Short Communication: Higher abundance of 2-dehydro-d-gluconate in the plasma of sub-fertile or infertile Bos taurus heifers.

Infertility or subfertility impacts approximately 5% and 15% of dairy and beef heifers (Bos taurus), respectively. Heifers that do not produce a calf within an optimum window of time have a significant negative impact on the profitability and sustainability of the cattle industry. Selection of heifers based on their fertility potential remains a challenge yet to be resolved. Here, we tested the hypothesis that heifers of different fertility potential have differing metabolome signatures in their plasma. We obtained blood from Bos taurus heifers at their first artificial insemination and processed the samples to separate the plasma. The heifers were classified based on their reproductive outcome as fertile (pregnant and delivered a calf after their first artificial insemination (AI)) or sub-fertile (Angus heifers: no pregnancy after two AI and exposure to a bull; Holstein heifers: no pregnancy by the third AI). We tested the relative abundance of 140 metabolites obtained from 22 heifers (Angus fertile n = 5, Angus sub-fertile n = 7, Holstein fertile N = 5, Holstein sub-fertile N = 5). The metabolite 2-Dehydro-D-gluconate (C6H10O7) was significantly more abundant in the plasma of sub-fertile heifers in both breeds (1.4-fold, false discovery rate <0.1). In the context that a small proportion of circulating metabolites in the plasma were quantified in this study, the results show that the metabolomic profile in the blood stream may be associated with heifer fertility potential.

Channel Expansion in the Ligand-Binding Domain of the Glucocorticoid Receptor Contributes to the Activity of Highly Potent Glucocorticoid Analogues.

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR) and are commonly used as anti-inflammatory and immunosuppressant medications. Chronic GC use has been linked with unwanted complications such as steroid-induced diabetes mellitus (SIDM), although the mechanisms for these effects are not completely understood. Modification of six GC parent molecules with 2-mercaptobenzothiazole resulted in consistently less promoter activity in transcriptional activation assays using a 3xGRE reporter construct while constantly reducing inflammatory pathway activity. The most selective candidate, DX1, demonstrated a significant reduction (87%) in transactivation compared to commercially available dexamethasone. DX1 also maintained 90% of the anti-inflammatory potential of dexamethasone while simultaneously displaying a reduced toxicity profile. Additionally, two novel and highly potent compounds, DX4 and PN4, were developed and shown to elicit similar mRNA expression at attomolar concentrations that dexamethasone exhibits at nanomolar dosages. To further explain these results, Molecular Dynamic (MD) simulations were performed to examine structural changes in the ligand-binding domain of the glucocorticoid receptor in response to docking with the top ligands. Differing interactions with the transcriptional activation function 2 (AF-2) region of the GR may be responsible for lower transactivation capacity in DX1. DX4 and PN4 lose contact with Arg611 due to a key interaction changing from a stronger hydrophilic to a weaker hydrophobic one, which leads to the formation of an unoccupied channel at the location of the deacylcortivazol (DAC)-expanded binding pocket. These findings provide insights into the structure-function relationships important for regulating anti-inflammatory activity, which has implications for clinical utility.

Pharmacological inhibition of lipolysis prevents adverse metabolic outcomes during glucocorticoid administration.

OBJECTIVE: Glucocorticoids are one of the most commonly prescribed classes of anti-inflammatory drugs; however, chronic treatment promotes iatrogenic (drug-induced) diabetes. As part of their physiological role, glucocorticoids stimulate lipolysis to spare glucose. We hypothesized that persistent stimulation of lipolysis during glucocorticoid therapy plays a causative role in the development of iatrogenic diabetes. METHODS: Male C57BL/6J mice were given 100 µg/mL corticosterone (Cort) in the drinking water for two weeks and were fed either normal chow (TekLad 8640) or the same diet supplemented with an adipose triglyceride lipase inhibitor (Atglistatin - 2  g/kg diet) to inhibit the first step of lipolysis. RESULTS: Herein, we report for the first time that glucocorticoid administration promotes a unique state of substrate excess and energetic overload in skeletal muscle that primarily results from the rampant mobilization of endogenous fuels. Inhibiting lipolysis protected mice from Cort-induced gains in fat mass, excess ectopic lipid accrual, hyperinsulinemia, and hyperglycemia. The role lipolysis plays in Cort-mediated pathology appears to differ between tissues. Within skeletal muscle, Cort-induced lipolysis facilitated diversion of glucose-derived carbons toward the pentose phosphate and hexosamine biosynthesis pathways but contributed to <3% of the Cort-induced genomic adaptations. In contrast, Cort stimulation of lipolysis accounted for ∼35% of the genomic changes in the liver but had minimal impact on hepatic metabolites reported. CONCLUSIONS: These data support the idea that activation of lipolysis plays a causal role in the progression toward iatrogenic diabetes during glucocorticoid therapy with differential impact on skeletal muscle and liver.

