Oxaloacetate Research Articles

Re-designing Escherichia coli for high-yield production of β-alanine by metabolic engineering

β-Alanine is the only natural β-amino acid and an important precursor for pantothenic acid (vitamin B5) synthesis. In this work, to improve the β-alanine fermentative production, collaborative modifications mainly focused on aspartate ammonia-lyase (aspA), phosphoenolpyruvate (PEP) – pyruvate (PYR) – oxaloacetate (OAA) – L-aspartate nodes, glucose uptake system, and β-alanine uptake system were performed, including knock out of aspA, reducing the consumption of central metabolic nodes PEP and PYR, enforcement of the non-PEP-dependent transferase system (non-PTS), inactivation of gluconeogenesis through deleting pck gene, and disruption of β-alanine uptake system. As a result, a significantly enhanced β-alanine accumulation was achieved. The β-alanine titer was increased to 6.67 g/L (final strain B15) from 2.46 g/L (original strain B1) in shake flask fermentation, and a titer of 41.12 g/L was achieved in 5-L fermentor fermentation. With the presence of succinic acid (0.25 g/L), the β-alanine titer eventually reached 52.61 g/L at 72 h, and a high productivity (0.73 g/L/h) and yield (0.268 g/g glucose) were also obtained. The yield was relatively higher compared with the reported levels and the B15 exhibited a remarkable fermentation stability over a long period. This result laid foundation for industrialization of β-alanine fermentative production.

Directional regulation of cytosolic PEPCK catalysis is mediated by competitive binding of anions

Phosphoenolpyruvate carboxykinase (PEPCK) is a well-characterized enzyme involved in primary glucose metabolism, responsible for catalyzing one of the key steps of gluconeogenesis. It is well demonstrated that PEPCK can efficiently catalyze the reversible interconversion of oxaloacetic acid (OAA) to phosphoenolpyruvate (PEP) in vitro, but the enzyme is typically ascribed a metabolic role that requires preferential catalysis in the direction of PEP synthesis in vivo. Here we present structural and functional data that demonstrate the preferential synthesis of PEP from OAA catalyzed by PEPCK in vivo is facilitated by anion-mediated enzyme inhibition that reduces enzyme activity more significantly in the direction of OAA synthesis than in the direction of PEP synthesis. From our studies we conclude that the specific binding of small, ubiquitous anions like chloride, present in millimolar concentrations under normal cellular conditions allows for metabolic control by restricting PEPCK to function in the direction of PEP synthesis.

Cordycepin exhibits anti-fatigue effect via activating TIGAR/SIRT1/PGC-1α signaling pathway

Fatigue, a most commonly sub-health condition, may cause people more susceptible to many diseases. Cordycepin, a principal active ingredient from Cordyceps militaris, exerts various pharmacological activities including anti-diabetes, anti-inflammatory, immunomodulatory and antioxidant effects. However, the anti-fatigue effect of cordycepin and specific mechanism remained unclear. This study aimed to investigate the beneficial effect of cordycepin on physical fatigue and elucidate the potential mechanism. 20 mg/kg, 40 mg/kg of cordycepin and 500 mg/kg taurine were respectively treated to mice for 28 days before weight-loaded swimming test. The results revealed that cordycepin significantly prolonged the weight-loaded swimming time of mice. Meanwhile, cordycepin decreased the levels of lactic acid, blood uric nitrogen, and malondialdehyde, and increased the contents of superoxide dismutase, glutathione, nicotinamide adenine dinucleotide phosphate, hepatic glycogen, muscle glycogen and ATP. The metabolomic study by GC-MS showed that eight biomarkers were found in livers, including L-lactic acid, L-asparagine, 3-phosphoglyceric acid, inosine, D-galactose, L-tyrosine, glyceric acid and L-threonine. There were seven biomarkers in gastrocnemius, including D-ribose-5-phosphate, acetic acid, propionic acid, butyric acid, palmitic acid, oxaloacetic acid and citric acid. The results of metabolomics indicated that cordycepin might relieve fatigue by regulating energy metabolism and pentose phosphate pathway. Furthermore, we found cordycepin significantly enhanced the protein levels of TIGAR, SIRT1, PGC-1α, NRF1 and TFAM in gastrocnemius of weight-loaded swimming mice. Taken together, the present study demonstrated that cordycepin possessed an anti-fatigue effect via activating TIGAR/SIRT1/PGC-1α signaling pathway. Our study indicated that cordycepin may be a potentially efficient candidate for fatigue.

