The nonvesicular sterol transporter Aster-C plays a minor role in whole body cholesterol balance.

Introduction: The Aster-C protein (encoded by the Gramd1c gene) is an endoplasmic reticulum (ER) resident protein that has been reported to transport cholesterol from the plasma membrane to the ER. Although there is a clear role for the closely-related Aster-B protein in cholesterol transport and downstream esterification in the adrenal gland, the specific role for Aster-C in cholesterol homeostasis is not well understood. Here, we have examined whole body cholesterol balance in mice globally lacking Aster-C under low or high dietary cholesterol conditions. Method: Age-matched Gramd1c +/+ and Gramd1c -/- mice were fed either low (0.02%, wt/wt) or high (0.2%, wt/wt) dietarycholesterol and levels of sterol-derived metabolites were assessed in the feces, liver, and plasma. Results: Compared to wild type controls (Gramd1c +/+) mice, mice lackingGramd1c (Gramd1c -/-) have no significant alterations in fecal, liver, or plasma cholesterol. Given the potential role for Aster C in modulating cholesterol metabolism in diverse tissues, we quantified levels of cholesterol metabolites such as bile acids, oxysterols, and steroid hormones. Compared to Gramd1c +/+ controls, Gramd1c -/- mice had modestly reduced levels of select bile acid species and elevated cortisol levels, only under low dietary cholesterol conditions. However, the vast majority of bile acids, oxysterols, and steroid hormones were unaltered in Gramd1c -/- mice. Bulk RNA sequencing in the liver showed that Gramd1c -/- mice did not exhibit alterations in sterol-sensitive genes, but instead showed altered expression of genes in major urinary protein and cytochrome P450 (CYP) families only under low dietary cholesterol conditions. Discussion: Collectively, these data indicate nominal effects of Aster-C on whole body cholesterol transport and metabolism under divergent dietary cholesterol conditions. These results strongly suggest that Aster-C alone is not sufficient to control whole body cholesterol balance, but can modestly impact circulating cortisol and bile acid levels when dietary cholesterol is limited.

Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1ß protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

Standards for fecal microbiota transplant: Tools and therapeutic advances.

Fecal microbiota transplantation (FMT) has been demonstrated to be efficacious in preventing recurrent Clostridioides difficile (C. difficile) infections, and is being investigated for treatment of several other diseases including inflammatory bowel disease, cancer, obesity, liver disease, and diabetes. To speed up the translation of FMT into clinical practice as a safe and standardized therapeutic intervention, additional evidence-based technical and regulatory guidance is needed. To this end in May of 2022, the International Alliance for Biological Standardization (IABS) and the BIOASTER Microbiology Technology Institute hosted a second webinar to discuss key issues still impeding the advancement and standardization of FMT. The goal of this two-day webinar was to provide a forum for scientific experts to share and discuss data and key challenges with one another. Discussion included a focus on the evaluation of safety, efficacy, clinical trial design, reproducibility and accuracy in obtained microbiome measurements and data reporting, and the potential for standardization across these areas. It also focused on increasing the application potential and visibility of FMT beyond treating C. difficile infections.

Increased glucose availability sensitizes pancreatic cancer to chemotherapy.

Pancreatic Ductal Adenocarcinoma (PDAC) is highly resistant to chemotherapy. Effective alternative therapies have yet to emerge, as chemotherapy remains the best available systemic treatment. However, the discovery of safe and available adjuncts to enhance chemotherapeutic efficacy can still improve survival outcomes. We show that a hyperglycemic state substantially enhances the efficacy of conventional single- and multi-agent chemotherapy regimens against PDAC. Molecular analyses of tumors exposed to high glucose levels reveal that the expression of GCLC (glutamate-cysteine ligase catalytic subunit), a key component of glutathione biosynthesis, is diminished, which in turn augments oxidative anti-tumor damage by chemotherapy. Inhibition of GCLC phenocopies the suppressive effect of forced hyperglycemia in mouse models of PDAC, while rescuing this pathway mitigates anti-tumor effects observed with chemotherapy and high glucose.