Bone Metabolite Profile Differs between Normal and Femur Head Necrosis (FHN/BCO)-Affected Broilers: Implications for Dysregulated Metabolic Cascades in FHN Pathophysiology.

Femur head necrosis (FHN), also known as bacterial chondronecrosis with osteomyelitis (BCO), has remained an animal welfare and production concern for modern broilers regardless of efforts to select against it in primary breeder flocks. Characterized by the bacterial infection of weak bone, FHN has been found in birds without clinical lameness and remains only detectable via necropsy. This presents an opportunity to utilize untargeted metabolomics to elucidate potential non-invasive biomarkers and key causative pathways involved in FHN pathology. The current study used ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS) and identified a total of 152 metabolites. Mean intensity differences at p < 0.05 were found in 44 metabolites, with 3 significantly down-regulated and 41 up-regulated in FHN-affected bone. Multivariate analysis and a partial least squares discriminant analysis (PLS-DA) scores plot showed the distinct clustering of metabolite profiles from FHN-affected vs. normal bone. Biologically related molecular networks were predicted using an ingenuity pathway analysis (IPA) knowledge base. Using a fold-change cut off of -1.5 and 1.5, top canonical pathways, networks, diseases, molecular functions, and upstream regulators were generated using the 44 differentially abundant metabolites. The results showed the metabolites NAD+, NADP+, and NADH to be downregulated, while 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and histamine were significantly increased in FHN. Ascorbate recycling and purine nucleotides degradation were the top canonical pathways, indicating the potential dysregulation of redox homeostasis and osteogenesis. Lipid metabolism and cellular growth and proliferation were some of the top molecular functions predicted based on the metabolite profile in FHN-affected bone. Network analysis showed significant overlap across metabolites and predicted upstream and downstream complexes, including AMP-activated protein kinase (AMPK), insulin, collagen type IV, mitochondrial complex, c-Jun N-terminal kinase (Jnk), extracellular signal-regulated kinase (ERK), and 3ß-hydroxysteroid dehydrogenase (3ß HSD). The qPCR analysis of relevant factors showed a significant decrease in AMPKα2 mRNA expression in FHN-affected bone, supporting the predicted downregulation found in the IPA network analysis. Taken as a whole, these results demonstrate a shift in energy production, bone homeostasis, and bone cell differentiation that is distinct in FHN-affected bone, with implications for how metabolites drive the pathology of FHN.

Renal histaminergic system and acute effects of histamine receptor 2 blockade on renal damage in the Dahl salt-sensitive rat.

Histamine is involved in the regulation of immune response, vasodilation, neurotransmission, and gastric acid secretion. Although elevated histamine levels and increased expression of histamine metabolizing enzymes have been reported in renal disease, there is a gap in knowledge regarding the mechanisms of histamine-related pathways in the kidney. We report here that all four histamine receptors as well as enzymes responsible for the metabolism of histamine are expressed in human and rat kidney tissues. In this study, we hypothesized that the histaminergic system plays a role in salt-induced kidney damage in the Dahl salt-sensitive (DSS) rat, a model characterized with inflammation-driven renal lesions. To induce renal damage related to salt sensitivity, DSS rats were challenged with 21 days of a high-salt diet (4% NaCl); normal-salt diet (0.4% NaCl)-fed rats were used as a control. We observed lower histamine decarboxylase and higher histamine N-methyltransferase levels in high-salt diet-fed rats, indicative of a shift in histaminergic tone; metabolomics showed higher histamine and histidine levels in the kidneys of high-salt diet-fed rats, whereas plasma levels for both compounds were lower. Acute systemic inhibition of histamine receptor 2 in the DSS rat revealed that it lowered vasopressin receptor 2 in the kidney. In summary, we established here the existence of the local histaminergic system, revealed a shift in the renal histamine balance during salt-induced kidney damage, and provided evidence that blockage of histamine receptor 2 in the DSS rat affects water balance and urine concentrating mechanisms.NEW & NOTEWORTHY Histamine is a nitrogenous compound crucial for the inflammatory response. The knowledge regarding the renal effects of histamine is very limited. We showed that renal epithelia exhibit expression of the components of the histaminergic system. Furthermore, we revealed that there was a shift in the histaminergic tone in salt-sensitive rats when they were challenged with a high-salt diet. These data support the notion that histamine plays a role in renal epithelial physiological and pathophysiological functions.

Dual stable isotopes enhance lipidomic studies in bacterial model organism Enterococcus faecalis.

