tRNA Biosynthesis Research Articles

Nitidine Chloride Alleviates Hypoxic Stress via PINK1-Parkin-Mediated Mitophagy in the Mammary Epithelial Cells of Milk Buffalo.

Hypoxia in the mammary gland epithelial cells of milk buffalo (BMECs) can affect milk yield and composition, and it can even cause metabolic diseases. Nitidine chloride (NC) is a natural alkaloid with antioxidant properties that can scavenge excessive reactive oxygen species (ROS). However, the effect of NC on the hypoxic injury of BMECs and its molecular mechanisms are still unknown. Here, an immunofluorescence assay, transmission electron microscopy (TEM), and flow cytometry, combined with untargeted metabolomics, were used to investigate the protective effect of NC on hypoxic stress injury in BMECs. It was found that NC can significantly reduce cell activity (p < 0.05) and inhibit cellular oxidative stress (p < 0.05) and cell apoptosis (p < 0.05). A significant decrease in mitophagy mediated by the PINK1-Parkin pathway was observed after NC pretreatment (p < 0.05). In addition, a metabolic pathway enrichment analysis demonstrated that the mechanisms of NC against hypoxic stress may be related to the downregulation of pathways involving aminoacyl tRNA biosynthesis; arginine and proline metabolism; glycine, serine, and threonine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis; and phenylalanine metabolism. Thus, NC has a protective effect on hypoxic mitochondria, and it can regulate amino acid metabolism in response to hypoxic stress. The present study provides a reference for the application of nitidine chloride to regulate the mammary lactation function of milk buffalo.

Graphene quantum dots disrupt the mitochondrial potential of Trypanosoma brucei by interacting with the p18 subunit of ATP synthase F1 after endocytosis via the VSG recycling pathway

HypothesisTrypanosomiasis is one of the main threats to human and animal health in African countries. Trypanosoma brucei can evade the host immune recognition by rapidly altering its variant surface glycoprotein (VSG). The ATP synthase F1 subunit of the parasite exhibits extremely low similarity to that of its mammalian hosts, hypothetically making it an ideal target for the development of novel therapeutics. ExperimentsGraphene quantum dots (GQDs) were synthesized, and their adhesion to T. brucei surface and internalization was observed microscopically. The activity of ATP synthase and mitochondrial membrane potential of T. brucei were measured after exposure to GQDs. Proteomics, biolayer interferometry, and molecular dynamic simulations were utilized to evaluate the interaction between GQDs with the target proteins. FindingsGQDs specifically adhered to the VSG of T. brucei and were conveyed inside the parasite via the VSG internalization pathway. The GQDs promoted intracellular ROS production, interacted with, and inhibited the activity of the p18 subunit of ATP synthase, disrupted parasite mitochondrial membrane potential. Additionally, the GQDs caused a decrease in aminoacyl – tRNA biosynthesis, and upregulated RNA and protein degradation pathways. The findings of this study offer a novel avenue for the target-oriented discovery of anti-trypanosome drugs.

Mapping the proteomic landscape and anti-inflammatory role of Streptococcus parauberis extracellular vesicles

Bacterial extracellular vesicles (BEVs) are nanoscale membrane-bound structures involved in intercellular communication and transport of bioactive molecules. In this study, we described the proteomic insight and anti-inflammatory activity of Streptococcus parauberis BEVs (SpEVs). Proteomics analysis of SpEVs identified 6209 distinct peptides and 1039 proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated enrichment in pathways related to the biosynthesis of aminoacyl tRNA, amino acids, and secondary metabolites. Based on the predicted protein-protein interactions, we discovered key immunological proteins such as IL12A, IL12B, IL8, CD28, and NF-κB between SpEVs and human proteins. Functionally, SpEVs exhibit strong anti-inflammatory activity in LPS-stimulated Raw 264.7 cells by reducing the production of key inflammatory mediators. These include nitric oxide (NO), reactive oxygen species (ROS), inflammatory cytokines such as TNFα and IL6, as well as inflammation-related proteins like inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). qRT-PCR and immunoblotting results clearly indicate that SpEVs modulate the NF-κB and MAPK pathways to induce anti-inflammatory activity. Furthermore, in vivo experiments with zebrafish larvae demonstrated that SpEVs treatment reduced the NO and ROS production with minimal cell mortality. Finally, we validated the anti-inflammatory activity of SpEVs in vivo by systematically assessing the inhibition of NO production, reduction in ROS generation, prevention of cell death, and modulation of NF-κB and MAPK signaling pathways. In conclusion, SpEVs contain rich in unique proteins that play crucial roles in mediating anti-inflammatory effects.