Cancer-associated fibroblast-secreted glucosamine alters the androgen biosynthesis program in prostate cancer via HSD3B1 upregulation.

After androgen deprivation, prostate cancer frequently becomes castration resistant (CRPC), with intratumoral androgen production from extragonadal precursors that activate the androgen receptor pathway. 3ß-Hydroxysteroid dehydrogenase-1 (3ßHSD1) is the rate-limiting enzyme for extragonadal androgen synthesis, which together lead to CRPC. Here, we show that cancer-associated fibroblasts (CAFs) increased epithelial 3ßHSD1 expression, induced androgen synthesis, activated the androgen receptor, and induced CRPC. Unbiased metabolomics revealed that CAF-secreted glucosamine specifically induced 3ßHSD1. CAFs induced higher GlcNAcylation in cancer cells and elevated expression of the transcription factor Elk1, which induced higher 3ßHSD1 expression and activity. Elk1 genetic ablation in cancer epithelial cells suppressed CAF-induced androgen biosynthesis in vivo. In patient samples, multiplex fluorescent imaging showed that tumor cells expressed more 3ßHSD1 and Elk1 in CAF-enriched areas compared with CAF-deficient areas. Our findings suggest that CAF-secreted glucosamine increases GlcNAcylation in prostate cancer cells, promoting Elk1-induced HSD3B1 transcription, which upregulates de novo intratumoral androgen synthesis to overcome castration.

5-Hydroxyeicosatetraenoic Acid Controls Androgen Reduction in Diverse Types of Human Epithelial Cells.

Androgens regulate broad physiologic and pathologic processes, including external genitalia development, prostate cancer progression, and anti-inflammatory effects in both cancer and asthma. In prostate cancer, several lines of evidence have implicated dietary and endogenous fatty acids in cell invasion, angiogenesis, and treatment resistance. However, the role of fatty acids in steroidogenesis and the mechanisms by which alterations in this pathway occur are not well understood. Here, we show that, of a panel of fatty acids tested, arachidonic acid and its specific metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) regulate androgen metabolism. Arachidonic acid is metabolized to 5-HETE and reduces androgens by inducing aldo-keto reductase (AKR) family members AKR1C2 and AKR1C3 expression in human prostate, breast, and lung epithelial cells. Finally, we provide evidence that these effects require the expression of the antioxidant response sensor, nuclear factor erythroid 2-related factor 2 (Nrf2). Our findings identify an interconnection between conventional fatty acid metabolism and steroid metabolism that has broad relevance to androgen physiology and inflammatory regulation.

Crohn's disease in endoscopic remission, obesity, and cases of high genetic risk demonstrates overlapping shifts in the colonic mucosal-luminal interface microbiome.