Dual stable isotope probes of deuterium oxide and 13C fatty acid were demonstrated to probe the lipid biosynthesis cycle of a Gram-positive bacterium Enterococcus faecalis. As external nutrients and carbon sources often interact with metabolic processes, the use of dual-labeled isotope pools allowed for the simultaneous investigation of both exogenous nutrient incorporation or modification and de novo biosynthesis. Deuterium was utilized to trace de novo fatty acid biosynthesis through solvent-mediated proton transfer during elongation of the carbon chain while 13C-fatty acids were utilized to trace exogenous nutrient metabolism and modification through lipid synthesis. Ultra-high-performance liquid chromatography high-resolution mass spectrometry identified 30 lipid species which incorporated deuterium and/or 13C fatty acid into the membrane. Additionally, MS2 fragments of isolated lipids identified acyl tail position confirming enzymatic activity of PlsY in the incorporation of the 13C fatty acid into membrane lipids.

Metabolite abundance in bovine preovulatory follicular fluid is influenced by follicle developmental progression post estrous onset in cattle.

Introduction: Preovulatory follicle response to the luteinizing hormone (LH) surge leads to metabolic, molecular, and functional changes in the oocyte and somatic follicular cells from the onset of estrus to ovulation. Follicular fluid contains metabolites, miRNAs, proteins, and hormones that are byproducts of follicular metabolism and support cellular processes of oocyte, cumulus, and granulosa constituents. Numerous studies have highlighted the importance of follicular fluid composition to support fertility, but critical gaps exist toward understanding dynamic modifications in the follicular fluid metabolome from estrous onset to ovulation. The hypothesis was that abundance of follicular fluid metabolites is dependent on follicle progression post LH surge and variability in follicular fluid metabolome profiles indicate key processes required for preparation of the follicle and oocyte for optimal fertility. The objective was to generate preovulatory follicular fluid metabolome profiles and discern differences in the metabolome of preovulatory follicular fluid samples collected at onset of estrus, 11 h post estrous onset, and 18 h post estrous onset. Methods: Estrus was synchronized in non-lactating Jersey cows (n=40) and follicular fluid was collected immediately after the first observed standing mount (hr 0) or at approximately h 11 or 18 after the first standing mount. Ultra-High-Performance Liquid Chromatography-High Resolution Mass Spectrometry was performed on preovulatory follicular fluid samples (n = 9 collected at hr 0, 9 at h 11, and 10 at h 18) and a multiple linear model was performed to determine if time post estrous onset impacted metabolite abundance. Results: Metabolites influenced by time post estrous onset were tested for enrichment in KEGG pathways. Ninety metabolites were identified in follicular fluid samples. Twenty metabolites differed in abundance among timepoints post estrous onset (p ≤ 0.05). Pathways corresponding to amino acid and energy metabolism were enriched with metabolites impacted by time post estrous onset (FDR ≤ 0.10). Discussion: Results from the current study indicate early response to the LH surge to increase bioavailability of amino acids and metabolites used by the cumulus and granulosa cells for energy production and shuttled into the oocyte to support meiotic maturation. Such metabolites may later be used by the ovulatory follicle for protein production.

Reduction of Plasma BCAAs following Roux-en-Y Gastric Bypass Surgery Is Primarily Mediated by FGF21.

Type 2 diabetes (T2D) is a challenging health concern worldwide. A lifestyle intervention to treat T2D is difficult to adhere, and the effectiveness of approved medications such as metformin, thiazolidinediones (TZDs), and sulfonylureas are suboptimal. On the other hand, bariatric procedures such as Roux-en-Y gastric bypass (RYGB) are being recognized for their remarkable ability to achieve diabetes remission, although the underlying mechanism is not clear. Recent evidence points to branched-chain amino acids (BCAAs) as a potential contributor to glucose impairment and insulin resistance. RYGB has been shown to effectively lower plasma BCAAs in insulin-resistant or T2D patients that may help improve glycemic control, but the underlying mechanism for BCAA reduction is not understood. Hence, we attempted to explore the mechanism by which RYGB reduces BCAAs. To this end, we randomized diet-induced obese (DIO) mice into three groups that underwent either sham or RYGB surgery or food restriction to match the weight of RYGB mice. We also included regular chow-diet-fed healthy mice as an additional control group. Here, we show that compared to sham surgery, RYGB in DIO mice markedly lowered serum BCAAs most likely by rescuing BCAA breakdown in both liver and white adipose tissues. Importantly, the restored BCAA metabolism following RYGB was independent of caloric intake. Fasting insulin and HOMA-IR were decreased as expected, and serum valine was strongly associated with insulin resistance. While gut hormones such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are postulated to mediate various surgery-induced metabolic benefits, mice lacking these hormonal signals (GLP-1R/Y2R double KO) were still able to effectively lower plasma BCAAs and improve glucose tolerance, similar to mice with intact GLP-1 and PYY signaling. On the other hand, mice deficient in fibroblast growth factor 21 (FGF21), another candidate hormone implicated in enhanced glucoregulatory action following RYGB, failed to decrease plasma BCAAs and normalize hepatic BCAA degradation following surgery. This is the first study using an animal model to successfully recapitulate the RYGB-led reduction of circulating BCAAs observed in humans. Our findings unmasked a critical role of FGF21 in mediating the rescue of BCAA metabolism following surgery. It would be interesting to explore the possibility of whether RYGB-induced improvement in glucose homeostasis is partly through decreased BCAAs.