Study on Shenbao Tablet in Treating Kidney-yang Deficiency Syndromebased on Metabolomics.

The aim of this study was to elucidate the mechanism of action of Shenbao tablets using metabolomics approach. Kidney-Yang deficiency is a common syndrome type in traditional Chinese Medicine (TCM) syndrome typology, closely related to disorders of multiple metabolic pathways and is the root cause and underlying syndrome type of many diseases. Shenbao tablets can significantly improve the main symptoms of kidney yang deficiency syndrome, but the mechanism of action of Shenbao tablets on kidney yang deficiency syndrome is still unknown. The rats were intraperitoneally injected with hydrocortisone once a day for 40 days to simulate the syndrome. Traditional pharmacodynamic indicators (body mass, biochemical indicators and pathology) were used to evaluate the efficacy of the medicine. Serum, urine and feces were collected from rats. UPLC/MS metabolomics method was used to study the overall metabolic profile of serum, while GC/MS metabolomics method was used to study the metabolic spectrum of urine and feces. Results showed that the syndrome was significantly improved in the treatment group, and obvious metabolic disorders were observed in rats with the syndrome, with 47 potential biomarkers identified. Pathway analysis showed that nicotinate and nicotinamide metabolism, glycine, serine and trione metabolism, aminoacyl tRNA biosynthesis, glycoxylate and dicarboxylate metabolism were the major ways for Shenbao tablet to improve kidney-yang deficiency syndrome. The mechanism of action of Shenbao tablet in improving the syndrome involves the regulation of energy metabolism, amino acid metabolism, bile acid metabolism, fatty acid metabolism and intestinal microorganisms. This work shows that metabolomics is a promising tool for studying the essence of syndrome theory in TCM and the mechanisms of TCM.

Arbuscular Mycorrhizal Fungi Regulate Lipid and Amino Acid Metabolic Pathways to Promote the Growth of Poncirus trifoliata (L.) Raf.

Arbuscular mycorrhizal (AM) fungi can enhance the uptake of soil nutrients and water by citrus, promoting its growth. However, the specific mechanisms underlying the action of AM fungi in promoting the growth of citrus were not fully elucidated. This study aimed to explore the role of AM fungi Funneliformis mosseae in the regulatory mechanisms of P. trifoliata growth. Pot experiments combined with non-targeted metabolomics methods were used to observe the growth process and changes in metabolic products of P. trifoliata under the conditions of F. mosseae inoculation. The results showed that F. mosseae could form an excellent symbiotic relationship with P. trifoliata, thereby enhancing the utilization of soil nutrients and significantly promoting its growth. Compared with the control, the plant height, stem diameter, number of leaves, and aboveground and underground dry weight in the F. mosseae inoculation significantly increased by 2.57, 1.29, 1.57, 4.25, and 2.78 times, respectively. Moreover, the root system results confirmed that F. mosseae could substantially promote the growth of P. trifoliata. Meanwhile, the metabolomics data indicated that 361 differential metabolites and 56 metabolic pathways were identified in the roots of P. trifoliata and were inoculated with F. mosseae. This study revealed that the inoculated F. mosseae could participate in ABC transporters by upregulating their participation, glycerophospholipid metabolism, aminoacyl tRNA biosynthesis, tryptophan metabolism and metabolites from five metabolic pathways of benzoxazinoid biosynthesis [mainly enriched in lipid (39.50%) and amino acid-related metabolic pathways] to promote the growth of P. trifoliata.

PRDX6 contributes to selenocysteine metabolism and ferroptosis resistance.

