuttle concentration resulted in a linear increase of the current density but for economic reasons we chose 37 nM riboflavin as suitable working concentration for further experiments The effect of direct riboflavin addition on average current density in BES with 0 Error bars represent the standard deviations from means of samples taken in independent triplicates The next step was to transfer the effect of soluble riboflavin on the current density to a recyclable shuttle that can be extracted easily from the medium. Therefore, riboflavin was coupled to magnetic beads via organic spacers of different chain lengths: short (C4), medium (C11), and long (C40). The number of C-atoms in the spacer had a strong impact on the current density in the BES (Figure 2) Short spacers resulted in the highest current densities [7.5 ( ± 0.76) μA/cm2] With increasing number of C-atoms between riboflavin and the magnetic beads Medium spacers exhibited a current of 4.6 (±0.22) μA/cm2 and long spacers of 3.1 ± 0.31 μA/cm2 Glycine-coupled beads served as control and produced a slightly higher amount of current compared to the experiment without addition of riboflavin or magnetic beads (4.6 ± 0.47 μA/cm2) addition of riboflavin-coupled beads to the anodic chamber resulted in a 2.4-fold increase of the average current density compared to experiment without magnetic beads whereas the glycine-coupled control only led to a minor increase (1.4-fold) Impact of riboflavin coupled beads on current density in BES Three different linker molecules characterized by short (C4) and long (C40) chain length were placed between beads and the riboflavin molecule The control experiment was conducted without addition of riboflavin or magnetic beads; C4/C11/C40 represent the addition of riboflavin-coupled beads with the respective linker chain lengths; glycine instead of a riboflavin molecule was added to the C4 linker in the glycine-coupled control beads The concentration of riboflavin was 37 nM in all experiments (B) Average current density after 46 h of operation We also analyzed the recovery rate and riboflavin concentration of all beads after the experiment and could measure for all setups very good recovery efficiencies of up to 90% (Figure 3) The stability of the riboflavin-coupled beads was analyzed via BES experiments in which the beads were recycled from the anode compartment and then reused in a fresh BES setup The experiment was repeated three times and no significant change in the current density could be detected (data not shown) Recovery efficiencies of the beads and riboflavin content before and after batch experiments C4/C11/C40 represent riboflavin-coupled beads with the respective chain lengths Gray bars show the normalization to the beads number striped bars to riboflavin content before the experiment and after the recovery process so that a binding between riboflavin and OMCs should result in a co-elution of cells with the beads microscopic analysis of all preparations exhibited no differences between wild type and ΔOMC cells (data not shown) which indicates that at least under the chosen conditions riboflavin does not seem to bind to the OMCs We compared total cell numbers in the different experiments and quantified the number of planktonic vs. anode-attached cells to investigate the effect of riboflavin and magnetic beads on the cell distribution in the anode chamber in further detail. Therefore, qPCR analysis was conducted to quantify the number of planktonic and biofilm-attached cells in the BES containing soluble riboflavin, riboflavin coupled to C4-beads and glycine-coupled control beads (Figure 4) the number of planktonic cells was comparable both with and without riboflavin the cell number increased significantly (p = 0.021) and 6.3-fold more cells could be detected compared to BES without the addition of riboflavin this correlates well with the measured current increase Adding riboflavin-coupled beads to the BES also influences the distribution of the cells As seen with riboflavin as soluble compound significantly more cells attached to the anode (p = 0.042) whereas with glycine-coupled beads cell adhesion was positively affected but with rather high standard deviations which rendered this effect to be not significant (p = 0.277) the increase in anode attached cells correlates roughly with the stimulating effect of the beads on current production it can be assumed that there is a correlation of cells on the anode and current production and that this correlation is driven by riboflavin (either coupled to beads or as free compound) whereas the beads per se affect biofilm growth at least not as robustly Comparison of the total cell number determined via qPCR in BES with the addition of riboflavin glycine-coupled control beads and riboflavin-coupled beads with a linker length of 4 carbon atoms The riboflavin-coupled beads were added so that the overall riboflavin concentration was equal to the experiment with free riboflavin (37 nM) The control experiment was conducted without addition of riboflavin or magnetic beads Gray bars indicate planktonic cells; striped bars biofilm cells even the addition of glycine-coupled beads leads to an increased GFP signal on the ITO WE magnetic beads coupled with riboflavin gave an even higher fluorescence signal indicating a promoting effect on the biofilm formation under anode respiring conditions (A) Nyquist presentation of EIS measurements after 24 h of flow cell operation (B) Bode plot and phase angle of the EIS measurements of flow cells without beads flow cells without beads are depicted in gray squares flow cells with glycine-coupled control beads in orange circles and flow cells with riboflavin-coupled beads in blue triangles Points represent average values of independent triplicates; arrows indicate the corresponding axis The inset in (A) shows an equivalent electrical circuit for the BES flow cell; RCT: charge transfer resistance; RU: solution resistance; CPEDL: double layer constant phase element (C) Fluorescence microscopy images of gfp-expressing MR-1 on ITO glass anodes in experiments without beads (1) with glycine-coupled control beads (2) and with riboflavin-coupled beads (3) after 24 h of incubation Circular areas without cells are due to the position of magnetic beads In this study, an increased current density in MR-1-inoculated BESs was achieved by adding a recyclable electron shuttle composed of riboflavin-coupled magnetic