Microbial survival mechanisms within serpentinizing Mariana forearc sediments.

Marine deep subsurface sediment is often a microbial environment under energy-limited conditions. However, microbial life has been found to persist and even thrive in deep subsurface environments. The Mariana forearc represents an ideal location for determining how microbial life can withstand extreme conditions including pH 10-12.5 and depleted nutrients. The International Ocean Discovery Program Expedition 366 to the Mariana Convergent Margin sampled three serpentinizing seamounts located along the Mariana forearc chain with elevated concentrations of methane, hydrogen, and sulfide. Across all three seamount summits, the most abundant transcripts were for cellular maintenance such as cell wall and membrane repair, and the most abundant metabolic pathways were the Entner-Doudoroff pathway and tricarboxylic acid cycle. At flank samples, sulfur cycling involving taurine assimilation dominated the metatranscriptomes. The in situ activity of these pathways was supported by the detection of their metabolic intermediates. All samples had transcripts from all three domains of Bacteria, Archaea, and Eukarya, dominated by Burkholderiales, Deinococcales, and Pseudomonales, as well as the fungal group Opisthokonta. All samples contained transcripts for aerobic methane oxidation (pmoABC) and denitrification (nirKS). The Mariana forearc microbial communities show activity not only consistent with basic survival mechanisms, but also coupled metabolic reactions.

Metabolome patterns identify active dechlorination in bioaugmentation consortium SDC-9™.

Ultra-high performance liquid chromatography-high-resolution mass spectrometry (UPHLC-HRMS) is used to discover and monitor single or sets of biomarkers informing about metabolic processes of interest. The technique can detect 1000's of molecules (i.e., metabolites) in a single instrument run and provide a measurement of the global metabolome, which could be a fingerprint of activity. Despite the power of this approach, technical challenges have hindered the effective use of metabolomics to interrogate microbial communities implicated in the removal of priority contaminants. Herein, our efforts to circumvent these challenges and apply this emerging systems biology technique to microbiomes relevant for contaminant biodegradation will be discussed. Chlorinated ethenes impact many contaminated sites, and detoxification can be achieved by organohalide-respiring bacteria, a process currently assessed by quantitative gene-centric tools (e.g., quantitative PCR). This laboratory study monitored the metabolome of the SDC-9™ bioaugmentation consortium during cis-1,2-dichloroethene (cDCE) conversion to vinyl chloride (VC) and nontoxic ethene. Untargeted metabolomics using an UHPLC-Orbitrap mass spectrometer and performed on SDC-9™ cultures at different stages of the reductive dechlorination process detected ~10,000 spectral features per sample arising from water-soluble molecules with both known and unknown structures. Multivariate statistical techniques including partial least squares-discriminate analysis (PLSDA) identified patterns of measurable spectral features (peak patterns) that correlated with dechlorination (in)activity, and ANOVA analyses identified 18 potential biomarkers for this process. Statistical clustering of samples with these 18 features identified dechlorination activity more reliably than clustering of samples based only on chlorinated ethene concentration and Dhc 16S rRNA gene abundance data, highlighting the potential value of metabolomic workflows as an innovative site assessment and bioremediation monitoring tool.

Metabolomic Profiles in Starved Light Breed Horses during the Refeeding Process.

The large population of emaciated horses continues to be an issue troubling the equine industry. However, little is known regarding the collection of equine metabolites (metabolome) during a malnourished state and the changes that occur throughout nutritional rehabilitation. In this study, ten emaciated horses underwent a refeeding process, during which blood samples were collected for a blood chemistry panel and metabolomics analysis via ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Significant differences among blood chemistry analytes and metabolite abundance during the critical care period (CCP; Days 1-10 of rehabilitation) and the recovery period (RP; the remainder of the rehabilitation process) were observed. Potentially toxic compounds, analytes related to liver, kidney, and muscle function, as well as energy-related metabolites were altered during the refeeding process. The combination of blood chemistry and metabolomics analyses on starved equine during rehabilitation provide vital biological insight and evidence that the refeeding process has a significant impact on the equine metabolome.