Selenocysteine (Sec) metabolism is crucial for cellular function and ferroptosis prevention and has traditionally been thought to begin with the uptake of the Sec carrier selenoprotein P (SELENOP). Following uptake, Sec released from SELENOP undergoes metabolisation via selenocysteine lyase (SCLY), producing selenide, a substrate used by selenophosphate synthetase 2 (SEPHS2), which provides the essential selenium donor - selenophosphate - for the biosynthesis of the selenocysteine tRNA. Here, we report the discovery of an alternative pathway mediating Sec metabolisation that is independent of SCLY and mediated by peroxiredoxin 6 (PRDX6). Mechanistically, we demonstrate that PRDX6 can readily react with selenide and interact with SEPHS2, potentially acting as a selenium delivery system. Moreover, we demonstrate the presence and functional significance of this alternative route in cancer cells where we reveal a notable association between elevated expression of PRDX6 with a highly aggressive neuroblastoma subtype. Altogether, our study sheds light on a previously unrecognized aspect of Sec metabolism and its implications in ferroptosis, offering new avenues for therapeutic exploitation.

Bee Venom Toxic Effect on MDA-MB-231 Breast Cancer Cells and Caenorhabditis Elegans.

Bee venom has therapeutics and pharmacological properties. Further toxicological studies on animal models are necessary due to the severe allergic reactions caused by this product. Here, Caenorhabditis elegans was used as an in vivo toxicity model, while breast cancer cells were used to evaluate the pharmacological benefits. The bee venom utilized in this research was collected from Apis mellifera species found in Northeast Brazil. The cytotoxicity caused by bee venom was measured by MTT assay on MDA-MB-231 and J774 A.1 cells during 24 - 72 hours of exposure. C. elegans at the L4 larval stage were exposed for three hours to M9 buffer or bee venom. Survival, behavioral parameters, reproduction, DAF-16 transcription factor translocation, the expression of superoxide dismutase (SOD), and metabolomics were analyzed. Bee venom suppressed the growth of MDA-MB-231 cancer cells and exhibited cytotoxic effects on macrophages. Also, decreased C. elegans survival impacted its behaviors by decreasing C. elegans feeding behavior, movement, and reproduction. Bee venom did not increase the expression of SOD-3, but it enhanced DAF-16 translocation from the cytoplasm to the nucleus. C. elegans metabolites differed after bee venom exposure, primarily related to aminoacyl- tRNA biosynthesis, glycine, serine and threonine metabolism, and sphingolipid and purine metabolic pathways. Our findings indicate that exposure to bee venom resulted in harmful effects on the cells and animal models examined. Thus, due to its potential toxic effect and induction of allergic reactions, using bee venom as a therapeutic approach has been limited. The development of controlled-release drug strategies to improve this natural product's efficacy and safety should be intensified.

Therapeutic effect and mechanism of Jingfang Granules on acute lung injury based on intestinal flora and fecal metabolomics

This study aims to elucidate the therapeutic effect and mechanism of Jingfang Granules on acute lung injury, and to investigate the regulatory effect of Jingfang Granules on the metabolic disorders of endogenous metabolites in feces and the homeostasis of intestinal microbiota in acute lung injury, mice were randomly divided into a sham group, a model group, and a Jingfang Granules group. After modeling, the mice were continuously administered for 6 days. Using ultra-high performance liquid chromatography quadrupole/electrostatic field orbital trap high-resolution mass spectrometry(UHPLC-HESI-QE-Orbitrap-MS/MS) metabolomics technology and 16S rRNA high-throughput sequencing technology, changes in endogenous small molecule substances and gut microbiota in mouse intestines were determined, and potential biomarkers were identified. The results showed that Jingfang Granules can regulate 11 biomarkers, including L-glutamic acid, succinic acid, arachidonic acid, linoleic acid, linolenic acid, phenylalanine, sphingosine, 2-hydroxy-2-methyl butyric acid, pyruvate, tryptophan, and palmitic acid. Metabolic pathway analysis was conducted on these 11 biomarkers using the online software MetaboAnalyst, identifying potential major metabolic pathways. Among them, a total of 10 metabolic pathways are closely related to the treatment of acute lung injury with Jingfang Granules, including alanine, aspartate and glutamate metabolism, aminoacyl-tRNA biosynthesis, citrate cycle(TCA cycle), alyoxylate and dicarboxylate metabolism, arginine and proline metabolism, linoleic acid metabolism and linolenic acid metabolism, nitrogen metabolism, D-glutamine and D-gluta-matemetabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism. The results of gut microbiota showed significant differences in bacteria, mainly including Bacteroides, Akkermansia, Lachnospiraceae＿NK4A136＿group, Lachnochlostridium, and Klebsiella. Spearman analysis confirms that Akkermansia and Lachnospiraceae＿NK4A136＿group is a significant positive correlation between the abundance of succinic acid, arachidonic acid, linolenic acid, linoleic acid, butyric acid, and pyruvate in the group; Bacteroides, Klebsiella, Lachnochlostrium are significantly positively correlated with the abundance of L-glutamic acid, phenylalanine, and sphingosine. The above results indicate that the therapeutic effect of Jingfang Granules on acute lung injury is achieved by improving the imbalance of gut microbiota in mice with acute lung injury, balancing the metabolism of alanine, biosynthesis of aminoacyl tRNA, aspartic acid, glutamate, tricarboxylic acid cycle, biosynthesis of phenylalanine, tyrosine, tryptophan, and metabolism of linoleic acid.