beads. From an applied point of view the concentration of endogenous shuttles is usually rather low and exogenous addition of it is expensive and in some cases even environmentally toxic (Rinaldi et al., 2008) permanent washout takes place in continuous cultivation systems a shuttle that can be recycled as the here presented riboflavin-coupled magnetic beads would be advantageous shuttles were either added to the medium or produced endogenously by microorganisms the redox mediator has to be added continuously to avoid performance decrease due to washout the beneficial effect of riboflavin can be recovered by magnetic separation and recycled with very good recovery efficiencies of up to 90% This makes the process of adding bead-coupled riboflavin to BES much more sustainable efficient and economical compared to the external addition or endogenous overproduction of the soluble compound it does not seem to be necessary that the riboflavin is freely diffusible as the effect was detected also with riboflavin coupled to beads with a diameter of 25 μm the effect of riboflavin on biofilm and current production was higher when it was added in its free form A possible factor for this might be that riboflavin coupled to the beads is not as accessible as the free compound This limited accessibility might also be the reason why we could not detect a binding of cells to the beads as a result of outer membrane cytochrome expression Impedance spectroscopy revealed that the addition of beads decreases the impedance by more than 50% This decrease is mostly connected to the charge transfer resistance which is seen in the Nyquist plot as the beginning of a classical charge transfer semi-circle non-electroactive) did not show any significant difference in the charge transfer resistance Since the beads are made out of iron oxide and covered by a presumably permeable agarose coating it is possible that they are conductive themselves and can reduce the resistance of the anode by acting as an extension of the electrode thereby extending the electroactive surface area and lowering the overall impedance the covalently attached compound (riboflavin and glycine respectively) does not seem to affect the electrochemical properties of the bioanode the microscopic images indicate an increased biofilm production caused by the addition of beads to the system and regarding biofilm density riboflavin-coupled beads clearly outperform glycine-coupled ones This enhanced cell density is reflected in the qPCR data where riboflavin-coupled beads also exhibits a boosting effect on the number of anode-attached cells It is possible that the effect of the beads on a BES is dual: an improved conductivity leads to decreased resistance and by this more effective EET and current production and (as stated above) riboflavin itself not only influences the electron transfer to the anode but also plays a role in biofilm production and/or formation KS-R wrote the manuscript with support from DR and JG RU and DH supervised the electrochemistry part Outer membrane cytochromes/flavin interactions in Shewanella spp.-A molecular perspective In situ monitoring of Shewanella oneidensis MR-1 biofilm growth on gold electrodes by using a Pt microelectrode Detection and Assay of Vitamin B-2 (Riboflavin) in Alkaline Borate Buffer with UV/Visible Spectrophotometry Extracellular reduction of solid electron acceptors by Shewanella oneidensis Electricity production by Geobacter sulfurreducens attached to electrodes CrossRef Full Text | Google Scholar Shuttling happens: soluble flavin mediators of extracellular electron transfer in Shewanella Outer-membrane cytochrome-independent reduction of extracellular electron acceptors in Shewanella oneidensis Acetoin production via unbalanced fermentation in Shewanella oneidensis Nature’s conductors: what can microbial multi-heme cytochromes teach us about electron transport and biological energy conversion Electron transfer process in microbial electrochemical 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Katrin Sturm-Richter, a2F0cmluLnN0dXJtLXJpY2h0ZXJAa2l0LmVkdQ== Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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. Volume 13 - 2022 | https://doi.org/10.3389/fpsyt.2022.819607 This article is part of the Research TopicThe Development of Biomarkers in PsychiatryView all 11 articles Major depressive disorder (MDD) is a widespread common disorder there are no easy and frequent to use non-invasive biomarkers that could guide the diagnosis and treatment of MDD The aim of this study was to investigate whether there are different mass concentrations of volatile organic compounds (VOCs) in the exhaled breath between patients with MDD and healthy controls patients with MDD according to DSM-V and healthy subjects were investigated VOCs contained in the breath were collected immediately after awakening and after 60 min in a respective breath sample and measured using PRT-MS (proton-transfer-reaction mass spectrometry) and 90 were significantly decreased in patients with MDD compared with healthy controls changes during the time in mass concentrations of m/z 93 and 69 significantly differed between groups Differentiation between groups was possible with an AUCs of 0.80–0.94 in ROC analyses VOCs differed between patients and controls might be a promising tool for future studies Altered masses are conceivable with energy metabolism in a variety of biochemical processes and involvement of the brain–gut–lung–microbiome axis The aforementioned measures currently under investigation are invasive blood tests except the test for cortisol when measured in saliva is unpleasant to collect for the participants It is not convenient for patients to give numerous blood samples throughout the day or even over several days in order to follow the disease course longitudinally functional neuroimaging is expensive and time consuming and it is not feasible to scan patients several times as a routine procedure A non-invasive longitudinal measurement would certainly increase the information on individual disease characteristics and the dynamics of these during the disease in patients new non-invasive markers are highly needed no study exists that investigated whether VOCs in breath differ between patients with MDD and healthy controls although metabolic changes are well known in patients with MDD The aim of this study was therefore primarily to identify potential