Body Mass Index (BMI) Impacts Soil Chemical and Microbial Response to Human Decomposition.

Microorganisms are key decomposers of vertebrate mortalities, breaking down body tissues and impacting decomposition progress. During human decomposition, both extrinsic environmental factors and intrinsic cadaver-related factors have the potential to impact microbial decomposers either directly or indirectly via altered physical or chemical conditions. While extrinsic factors (e.g., temperature, humidity) explain some variation in microbial response during human decomposition in terrestrial settings, recent work has noted that even under the same environmental conditions, individuals can have different decomposition patterns, highlighting the potential for intrinsic factors to impact microbial decomposers. The goal of this study was to investigate the effects of several intrinsic factors (age, sex, diseases at time of death, and body mass index [BMI]) on chemical and microbial changes in decomposition-impacted soils. In a field study conducted at the University of Tennessee Anthropology Research Facility, soils were collected from the decomposition-impacted area surrounding 19 deceased human individuals through the end of active decomposition. Soil physicochemical parameters were measured, and microbial (bacterial and fungal) communities were assessed via amplicon sequencing. BMI was shown to explain some variation in soil pH and microbial response to human decomposition. Hierarchical linear mixed (HLM) effects models revealed that BMI category significantly explained variation in pH response within decomposition-impacted soils over time (HLM F = 9.647; P < 0.001). Additionally, the relative abundance of soil Saccharomycetes in decomposition soils under underweight donors displayed little to no changes (mean maximum change in relative abundance, +6.6%), while all other BMI categories displayed an increased relative abundance of these organisms over time (normal, +50.6%; overweight, +64.4%; and obese, +64.6%) (HLM F = 3.441; P = 0.11). Together, these results reveal intrinsic factors influencing decomposition patterns, especially within the soil environment, and suggest BMI is an important factor for controlling decomposition processes. IMPORTANCE This work begins to address questions about interindividual variation in vertebrate decomposition attributed to intrinsic factors, that is, properties of the carcass or cadaver itself. Most research on factors affecting decomposition has focused on the extrinsic environment, such as temperature or humidity. While these extrinsic factors do explain some variation in decomposition patterns, interindividual variability is still observed. Understanding how intrinsic factors influence microbial decomposers will help reveal the ecological impacts of decomposition. This work also has forensic applications, as soil chemical and biological changes have been suggested as indicators of postmortem interval. We reveal factors that explain variation in the decomposition environment that should be considered in these estimates. This is particularly important as we consider the implications of variations in human populations due to diet, age, BMI, disease, toxicological loading, etc. on forensic investigations dealing with decomposing remains.

Stereoisomers of an Aryl Pyrazole Glucocorticoid Receptor Agonist Scaffold Elicit Differing Anti-inflammatory Responses.

Glucocorticoids (GCs) are heavily prescribed to control inflammation in various human diseases; however, side effects associated with GCs are well documented and lead to serious metabolic and immunological complications with long-term use. The paradigm for GC function includes two well described modes of activity: dimer formation of the glucocorticoid receptor (GR) promotes transactivation, while monomeric interaction with co-regulators promotes transrepression. Previously, a set of aryl pyrazole-derived glucocorticoid receptor agonists (APGRAs) with potency rivaling current commercially available glucocorticoids were described. In this study, a further series of existing and novel stereopure APGRAs were thoroughly examined for biological activity and evaluated for structure-activity relationships (SARs). The si isomers with an upward OH moiety were ∼70% more active on average than the re isomers. Additionally, AP13 was found to elicit 79% transrepression of dexamethasone while eliciting less than half the transactivation response in 832/13 cells, a rat insulinoma cell line.

Growth Substrate and Prophage Induction Collectively Influence Metabolite and Lipid Profiles in a Marine Bacterium.