434 Investigating the metabolic-inflammatory mechanisms of cachexia symptoms in head and neck cancer patient plasma via multiomics integration of the metabolome, lipidome, and inflammation cytokines

OBJECTIVES/GOALS: Cachexia is the involuntary and irreversible loss of muscle and fat and is a major cause of morbidity and mortality in head and neck cancer (HNC). It remains a poorly understood disease diagnosed by weight loss and a confluence of symptoms. We explored the metabolic and inflammatory mechanisms of cachexia symptoms via an multiomics network algorithm. METHODS/STUDY POPULATION: Prior to chemoradiotherapy, HNC subjects completed questionnaires and donated blood for untargeted (metabolites) and targeted (lipids and cytokines) assays. Metabolites and lipids were measured by liquid chromatography mass spectrometry. Cytokines were measured by multiplex assays. We plotted a multiomics network graph by estimating partial least squares correlations amongst metabolites, lipids, cytokines, and common cachexia symptoms—max percent weight loss over 1 year, baseline BMI, fatigue, performance, albumin, hemoglobin, and white blood cell count. To interpret the network, an algorithm identified highly correlated clusters of metabolites-lipids-cytokines-symptoms representing possible biological relatedness, which were functionally annotated via metabolic enrichment analysis. RESULTS/ANTICIPATED RESULTS: In 123 subjects (59 years of age, 72% male, 84% white, avg weight loss of 13%), we analyzed 186 metabolites, 54 lipids, 7 cytokines and 7 cachexia symptoms. We required a correlation &gt;0.25 and P-value &lt;.05 to be included in the network graph, resulting in 323 connections and 3 identified clusters. Max weight loss and baseline BMI were in a cluster enriched by unsaturated fatty acid biosynthesis (P&lt;.0001) and arachidonic acid (P=.01) metabolic pathways but not linked to inflammation cytokines. The five other cachexia symptoms were in a cluster with 4 cytokines (C-reactive protein, interleukin 6, IL10, IL1, Tumor necrosis factor receptor 2) and enriched by aminoacyl tRNA (P&lt;.01) and valine biosynthesis (P=.02). We observed no meaningful differences when we stratified the analysis by human papillomavirus. DISCUSSION/SIGNIFICANCE: Cachexia symptoms in head and neck cancer may be linked to specific metabolic dysregulation—weight loss and BMI were linked to fatty acids; fatigue, anemia and others were linked to amino acids and inflammation. This information may allow for the recognition of a cachexic-metabolic subtype or provide novel targets for metabolic intervention.

1H-NMR-based metabolomics reveals the preventive effect of Enteromorpha prolifera polysaccharides on diabetes in Zucker diabetic fatty rats.

The primary objective of this investigation was to explore the beneficial impacts of Enteromorpha prolifera polysaccharide (EP) on dysglycemia in Zucker diabetic fatty (ZDF) rats, while also shedding light on its potential mechanism using 1H-NMR-based metabolomics. The results demonstrated a noteworthy reduction in fasting blood glucose (FBG, 46.3%), fasting insulin (50.17%), glycosylated hemoglobin A1c (HbA1c, 44.1%), and homeostatic model assessment of insulin resistance (HOMA-IR, 59.75%) following EP administration, while the insulin sensitivity index (ISI, 19.6%) and homeostatic model assessment of β-cell function (HOMA-β, 2.5-fold) were significantly increased. These findings indicate that EP enhances β-cell function, increases insulin sensitivity, and improves insulin resistance caused by diabetes. Moreover, EP significantly reduced serum lipid levels, suggesting improvement of dyslipidemia. Through the analysis of serum metabolomics, 17 metabolites were found to be altered in diabetic rats, 14 of which were upregulated and 3 of which were downregulated. Notably, the administration of EP successfully reversed the abnormal levels of 9 out of the 17 metabolites. Pathway analysis further revealed that EP treatment partially restored metabolic dysfunction, with notable effects observed in valine, leucine, and isoleucine metabolism; aminoacyl-transfer RNA (tRNA) biosynthesis; and ketone body metabolism. These findings collectively indicate the potential therapeutic efficacy of EP in preventing glycemic abnormalities and improving insulin resistance. Thus, EP holds promise as a valuable treatment option for individuals with diabetes.