BACKGROUND: Crohn's disease (CD) patients demonstrate distinct intestinal microbial compositions and metabolic characteristics compared to unaffected controls. However, the impact of inflammation and underlying genetic risk on these microbial profiles and their relationship to disease phenotype are unclear. We used lavage sampling to characterize the colonic mucosal-luminal interface (MLI) microbiome of CD patients in endoscopic remission and unaffected controls relative to obesity, disease genetics, and phenotype. METHODS: Cecum and sigmoid colon were sampled from 110 non-CD controls undergoing screening colonoscopy who were stratified by body mass index and 88 CD patients in endoscopic remission (396 total samples). CD polygenic risk score (GRS) was calculated using 186 known CD variants. MLI pellets were analyzed by 16S ribosomal RNA gene sequencing, and supernatants by untargeted liquid chromatography-mass spectrometry. RESULTS: CD and obesity were each associated with decreased cecal and sigmoid MLI bacterial diversity and distinct bacterial composition compared to controls, including expansion of Escherichia/Shigella. Cecal and sigmoid dysbiosis indices for CD were significantly greater in obese controls than non-overweight controls. CD, but not obesity, was characterized by altered biogeographic relationship between the sigmoid and cecum. GRS was associated with select taxonomic shifts that overlapped with changes seen in CD compared to controls including Fusobacterium enrichment. Stricturing or penetrating Crohn's disease behavior was characterized by lower MLI bacterial diversity and altered composition, including reduced Faecalibacterium, compared to uncomplicated CD. Taxonomic profiles including reduced Parasutterella were associated with clinical disease progression over a mean follow-up of 3.7 years. Random forest classifiers using MLI bacterial abundances could distinguish disease state (area under the curve (AUC) 0.93), stricturing or penetrating Crohn's disease behavior (AUC 0.82), and future clinical disease progression (AUC 0.74). CD patients showed alterations in the MLI metabolome including increased cholate:deoxycholate ratio compared to controls. CONCLUSIONS: Obesity, CD in endoscopic remission, and high CD genetic risk have overlapping colonic mucosal-luminal interface (MLI) microbiome features, suggesting a shared microbiome contribution to CD and obesity which may be influenced by genetic factors. Microbial profiling during endoscopic remission predicted Crohn's disease behavior and progression, supporting that MLI sampling could offer unique insight into CD pathogenesis and provide novel prognostic biomarkers.

Gut microbe-targeted choline trimethylamine lyase inhibition improves obesity via rewiring of host circadian rhythms.

Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here, we show that gut microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (Lepob/ob). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake but is instead associated with alterations in the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that gut microbial CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have potential as anti-obesity therapeutics.

Bridging the Gap between Analytical and Microbial Sciences in Microbiome Research.

Metabolites from the microbiome influence human, animal, and environmental health, but the diversity and functional roles of these compounds have only begun to be elucidated. Comprehensively characterizing these molecules are significant challenges, as it requires expertise in analytical methods, such as mass spectrometry and nuclear magnetic resonance spectroscopy, skills that not many traditional microbiologists or microbial ecologists possess. This creates a gap between microbiome scientists that want to understand the role of microbial metabolites in microbiome systems and the skills required to generate and interpret complex metabolomics data sets. To bridge this gap, microbiome scientists should engage analytical chemists to best understand the underlying chemical principles of the data. Conversely, analytical scientists are encouraged to engage with microbiome scientists to better understand the biological questions being asked with metabolomics and to best communicate its intricacies. Better communication across the chemistry/biology disciplines will further reveal the "dark matter" within microbiomes that maintain healthy humans and environments.

Metabolic Profiling of Skeletal Muscle During Ex-Vivo Normothermic Limb Perfusion.