Bacterial growth substrates influence a variety of biological functions, including the biosynthesis and regulation of lipid intermediates. The extent of this rewiring is not well understood nor has it been considered in the context of virally infected cells. Here, we used a one-host-two-temperate phage model system to probe the combined influence of growth substrate and phage infection on host carbon and lipid metabolism. Using untargeted metabolomics and lipidomics, we reported the detection of a suite of metabolites and lipid classes for two Sulfitobacter lysogens provided with three growth substrates of differing complexity and nutrient composition (yeast extract/tryptone [complex], glutamate and acetate). The growth medium led to dramatic differences in the detectable intracellular metabolites, with only 15% of 175 measured metabolites showing overlap across the three growth substrates. Between-strain differences were most evident in the cultures grown on acetate, followed by glutamate then complex medium. Lipid distribution profiles were also distinct between cultures grown on different substrates as well as between the two lysogens grown in the same medium. Five phospholipids, three aminolipid, and one class of unknown lipid-like features were identified. Most (≥94%) of these 75 lipids were quantifiable in all samples. Metabolite and lipid profiles were strongly determined by growth medium composition and modestly by strain type. Because fluctuations in availability and form of carbon substrates and nutrients, as well as virus pressure, are common features of natural systems, the influence of these intersecting factors will undoubtedly be imprinted in the metabolome and lipidome of resident bacteria. IMPORTANCE Community-level metabolomics approaches are increasingly used to characterize natural microbial populations. These approaches typically depend upon temporal snapshots from which the status and function of communities are often inferred. Such inferences are typically drawn from lab-based studies of select model organisms raised under limited growth conditions. To better interpret community-level data, the extent to which ecologically relevant bacteria demonstrate metabolic flexibility requires elucidation. Herein, we used an environmentally relevant model heterotrophic marine bacterium to assess the relationship between growth determinants and metabolome. We also aimed to assess the contribution of phage activity to the host metabolome. Striking differences in primary metabolite and lipid profiles appeared to be driven primarily by growth regime and, secondarily, by phage type. These findings demonstrated the malleable nature of metabolomes and lipidomes and lay the foundation for future studies that relate cellular composition with function in complex environmental microbial communities.

Pseudomonas sp. Strain 273 Incorporates Organofluorine into the Lipid Bilayer during Growth with Fluorinated Alkanes.

Anthropogenic organofluorine compounds are recalcitrant, globally distributed, and a human health concern. Although rare, natural processes synthesize fluorinated compounds, and some bacteria have evolved mechanisms to metabolize organofluorine compounds. Pseudomonas sp. strain 273 grows with 1-fluorodecane (FD) and 1,10-difluorodecane (DFD) as carbon sources, but inorganic fluoride release was not stoichiometric. Metabolome studies revealed that this bacterium produces fluorinated anabolites and phospholipids. Mass spectrometric fatty acid profiling detected fluorinated long-chain (i.e., C12-C19) fatty acids in strain 273 cells grown with FD or DFD, and lipidomic profiling determined that 7.5 ± 0.2 and 82.0 ± 1.0% of the total phospholipids in strain 273 grown with FD or DFD, respectively, were fluorinated. The detection of the fluorinated metabolites and macromolecules represents a heretofore unrecognized sink for organofluorine, an observation with consequences for the environmental fate and transport of fluorinated aliphatic compounds.

Preovulatory serum estradiol concentration is positively associated with oocyte ATP and follicular fluid metabolite abundance in lactating beef cattle.

Cattle induced to ovulate a small, physiologically immature preovulatory follicle had reduced oocyte developmental competence that resulted in decreased embryo cleavage and day 7 embryo quality compared with animals induced to ovulate a more advanced follicle. RNA-sequencing was performed on oocytes and their corresponding cumulus cells approximately 23 h after gonadotropin-releasing hormone (GnRH) administration to induce the preovulatory gonadotropin surge suggested reduced capacity for glucose metabolism and oxidative phosphorylation in the cumulus cells and oocytes from follicles ≤11.7 mm, respectively. We hypothesized that induced ovulation of a small, physiologically immature preovulatory follicle results in a suboptimal follicular microenvironment and reduced oocyte metabolic capacity. We performed a study with the objective to determine the impact of preovulatory follicle diameter and serum estradiol concentration at GnRH administration on oocyte metabolic competence and follicular fluid metabolome profiles. We synchronized the development of a preovulatory follicle and collected the follicle contents via transvaginal aspiration approximately 19 h after GnRH administration in lactating beef cows (n = 319). We determined ATP levels and mitochondrial DNA (mtDNA) copy number in 110 oocytes and performed ultra-high-performance liquid chromatography-high resolution mass spectrometry metabolomic studies on 45 follicular fluid samples. Intraoocyte ATP and the amount of ATP produced per mtDNA copy number were associated with serum estradiol concentration at GnRH and time from GnRH administration to follicle aspiration (P < 0.05). mtDNA copy number was not related to follicle diameter at GnRH, serum estradiol concentration at GnRH, or any potential covariates (P > 0.10). We detected 90 metabolites in the aspirated follicular fluid. We identified 22 metabolites associated with serum estradiol concentration at GnRH and 63 metabolites associated with follicular fluid progesterone concentration at the time of follicle aspiration (FDR < 0.10). Pathway enrichment analysis of significant metabolites suggested altered proteinogenesis, citric acid cycle, and pyrimidine metabolism in follicles of reduced estrogenic capacity pre-gonadotropin surge or reduced progesterone production by the time of follicle aspiration.