Plasma metabolomic differences in early-onset compared to average-onset colorectal cancer

Deleterious effects of environmental exposures may contribute to the rising incidence of early-onset colorectal cancer (eoCRC). We assessed the metabolomic differences between patients with eoCRC, average-onset CRC (aoCRC), and non-CRC controls, to understand pathogenic mechanisms. Patients with stage I–IV CRC and non-CRC controls were categorized based on age ≤ 50 years (eoCRC or young non-CRC controls) or ≥ 60 years (aoCRC or older non-CRC controls). Differential metabolite abundance and metabolic pathway analyses were performed on plasma samples. Multivariate Cox proportional hazards modeling was used for survival analyses. All P values were adjusted for multiple testing (false discovery rate, FDR P < 0.15 considered significant). The study population comprised 170 patients with CRC (66 eoCRC and 104 aoCRC) and 49 non-CRC controls (34 young and 15 older). Citrate was differentially abundant in aoCRC vs. eoCRC in adjusted analysis (Odds Ratio = 21.8, FDR P = 0.04). Metabolic pathways altered in patients with aoCRC versus eoCRC included arginine biosynthesis, FDR P = 0.02; glyoxylate and dicarboxylate metabolism, FDR P = 0.005; citrate cycle, FDR P = 0.04; alanine, aspartate, and glutamate metabolism, FDR P = 0.01; glycine, serine, and threonine metabolism, FDR P = 0.14; and amino-acid t-RNA biosynthesis, FDR P = 0.01. 4-hydroxyhippuric acid was significantly associated with overall survival in all patients with CRC (Hazards ratio, HR = 0.4, 95% CI 0.3–0.7, FDR P = 0.05). We identified several unique metabolic alterations, particularly the significant differential abundance of citrate in aoCRC versus eoCRC. Arginine biosynthesis was the most enriched by the differentially altered metabolites. The findings hold promise in developing strategies for early detection and novel therapies.

Analysis of the relationship of refractory gout between potential biomarkers and diet structure and lifestyle based on 1H-NMR.

We investigated the different life styles among the diet structures and exercise patterns of 100 patients with refractory gout and 79 healthy volunteers; of these, we selected 39 patients and 20 healthy volunteers for serum proton magnetic resonance (1H-NMR) metabolic network detection. We determined the potential biomarkers of refractory gout and attempted to explore the relation between potential biomarkers and diet structures and exercise patterns. The study employed a questionnaire survey to analyze diet structures and exercise patterns from 100 patients of refractory gout and 79 healthy volunteers. At the same time, using 1H-NMR metabolic technology to analyze the metabolites present in the serum samples obtained from 39 patients of refractory gout (group B) and 20 healthy subjects (group A). Employing MestReNova (Version 8.0.1) to analyze the metabolitesmaps, collecting the NMR results, further importing into SIMCA-P+ 14.0 software (Umetrics, Sweden) for principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) statistical analysis. Combining patterns recognition and multivariate statistics, potential biomarkers were searched. Other experimental data, including creatinine and adiponectin, were counted by the SPSS21.0. The measurement data were expressed by X ± S and t test. The counting data were expressed in percent and performed by X2 test. Our results revealed that patients with gout tended to be obese, and there were differences in their lifestyle with exercise, sleep, and smoking, as well as in their preference for fructose drinks, alcohol, and total and structural distribution of meat, milk, eggs, and so on when compared with the healthy volunteers. Importantly, we found the adiponectin in the gout group was lower as compared to the healthy group. Further, metabolomics in combination with KEGG analysis revealed that the biosynthesis of aminoacyl tRNA, biosynthesis of valine, leucine, and isoleucine, metabolism of alanine, aspartic, and glutamate, metabolism of glycine, serine, and threonine, phenylalanine, glycolysis/gluconeogenesis, ketone body synthesis and degradation, metabolism of D-glutamine, citric acid cycle (TCA cycle), triglyceride metabolism, and others could be used as specific biomarkers of this disease. Recurrent refractory gout and formation of tophus may be related to the diet structures and lifestyles between the patients and the healthy people, and their abnormal metabolic network may be related to the disorder of mitochondrial energy metabolism, which further results in abnormal metabolism of glucose, lipids, amino acids, and deposition of uric acid in joints, peripheral connective tissue, and kidney, inducing an inflammatory response.