INTRODUCTION: Ex vivo normothermic limb perfusion (EVNLP) provides several advantages for the preservation of limbs following amputation: the ability to maintain oxygenation and temperature of the limb close to physiological values, a perfusion solution providing all necessary nutrients at optimal concentrations, and the ability to maintain physiological pH and electrolytes. However, EVNLP cannot preserve the organ viability infinitely. We identified evidence of mitochondrial injury (swelling, elongation, and membrane disruption) after 24 hours of EVNLP of human upper extremities. The goal of this study was to identify metabolic derangements in the skeletal muscle during EVNLP. MATERIALS AND METHODS: Fourteen human upper extremities were procured from organ donors after family consent. Seven limbs underwent EVNLP for an average of 41.6 ± 9.4 hours, and seven contralateral limbs were preserved at 4°C for the same amount of time. Muscle biopsies were performed at 24 hours of perfusion, both from the EVNLP and control limbs. Perturbations in the metabolic profiles of the muscle during EVNLP were determined via untargeted liquid chromatography-mass spectrometry (MS) operated in positive and negative electrospray ionization modes, over a mass range of 50 to 750 Da. The data were deconvoluted using the XCMS software and further statistically analyzed using the in-house statistical package, MetaboLyzer. Putative identification of metabolites using exact mass within ±7 ppm mass error and MS/MS spectral matching to the mzCloud spectral library were performed via Compound Discoverer v.2.1 (Thermo Scientific, Fremont, CA, USA). We further validated the identity of candidate metabolites by matching the fragmentation pattern of these metabolites to those of their reference pure chemicals. A nonparametric Mann-Whitney U-test was used to compare EVNLP and control group spectral features. Differences were considered significantly different when P-value < 0.05. RESULTS: We detected over 13,000 spectral features of which 58 met the significance criteria with biologically relevant putative identifications. Furthermore we were able to confirm the identities of the ions taurine (P-value: 0.002) and tryptophan (P-value: 0.002), which were among the most significantly perturbed ions at 24 hours between the experimental and control groups. Metabolites belonging to the following pathways were the most perturbed at 24 hours: neuroactive ligand-receptor interaction (P-values: 0.031 and 0.036) and amino acid metabolism, including tyrosine and tryptophan metabolism (P-values: 0.015, 0.002, and 0.017). Taurine abundance decreased and tryptophan abundance increased at 24 hours. Other metabolites also identified at 24 hours included phenylalanine, xanthosine, and citric acid (P-values: 0.002, 0.002, and 0.0152). DISCUSSION: This study showed presence of active metabolism during EVNLP and metabolic derangement toward the end of perfusion, which correlated with detection of altered mitochondrial structure, swelling, and elongation.

Serum Metabolomic Alterations Associated with Cesium-137 Internal Emitter Delivered in Various Dose Rates.

Our laboratory and others have use radiation metabolomics to assess responses in order to develop biomarkers reflecting exposure and level of injury. To expand the types of exposure and compare to previously published results, metabolomic analysis has been carried out using serum samples from mice exposed to 137Cs internal emitters. Animals were injected intraperitoneally with 137CsCl solutions of varying radioactivity, and the absorbed doses were calculated. To determine the dose rate effect, serum samples were collected at 2, 3, 5, 7, and 14 days after injection. Based on the time for each group receiving the cumulative dose of 4 Gy, the dose rate for each group was determined. The dose rates analyzed were 0.16 Gy/day (low), 0.69 Gy/day (medium), and 1.25 Gy/day (high). The results indicated that at a cumulative dose of 4 Gy, the low dose rate group had the least number of statistically significantly differential spectral features. Some identified metabolites showed common changes for different dose rates. For example, significantly altered levels of oleamide and sphingosine 1-phosphate were seen in all three groups. On the other hand, the intensity of three amino acids, Isoleucine, Phenylalanine and Arginine, significantly decreased only in the medium dose rate group. These findings have the potential to be used in assessing the exposure and the biological effects of internal emitters.

The ABRF Metabolomics Research Group 2016 Exploratory Study: Investigation of Data Analysis Methods for Untargeted Metabolomics.

Lack of standardized applications of bioinformatics and statistical approaches for pre- and postprocessing of global metabolomic profiling data sets collected using high-resolution mass spectrometry platforms remains an inadequately addressed issue in the field. Several publications now recognize that data analysis outcome variability is caused by different data treatment approaches. Yet, there is a lack of interlaboratory reproducibility studies that have looked at the contribution of data analysis techniques toward variability/overlap of results. The goal of our study was to identify the contribution of data pre- and postprocessing methods on metabolomics analysis results. We performed urinary metabolomics from samples obtained from mice exposed to 5 Gray of external beam gamma rays and those exposed to sham irradiation (control group). The data files were made available to study participants for comparative analysis using commonly used bioinformatics and/or biostatistics approaches in their laboratories. The participants were asked to report back the top 50 metabolites/features contributing significantly to the group differences. Herein we describe the outcome of this study which suggests that data preprocessing is critical in defining the outcome of untargeted metabolomic studies.

