M cell maturation and cDC activation determine the onset of adaptive immune priming in the neonatal Peyer's patch.

Early-life immune development is critical to long-term host health. However, the mechanisms that determine the pace of postnatal immune maturation are not fully resolved. Here, we analyzed mononuclear phagocytes (MNPs) in small intestinal Peyer's patches (PPs), the primary inductive site of intestinal immunity. Conventional type 1 and 2 dendritic cells (cDC1 and cDC2) and RORgt+ antigen-presenting cells (RORgt+ APC) exhibited significant age-dependent changes in subset composition, tissue distribution, and reduced cell maturation, subsequently resulting in a lack in CD4+ T cell priming during the postnatal period. Microbial cues contributed but could not fully explain the discrepancies in MNP maturation. Type I interferon (IFN) accelerated MNP maturation but IFN signaling did not represent the physiological stimulus. Instead, follicle-associated epithelium (FAE) M cell differentiation was required and sufficient to drive postweaning PP MNP maturation. Together, our results highlight the role of FAE M cell differentiation and MNP maturation in postnatal immune development.

A dual-function RNA balances carbon uptake and central metabolism in Vibrio cholerae.

Bacterial small RNAs (sRNAs) are well known to modulate gene expression by base pairing with trans-encoded transcripts and are typically non-coding. However, several sRNAs have been reported to also contain an open reading frame and thus are considered dual-function RNAs. In this study, we discovered a dual-function RNA from Vibrio cholerae, called VcdRP, harboring a 29 amino acid small protein (VcdP), as well as a base-pairing sequence. Using a forward genetic screen, we identified VcdRP as a repressor of cholera toxin production and link this phenotype to the inhibition of carbon transport by the base-pairing segment of the regulator. By contrast, we demonstrate that the VcdP small protein acts downstream of carbon transport by binding to citrate synthase (GltA), the first enzyme of the citric acid cycle. Interaction of VcdP with GltA results in increased enzyme activity and together VcdR and VcdP reroute carbon metabolism. We further show that transcription of vcdRP is repressed by CRP allowing us to provide a model in which VcdRP employs two different molecular mechanisms to synchronize central metabolism in V. cholerae.

Deconstructing Methanosarcina acetivorans into an acetogenic archaeon.

The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide-dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.

Sulfur and methane oxidation by a single microorganism.

Natural and anthropogenic wetlands are major sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic bacteria at the oxic-anoxic interface, a zone of intense redox cycling of carbon, sulfur, and nitrogen compounds. Here, we report on the isolation of an aerobic methanotrophic bacterium, 'Methylovirgula thiovorans' strain HY1, which possesses metabolic capabilities never before found in any methanotroph. Most notably, strain HY1 is the first bacterium shown to aerobically oxidize both methane and reduced sulfur compounds for growth. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are responsible for methane and methanol oxidation, respectively. Various pathways for respiratory sulfur oxidation were present, including the Sox-rDsr pathway and the S4I system. Strain HY1 employed the Calvin-Benson-Bassham cycle for CO2 fixation during chemolithoautotrophic growth on reduced sulfur compounds. Proteomic and microrespirometry analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of the respective substrates. Methane and thiosulfate could therefore be independently or simultaneously oxidized. The discovery of this versatile bacterium demonstrates that methanotrophy and thiotrophy are compatible in a single microorganism and underpins the intimate interactions of methane and sulfur cycles in oxic-anoxic interface environments.

Prebiotic diet changes neural correlates of food decision-making in overweight adults: a randomised controlled within-subject cross-over trial.

