Proteomic and transcriptomic analysis of selenium utilization in Methanococcus maripaludis.

Methanococcus maripaludis utilizes selenocysteine- (Sec-) containing proteins (selenoproteins), mostly active in the organism's primary energy metabolism, methanogenesis. During selenium depletion, M. maripaludis employs a set of enzymes containing cysteine (Cys) instead of Sec. The genes coding for these Sec-/Cys-containing isoforms were the only genes known of which expression is influenced by the selenium status of the cell. Using proteomics and transcriptomics, approx. 7% and 12%, respectively, of all genes/proteins were found differentially expressed/synthesized in response to the selenium supply. Some of the genes identified involve methanogenesis, nitrogenase functions, and putative transporters. An increase of transcript abundance for putative transporters under selenium depletion indicated the organism's effort to tap into alternative sources of selenium. M. maripaludis is known to utilize selenite and dimethylselenide as selenium sources. To expand this list, a selenium-responsive reporter strain was assessed with nine other, environmentally relevant selenium species. While the effect of some was very similar to that of selenite, others were effectively utilized at lower concentrations. Conversely, selenate and seleno-amino acids were only utilized at unphysiologically high concentrations and two compounds were not utilized at all. To address the role of the selenium-regulated putative transporters, M. maripaludis mutant strains lacking one or two of the putative transporters were tested for the capability to utilize the different selenium species. Of the five putative transporters analyzed by loss-of-function mutagenesis, none appeared to be absolutely required for utilizing any of the selenium species tested, indicating they have redundant and/or overlapping specificities or are not dedicated selenium transporters. IMPORTANCE: While selenium metabolism in microorganisms has been studied intensively in the past, global gene expression approaches have not been employed so far. Furthermore, the use of different selenium sources, widely environmentally interconvertible via biotic and abiotic processes, was also not extensively studied before. Methanococcus maripaludis JJ is ideally suited for such analyses, thanks to its known selenium usage and available genetic tools. Thus, an overall view on the selenium regulon of M. maripaludis was obtained via transcriptomic and proteomic analyses, which inspired further experimentation. This led to demonstrating the use of selenium sources M. maripaludis was previously not known to employ. Also, an attempt-although so far unsuccessful-was made to pinpoint potential selenium transporter genes, in order to deepen our understanding of trace element utilization in this important model organism.

Triclosan and its alternatives, especially chlorhexidine, modulate macrophage immune response with distinct modes of action.

Since European regulators restricted the use of bacteriocidic triclosan (TCS), alternatives for TCS are emerging. Recently, TCS has been shown to reprogram immune metabolism, trigger the NLRP3 inflammasome, and subsequently the release of IL-1ß in human macrophages, but data on substitutes is scarce. Hence, we aimed to examine the effects of TCS compared to its alternatives at the molecular level in human macrophages. LPS-stimulated THP-1 macrophages were exposed to TCS or its substitutes, including benzalkonium chloride, benzethonium chloride, chloroxylenol, chlorhexidine (CHX) and cetylpyridinium chloride, with the inhibitory concentration (IC10-value) of cell viability to decipher their mode of action. TCS induced the release of the pro-inflammatory cytokine TNF and high level of IL-1ß, suggesting the activation of the NLRP3-inflammasome, which was confirmed by non-apparent IL-1ß under the NLRP3-inhibitor MCC950 treatment d. While IL-6 release was reduced in all treatments, the alternative CHX completely abolished the release of all investigated cytokines. To unravel the underlying molecular mechanisms, we used untargeted LC-MS/MS-based proteomics. TCS and CHX showed the strongest cellular response at the protein and signalling pathway level, whereby pathways related to metabolism, translation, cellular stress and migration were mainly affected but to different proposed modes of action. TCS inhibited mitochondrial electron transfer and affected phagocytosis. In contrast, in CHX-treated cells, the translation was arrested due to stress conditions, resulting in the formation of stress granules. Mitochondrial (e.g. ATP5F1D, ATP5PB, UQCRQ) and ribosomal (e.g. RPL10, RPL35, RPS23) proteins were revealed as putative key drivers. Furthermore, we have demonstrated the formation of podosomes by CHX, potentially involved in ECM degradation. Our results exhibit modulation of the immune response in macrophages by TCS and its substitutes and illuminated underlying molecular effects. These results illustrate critical processes involved in the modulation of macrophages' immune response by TCS and its alternatives, providing information essential for hazard assessment.

Oxidation is an underappreciated post-translational modification in the regulation of immune responses associated with changes in phosphorylation.