VOCs in breath that differ between patients and controls we were interested to investigate the change of breath gas markers during the awakening time since the awakening time was previously under focus of cortisol research in depression we were interested to examine whether breath gas analysis might have the potential to differentiate between patients and controls and to investigate if multiple measurements over the first hour after awakening might improve classification we tested for the influence of hospital environment and medication on the detected mass concentrations From our psychiatric service 26 patients with DSM-V MDD diagnosis and 25 healthy controls were recruited in a prospective design All the patients were in therapy either as inpatients or outpatients and at time of investigation were mildly to severely depressed Inclusion criteria for patients were MDD according to DSM-V and age between 18 and 65 years The latter applied to healthy controls too who were recruited from the local community matched for age and sex to the patients included into the study Exclusion criteria for participants were a known alcohol or drug dependency Neurological disorders and diseases affecting the brain function were further excluded (e.g. hypo- or hyperthyroidism) and also acute internal disorders Our participants reported no acute gastrointestinal disease for the patients a current psychotic disorder depressive episodes with psychotic symptoms other psychiatric axis 1 disorders were excluded For healthy controls any history of a psychiatric disorder was an exclusion criterion All the patients and controls had to give written informed consent to participate in the study All the subjects were investigated clinically and diagnosis were confirmed using the SCID diagnostic interview (13) by an additional independent investigator. Moreover, they have been examined using standardized questionnaires for psychiatric symptoms: Hamilton Depression Scale (HAMD) (14), Clinical Global Impression (CGI) (15), and Beck Depression Inventory (BDI) (16) The current medication and also the medication during the last 2 weeks before study inclusion were documented for the group of patients receiving antidepressants the past clinical course was assessed from interviews and case notes within the psychiatric services in particular to stage the recurrence of the disease previous other doctors’ reports were collected as exact as possible treatment history for medications and psychotherapy were obtained We measured the VOC concentrations as counts per seconds in the breath gas of all the participants (patients as well as controls) with Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) Awakening was defined when participants got out of the bed in the morning All the breath gas collection was carried out after awakening in patients and controls on an empty stomach to avoid contamination with acute food intake or smoking effects Participants were instructed to sit calm for 2 min and to breath normally and then to start breath gas sampling Since breath gas was collected at awakening which are special tubes for collecting air and breath No further preparation such as freezing or making use of condensation is necessary For better detection of the exact substances we carried out six probes with the PTR-MS system as well as a more sophisticated system based on a time-of-flight (TOF) analyzer unit The sensitivity and specificity of such a system is by far better than the quadrupole-based system Smoking influences some of the VOCs. In the smokers benzene, xylene isomers, styrene, ethylbenzene, acetonitrile, and furan derivatives and also hydrocarbons have been detected in exhaled breath (17) With regards to smoking the patients and test persons behavior was documented and moreover it can clearly be detected with breath gas analysis and the VOCs concerned were excluded from the analysis we selected those VOCs that are already known from breath research and are not related to smoking We determined an effect-size for group differences between patients and controls as d = 1.37 for comparison of single values and an effect size of 15 per group we intended thus to recruit 25 patients and controls to allow also for covariates to be included in the analyses Group differences between patients and controls and interactive effects between groups and time (during awakening) were analyzed using general linear models for repeated measures Correction for multiple comparison was applied using false discovery rate (FDR) and significance was assumed with p = 0.05 Cliffs Delta statistics was used to indicate effect sizes for differences between patients and controls irrespective of distribution of values Effects of antidepressant medication and the clinical environment (inpatients and outpatients) were analyzed further a random forest analysis for feature extraction was used to cope with the structure of the test set and the measured values the data set was divided into ten randomly chosen training and validation data sets in the ratio of 75 and 25% The algorithm tries to keep the number of patients and healthy subjects in each randomly chosen data set equal In each validation of the trained random forest model the importance of each measured value is calculated using the permutation method calculated importance values were summed for each measured mass the five masses with the highest summed importance values were then used for logistic regression Based on the obtained formulas for combination of markers we obtained the predicted values for the model in the test sample These models were available to our statistician The obtained results and reference standards were confirmed in the validation sample A ROC analysis was then carried out to identify sensitivity and accuracy for discrimination between patients with MDD and healthy controls There are no significant differences between patients and controls in terms of gender, age, BMI, alcohol consumption and education (Table 1) patients with MDD show significant higher depression severity compared to healthy controls but did not survive false discovery rate (FDR) correction: m/z 42 was increased in patients with MDD compared with healthy controls [F(1/50) = 8.3 Decreased in patients with MDD compared with healthy controls were m/z 70 [F(1/50) = 5.3 Time course and differences between groups for markers 88 Moreover, different time courses during the awakening for the