OBJECTIVE: Animal studies suggest that prebiotic, plant-derived nutrients could improve homoeostatic and hedonic brain functions through improvements in microbiome-gut-brain communication. However, little is known if these results are applicable to humans. Therefore, we tested the effects of high-dosed prebiotic fibre on reward-related food decision-making in a randomised controlled within-subject cross-over study and assayed potential microbial and metabolic markers. DESIGN: 59 overweight young adults (19 females, 18-42 years, body mass index 25-30 kg/m2) underwent functional task MRI before and after 14 days of supplementary intake of 30 g/day of inulin (prebiotics) and equicaloric placebo, respectively. Short chain fatty acids (SCFA), gastrointestinal hormones, glucose/lipid and inflammatory markers were assayed in fasting blood. Gut microbiota and SCFA were measured in stool. RESULTS: Compared with placebo, participants showed decreased brain activation towards high-caloric wanted food stimuli in the ventral tegmental area and right orbitofrontal cortex after prebiotics (preregistered, family wise error-corrected p <0.05). While fasting blood levels remained largely unchanged, 16S-rRNA sequencing showed significant shifts in the microbiome towards increased occurrence of, among others, SCFA-producing Bifidobacteriaceae, and changes in >60 predicted functional signalling pathways after prebiotic intake. Changes in brain activation correlated with changes in Actinobacteria microbial abundance and associated activity previously linked with SCFA production, such as ABC transporter metabolism. CONCLUSIONS: In this proof-of-concept study, a prebiotic intervention attenuated reward-related brain activation during food decision-making, paralleled by shifts in gut microbiota. TRIAL REGISTRATION NUMBER: NCT03829189.

Metabolomics in human SGBS cells as new approach method for studying adipogenic effects: Analysis of the effects of DINCH and MINCH on central carbon metabolism.

Growing evidence suggests that exposure to certain metabolism-disrupting chemicals (MDCs), such as the phthalate plasticizer DEHP, might promote obesity in humans, contributing to the spread of this global health problem. Due to the restriction on the use of phthalates, there has been a shift to safer declared substitutes, including the plasticizer diisononyl-cyclohexane-1,2-dicarboxylate (DINCH). Notwithstanding, recent studies suggest that the primary metabolite monoisononyl-cyclohexane-1,2-dicarboxylic acid ester (MINCH), induces differentiation of human adipocytes and affects enzyme levels of key metabolic pathways. Given the lack of methods for assessing metabolism-disrupting effects of chemicals on adipose tissue, we used metabolomics to analyze human SGSB cells exposed to DINCH or MINCH. Concentration analysis of DINCH and MINCH revealed that uptake of MINCH in preadipocytes was associated with increased lipid accumulation during adipogenesis. Although we also observed intracellular uptake for DINCH, the solubility of DINCH in cell culture medium was limited, hampering the analysis of possible effects in the µM concentration range. Metabolomics revealed that MINCH induces lipid accumulation similar to peroxisome proliferator-activated receptor gamma (PPARG)-agonist rosiglitazone through upregulation of the pyruvate cycle, which was recently identified as a key driver of de novo lipogenesis. Analysis of the metabolome in the presence of the PPARG-inhibitor GW9662 indicated that the effect of MINCH on metabolism was mediated at least partly by a PPARG-independent mechanism. However, all effects of MINCH were only observed at high concentrations of 10 µM, which are three orders of magnitudes higher than the current concentrations of plasticizers in human serum. Overall, the assessment of the effects of DINCH and MINCH on SGBS cells by metabolomics revealed no adipogenic potential at physiologically relevant concentrations. This finding aligns with previous in vivo studies and supports the potential of our method as a New Approach Method (NAM) for the assessment of adipogenic effects of environmental chemicals.

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.

Bacterial Dehydrogenases Facilitate Oxidative Inactivation and Bioremediation of Chloramphenicol.

Antimicrobial resistance represents a major threat to human health and knowledge of the underlying mechanisms is therefore vital. Here, we report the discovery and characterization of oxidoreductases that inactivate the broad-spectrum antibiotic chloramphenicol via dual oxidation of the C3-hydroxyl group. Accordingly, chloramphenicol oxidation either depends on standalone glucose-methanol-choline (GMC)-type flavoenzymes, or on additional aldehyde dehydrogenases that boost overall turnover. These enzymes also enable the inactivation of the chloramphenicol analogues thiamphenicol and azidamfenicol, but not of the C3-fluorinated florfenicol. Notably, distinct isofunctional enzymes can be found in Gram-positive (e. g., Streptomyces sp.) and Gram-negative (e. g., Sphingobium sp.) bacteria, which presumably evolved their selectivity for chloramphenicol independently based on phylogenetic analyses. Mechanistic and structural studies provide further insights into the catalytic mechanisms of these biotechnologically interesting enzymes, which, in sum, are both a curse and a blessing by contributing to the spread of antibiotic resistance as well as to the bioremediation of chloramphenicol.