Disparate Metabolomics Data Reassembler: A Novel Algorithm for Agglomerating Incongruent LC-MS Metabolomics Datasets.

In the past decade, the field of LC-MS-based metabolomics has transformed from an obscure specialty into a major "-omics" platform for studying metabolic processes and biomolecular characterization. However, as a whole the field is still very fractured, as the nature of the instrumentation and the information produced by the platform essentially creates incompatible "islands" of datasets. This lack of data coherency results in the inability to accumulate a critical mass of metabolomics data that has enabled other -omics platforms to make impactful discoveries and meaningful advances. As such, we have developed a novel algorithm, called Disparate Metabolomics Data Reassembler (DIMEDR), which attempts to bridge the inconsistencies between incongruent LC-MS metabolomics datasets of the same biological sample type. A single "primary" dataset is postprocessed via traditional means of peak identification, alignment, and grouping. DIMEDR utilizes this primary dataset as a progenitor template by which data from subsequent disparate datasets are reassembled and integrated into a unified framework that maximizes spectral feature similarity across all samples. This is accomplished by a novel procedure for universal retention time correction and comparison via identification of ubiquitous features in the initial primary dataset, which are subsequently utilized as endogenous internal standards during integration. For demonstration purposes, two human and two mouse urine metabolomics datasets from four unrelated studies acquired over 4 years were unified via DIMEDR, which enabled meaningful analysis across otherwise incomparable and unrelated datasets.

Fabric Phase Sorptive Extraction-A Metabolomic Preprocessing Approach for Ionizing Radiation Exposure Assessment.

The modern application of mass spectrometry-based metabolomics to the field of radiation assessment and biodosimetry has allowed for the development of prompt biomarker screenings for radiation exposure. Our previous work on radiation assessment, in easily accessible biofluids (such as urine, blood, saliva), has revealed unique metabolic perturbations in response to radiation quality, dose, and dose rate. Nevertheless, the employment of swift injury assessment in the case of a radiological disaster still remains a challenge as current sample processing can be time consuming and cause sample degradation. To address these concerns, we report a metabolomics workflow using a mass spectrometry-compatible fabric phase sorptive extraction (FPSE) technique. FPSE employs a matrix coated with sol-gel poly(caprolactone-b-dimethylsiloxane-b-caprolactone) that binds both polar and nonpolar metabolites in whole blood, eliminating serum processing steps. We confirm that the FPSE preparation technique combined with liquid chromatography-mass spectrometry can distinguish radiation exposure markers such as taurine, carnitine, arachidonic acid, α-linolenic acid, and oleic acid found 24 h after 8 Gy irradiation. We also note the effect of different membrane fibers on both metabolite extraction efficiency and the temporal stabilization of metabolites in whole blood at room temperature. These findings suggest that the FPSE approach could work in future technology to triage irradiated individuals accurately, via biomarker screening, by providing a novel method to stabilize biofluids between collection and sample analysis.

Paper-based platform for detection by hybridization using intrinsically labeled fluorescent oligonucleotide probes on quantum dots.

A paper-based platform was investigated in which the selective detection of oligonucleotide targets by hybridization was accomplished via the enhancement of fluorescence emission from intrinsically labeled DNA probes that were immobilized on the surface of quantum dots (QDs). Multiple copies of a derivative of thiazole orange, an intercalating dye known to form non-emissive dimers, were conjugated to single-stranded oligonucleotide probes. Dimerization resulted in the formation of H-aggregates where excitonic interactions led to the suppression of fluorescence. The hybridization of the oligonucleotide probe with a complementary target resulted in the enhancement of fluorescence emission as the dimers dissociated and the dyes preferentially intercalated with the duplex. The detection of oligonucleotide targets using this configuration eliminated the need for labeling the target strands, and fluorescence intensity was proportional to the extent of hybridization. In addition, the dye molecules were excited using Foerster Resonance Energy Transfer (FRET) from QD donors, which resulted in improved selectivity and allowed for ratiometric detection. A solution-phase hybridization assay based on similar operational principles has been previously reported, and this new work investigated the advantages offered for this transduction scheme using paper-based solid-phase substrates. QD-probe conjugates were immobilized in sufficient density on the paper matrix to provide for multiple-donor-multiple-acceptor interactions that resulted in a 20-fold enhancement of acceptor emission compared to the solution-based assay, providing a limit of detection of 0.1 pmol. The paper-based assay provided for the reduction of the time needed for sample preparation and data acquisition, demonstrated that transduction was possible in a complex matrix (goat serum) without compromising on the performance observed in buffer solution, and that oligonucleotides generated from standard PCR amplification could be detected.