concentrations of the m/z 93 [F(1/50) = 5.2, p = 0.007 with assumed sphericity] and 69 [F(1/50) = 2.6, p = 0.091 with Greenhouse–Geisser correction] were detected between patients with MDD and controls (Supplementary Figure 1) The difference between m/z 93 at 30 min minus baseline was significantly higher for patients with MDD compared with the controls [F(1/50 = 13.5 The difference between m/z 69 at 60 min minus 30 min is significantly different between groups as well [F(1/50) = 4.6 Random forest analyses indicate the masses most important for differentiating between patients and controls Shown is sensitivity and 1-specificity for the combination of markers in the validation sample First m/z 74 was calculated alone (1x Vars) then subsequently the other important markers are added showing an increase up to the first three markers Adding the marker m/z 69 showed very good classification. The classification was as following: AUC = 0.96 in the test sample and AUC = 0.91 in the validation sample (Supplementary Figure 2) we tested whether breath gas sampling in the hospital for inpatients differed from breath gas sampling at home considering depression severity the six patients being in outpatient therapy were compared with the 19 patients in the hospital No significant difference was found in the aforementioned significant different breath gas markers Levels of m/z 74 were significantly lower in patients sampling at home compared to those sampling in the hospital [F(1/24) = 6.2 No significant difference in concentration of these VOCs was detected between patients on antidepressants compared with those without antidepressants Endogenous VOCs provide a plethora of information regarding the metabolic processes This is to our knowledge the first study using breath gas analysis to explore potential signatures for supporting diagnostics in MDD oral microbiome and not only gut or lung microbiome might need to be considered when locating origins of altered VOCs This would argue that reduced physical activity might be a contributing factor to butyric acid in breath further studies are necessary to study the effect on exercise butyric acid in patients with MDD this might be different due to pulmonary ventilation where increases of isoprene have been detected and m/z 94 were not found to be associated with exercise so far It could also be proven for isoprene that it can be measured in increased concentration in the air breathed during physical activity. Here, it could be proven that the measured concentration also depends on the lung blood flow. Endogenous production is not increased during increased activity (28) M/z 93 was significantly increased in MDD compared with the controls and significantly increased during the awakening response in patients more than in the healthy controls. This mass belongs to toluene (29), which is predominantly used as an industrial feedstock. Its exposure was previously found to be associated with cognitive dysfunctioning in the neuropsychological tests (30) diagnostics are still based on clinical interviews psychiatric history and assessment of psychopathology Breath gas analysis has the advantage against blood analyses or functional MRI that it is a non-invasive Measurement in patients with MDD was well-tolerated and is feasible also over several time points during the day during the awakening period like in our study subjects were comfortable with the procedure even when they were severely depressed and were not stressed by it Breath gas analysis did show a very good accuracy in predicting patients with MDD vs healthy subjects in the test as well as the validation sample An AUC of 0.93 for a three-factor solution seems to be promising for the next step in studying these signatures in the larger clinical studies including also other diagnoses the sample size was small thus limiting the generalizability of the findings the sample size needs to be extended toward more outpatients treated in the community and patients in remission as well as longitudinal assessments during the disease course This will allow to explore whether the markers are trait or state dependent antidepressant medication seems not to have a major effect it should then be tested whether this might be the case for all the antidepressant classes The type of diet can have an impact on metabolites excreted these effects can be seen in exhaled breath the exact components of the diet were not explicitly asked about nutrition needs to be taken into consideration we are not aware of any of the masses we found to be associated with nutrition but given the limited number of existing studies it seems to be important to control for nutrition breath gas analysis was carried out in patients and controls at awakening and the hour afterward on an empty stomach patients were more often smokers than non-smokers Although VOCs known to be associated with smoking were excluded from analyses in a larger sample the effect of smoking needs to be investigated the present study shows that breath gas analysis might be an interesting method to investigate the metabolic profile in patients with major depressive disorder (MDD) The raw data supporting the conclusions of this article will be made available by the authors The studies involving human participants were reviewed and approved by Ethics Committee of medical faculty of Otto von Guericke University Magdeburg The patients/participants provided their written informed consent to participate in this study HD: analysis and interpretation of the data and revising the manuscript LG and DG: interpretation of the data and revising the manuscript All authors agreed to be accountable for all aspects of the work ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated TF received funding from the European Union Horizon 2020 Programme within the project Deep-Learning and HPC to boost biomedical applications for health (DeepHealth): Grant number: 825111 The authors obtained a patent for breath gas analysis in major depressive disorder there is no embargo for the publication any more TF received fees for presentation from Janssen-Cilag The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyt.2022.819607/full#supplementary-material PubMed Abstract | CrossRef Full Text | Google Scholar Treatment resistant 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Thomas Frodl, dGZyb2RsQHVrYWFjaGVuLmRl This website is using a security service to protect itself from online attacks. The action you just performed triggered the security solution. There are several actions that could trigger this block including submitting a certain word or phrase, a SQL command or malformed data. You can email the site owner to let them know you were blocked. Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page. Microbiological Chemistry and Geomicrobiology Volume 11 - 2020 | https://doi.org/10.3389/fmicb.2020.00815 This article is part of the Research TopicElectromicrobiology – From Electrons To EcosystemsView all 14 articles Microbial electrochemical technologies (METs) have emerged in recent years as a promising alternative green source of energy with microbes consuming organic matter to produce energy or valuable byproducts It is the ability of performing extracellular electron transfer that allows these microbes to exchange electrons with an electrode in these systems The low levels of current achieved have been the limiting factor for the large-scale application of METs Shewanella oneidensis MR-1 is one of the most studied electroactive organisms regarding extracellular electron transfer and it has been shown that biofilm formation is a key factor for current generation The transcription factor bolA has been identified as a central player in biofilm formation in other organisms with its overexpression leading to increased biofilm In this work we explore the effect of this gene in biofilm formation and current production by S Our results demonstrate that an increased biofilm formation and consequent current generation was achieved by the overexpression of this gene This information is crucial to optimize electroactive organisms toward their practical application in METs highlighting the potential of EAB manipulation to optimize BES The bolA gene, initially identified as a transcriptional regulator for cell shape in Escherichia coli (Aldea et al., 1988), was recently shown to be responsible for promoting biofilm development by targeting several genes encoding proteins related to this process (Dressaire et al., 2015). This gene is present in numerous electroactive organisms (Supplementary Table S1) we report the effect of the bolA gene in the development of S oneidensis MR-1 biofilm and the consequences for current density production in BES We demonstrate that the bolA gene increases both current generation in BES and biofilm formation opening the way for future regulation studies focusing on this gene This work is a step forward towards a clear understanding of the genes that regulate biofilm formation in S This knowledge is of great importance towards the optimization of current production in BES paving the way for their commercial application Bacterial strains were cultivated overnight in LB supplemented with kanamycin (50 μg/mL) at 30°C with 150 rpm agitation. For the growth curves, cells were diluted in M4 medium (Delgado et al., 2019) supplemented with kanamycin (50 μg/mL) to a starting OD600 of 0.07 200 μL were then transferred to individual wells of a polystyrene flat bottom 96-well plate (Sarstedt) and allowed to grow at 30°C without stirring The cell density was measured at 600 nm every 30 min for 46 h using a Multiskan Sky Microplate Spectrophotometer (Thermo ScientificTM) The experiment was conducted using twelve replicates and the mean and standard deviation were calculated using MS Excel Planktonic cells were harvested from the cultures after the growth curves (46 h of growth) Samples were observed in slides coated with 1.5% (wt/vol) agarose film and enclosed with a cover glass Images were acquired on a Leica DM 6000B upright microscope equipped with an Andor iXon 885 EMCCD camera and controlled with the MetaMorph V5.8 software using the 100 × 1.4 NA oil immersion objective plus a 1.6× optvar Images were processed using ImageJ software Growth and analysis of static biofilms under microoxic conditions were measured using crystal violet staining on the 96-well plate resulting from the growth curve experiment according to (Dressaire et al., 2015) Each well was cleaned three times with 200 μL water and then treated with 200 μL 0.1% crystal violet for 15 min The plate was then washed three times with water to remove the excess of crystal violet and dried at 65°C for 15 min 200 μL of 96% ethanol were added to each well and incubated for 15 min at room temperature and the optical density of each well was measured at 570 nm using a Multiskan Sky Microplate Spectrophotometer (Thermo ScientificTM) The ratio of biofilm development to planktonic growth was calculated using the cell density (OD570/OD600) An unpaired t-test was used to determine significance of the data The bioelectrochemical experiments were conducted in triplicates using a single chamber BES with a working volume of 270 mL (Bursa et al., 2017) Germany) were used as working and counter electrode material 0.199 V vs standard hydrogen electrode (SHE)] (Sensortechnik Meinsberg the working electrode was rinsed with isopropanol cells were harvested from the preculture by centrifugation (7 min 6000 × g) and washed 3 times with M4 medium containing neither electron donor nor electron acceptor Cells were then resuspended to a starting OD600 of 0.07 in M4 medium containing 70 mM lactate and kanamycin (50 μg/mL) the working electrode was poised to 0 mV vs SHE and current was monitored for 46 h BES were incubated at 30°C and constantly flushed with N2 gas in order to ensure anoxic conditions the medium was continuously agitated using a magnetic stirrer The OD600 was measured in the beginning and after the 46 h of the BES experiments and showed that the planktonic growth was negligible (ΔOD600 < 0.02) a standard curve using biological triplicates of the bacterial strains in six different dilutions was established the cells were counted in two different dilutions (Neubauer chamber improved cell counts of isolated DNA sample were determined Bacterial growth profile of the different strains was monitored for 46 h under microoxic conditions to evaluate the effect of bolA (Figure 1A). There were no significant differences detected in the growth of the strains. Furthermore, the microscopic images taken to explore differences in cell shape show that no morphological differences exist between the strains at this scale (Figure 1B) oneidensis MR-1 strains obtained in the 96 well-plate: Black - WT; Brown – WT+; Green – ΔbolA; Blue – ΔbolA+ Cells were cultured for 46 h under microaerobic