Incorporation of a fixed-time artificial insemination protocol results in improved reproductive management and genetics of the beef herd. However, a subset of animals exposed to such protocols will not display estrus prior to insemination. Behavioral estrus is indicative of the preovulatory follicle's physiological maturity and is essential for both the production of an oocyte with optimal developmental competence and preparation of the maternal environment for pregnancy establishment. Animals that do not display estrus prior to insemination and are induced to ovulate a physiologically less advanced follicle have reduced oocyte developmental competence that leads to reduced embryo cleavage rates, embryo quality, and pregnancy rates. This study investigated the impacts of reduced follicle maturity at the initiation of ovulation on the energy production capacity of the oocyte as well as follicular fluid metabolic composition. Results from this study demonstrated that follicle maturity, indicated by increased serum estradiol concentration at the initiation of ovulation, resulted in increased ATP within the oocyte as well as an increased level of metabolites involved in glucose metabolism in the follicular fluid. Increased energy production ability in the oocytes from more mature follicles could contribute to the increased cleavage rates and embryo quality seen in previous studies.

Duodenal Metabolic Profile Changes in Heat-Stressed Broilers.

Heat stress (HS) is devastating to poultry production sustainability worldwide. In addition to its adverse effects on growth, welfare, meat quality, and mortality, HS alters the gut integrity, leading to dysbiosis and leaky gut syndrome; however, the underlying mechanisms are not fully defined. Here, we used a high-throughput mass spectrometric metabolomics approach to probe the metabolite profile in the duodenum of modern broilers exposed to acute (AHS, 2 h) or chronic cyclic (CHS, 8 h/day for 2 weeks) HS in comparison with thermoneutral (TN) and pair-fed birds. Ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) identified a total of 178 known metabolites. The trajectory analysis of the principal component analysis (PCA) score plots (both 2D and 3D maps) showed clear separation between TN and each treated group, indicating a unique duodenal metabolite profile in HS birds. Within the HS groups, partial least squares discriminant analysis (PLS-DA) displayed different clusters when comparing metabolite profiles from AHS and CHS birds, suggesting that the metabolite signatures were also dependent on HS duration. To gain biologically related molecule networks, the above identified duodenal metabolites were mapped into the Ingenuity Pathway Analysis (IPA) knowledge-base and analyzed to outline the most enriched biological functions. Several common and specific top canonical pathways were generated. Specifically, the adenosine nucleotide degradation and dopamine degradation pathways were specific for the AHS group; however, the UDP-D-xylose and UDP-D-glucuronate biosynthesis pathways were generated only for the CHS group. The top diseases enriched by the IPA core analysis for the DA metabolites, including cancer, organismal (GI) injury, hematological, cardiovascular, developmental, hereditary, and neurological disorders, were group-specific. The top altered molecular and cellular functions were amino acid metabolism, molecular transport, small molecule biochemistry, protein synthesis, cell death and survival, and DNA damage and repair. The IPA-causal network predicted that the upstream regulators (carnitine palmitoyltransferase 1B, CPT1B; histone deacetylase 11, HDAC11; carbonic anhydrase 9, CA9; interleukin 37, IL37; glycine N-methyl transferase, GNMT; GATA4) and the downstream mediators (mitogen-activated protein kinases, MAPKs; superoxide dismutase, SOD) were altered in the HS groups. Taken together, these data showed that, independently of feed intake depression, HS induced significant changes in the duodenal metabolite profile in a duration-dependent manner and identified a potential duodenal signature for HS.

Preovulatory follicular fluid and serum metabolome profiles in lactating beef cows with thin, moderate, and obese body condition.

Extremes in body condition reduce fertility and overall productivity in beef cattle herds, due in part to altered systemic metabolic conditions that influence the intrafollicular and uterine environment. Follicular fluid and serum metabolome profiles are influenced by body composition in women and dairy cattle; however, such information is lacking in beef cattle. We hypothesized that body condition score (BCS)-related alterations in the metabolome of preovulatory follicular fluid and serum may influence oocyte maturation while impacting the oviductal or uterine environment. Therefore, we performed a study with the objective to determine the relationship between BCS and the metabolome of follicular fluid and serum in lactating beef cattle. We synchronized the development of a preovulatory follicle in 130 cows of varying BCS. We collected blood and performed transvaginal follicle aspirations to collect follicular fluid from the preovulatory follicle ~18 h after gonadotropin-releasing hormone administration to stimulate the preovulatory gonadotropin surge. We then selected follicular fluid and serum samples from cows with BCS 4 (Thin; n = 14), BCS 6 (Moderate; n = 18), or BCS >8 (Obese; n = 14) for ultra-high performance liquid chromatography-high resolution mass spectrometry. We identified differences in the follicular fluid or serum of thin, moderate, and obese animals based on multiple linear regression. MetaboAnalyst 5.0 was used for enrichment analysis of significant metabolites. We identified 38 metabolites in follicular fluid and 49 metabolites in serum. There were no significant differences in follicular fluid metabolite content among BCS classifications. There were 5, 22, and 1 serum metabolites differentially abundant between thin-obese, moderate-thin, and moderate-obese classifications, respectively (false discovery rate [FDR] < 0.10). These metabolites were enriched in multiple processes including "arginine biosynthesis," "arginine/proline metabolism," and "D-glutamine/D-glutamate metabolism" (FDR < 0.04). Pathways enriched with serum metabolites associated with BCS indicate potentially increased reactive oxygen species (ROS) in serum of thin cows. ROS crossing the blood follicular barrier may negatively impact the oocyte during oocyte maturation and contribute to the reduced pregnancy rates observed in thin beef cows.