Metabolomic alterations associated with Behçet's disease.

BACKGROUND: The diagnosis of Behçet's disease (BD) remains challenging due to the lack of diagnostic biomarkers. This study aims to identify potential serum metabolites associated with BD and its disease activity. METHODS: Medical records and serum samples of 24 pretreated BD patients, 12 post-treated BD patients, and age-matched healthy controls (HC) were collected for metabolomics and lipidomics profiling using UPLC-QTOF-MS and UPLC-QTOF-MSE approaches. Additionally, serum samples from an independent cohort of BD patients, disease controls including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Takayasu's arteritis (TA), Crohn's disease (CD) patients, and HC were collected for further validation of two potential biomarkers using UPLC-QTOFMS analysis. RESULTS: Unsupervised principal component analysis (PCA) showed a clear separation of metabolomics profiles of BD patients from HC. Statistical analysis of the data revealed differential metabolites between BD patients and HC. The serum levels of some phosphatidylcholines (PCs) were found to be significantly lower in BD patients, while the levels of several polyunsaturated fatty acids (PUFAs) were increased markedly in the BD group compared with HC. Furthermore, the serum level of two omega-6 PUFAs, linoleic acid (LA) and arachidonic acid (AA), were dramatically decreased in patients with remission. A validation cohort confirmed that the serum LA and AA levels in BD patients were significantly higher than those in HC and patients with RA, SLE, TA, and CD. In addition, receiver operating characteristic (ROC) analysis indicated good sensitivity and specificity. CONCLUSIONS: The serum metabolomics profiles in BD patients are altered. Serum LA and AA are promising diagnostic biomarkers for BD.

Microbial, metabolomic, and immunologic dynamics in a relapsing genetic mouse model of colitis induced by T-synthase deficiency.

Intestinal dysbiosis is thought to confer susceptibility to inflammatory bowel disease (IBD), but it is unknown whether dynamic changes in the microbiome contribute to fluctuations in disease activity. We explored this question using mice with intestine-specific deletion of C1galt1 (also known as T-synthase) (Tsyn mice). These mice develop spontaneous microbiota-dependent colitis with a remitting/relapsing course due to loss of mucin core-1 derived O-glycans. 16S rRNA sequencing and untargeted metabolomics demonstrated age-specific perturbations in the intestinal microbiome and metabolome of Tsyn mice compare with littermate controls at weeks 3 (disease onset), 5 (during remission), and 9 (after relapse). Colitis remission corresponded to increased levels of FoxP3+RORγt+CD4+ T cells in the colonic lamina propria that were positively correlated with operational taxonomic units (OTUs) in the S24-7 family and negatively correlated with OTUs in the Clostridiales order. Relapse was characterized by marked expansion of FoxP3-RORγt+CD4+ T cells expressing IFNγ and IL17A, which were associated with Clostridiales OTUs distinct from those negatively correlated with FoxP3+RORγt+CD4+ T cells. Our findings suggest that colitis remission and relapse in the Tsyn model may reflect alterations in the microbiome due to reduced core-1 O-glycosylation that shift the balance of regulatory and pro-inflammatory T cell subsets. We investigated whether genetic variation in C1galt1 correlated with the microbiome in a cohort of 78 Crohn's disease patients and 101 healthy controls. Polymorphisms near C1galt1 (rs10486157) and its molecular chaperone, Cosmc (rs4825729), were associated with altered composition of the colonic mucosal microbiota, supporting the relevance of core-1 O-glycosylation to host regulation of the microbiome.