Although macrophages are known to be affected by their redox status, oxidation is not yet a well-recognized post-translational modification (PTM) in regulating macrophages and immune cells in general. While it has been described that the redox status of single cysteines in specific proteins is relevant for macrophage functions, global oxidation information is scarce. Hence, we globally assessed the impact of oxidation on macrophage activation using untargeted proteomics and PTM-omics. We exposed THP-1 macrophages to lipopolysaccharide (LPS) for 4 h and 24 h and applied a sequential iodoTMT labeling approach to get information on overall oxidation as well as reversible oxidation of cysteines. Thus, we identified 10452 oxidation sites, which were integratively analyzed with 5057 proteins and 7148 phosphorylation sites to investigate their co-occurance with other omics layers. Based on this integrative analysis, we found significant upregulation of several immune-related pathways, e.g. toll-like receptor 4 (TLR4) signaling, for which 19 proteins, 7 phosphorylation sites, and 39 oxidation sites were significantly affected, highlighting the relevance of oxidations in TLR4-induced macrophage activation. Co-regulation of oxidation and phosphorylation was observed, as evidenced by multiply modified proteins related to inflammatory pathways. Additionally, we observed time-dependent effects, with differences in the dynamics of oxidation sites compared to proteins and phosphorylation sites. Overall, this study highlights the importance of oxidation in regulating inflammatory processes and provides a method that can be readily applied to study the cellular redoxome globally.

The effect of polyphenols on DNA methylation-assessed biological age attenuation: the DIRECT PLUS randomized controlled trial.

BACKGROUND: Epigenetic age is an estimator of biological age based on DNA methylation; its discrepancy from chronologic age warrants further investigation. We recently reported that greater polyphenol intake benefitted ectopic fats, brain function, and gut microbiota profile, corresponding with elevated urine polyphenols. The effect of polyphenol-rich dietary interventions on biological aging is yet to be determined. METHODS: We calculated different biological aging epigenetic clocks of different generations (Horvath2013, Hannum2013, Li2018, Horvath skin and blood2018, PhenoAge2018, PCGrimAge2022), their corresponding age and intrinsic age accelerations, and DunedinPACE, all based on DNA methylation (Illumina EPIC array; pre-specified secondary outcome) for 256 participants with abdominal obesity or dyslipidemia, before and after the 18-month DIRECT PLUS randomized controlled trial. Three interventions were assigned: healthy dietary guidelines, a Mediterranean (MED) diet, and a polyphenol-rich, low-red/processed meat Green-MED diet. Both MED groups consumed 28 g walnuts/day (+ 440 mg/day polyphenols). The Green-MED group consumed green tea (3-4 cups/day) and Mankai (Wolffia globosa strain) 500-ml green shake (+ 800 mg/day polyphenols). Adherence to the Green-MED diet was assessed by questionnaire and urine polyphenols metabolomics (high-performance liquid chromatography quadrupole time of flight). RESULTS: Baseline chronological age (51.3 ± 10.6 years) was significantly correlated with all methylation age (mAge) clocks with correlations ranging from 0.83 to 0.95; p < 2.2e - 16 for all. While all interventions did not differ in terms of changes between mAge clocks, greater Green-Med diet adherence was associated with a lower 18-month relative change (i.e., greater mAge attenuation) in Li and Hannum mAge (beta = - 0.41, p = 0.004 and beta = - 0.38, p = 0.03, respectively; multivariate models). Greater Li mAge attenuation (multivariate models adjusted for age, sex, baseline mAge, and weight loss) was mostly affected by higher intake of Mankai (beta = - 1.8; p = 0.061) and green tea (beta = - 1.57; p = 0.0016) and corresponded with elevated urine polyphenols: hydroxytyrosol, tyrosol, and urolithin C (p < 0.05 for all) and urolithin A (p = 0.08), highly common in green plants. Overall, participants undergoing either MED-style diet had ~ 8.9 months favorable difference between the observed and expected Li mAge at the end of the intervention (p = 0.02). CONCLUSIONS: This study showed that MED and green-MED diets with increased polyphenols intake, such as green tea and Mankai, are inversely associated with biological aging. To the best of our knowledge, this is the first clinical trial to indicate a potential link between polyphenol intake, urine polyphenols, and biological aging. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03020186.

A polyphenol-rich green Mediterranean diet enhances epigenetic regulatory potential: the DIRECT PLUS randomized controlled trial.