Protective Effect of Artocarpus heterophyllus Lam. (Jackfruit) Polysaccharides on Liver Injury Induced by Cyclophosphamide in Mice.

In recent years, Artocarpus heterophyllus Lam. (jackfruit) polysaccharides (namely JFP-Ps) have attracted much attention due to their multiple biological activities. This study aimed to explore the protective effects and the underlying mechanisms of JFP-Ps on cyclophosphamide (Cp)-induced liver damage. The protective effect of JFP-Ps was evaluated using HE staining, antioxidant testing, enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), Western blot and ultra-performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) metabolomics analysis. The results showed that Cp caused pathological liver damage, activated oxidative stress and downregulated cytokine expression, while JFP-Ps treatment was found to exert antioxidant effects and play immune regulatory roles through mitogen-activated protein kinase/nuclear factor-κB (MAPK/NF-κB) related inflammation and cell apoptosis pathways to protect the Cp-induced liver injury. Metabolomic results showed that the liver-protective effects of JFP-Ps were mainly related to aminoacyl transfer ribonucleic acid (tRNA) biosynthesis, sphingolipid metabolism, purine metabolism and the citrate cycle. These results indicate that JFP-Ps have great potential application in alleviating liver injury.

The Mechanism of Hepatic Encephalopathy Induced by Thioacetamide Based on Metabolomics and Proteomics: A Preliminary Study.

Hepatic encephalopathy (HE) is a central nervous system dysfunction syndrome caused by acute and chronic liver failure or various portal systemic shunt disorders. HE arises from metabolic disorder and excludes other known types of encephalopathy. HE is a major cause of death in people with liver disease. Early diagnosis and timely treatment are key to improving HE prognosis. Herein, we established a model of HE and performed metabolomics to identify 50 significantly differential metabolites between the HE group and control group. The main metabolic pathways associated with these differential metabolites were the purine metabolism, pyrimidine metabolism, aminoacyl tRNA biosynthesis, and glucose metabolism. Through proteomics analysis, we identified 226 significantly differential proteins (52 up-regulated and 174 down-regulated). The main (Kyoto Encyclopedia of Genes and Genomes) enrichment pathways were the Staphylococcus aureus infection, vitamin digestion and absorption, and complement and coagulation cascades. Through the conjoint analysis of proteomics and metabolomics, the differentially present proteins and metabolites were found to be involved in vitamin digestion and absorption, and ferroptosis pathways. In HE, malondialdehyde was significantly elevated, but glutathione was significantly diminished, and the redox balance was destroyed, thus leading to changes in proteins' levels associated with the ferroptosis pathway. In conclusion, this study preliminarily explored the molecular and metabolic mechanisms underlying HE.

TMT-based quantitative proteomics reveals the nutritional and stress resistance functions of anaerobic fungi in yak rumen during passage at different time intervals