Regulation of Cytochrome P450 2B10 (CYP2B10) Expression in Liver by Peroxisome Proliferator-activated Receptor-ß/δ Modulation of SP1 Promoter Occupancy.

Alcoholic liver disease is a pathological condition caused by overconsumption of alcohol. Because of the high morbidity and mortality associated with this disease, there remains a need to elucidate the molecular mechanisms underlying its etiology and to develop new treatments. Because peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) modulates ethanol-induced hepatic effects, the present study examined alterations in gene expression that may contribute to this disease. Chronic ethanol treatment causes increased hepatic CYP2B10 expression inPparß/δ+/+ mice but not in Pparß/δ-/- mice. Nuclear and cytosolic localization of the constitutive androstane receptor (CAR), a transcription factor known to regulate Cyp2b10 expression, was not different between genotypes. PPARγ co-activator 1α, a co-activator of both CAR and PPARß/δ, was up-regulated in Pparß/δ+/+ liver following ethanol exposure, but not in Pparß/δ-/- liver. Functional mapping of the Cyp2b10 promoter and ChIP assays revealed that PPARß/δ-dependent modulation of SP1 promoter occupancy up-regulated Cyp2b10 expression in response to ethanol. These results suggest that PPARß/δ regulates Cyp2b10 expression indirectly by modulating SP1 and PPARγ co-activator 1α expression and/or activity independent of CAR activity. Ligand activation of PPARß/δ attenuates ethanol-induced Cyp2b10 expression in Pparß/δ+/+ liver but not in Pparß/δ-/- liver. Strikingly, Cyp2b10 suppression by ligand activation of PPARß/δ following ethanol treatment occurred in hepatocytes and was mediated by paracrine signaling from Kupffer cells. Combined, results from the present study demonstrate a novel regulatory role of PPARß/δ in modulating CYP2B10 that may contribute to the etiology of alcoholic liver disease.

The structure, kinetics and interactions of the ß-carboxysomal ß-carbonic anhydrase, CcaA.

CcaA is a ß-carbonic anhydrase (CA) that is a component of the carboxysomes of a subset of ß-cyanobacteria. This protein, which has a characteristic C-terminal extension of unknown function, is recruited to the carboxysome via interactions with CcmM, which is itself a γ-CA homolog with enzymatic activity in many, but not all cyanobacteria. We have determined the structure of CcaA from Synechocystis sp. PCC 6803 at 1.45 Å. In contrast with the dimer-of-dimers organization of most bacterial ß-CAs, or the loose dimer-of-dimers-of-dimers organization found in the plant enzymes, CcaA shows a well-packed trimer-of-dimers organization. The proximal part of the characteristic C-terminal extension is ordered by binding at a site that passes through the two-fold symmetry axis shared with an adjacent dimer; as a result, only one of a pair of converging termini can be ordered at any given time. Docking in Rosetta failed to find well-packed solutions, indicating that formation of the CcaA/CcmM complex probably requires significant backbone movements in at least one of the binding partners. Surface plasmon resonance experiments showed that CcaA forms a complex with CcmM with sub-picomolar affinity, with contributions from residues in CcmM's αA helix and CcaA's C-terminal tail. Catalytic characterization showed CcaA to be among the least active ß-CAs characterized to date, with activity comparable with the γ-CA, CcmM, it either complements or replaces. Intriguingly, the C-terminal tail appears to partly inhibit activity, possibly indicating a role in minimizing the activity of unencapsulated enzyme.