A comparative proteomics analysis of four contact allergens in THP-1 cells shows distinct alterations in key metabolic pathways.

Allergic contact dermatitis (ACD) is the predominant form of immunotoxicity in humans. The sensitizing potential of chemicals can be assessed in vitro. However, a better mechanistic understanding could improve the current OECD-validated test battery. The aim of this study was to get insights into toxicity mechanisms of four contact allergens, p-benzoquinone (BQ), 2,4-dinitrochlorobenzene (DNCB), p-nitrobenzyl bromide (NBB) and NiSO4, by analyzing differential proteome alterations in THP-1 cells using two common proteomics workflows, stable isotope labeling by amino acids in cell culture (SILAC) and label-free quantification (LFQ). Here, SILAC was found to deliver more robust results. Overall, the four allergens induced similar responses in THP-1 cells, which underwent profound metabolic reprogramming, including a striking upregulation of the TCA cycle accompanied by pronounced induction of the Nrf2 oxidative stress response pathway. The magnitude of induction varied between the allergens with DNCB and NBB being most potent. A considerable overlap between transcriptome-based signatures of the GARD assay and the proteins identified in our study was found. When comparing the results of this study to a previous proteomics study in human primary monocyte-derived dendritic cells, we found a rather low share in regulated proteins. However, on pathway level, the overlap was high, indicating that affected pathways rather than single proteins are more eligible to investigate proteomic changes induced by contact allergens. Overall, this study confirms the potential of proteomics to obtain a profound mechanistic understanding, which may help improving existing in vitro assays for skin sensitization.

Applying genomics in regulatory toxicology: a report of the ECETOC workshop on omics threshold on non-adversity.

In a joint effort involving scientists from academia, industry and regulatory agencies, ECETOC's activities in Omics have led to conceptual proposals for: (1) A framework that assures data quality for reporting and inclusion of Omics data in regulatory assessments; and (2) an approach to robustly quantify these data, prior to interpretation for regulatory use. In continuation of these activities this workshop explored and identified areas of need to facilitate robust interpretation of such data in the context of deriving points of departure (POD) for risk assessment and determining an adverse change from normal variation. ECETOC was amongst the first to systematically explore the application of Omics methods, now incorporated into the group of methods known as New Approach Methodologies (NAMs), to regulatory toxicology. This support has been in the form of both projects (primarily with CEFIC/LRI) and workshops. Outputs have led to projects included in the workplan of the Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) group of the Organisation for Economic Co-operation and Development (OECD) and to the drafting of OECD Guidance Documents for Omics data reporting, with potentially more to follow on data transformation and interpretation. The current workshop was the last in a series of technical methods development workshops, with a sub-focus on the derivation of a POD from Omics data. Workshop presentations demonstrated that Omics data developed within robust frameworks for both scientific data generation and analysis can be used to derive a POD. The issue of noise in the data was discussed as an important consideration for identifying robust Omics changes and deriving a POD. Such variability or "noise" can comprise technical or biological variation within a dataset and should clearly be distinguished from homeostatic responses. Adverse outcome pathways (AOPs) were considered a useful framework on which to assemble Omics methods, and a number of case examples were presented in illustration of this point. What is apparent is that high dimension data will always be subject to varying processing pipelines and hence interpretation, depending on the context they are used in. Yet, they can provide valuable input for regulatory toxicology, with the pre-condition being robust methods for the collection and processing of data together with a comprehensive description how the data were interpreted, and conclusions reached.