BACKGROUND: The capacity of a polyphenol-enriched diet to modulate the epigenome in vivo is partly unknown. Given the beneficial metabolic effects of a Mediterranean (MED) diet enriched in polyphenols and reduced in red/processed meat (green-MED), as previously been proven by the 18-month DIRECT PLUS randomized controlled trial, we analyzed the effects of the green-MED diet on methylome and transcriptome levels to highlight molecular mechanisms underlying the observed metabolic improvements. METHODS: Our study included 260 participants (baseline BMI = 31.2 kg/m2, age = 5 years) of the DIRECT PLUS trial, initially randomized to one of the intervention arms: A. healthy dietary guidelines (HDG), B. MED (440 mg polyphenols additionally provided by walnuts), C. green-MED (1240 mg polyphenols additionally provided by walnuts, green tea, and Mankai: green duckweed shake). Blood methylome and transcriptome of all study subjects were analyzed at baseline and after completing the 18-month intervention using Illumina EPIC and RNA sequencing technologies. RESULTS: A total of 1573 differentially methylated regions (DMRs; false discovery rate (FDR) < 5 %) were found in the green-MED compared to the MED (177) and HDG (377) diet participants. This corresponded to 1753 differentially expressed genes (DEGs; FDR < 5 %) in the green-MED intervention compared to MED (7) and HDG (738). Consistently, the highest number (6 %) of epigenetic modulating genes was transcriptionally changed in subjects participating in the green-MED intervention. Weighted cluster network analysis relating transcriptional and phenotype changes among participants subjected to the green-MED intervention identified candidate genes associated with serum-folic acid change (all P < 1 × 10-3) and highlighted one module including the KIR3DS1 locus, being negatively associated with the polyphenol changes (e.g. P < 1 × 10-4), but positively associated with the MRI-assessed superficial subcutaneous adipose area-, weight- and waist circumference- 18-month change (all P < 0.05). Among others, this module included the DMR gene Cystathionine Beta-Synthase, playing a major role in homocysteine reduction. CONCLUSIONS: The green-MED high polyphenol diet, rich in green tea and Mankai, renders a high capacity to regulate an individual's epigenome. Our findings suggest epigenetic key drivers such as folate and green diet marker to mediate this capacity and indicate a direct effect of dietary polyphenols on the one­carbon metabolism.

Biomarkers of hepatocellular synthesis in patients with decompensated cirrhosis.

BACKGROUND AND AIM: Since hepatocytes produce majority of serum proteins, patients with cirrhosis display substantial alterations in the serum proteome. The aim of the current study was to characterize these changes and to study the prognostic utility of hepatocellular proteins available in routine clinical testing. METHODS: Sera from 29 healthy controls and 43 patients with cirrhosis were subjected to untargeted proteomic analysis. Unsupervised hierarchical clustering was performed with Perseus software and R. Ingenuity pathway analysis (IPA) suggested upstream regulators that were validated in liver tissues. The behavior and prognostic usefulness of selected biomarkers was investigated in 61 controls and 285 subjects with decompensated cirrhosis. RESULTS: Proteomics uncovered 65 and 16 hepatocellular serum proteins that are significantly downregulated or upregulated in patients with cirrhosis vs. controls. Hierarchical clustering revealed two main clusters and six sub-clusters. IPA identified HNF4α and IL-6 as the two major upstream regulators that were confirmed by hepatic gene expression analyses. Among pseudocholinesterase, transferrin, transthyretin, albumin, and apolipoprotein AI (Apo-AI), Apo-AI was the best predictor of 90-days transplant-free survival (AUROC 0.678; p = 0.0001) and remained an independent predictor in multivariable Cox independently of the presence of acute-on-chronic liver failure. CONCLUSION: Our study reveals cirrhosis-associated changes in hepatocellular serum proteins and underlying transcription factors. Serum apolipoprotein AI may constitute a useful prognostic adjunct in patients with decompensated cirrhosis.

Cocoa seeds and chocolate products interaction with gut microbiota; mining microbial and functional biomarkers from mechanistic studies, clinical trials and 16S rRNA amplicon sequencing.

In recent years, gut microbiome has evolved as a focal point of interest with growing recognition that a well-balanced gut microbiota is highly relevant to an individual's health status. The present review provides a mechanistic insight on the effects of cocoa chemicals on the gut microbiome and further reveals in silico biomarkers, taxonomic and functional features that distinguish gut microbiome of cocoa consumers and controls by using 16S rRNA gene sequencing data. The polyphenols in cocoa can change the gut microbiota either by inhibiting the growth of pathogenic bacteria in the gut such as Clostridium perfringens or by increasing the growth of beneficial microbiota in the gut such as Lactobacillus and Bifidobacterium. This paper demonstrates the holistic effect of gut microbiota on cocoa chemicals and how it impacts human health. We present herein the first comprehensive review and analysis of how raw and roasted cocoa and its products can specifically influence gut homeostasis, and likewise, how microbiota metabolizes cocoa chemicals. In addition to that, our 16S rRNA amplicon sequencing analysis revealed that the flavone and flavonols metabolism, aminobenzoate degradation and fatty acid elongation pathways represent the three most important signatures of microbial functions associated with cocoa consumption.