ObjectivesAnaerobic fungi are critical for nutrient digestion in the yak rumen. Although studies have reported the effects of passage at different time intervals on the community structure of yak rumen anaerobic fungi, it is unknown whether passage culture at different time intervals affects the microbial proteins of rumen anaerobic fungi and their functions. MethodsMycelium was obtained using the anaerobic continuous batch culture (CBC) of yak rumen fluid at intervals of 3 d, 5 d and 7 d. Quantitative analysis of fungal proteins and functional analysis was performed using tandem mass tagging (TMT) and bioinformatics. ResultsA total of 56 differential proteins (DPs) were found in 5 d vs. 3 d and 7 d vs. 3 d. Gene ontology (GO) enrichment indicated that the up-regulated proteins were mainly involved in biological regulation, cellular process, metabolic process, macromolecular complex, membrane, cell part, organelle, binding, catalytic activity and transporter activity. The downregulated proteins were mainly enriched in metabolic process, cell part, binding and catalytic activity. Furthermore, the downregulated proteins in 7 d vs. 3 d were related to membrane and organelle. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results indicated that DPs were enriched in 14 pathways in 5 d vs. 3 d and 7 d vs. 3 d, mainly including terpenoid backbone biosynthesis, alaine, aspartate and glutamate metabolism, arginine biosynthesis, hypotaurine, cyanoamino acid, glutathione, β-alanine, pyrimidine, purine, galactose and propanate metabolism, steroid biosynthesis, ribosome biogenesis in eukaryotes and aminoacyl tRNA biosynthesis. The DPs were enriched in only 2 pathways in 5 d vs 3 d, lysine biosynthesis and cysteine and methionine metabolism. N-glycan biosynthesis and retinol metabolism are only found in the metabolism of DPs in 7 d vs 3 d. ConclusionsYak rumen anaerobic fungal proteins are involved in nutrition and stress tolerance during passage at different time intervals.

An atypical GdpP enzyme linking cyclic nucleotide metabolism to osmotic tolerance and gene regulation in Mycoplasma bovis

Nucleotide second messengers play an important role in bacterial adaptation to environmental changes. Recent evidence suggests that some of these regulatory molecular pathways were conserved upon the degenerative evolution of the wall-less mycoplasmas. We have recently reported the occurrence of a phosphodiesterase (PDE) in the ruminant pathogen Mycoplasma bovis, which was involved in c-di-AMP metabolism. In the present study, we demonstrate that the genome of this mycoplasma species encodes a PDE of the GdpP family with atypical DHH domains. Characterization of M. bovis GdpP (MbovGdpP) revealed a multifunctional PDE with unusual nanoRNase and single-stranded DNase activities. The alarmone ppGpp was found unable to inhibit c-di-NMP degradation by MbovGdpP but efficiently blocked its nanoRNase activity. Remarkably, MbovGdpP was found critical for the osmotic tolerance of M. bovis under K+ and Na+ conditions. Transcriptomic analyses further revealed the biological importance of MbovGdpP in tRNA biosynthesis, pyruvate metabolism, and several steps in genetic information processing. This study is an important step in understanding the role of PDE and nucleotide second messengers in the biology of a minimal bacterial pathogen.

Transcriptome and metabolome profiling to elucidate the mechanism underlying the poor growth of Streptococcus suis serotype 2 after orphan response regulator CovR deletion.

The deletion of orphan response regulator CovR reduces the growth rate of Streptococcus suis serotype 2 (S. suis 2). In this study, metabolome and transcriptome profiling were performed to study the mechanisms underlying the poor growth of S. suis 2 caused by the deletion of orphan response regulator CovR. By comparing S. suis 2 (ΔcovR) and S. suis 2 (SC19), 146 differentially accumulated metabolites (upregulated: 83 and downregulated: 63) and 141 differentially expressed genes (upregulated: 86 and downregulated: 55) were identified. Metabolome and functional annotation analysis revealed that the growth of ΔcovR was inhibited by the imbalance aminoacyl tRNA biosynthesis (the low contents of L-lysine, L-aspartic acid, L-glutamine, and L-glutamic acid, and the high content of L-methionine). These results provide a new insight into the underlying poor growth of S. suis 2 caused by the deletion of orphan response regulator CovR. Metabolites and candidate genes regulated by the orphan response regulator CovR and involved in the growth of S. suis 2 were reported in this study.

Integrated transcriptomic and metabolomic analysis reveals the response of pearl oyster (Pinctada fucata martensii) to long-term hypoxia