Extremes in body condition affect fertility and pregnancy outcomes in beef cows. Much research has been done in women and dairy cows to evaluate body condition's effect on oocyte and embryo quality, pregnancy rates, and pregnancy outcomes. However, little work of this type has been done in beef cows and most studies do not focus on the preovulatory time period. The preovulatory time period is an essential time for the oocyte, as final stages of prematuration and the completion of oocyte maturation take place in the peri-ovulatory follicle. The follicular fluid provides the microenvironment for oocyte maturation and exchanges substances with maternal circulation at the blood follicular barrier. Alterations in maternal circulation due to extremes in body condition may pass into the follicular fluid and affect the oocyte during the preovulatory time period. Such conditions may contribute to the reduced fertility seen in beef cows with extreme body condition.

A Surface Exposed, Two-Domain Lipoprotein Cargo of a Type XI Secretion System Promotes Colonization of Host Intestinal Epithelia Expressing Glycans.

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISSOMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded ß-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISSOMP. The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a ß-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISSOMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N-acetylglucosamine or N-acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS.

Apex Predator Nematodes and Meso-Predator Bacteria Consume Their Basal Insect Prey through Discrete Stages of Chemical Transformations.

Microbial symbiosis drives physiological processes of higher-order systems, including the acquisition and consumption of nutrients that support symbiotic partner reproduction. Metabolic analytics provide new avenues to examine how chemical ecology, or the conversion of existing biomass to new forms, changes over a symbiotic life cycle. We applied these approaches to the nematode Steinernema carpocapsae, its mutualist bacterium, Xenorhabdus nematophila, and the insects they infect. The nematode-bacterium pair infects, kills, and reproduces in an insect until nutrients are depleted. To understand the conversion of insect biomass over time into either nematode or bacterium biomass, we integrated information from trophic, metabolomic, and gene regulation analyses. Trophic analysis established bacteria as meso-predators and primary insect consumers. Nematodes hold a trophic position of 4.6, indicative of an apex predator, consuming bacteria and likely other nematodes. Metabolic changes associated with Galleria mellonella insect bioconversion were assessed using multivariate statistical analyses of metabolomics data sets derived from sampling over an infection time course. Statistically significant, discrete phases were detected, indicating the insect chemical environment changes reproducibly during bioconversion. A novel hierarchical clustering method was designed to probe molecular abundance fluctuation patterns over time, revealing distinct metabolite clusters that exhibit similar abundance shifts across the time course. Composite data suggest bacterial tryptophan and nematode kynurenine pathways are coordinated for reciprocal exchange of tryptophan and NAD+ and for synthesis of intermediates that can have complex effects on bacterial phenotypes and nematode behaviors. Our analysis of pathways and metabolites reveals the chemistry underlying the recycling of organic material during carnivory. IMPORTANCE The processes by which organic life is consumed and reborn in a complex ecosystem were investigated through a multiomics approach applied to the tripartite Xenorhabdus bacterium-Steinernema nematode-Galleria insect symbiosis. Trophic analyses demonstrate the primary consumers of the insect are the bacteria, and the nematode in turn consumes the bacteria. This suggests the Steinernema-Xenorhabdus mutualism is a form of agriculture in which the nematode cultivates the bacterial food sources by inoculating them into insect hosts. Metabolomics analysis revealed a shift in biological material throughout progression of the life cycle: active infection, insect death, and conversion of cadaver tissues into bacterial biomass and nematode tissue. We show that each phase of the life cycle is metabolically distinct, with significant differences including those in the tricarboxylic acid cycle and amino acid pathways. Our findings demonstrate that symbiotic life cycles can be defined by reproducible stage-specific chemical signatures, enhancing our broad understanding of metabolic processes that underpin a three-way symbiosis.