The effect of high-polyphenol Mediterranean diet on visceral adiposity: the DIRECT PLUS randomized controlled trial.

BACKGROUND: Mediterranean (MED) diet is a rich source of polyphenols, which benefit adiposity by several mechanisms. We explored the effect of the green-MED diet, twice fortified in dietary polyphenols and lower in red/processed meat, on visceral adipose tissue (VAT). METHODS: In the 18-month Dietary Intervention Randomized Controlled Trial PoLyphenols UnproceSsed (DIRECT-PLUS) weight-loss trial, 294 participants were randomized to (A) healthy dietary guidelines (HDG), (B) MED, or (C) green-MED diets, all combined with physical activity. Both isocaloric MED groups consumed 28 g/day of walnuts (+ 440 mg/day polyphenols). The green-MED group further consumed green tea (3-4 cups/day) and Wolffia globosa (duckweed strain) plant green shake (100 g frozen cubes/day) (+ 800mg/day polyphenols) and reduced red meat intake. We used magnetic resonance imaging (MRI) to quantify the abdominal adipose tissues. RESULTS: Participants (age = 51 years; 88% men; body mass index = 31.2 kg/m2; 29% VAT) had an 89.8% retention rate and 79.3% completed eligible MRIs. While both MED diets reached similar moderate weight (MED: - 2.7%, green-MED: - 3.9%) and waist circumference (MED: - 4.7%, green-MED: - 5.7%) loss, the green-MED dieters doubled the VAT loss (HDG: - 4.2%, MED: - 6.0%, green-MED: - 14.1%; p < 0.05, independent of age, sex, waist circumference, or weight loss). Higher dietary consumption of green tea, walnuts, and Wolffia globosa; lower red meat intake; higher total plasma polyphenols (mainly hippuric acid), and elevated urine urolithin A polyphenol were significantly related to greater VAT loss (p < 0.05, multivariate models). CONCLUSIONS: A green-MED diet, enriched with plant-based polyphenols and lower in red/processed meat, may be a potent intervention to promote visceral adiposity regression. TRIAL REGISTRATION: ClinicalTrials.gov , NCT03020186.

Glyphosate differentially affects the allergic immune response across generations in mice.

Exposure to environmental pollutants via food, particularly during the prenatal and early postnatal periods, has been linked to adverse effects on the immune system. Among these pollutants, the widely used pesticide glyphosate has been associated with endocrine disruption, autism, and cancer. Occupational high exposure to glyphosate has also been shown to influence immune function and exacerbate allergic asthma. However, there are no studies investigating the effect of a common low-dose glyphosate exposure on the allergic immune response - neither directly nor across generations. We therefore explored the impact of oral low-dose glyphosate exposure (0.5 and 50 mg/kg body weight/day) on airway inflammation in dams (F0) and the offspring (F1 and F2 generations) using a murine multi-generational asthma model. While exposure to 50 mg/kg glyphosate induced a mild eosinophilic infiltration in the bronchoalveolar lavage and TH2 cytokine production in the dams, the F1 offspring developed a reduced immune response after maternal exposure to 0.5 mg/kg glyphosate. In particular, decreased lung inflammation, HDM-specific IgE levels, and asthma-relevant cytokine production were primarily observed in the female F1 offspring. However, not only the TH2 cytokines IL-13 and IL-5 but also the TH17 cytokine IL-17 and TH1 cytokine IFN-γ were reduced indicating a more general immunosuppressive function. Notably, the dampened immune response was no longer observed in the female F2 generation. Furthermore, female F1 offspring showed an increased abundance of bacteria in the gut, which have been associated with probiotic-mediated reduced allergic immune responses. Our results suggest a potential immunosuppressive effect of low-dose maternal glyphosate exposure in the F1 offspring that might be mediated by an altered microbiota composition. Further studies are needed to explore if this type of immune response modulation might also be associated with impairments in immune defense upon infectious diseases or even cancer pathology.

Functional changes of the gastric bypass microbiota reactivate thermogenic adipose tissue and systemic glucose control via intestinal FXR-TGR5 crosstalk in diet-induced obesity.