conditions in M4 medium and OD600 was measured every 30 min (B) Phase contrast microscope imaging of the different strains: 1 – WT; 2 – WT+; 3 – ΔbolA; 4 – ΔbolA+ Pictures were taken using agarose-coated slides and cultures resulting from the growth curves Cultures resulting from the growth curve were used to evaluate biofilm production by crystal violet staining (Figure 2A) The results show that the overexpression of bolA leads to an increase in biofilm production with significant differences (unpaired t-test p < 0.05) found between the WT and both the WT+ and ΔbolA+ The strain WT+ produces a 1.24-fold increase in the ratio of planktonic cells to biofilm relative to the WT while the strain ΔbolA+ produces a 1.26-fold increase Although the ΔbolA strain presents a slight increase in biofilm formation relative to the WT strain this difference is not significant Representation of the biofilm production by the different strains normalized by OD600 of the planktonic cultures (A) and mean current density produced by the different strains in BES (B) Black – WT; Brown – WT+; Green – ΔbolA; Blue – ΔbolA+ Biofilm formation was measured by crystal violet stain with cell cultures resulting from the growth under microaerobic conditions after 46 h incubation The potential of the anode in BES was poised to 0 vs SHE using an Ag/AgCl reference electrode Stars represent significant differences (unpaired t-test p < 0.05) A very similar effect can be observed in the ΔbolA+ strain The DNA deposited on the anodes of BES was isolated using qPCR and the DNA content was examined to evaluate if the increased current density of the bolA overexpressing strains correlates with cell attachment on the anode. Interestingly, the deletion of bolA leads to a 1.8-fold decreased amount of DNA on the anode (Figure 3) while the complementation of the bolA deletion shows almost the same amount of DNA on the anode as the WT No significant difference was observed between the WT and the bolA overexpressing strain (WT +) Amount of DNA on the anode relative to the wildtype determined by qPCR DNA was isolated from the anodes after BES experiments The bolA gene was originally identified as a transcriptional regulator for cell shape in E. coli, as its overexpression leads to round cell morphology (Aldea et al., 1988). BolA was demonstrated to promote biofilm development by targeting several genes encoding proteins related to this process, being described as a modulator for the switch between planktonic to sessile lifestyles (Vieira et al., 2004; Dressaire et al., 2015) In this work we addressed the effect of this gene in S. oneidensis MR-1, one of the most widely studied microorganism regarding extracellular electron transfer in BES. It has been demonstrated that, in these systems, an increase in biofilm thickness leads to an improvement in current generation (Yu et al., 2011; Yong et al., 2014; Liu et al., 2015; Zajdel et al., 2018; Freyman et al., 2019) Phylogenetic tree based on the alignment of the bolA sequence from the different electroactive Shewanella species Tree constructed by neighbor joining using CLC main workbench 8.1 software (Qiagen) Differences could also be observed in the biofilm formation with the overexpression and complemented strains producing an approximate 1.25-fold increase in biofilm production This result relates very well with the 1.2-fold increase in the mean current density observed upon expression of the bolA gene the amount of DNA deposited on the anode in the ΔbolA+ strain was higher than that of the ΔbolA As the qPCR detects both intracellular DNA and extracellular DNA that can be embedded in the biofilm matrix and help in extracellular electron transfer processes it is difficult to determine if the bolA gene promotes biofilm formation by increasing extracellular DNA The biofilm assay was conducted under microoxic conditions given the inability to detect biofilm formation using this protocol under anoxic conditions This could be attributed to the low optical densities obtained when growing S oneidensis MR-1 in minimal medium under anoxic condition in the 96-well plates in the case of oxic or microoxic conditions the biofilm is formed on the top of the well at the interface between the culture medium and oxygen which allows the cells to get both nutrients and the terminal electron acceptor (i.e. the oxygen is absent and the electrode acts as the terminal electron acceptor promoting biofilm development on its surface as well as the understanding of what processes bolA regulates would increase our knowledge in enhancing biofilm formation and consequently increase current production in BES The datasets generated for this study are available on request to the corresponding author AS and ME performed the experiments and carried out the data acquisition and analysis All authors were involved in the discussion of the results and in writing the manuscript This work was supported by Fundação para a Ciência e a Tecnologia (FCT) Portugal (Project PTDC/BIA-BQM/30176/2017 fellowship SFRH/BD/129067/2017 to AS) and by Cooperação Científica e Tecnológica FCT/DAAD It received funding by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular Estrutural e Celular) funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No This work was also partially supported by Portuguese Platform of BioImaging (PPBI) (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from Orçamento de Estado (OE) and by european funds from Fundo Europeu de Desenvolvimento Regional (FEDER) Arraiano for their support with 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Catarina M. Paquete, Y3BhcXVldGVAaXRxYi51bmwucHQ= Please enable JS and disable any ad blocker has opened new stations in North Rhine-Westphalia thereby increasing its number of employees in this German state by 150 to around 750 in total in the past two years The transport company announced that over the past four months it had begun operations at stations in Bergneustadt in Bergisches Land There are already FedEx branches in North Rhine-Westphalia in Dortmund Almost 600 team members are now employed by FedEx at the Central and Eastern Europe hub at the Cologne Bonn Airport The company employs more than 1,900 in Germany FedEx operates almost 50 stations across the countrywhich act as small distribution centers Shipments from regional customers are sorted and bundled here before being delivered to recipients in Germany “With the expansion in North