HumanMetagenomeDB: a public repository of curated and standardized metadata for human metagenomes.

Metagenomics became a standard strategy to comprehend the functional potential of microbial communities, including the human microbiome. Currently, the number of metagenomes in public repositories is increasing exponentially. The Sequence Read Archive (SRA) and the MG-RAST are the two main repositories for metagenomic data. These databases allow scientists to reanalyze samples and explore new hypotheses. However, mining samples from them can be a limiting factor, since the metadata available in these repositories is often misannotated, misleading, and decentralized, creating an overly complex environment for sample reanalysis. The main goal of the HumanMetagenomeDB is to simplify the identification and use of public human metagenomes of interest. HumanMetagenomeDB version 1.0 contains metadata of 69 822 metagenomes. We standardized 203 attributes, based on standardized ontologies, describing host characteristics (e.g. sex, age and body mass index), diagnosis information (e.g. cancer, Crohn's disease and Parkinson), location (e.g. country, longitude and latitude), sampling site (e.g. gut, lung and skin) and sequencing attributes (e.g. sequencing platform, average length and sequence quality). Further, HumanMetagenomeDB version 1.0 metagenomes encompass 58 countries, 9 main sample sites (i.e. body parts), 58 diagnoses and multiple ages, ranging from just born to 91 years old. The HumanMetagenomeDB is publicly available at https://webapp.ufz.de/hmgdb/.

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.

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.

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.

Bidirectional Role of NLRP3 During Acute and Chronic Cholestatic Liver Injury.

BACKGROUND AND AIMS: Cholestatic liver injury leads to cell death and subsequent inflammation and fibrosis. As shown for primary biliary cholangitis (PBC), the mechanisms and circuits between different cell death pathways leading to disease progression are incompletely defined. Common bile duct ligation (BDL) is a well-established murine model to mimic cholestatic liver injury. Here, we hypothesized that pyroptotic cell death by the Nucleotide-Binding Domain, Leucine-Rich-Containing Family, Pyrin Domain-Containing-3 (Nlrp3) inflammasome plays an essential role during human and murine cholestasis. APPROACH AND RESULTS: NLRP3 activation was analyzed in humans with cholestatic liver injury. Wild-type (WT) and Nlrp3-/- mice were subjected to BDL for 2 or 28 days. Chronic cholestasis in humans and mice is associated with NLRP3 activation and correlates with disease activity. Acute BDL in Nlrp3-deficient mice triggered increased inflammation as well as liver injury, associated with stronger apoptotic and necroptotic cell death. In contrast, NLRP3 deletion led to decreased liver injury and inflammation in chronic cholestasis. Moreover, bridging fibrosis was observed in WT, but not in NLRP3 knockout, mice 28 days after BDL. In contrast, lack of NLRP3 expression attenuated kidney injury and fibrosis after acute and chronic BDL. Importantly, administration of MCC950, an NLRP3 small molecule inhibitor, reduced BDL-induced disease progression in WT mice. CONCLUSIONS: NLRP3 activation correlates with disease activity in patients with PBC. NLRP3 has a differential role during acute and chronic cholestatic liver injury in contrast to kidney injury. Disease progression during chronic cholestasis can be targeted through small molecules and thus suggests a potential clinical benefit for humans, attenuating liver and kidney injury.

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.

One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window.

Reversible biological electron transfer usually occurs between redox couples at standard redox potentials ranging from +0.8 to -0.5 V. Dearomatizing benzoyl-CoA reductases (BCRs), key enzymes of the globally relevant microbial degradation of aromatic compounds at anoxic sites, catalyze a biological Birch reduction beyond the negative limit of this redox window. The structurally characterized BamBC subunits of class II BCRs accomplish benzene ring reduction at an active-site tungsten cofactor; however, the mechanism and components involved in the energetic coupling of endergonic benzene ring reduction have remained hypothetical. We present a 1-MDa, membrane-associated, Bam[(BC)2DEFGHI]2 complex from the anaerobic bacterium Geobacter metallireducens harboring 4 tungsten, 4 zinc, 2 selenocysteines, 6 FAD, and >50 FeS cofactors. The results suggest that class II BCRs catalyze electron transfer to the aromatic ring, yielding a cyclic 1,5-dienoyl-CoA via two flavin-based electron bifurcation events. This work expands our knowledge of energetic couplings in biology by high-molecular-mass electron bifurcating machineries.