BACKGROUND: Bariatric surgery remains the most effective therapy for adiposity reduction and remission of type 2 diabetes. Although different bariatric procedures associate with pronounced shifts in the gut microbiota, their functional role in the regulation of energetic and metabolic benefits achieved with the surgery are not clear. METHODS: To evaluate the causal as well as the inherent therapeutic character of the surgery-altered gut microbiome in improved energy and metabolic control in diet-induced obesity, an antibiotic cocktail was used to eliminate the gut microbiota in diet-induced obese rats after gastric bypass surgery, and gastric bypass-shaped gut microbiota was transplanted into obese littermates. Thorough metabolic profiling was combined with omics technologies on samples collected from cecum and plasma to identify adaptions in gut microbiota-host signaling, which control improved energy balance and metabolic profile after surgery. RESULTS: In this study, we first demonstrate that depletion of the gut microbiota largely reversed the beneficial effects of gastric bypass surgery on negative energy balance and improved glucolipid metabolism. Further, we show that the gastric bypass-shaped gut microbiota reduces adiposity in diet-induced obese recipients by re-activating energy expenditure from metabolic active brown adipose tissue. These beneficial effects were linked to improved glucose homeostasis, lipid control, and improved fatty liver disease. Mechanistically, these effects were triggered by modulation of taurine metabolism by the gastric bypass gut microbiota, fostering an increased abundance of intestinal and circulating taurine-conjugated bile acid species. In turn, these bile acids activated gut-restricted FXR and systemic TGR5 signaling to stimulate adaptive thermogenesis. CONCLUSION: Our results establish the role of the gut microbiome in the weight loss and metabolic success of gastric bypass surgery. We here identify a signaling cascade that entails altered bile acid receptor signaling resulting from a collective, hitherto undescribed change in the metabolic activity of a cluster of bacteria, thereby readjusting energy imbalance and metabolic disease in the obese host. These findings strengthen the rationale for microbiota-targeted strategies to improve and refine current therapies of obesity and metabolic syndrome. Video Abstract Bariatric Surgery (i.e. RYGB) or the repeated fecal microbiota transfer (FMT) from RYGB donors into DIO (diet-induced obesity) animals induces shifts in the intestinal microbiome, an effect that can be impaired by oral application of antibiotics (ABx). Our current study shows that RYGB-dependent alterations in the intestinal microbiome result in an increase in the luminal and systemic pool of Taurine-conjugated Bile acids (TCBAs) by various cellular mechanisms acting in the intestine and the liver. TCBAs induce signaling via two different receptors, farnesoid X receptor (FXR, specifically in the intestines) and the G-protein-coupled bile acid receptor TGR5 (systemically), finally resulting in metabolic improvement and advanced weight management. BSH, bile salt hydrolase; BAT brown adipose tissue.

Metabolic Profile and Metabolite Analyses in Extreme Weight Responders to Gastric Bypass Surgery.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) surgery belongs to the most frequently performed surgical therapeutic strategies against adiposity and its comorbidities. However, outcome is limited in a substantial cohort of patients with inadequate primary weight loss or considerable weight regain. In this study, gut microbiota composition and systemically released metabolites were analyzed in a cohort of extreme weight responders after RYGB. METHODS: Patients (n = 23) were categorized based on excess weight loss (EWL) at a minimum of two years after RYGB in a good responder (EWL 93 ± 4.3%) or a bad responder group (EWL 19.5 ± 13.3%) for evaluation of differences in metabolic outcome, eating behavior and gut microbiota taxonomy and metabolic activity. RESULTS: Mean BMI was 47.2 ± 6.4 kg/m2 in the bad vs. 26.6 ± 1.2 kg/m2 in the good responder group (p = 0.0001). We found no difference in hunger and satiety sensation, in fasting or postprandial gut hormone release, or in gut microbiota composition between both groups. Differences in weight loss did not reflect in metabolic outcome after RYGB. While fecal and circulating metabolite analyses showed higher levels of propionate (p = 0.0001) in good and valerate (p = 0.04) in bad responders, respectively, conjugated primary and secondary bile acids were higher in good responders in the fasted (p = 0.03) and postprandial state (GCA, p = 0.02; GCDCA, p = 0.02; TCA, p = 0.01; TCDCA, p = 0.02; GDCA, p = 0.05; GUDCA, p = 0.04; TLCA, p = 0.04). CONCLUSIONS: Heterogenous weight loss response to RYGB surgery separates from patients' metabolic outcome, and is linked to unique serum metabolite signatures post intervention. These findings suggest that the level of adiposity reduction alone is insufficient to assess the metabolic success of RYGB surgery, and that longitudinal metabolite profiling may eventually help us to identify markers that could predict individual adiposity response to surgery and guide patient selection and counseling.

Autophagopathies: from autophagy gene polymorphisms to precision medicine for human diseases.