The frequency at which organisms are exposed to hypoxic conditions in aquatic environments is increasing due to coastal eutrophication and global warming. To reveal the effects of long-term hypoxic stress on metabolic changes of pearl oyster, commonly known as Pinctada (Pinctada fucata martensii), the present study performed the integrated analysis of transcriptomics and metabolomics to investigate the global changes of genes and metabolites following 25 days hypoxia challenge. Transcriptome analysis detected 1108 differentially expressed genes (DEGs) between the control group and the hypoxia group. The gene ontology (GO) analysis of DEGs revealed that they are significantly enriched in functions such as “microtubule−based process”, “histone (H3–K4, H3–K27, and H4–K20) trimethylation”, “histone H4 acetylation”, “kinesin complex”, and “ATPase activity”, and KEGG pathway functions, such as “DNA replication”, “Apoptosis”, and “MAPK signaling pathways”. Metabolome analysis identified 68 significantly different metabolites from all identified metabolites, and associated with 25 metabolic pathways between the control and hypoxia groups. These pathways included aminoacyl−tRNA biosynthesis, arginine and proline metabolism, and phenylalanine metabolism. Our integrated analysis suggested that pearl oysters were subject to oxidative stress, apoptosis, immune inhibition, and neuronal excitability reduction under long-term hypoxic conditions. We also found a remarkable depression in a variety of biological functions under long-term hypoxia, including metabolic rates, biomineralization activities, and the repression of reorganization of the cytoskeleton and cell metabolism. These findings provide a basis for elucidating the mechanisms used by marine bivalves to cope with long-term hypoxic stress.

Metabolic Infrastructure of Pregnant Women with Fetuses Having Nervous System Abnormalities; Metabolomic Analysis

Central nervous system diseases are neurological disorders that affect the structure or function of the brain and spinal cord that make up the central nervous system. In this study, it was aimed to examine the impaired/altered metabolomic profiles of pregnant women carrying fetuses with nervous system abnormalities (NSA). The study group consisted of 30 normal pregnancies with normal fetuses (control group) and 8 pregnancies with fetuses having NSA (study group), as determined by prenatal screening and diagnosis as part of an antenatal care program. Metabolomic analyses were carried out using gas chromatography-mass spectrometry (GC-MS). GC-MS-based metabolomics analysis was able to identify 95 metabolites and 27 of them were statistically significant between the two groups (p&amp;lt;0.05). Moreover, the pathway analysis, performed with significantly altered metabolites, showed alteration in the alanine, aspartate, and glutamate metabolism, citrate cycle, aminoacyl t-RNA biosynthesis, and glutathione metabolism. Alanine, aspartate and glutamate metabolism, citrate cycle, aminoacyl t-RNA biosynthesis, and glutathione metabolism seem to be critical in the prenatal screening of NSAs. However, abnormality-specific studies are necessary for further recommendations.

739-P: Metabolome Alterations Associated with 12-Week Sitting Time Reduction among Sedentary, Postmenopausal Latinas

Background: Prolonged sitting is associated with health risk. The metabolic impact of replacing sitting time with standing is unknown. We explored metabolome changes after a 12-week standing intervention. Methods: Sedentary postmenopausal Latinas with overweight (N=254; 55+ years of age; BMI ≥25 kg/m2) were randomized to an increased standing intervention or a health education control group. Clinical, behavioral and fasting plasma measurements were collected at baseline and 12-weeks. Sitting, standing and stepping time was assessed objectively by a thigh-worn inclinometer (activPAL). Aqueous metabolites and carnitines were measured using LC-MS/MS. Results: Top tertile of intervention group Best Responders (N=43) was selected by criteria of change in mean sitting bout duration and total sitting time. Variable-Matched Controls (N=43) were selected from the control group using a random forest model trained to predict the Best-Responders. Best Responders changed sitting and standing time by -110.0 (11.0) and 104.6 (10.1) min, respectively, compared to the Matched Controls change of 7.1 (9.8) and -7.8 (9.0) min, respectively. A covariate-controlled GLM revealed significant group differences in change scores of 24 metabolites (FDR q = 0.048-2.1E-4) from baseline to 12-week follow-up. Enzyme enrichment predicted significant functional changes in enzymes involved in glutamate and histidine catabolism. Pathway analysis showed significant effects in 14 metabolites of aminoacyl tRNA biosynthesis, as well as 4 metabolites involved in phenylalanine, tyrosine, tryptophan, ubiquinone/terpenoid-quinone biosynthesis and beta-alanine metabolism (all p &amp;lt; 0.05). Metabolite change scores mapped to central carbon metabolism with significant elevation of 14 amino acids involved in pyruvate, acetoacetyl- and acetyl-CoA metabolism, and TCA cycle intermediate biosynthesis. Conclusion: Standing intervention results in broad amino acid metabolism alterations. Disclosure J.Patterson: None. P.Jasbi: None. H.Gu: None. M.Allison: None. C.Reuter: None. B.Rana: None. L.Natarajan: None. D.D.Sears: None. Funding American Heart Association (16SFRN28420000)