Widespread soil bacterium that oxidizes atmospheric methane.

The global atmospheric level of methane (CH4), the second most important greenhouse gas, is currently increasing by ∼10 million tons per year. Microbial oxidation in unsaturated soils is the only known biological process that removes CH4 from the atmosphere, but so far, bacteria that can grow on atmospheric CH4 have eluded all cultivation efforts. In this study, we have isolated a pure culture of a bacterium, strain MG08 that grows on air at atmospheric concentrations of CH4 [1.86 parts per million volume (p.p.m.v.)]. This organism, named Methylocapsa gorgona, is globally distributed in soils and closely related to uncultured members of the upland soil cluster α. CH4 oxidation experiments and 13C-single cell isotope analyses demonstrated that it oxidizes atmospheric CH4 aerobically and assimilates carbon from both CH4 and CO2 Its estimated specific affinity for CH4 (a0s) is the highest for any cultivated methanotroph. However, growth on ambient air was also confirmed for Methylocapsa acidiphila and Methylocapsa aurea, close relatives with a lower specific affinity for CH4, suggesting that the ability to utilize atmospheric CH4 for growth is more widespread than previously believed. The closed genome of M. gorgona MG08 encodes a single particulate methane monooxygenase, the serine cycle for assimilation of carbon from CH4 and CO2, and CO2 fixation via the recently postulated reductive glycine pathway. It also fixes dinitrogen and expresses the genes for a high-affinity hydrogenase and carbon monoxide dehydrogenase, suggesting that atmospheric CH4 oxidizers harvest additional energy from oxidation of the atmospheric trace gases carbon monoxide (0.2 p.p.m.v.) and hydrogen (0.5 p.p.m.v.).

A workflow to identify novel proteins based on the direct mapping of peptide-spectrum-matches to genomic locations.

BACKGROUND: Small Proteins have received increasing attention in recent years. They have in particular been implicated as signals contributing to the coordination of bacterial communities. In genome annotations they are often missing or hidden among large numbers of hypothetical proteins because genome annotation pipelines often exclude short open reading frames or over-predict hypothetical proteins based on simple models. The validation of novel proteins, and in particular of small proteins (sProteins), therefore requires additional evidence. Proteogenomics is considered the gold standard for this purpose. It extends beyond established annotations and includes all possible open reading frames (ORFs) as potential sources of peptides, thus allowing the discovery of novel, unannotated proteins. Typically this results in large numbers of putative novel small proteins fraught with large fractions of false-positive predictions. RESULTS: We observe that number and quality of the peptide-spectrum matches (PSMs) that map to a candidate ORF can be highly informative for the purpose of distinguishing proteins from spurious ORF annotations. We report here on a workflow that aggregates PSM quality information and local context into simple descriptors and reliably separates likely proteins from the large pool of false-positive, i.e., most likely untranslated ORFs. We investigated the artificial gut microbiome model SIHUMIx, comprising eight different species, for which we validate 5114 proteins that have previously been annotated only as hypothetical ORFs. In addition, we identified 37 non-annotated protein candidates for which we found evidence at the proteomic and transcriptomic level. Half (19) of these candidates have close functional homologs in other species. Another 12 candidates have homologs designated as hypothetical proteins in other species. The remaining six candidates are short (< 100 AA) and are most likely bona fide novel proteins. CONCLUSIONS: The aggregation of PSM quality information for predicted ORFs provides a robust and efficient method to identify novel proteins in proteomics data. The workflow is in particular capable of identifying small proteins and frameshift variants. Since PSMs are explicitly mapped to genomic locations, it furthermore facilitates the integration of transcriptomics data and other sources of genome-level information.

Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.