At a time when complex diseases affect globally 280 million people and claim 14 million lives every year, there is an urgent need to rapidly increase our knowledge into their underlying etiologies. Though critical in identifying the people at risk, the causal environmental factors (microbiome and/or pollutants) and the affected pathophysiological mechanisms are not well understood. Herein, we consider the variations of autophagy-related (ATG) genes at the heart of mechanisms of increased susceptibility to environmental stress. A comprehensive autophagy genomic resource is presented with 263 single nucleotide polymorphisms (SNPs) for 69 autophagy-related genes associated with 117 autoimmune, inflammatory, infectious, cardiovascular, neurological, respiratory, and endocrine diseases. We thus propose the term 'autophagopathies' to group together a class of complex human diseases the etiology of which lies in a genetic defect of the autophagy machinery, whether directly related or not to an abnormal flux in autophagy, LC3-associated phagocytosis, or any associated trafficking. The future of precision medicine for common diseases will lie in our ability to exploit these ATG SNP x environment relationships to develop new polygenetic risk scores, new management guidelines, and optimal therapies for afflicted patients.Abbreviations: ATG, autophagy-related; ALS-FTD, amyotrophic lateral sclerosis-frontotemporal dementia; ccRCC, clear cell renal cell carcinoma; CD, Crohn disease; COPD, chronic obstructive pulmonary disease; eQTL, expression quantitative trait loci; HCC, hepatocellular carcinoma; HNSCC, head and neck squamous cell carcinoma; GTEx, genotype-tissue expression; GWAS, genome-wide association studies; LAP, LC3-associated phagocytosis; LC3-II, phosphatidylethanolamine conjugated form of LC3; LD, linkage disequilibrium; LUAD, lung adenocarcinoma; MAF, minor allele frequency; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NSCLC, non-small cell lung cancer; OS, overall survival; PtdIns3K CIII, class III phosphatidylinositol 3 kinase; PtdIns3P, phosphatidylinositol-3-phosphate; SLE, systemic lupus erythematosus; SNPs, single-nucleotide polymorphisms; mQTL, methylation quantitative trait loci; ULK, unc-51 like autophagy activating kinase; UTRs, untranslated regions; WHO, World Health Organization.

Genes and enzymes involved in the biodegradation of the quaternary carbon compound pivalate in the denitrifying Thauera humireducens strain PIV-1.

Quaternary carbon-containing compounds exist in natural and fossil oil-derived products and are used in chemical and pharmaceutical applications up to industrial scale. Due to the inaccessibility of the quaternary carbon atom for a direct oxidative or reductive attack, they are considered as persistent in the environment. Here, we investigated the unknown degradation of the quaternary carbon-containing model compound pivalate (2,2-dimethyl-propionate) in the denitrifying bacterium Thauera humireducens strain PIV-1 (formerly Thauera pivalivorans). We provide multiple evidence for a pathway comprising the activation to pivalyl-CoA and the carbon skeleton rearrangement to isovaleryl-CoA. Subsequent reactions proceed similar to the catabolic leucine degradation pathway such as the carboxylation to 3-methylglutaconyl-CoA and the cleavage of 3-methyl-3-hydroxyglutaryl-CoA to acetyl-CoA and acetoacetate. The completed genome of Thauera humireducens strain PIV-1 together with proteomic data was used to identify pivalate-upregulated gene clusters including genes putatively encoding pivalate CoA ligase and adenosylcobalamin-dependent pivalyl-CoA mutase. A pivalate-induced gene encoding a putative carboxylic acid CoA ligase was heterologously expressed, and its highly enriched product exhibited pivalate CoA ligase activity. The results provide the first experimental insights into the biodegradation pathway of a quaternary carbon-containing model compound that serves as a blueprint for the degradation of related quaternary carbon-containing compounds.

Danger signal extracellular calcium initiates differentiation of monocytes into SPP1/osteopontin-producing macrophages.

The danger signal extracellular calcium is pathophysiologically increased in the synovial fluid of patients with rheumatoid arthritis (RA). Calcium activates the NLRP3-inflammasome via the calcium-sensing receptor in monocytes/macrophages primed by lipopolysaccharide, and this effect is mediated by the uptake of calciprotein particles (CPPs) formed out of calcium, phosphate, and fetuin-A. Aim of the study was to unravel the influence of calcium on monocytes when the priming signal is not present. Monocytes were isolated from the blood of healthy controls and RA patients. Macrophages were characterized using scRNA-seq, DNA microarray, and proteomics. Imaging flow cytometry was utilized to study intracellular events. Here we show that extracellular calcium and CPPs lead to the differentiation of monocytes into calcium-macrophages when the priming signal is absent. Additional growth factors are not needed, and differentiation is triggered by calcium-dependent CPP-uptake, lysosomal alkalization due to CPP overload, and TFEB- and STAT3-dependent increased transcription of the lysosomal gene network. Calcium-macrophages have a needle-like shape, are characterized by excessive, constitutive SPP1/osteopontin production and a strong pro-inflammatory cytokine response. Calcium-macrophages differentiated out of RA monocytes show a stronger manifestation of this phenotype, suggesting the differentiation process might lead to the pro-inflammatory macrophage response seen in the RA synovial membrane.