Rhine-Westphalia we want to boost both our proximity to customers and our flexibility,” says Carl Graham Operations at FedEx Express Central and Eastern 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digital access to quality FT journalism with expert analysis from industry leaders Complete digital access to quality analysis and expert insights complemented with our award-winning Weekend Print edition Terms & Conditions apply Discover all the plans currently available in your country See why over a million readers pay to read the Financial Times “There’s no such thing as bad weather — only the wrong clothes,” could have been written about Skye this is a place where sensibly attired islanders think nothing of hopping in their kayaks to glide past curious otters or hiking the famous mountain range in horizontal rain when a road bridge connecting the town of Broadford to Kyle of Lochalsh was built Skye has become an island that is also not an island its accessibility means it has surged in popularity with second-home owners who drive up from Glasgow and Edinburgh (four hours and 40 minutes from both cities) and open their car doors to a land Class of 2017: Regis High SchoolStatesman JournalGraduation ceremonies were at 1:30 p.m Stayton High School graduatesStatesman JournalGraduation ceremonies were at 7:30 p.m Put your prejudices aside and get ready to try the dairy-free vegan ice cream that just might be your new favorite at Frankie & Jo’s “Welcome to our new tradition of mindful ice cream,” says the website for Seattle’s new Frankie & Jo’s You’d be forgiven an eye-roll at the language appropriated from the realm of yoga or meditation: “We live and breathe the layered nuances of plant-based ice cream making so that your experience can be pure and delicious.” (What would an unintentional ice-cream experience be like Frances and Joanne (who were neither vegan nor gluten-free) The veganism-opposed might mount a protest: “Cream” is in “ice cream” for a reason — it’s not “ice plant” Linguists could calmly take issue: “New tradition” is an oxymoron And there may be some part of us all that screams: Can’t we just enjoy our ice cream Sunday-Thursday 2-10 p.m., Friday-Saturday 2-11 p.m.; 206-257-1676; frankieandjos.com cleansing breath and prepare to have your palate for Frankie & Jo’s ice cream is straight-up delicious Believe it or not — all dietary needs and political predilections aside — it could be your new favorite ice cream Frankie & Jo’s vanilla has a faint caramelly flavor from its brown cane sugar plus the taste of real vanilla bean; it’s unabashedly sweet lush and very ready for any berry to top it Is it slightly lighter than regular ice cream with maybe a little bit of a soft-serve texture to it It’s something to ponder while you eat it all up The cocoa-nib brownie mint was my instant favorite despite the fact that I don’t usually like stuff mixed into my ice cream and as such are a major achievement of gooey goodness; the cocoa nibs add welcome little explosions of bitter crunch; the mint is subtle Beet strawberry rose sorbet practically glows a bright crimson Electric-yellow gingered golden milk is even brighter its complex taste a love-it-or-hate-it punch of fresh turmeric root; fresh and candied ginger; cinnamon; cardamom; black pepper; and sea salt its spicy coconut richness will remind you of curry (I’d say skip the waffle cone; it’s made with oat flour and it ends up a little crumbly and overly healthy-tasting.) sprouts them overnight and makes nut milk from them the next day Most flavors also contain the nondairy lusciousness that is coconut milk — think of the smooth richness of a piña colada expensive Vitamix that sounds like an airplane taking off And she says the secret — the thing that makes it so much better than whatever other dairy-free ice cream you may have had the misfortune of trying — is what’s not there: no gums to create an artificially fluffy texture no stabilizers to keep the fat and liquid combined for an artificially prolonged shelf life The path to Frankie & Jo’s began with pastry chef Martin realizing she had a dairy allergy Martin started making her own dairy-free ice cream back then eventually approaching Brunson about sharing it with the world and “kind of admired each other from afar,” Brunson says “We weren’t best friends like we are now.” Their research and development included a trip to L.A shipments of more nondairy ice cream from around the country eating every kind available in the grocery-store freezer case Then came “experimenting a lot with the fact that ice cream is a formula of sugar and fat just like most baking … playing with those percentages to get a beautiful Brunson and Martin worked for a year and a half to get the recipes right and the shop open and the reception has been almost clamorously appreciative They’re so sincerely delighted that people like the result For those with allergies or diets of their own choice “It doesn’t feel like an afterthought — it’s specifically for them,” Brunson says to be trendy; neither of them are vegan themselves but they both respect “the idea that you don’t have to use animals to make beautiful food.” And those with a knee-jerk negative response might just come around with the first spoonful Stay secure and make sure you have the best reading experience possible by upgrading your browser Home / News / Article The Building and Construction Authority (BCA) issued orders to stop excavation works on a site in Birżebbugia just a few metres away from the archaeological heritage of Għar il-Friefet following the contractor’s failure to submit a commencement notice before starting works The development in question will consist of 178 apartment units five retail outlets and one office spaced out over three blocks four storeys high Ample evidence of the works that were carried out could be seen in the form of heavy machinery and recently disturbed soil on site works that may affect third parties adjacent to a site require a thorough assessment of any potential risks to nearby property with the contractors being responsible for submitting a commencement notice at least five days before the works start While the planning application (PA/2901/16) was already approved in March 2019 the contractors initiated works before completed geological studies were submitted for scrutiny The application lists Ansgar Gescher as the applicant on behalf of Scirocco Heights Ltd a real estate firm