The effect of a high-polyphenol Mediterranean diet (Green-MED) combined with physical activity on age-related brain atrophy: the Dietary Intervention Randomized Controlled Trial Polyphenols Unprocessed Study (DIRECT PLUS).

BACKGROUND: The effect of diet on age-related brain atrophy is largely unproven. OBJECTIVES: We aimed to explore the effect of a Mediterranean diet (MED) higher in polyphenols and lower in red/processed meat (Green-MED diet) on age-related brain atrophy. METHODS: This 18-mo clinical trial longitudinally measured brain structure volumes by MRI using hippocampal occupancy score (HOC) and lateral ventricle volume (LVV) expansion score as neurodegeneration markers. Abdominally obese/dyslipidemic participants were randomly assigned to follow 1) healthy dietary guidelines (HDG), 2) MED, or 3) Green-MED diet. All subjects received free gym memberships and physical activity guidance. Both MED groups consumed 28 g walnuts/d (+440 mg/d polyphenols). The Green-MED group consumed green tea (3-4 cups/d) and Mankai (Wolffia-globosa strain, 100 g frozen cubes/d) green shake (+800 mg/d polyphenols). RESULTS: Among 284 participants (88% men; mean age: 51 y; BMI: 31.2 kg/m2; APOE-ε4 genotype = 15.7%), 224 (79%) completed the trial with eligible whole-brain MRIs. The pallidum (-4.2%), third ventricle (+3.9%), and LVV (+2.2%) disclosed the largest volume changes. Compared with younger participants, atrophy was accelerated among those ≥50 y old (HOC change: -1.0% ± 1.4% compared with -0.06% ± 1.1%; 95% CI: 0.6%, 1.3%; P < 0.001; LVV change: 3.2% ± 4.5% compared with 1.3% ± 4.1%; 95% CI: -3.1%, -0.8%; P = 0.001). In subjects ≥ 50 y old, HOC decline and LVV expansion were attenuated in both MED groups, with the best outcomes among Green-MED diet participants, as compared with HDG (HOC: -0.8% ± 1.6% compared with -1.3% ± 1.4%; 95% CI: -1.5%, -0.02%; P = 0.042; LVV: 2.3% ± 4.7% compared with 4.3% ± 4.5%; 95% CI: 0.3%, 5.2%; P = 0.021). Similar patterns were observed among younger subjects. Improved insulin sensitivity over the trial was the parameter most strongly associated with brain atrophy attenuation (P < 0.05). Greater Mankai, green tea, and walnut intake and less red and processed meat were significantly and independently associated with reduced HOC decline (P < 0.05). Elevated urinary concentrations of the polyphenols urolithin-A (r = 0.24; P = 0.013) and tyrosol (r = 0.26; P = 0.007) were significantly associated with lower HOC decline. CONCLUSIONS: A Green-MED (high-polyphenol) diet, rich in Mankai, green tea, and walnuts and low in red/processed meat, is potentially neuroprotective for age-related brain atrophy.This trial was registered at clinicaltrials.gov as NCT03020186.

Multiomics reveal unique signatures of human epiploic adipose tissue related to systemic insulin resistance.

OBJECTIVE: Human white adipose tissue (AT) is a metabolically active organ with distinct depot-specific functions. Despite their locations close to the gastrointestinal tract, mesenteric AT and epiploic AT (epiAT) have only scarcely been investigated. Here, we aim to characterise these ATs in-depth and estimate their contribution to alterations in whole-body metabolism. DESIGN: Mesenteric, epiploic, omental and abdominal subcutaneous ATs were collected from 70 patients with obesity undergoing Roux-en-Y gastric bypass surgery. The metabolically well-characterised cohort included nine subjects with insulin sensitive (IS) obesity, whose AT samples were analysed in a multiomics approach, including methylome, transcriptome and proteome along with samples from subjects with insulin resistance (IR) matched for age, sex and body mass index (n=9). Findings implying differences between AT depots in these subgroups were validated in the entire cohort (n=70) by quantitative real-time PCR. RESULTS: While mesenteric AT exhibited signatures similar to those found in the omental depot, epiAT was distinct from all other studied fat depots. Multiomics allowed clear discrimination between the IS and IR states in all tissues. The highest discriminatory power between IS and IR was seen in epiAT, where profound differences in the regulation of developmental, metabolic and inflammatory pathways were observed. Gene expression levels of key molecules involved in AT function, metabolic homeostasis and inflammation revealed significant depot-specific differences with epiAT showing the highest expression levels. CONCLUSION: Multi-omics epiAT signatures reflect systemic IR and obesity subphenotypes distinct from other fat depots. Our data suggest a previously unrecognised role of human epiploic fat in the context of obesity, impaired insulin sensitivity and related diseases.

Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Mesenchymal stromal cells mitigate liver damage after extended resection in the pig by modulating thrombospondin-1/TGF-ß.

Post-surgery liver failure is a serious complication for patients after extended partial hepatectomies (ePHx). Previously, we demonstrated in the pig model that transplantation of mesenchymal stromal cells (MSC) improved circulatory maintenance and supported multi-organ functions after 70% liver resection. Mechanisms behind the beneficial MSC effects remained unknown. Here we performed 70% liver resection in pigs with and without MSC treatment, and animals were monitored for 24 h post surgery. Gene expression profiles were determined in the lung and liver. Bioinformatics analysis predicted organ-independent MSC targets, importantly a role for thrombospondin-1 linked to transforming growth factor-ß (TGF-ß) and downstream signaling towards providing epithelial plasticity and epithelial-mesenchymal transition (EMT). This prediction was supported histologically and mechanistically, the latter with primary hepatocyte cell cultures. MSC attenuated the surgery-induced increase of tissue damage, of thrombospondin-1 and TGF-ß, as well as of epithelial plasticity in both the liver and lung. This suggests that MSC ameliorated surgery-induced hepatocellular stress and EMT, thus supporting epithelial integrity and facilitating regeneration. MSC-derived soluble factor(s) did not directly interfere with intracellular TGF-ß signaling, but inhibited thrombospondin-1 secretion from thrombocytes and non-parenchymal liver cells, therewith obviously reducing the availability of active TGF-ß.

Proteomic Characterization of the Cellular Effects of AhR Activation by Microbial Tryptophan Catabolites in Endotoxin-Activated Human Macrophages.

Sensing microbial tryptophan catabolites by the aryl hydrocarbon receptor (AhR) plays a pivotal role in host-microbiome homeostasis by modulating the host immune response. Nevertheless, the involved cellular processes triggered by the metabolites are mainly unknown. Here, we analyzed proteomic changes in macrophages after treatment with the tryptophan metabolites indole-3-acetic acid (I3AA) or indole-3-aldehyde (IAld), as well as the prototypic exogenous AhR-ligand benzo(a)pyrene (BaP) in the absence and presence of lipopolysaccharide (LPS) to identify affected cellular processes and pathways. The AhR-ligands regulated metabolic and immunologic processes in dependency of LPS co-stimulation. All investigated ligands time-dependently enhanced fatty acid ß-oxidation. Differences due to the combination with LPS were observed for all three ligands. Additionally, oxidative phosphorylation was significantly increased by IAld and I3AA in a time and LPS-dependent manner. Immunoregulatory processes were affected in distinct ways. While BaP and I3AA up-regulated IL-8 signaling, IL-6 signaling was decreased by IAld. BaP decreased the inflammasome pathway. Thus, AhR-ligand-dependent regulations were identified, which may modulate the response of macrophages to bacterial infections, but also the commensal microbiota through changes in immune cell signaling and metabolic pathways that may also alter functionality. These findings highlight the relevance of AhR for maintaining microbial homeostasis and, consequently, host health.

Differences in the sperm metabolomes of smoking and nonsmoking men†.

Currently, spermiogram analysis is the most relevant method used to clarify the potential infertility of a couple. However, in some cases, the reasons for infertility remain obscure. Smoking is among the factors that have been described to adversely affect male fertility. Smoking increases oxidative stress and thus promotes various pathological processes. Comparative studies, particularly those on metabolomic changes in sperm and seminal plasma caused by smoking, have not yet been published. Thus, the present pilot study aimed at the mass spectrometric characterization of the metabolomes of specimens from both smoking and nonsmoking subjects and the comparison of the evaluated data in terms of sperm apoptosis and spermiogram parameters. The results provided evidence that the conventional spermiogram is not altered in smokers compared to nonsmokers. However, a more careful investigation of sperm cells by metabolomic profiling reveals profound effects of smoking on sperm: first, nitrogen oxide synthase, a marker of oxidative stress, is activated. Second, the uptake of fatty acids into sperm mitochondria is reduced, leading to an impaired energy supply. Third, phenylalanine hydroxylation and tryptophan degradation, which are both indications of altered tetrahydrobiopterin biosynthesis, are reduced. Moreover, flow cytometry approaches indicated increased sperm caspase-3 activity, a sign of apoptosis. The present study clearly shows the negative effects of smoking on semen quality. Especially for idiopathic cases, metabolomic profiling can help to shed light on male subfertility or infertility.