that lists Gescher as the sole shareholder on the Malta Business Registry The contractors on site both fall under the umbrella of Zahra Group a cave described by the case officer as “an underground cave of ecological A YouTube channel listed under the name of Pierre Farrugia uploaded recent footage of an excursion into the cave which is closed off to public access unless permission is obtained from the relevant authorities “Għar il-Friefet forms part of a greater area of the archaeological and geological area that includes Għar Dalam This cave is a Level 1 Area of Scientific Importance and a Grade B Area of Archaeological Importance,” the case officer’s report reads The development itself now remains in limbo as the BCA is expected to assess the latest method statements submitted by the applicant with its order to stop works until further notice who is the architect for Birżebbuġia’s local council wrote a damage report on behalf of one of the residents whose property might be affected by development works in the area When asked whether residents ought to worry about the development “the matter remains unclear” given that the latest method statements did not include the necessary geological studies to ensure third party property would not be affected “The reports need to determine whether there are fissures in the rocks and whether there is any potential for rocks to slide over each other and if so what measures need to be taken,” Cacopardo told The Shift The architect also spoke of how development on the site has been in the works since the 1970s pointing out that he had personally participated in the PA’s board meeting on the original development in the name of the local council “We had participated in the process and we had insisted on ensuring protection for Għar il-Friefet among other things,” Cacopardo told The Shift including where access is limited to trucks and vehicles to avoid damage as well as other conditions on controlling vibrations in the area to prevent any damage being caused to either people’s residences or Għar il-Friefet’s archaeological heritage,” he added Join our Corporate Democratic Responsibility Program The Shift is an independent online news platform committed to investigative journalism and the defence of press freedom cultural and social commentary from civil society We use cookies to enhance your browsing experience We use cookies to help you navigate efficiently and perform certain functions You will find detailed information about all cookies under each consent category below The cookies that are categorized as "Necessary" are stored on your browser as they are essential for enabling the basic functionalities of the site 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Advertisement cookies are used to provide visitors with customized advertisements based on the pages you visited previously and to analyze the effectiveness of the ad campaigns One person was killed in an accident on a German highway after he drove his truck into a cargo container filled with iron material that was left on the road The accident happened just past midnight on Thursday when the container dislodged from a truck being driven by a Dutch operator with no valid driver's license was later found to have a blood alcohol content of 0.74 per mille He was found about a half-kilometer further north of the accident site on Autobahn 31 near Gescher Münster Police said it was not known why the cargo separated from the truck His body was recovered once rescue workers were able to cut through the wreckage The four others who were hurt were aged 30 Portions of Autobahn 31 remained closed until about 2 p.m Police said the accident caused nearly 130 thousand euros in property damage A blood test was used to determine the amount of alcohol in the Dutch driver's system The legal limit in Germany is 0.5 per mille for most drivers and truck drivers are not allowed to have any alcohol in their system according to the European Transport Safety Council You are in: Leicester > People > Your Stories > Broccoli Juice from Braunstone A Leicester man with bladder cancer says his daily glass of broccoli juice is helping to prevent his cancer from spreading Ray Wiseman says that Cancer Research UK is now planning to look into the benefits of broccoli Ray Wiseman from Braunstone in Leicester was diagnosed with bladder cancer five and half years ago He's 79 years old and the prognosis wasn't good suggested that they should buy a juicing machine She told Ray that he should drink a glass of broccoli juice once a day as it would help build up the immunity in his bladder BBC Leicester's Bridget Blair went to meet Ray and Joan Wiseman at their home.. Help playing audio/video Broccoli juice isn't proven to help stop bladder cancer from spreading but Ray believes it is doing him some good: Ray and Joan wrote to The Daily Mail with the idea of telling other people about the positive effects of broccoli juice They weren't expecting the media frenzy it spawned with interviews on radio and television and in national newspapers Joan says they're a little old to cope with all the photo shoots and interviews but it'll be worth it if other people can be helped: "If all through this any people can become more aware of it "I would love to think that if anybody else could benefit from this "By all accounts Cancer [Research] UK are going to get in touch with us wondering if what we tell them about the recipe and everything would be of any use to their cancer sufferers." But is there any evidence behind the broccoli juice BBC Leicester's Chris Baxter spoke to Professor Andy Gescher from the University of Leicester.. Professor Gescher is investigating naturally occurring chemicals and their role in preventing cancer He says it could be true that broccoli juice has helped slow down Ray's cancer but there is no scientific proof that broccoli can help existing cancers yet However a connection between cancer and broccoli has been discussed before: it has long been suggested that certain constituents...that they may prevent cancer That has been shown in animal experiments." Chris Baxter and Breakfast show producer Kay Wright made some broccoli juice and gave it a try - listen to how they got on: 500mg broccoli2 carrots1 apple and a few drops of lemon juice Wash the fruit and vegetables and put them in a juicer add the lemon juice and put it in the fridge last updated: 12/09/2008 at 16:52created: 23/07/2008