Effective 5-aminolevulinic acid production via T7 RNA polymerase and RuBisCO equipped Escherichia coli W3110.

Chromosome-based engineering is a superior approach for gene integration generating a stable and robust chassis. Therefore, an effective amplifier, T7 RNA polymerase (T7RNAP) from bacteriophage, has been incorporated into Escherichia coli W3110 by site-specific integration. Herein, we performed the 5-aminolevulinic acid (5-ALA) production in four T7RNAP-equipped W3110 strains using recombinant 5-aminolevulinic synthase and further explored the metabolic difference in best strain. The fastest glucose consumption resulted in the highest biomass and the 5-ALA production reached to 5.5 g/L; thus, the least by-product of acetate was shown in RH strain in which T7RNAP was inserted at HK022 phage attack site. Overexpression of phosphoenolpyruvate (PEP) carboxylase would pull PEP to oxaloacetic acid in tricarboxylic acid cycle, leading to energy conservation and even no acetate production, thus, 6.53 g/L of 5-ALA was achieved. Amino acid utilization in RH deciphered the major metabolic flux in α-ketoglutaric acid dominating 5-ALA production. Finally, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were expressed for carbon dioxide recycling; a robust and efficient chassis toward low-carbon assimilation and high-level of 5-ALA production up to 11.2 g/L in fed-batch fermentation was established.

Insertion of 4-Demethylwyosine in tRNAPhe Catalyzed by the Radical S-Adenosyl-l-methionine Enzyme TYW1 Entails Oxidative Cleavage of Pyruvate to Form CO2.

The radical S-adenosyl-l-methionine (SAM) enzyme TYW1 catalyzes the condensation of C-2 and C-3 atoms of pyruvate with N-methylguanosine containing tRNAPhe to form 4-demethylwyosine (imG-14) modified tRNAPhe. The fate of C-1 is not known, and either formate or carbon dioxide (CO2) has been proposed. In this study, a coupled assay that transforms either CO2 or formate to oxaloacetate (OAA) was used to determine the fate of C-1. In the presence of [1-13C1]-pyruvate, 13C-enriched OAA was observed in a process that is concomitant with the formation of imG-14, under conditions that preferentially transform CO2 and not formate to OAA. These findings are discussed in the context of the cofactor content of TYW1 and a new role for the auxiliary cluster in catalyzing the oxidative cleavage of C-1-C-2 bond of pyruvate in the catalytic cycle of TYW1.

Glis1 and oxaloacetate in nucleus pulposus stromal cell somatic reprogramming and survival.

Regenerative medicine aims to repair degenerate tissue through cell refurbishment with minimally invasive procedures. Adipose tissue (FAT)-derived stem or stromal cells are a convenient autologous choice for many regenerative cell therapy approaches. The intervertebral disc (IVD) is a suitable target. Comprised of an inner nucleus pulposus (NP) and an outer annulus fibrosus (AF), the degeneration of the IVD through trauma or aging presents a substantial socio-economic burden worldwide. The avascular nature of the mature NP forces cells to reside in a unique environment with increased lactate levels, conditions that pose a challenge to cell-based therapies. We assessed adipose and IVD tissue-derived stromal cells through in vitro transcriptome analysis in 2D and 3D culture and suggested that the transcription factor Glis1 and metabolite oxaloacetic acid (OAA) could provide NP cells with survival tools for the harsh niche conditions in the IVD.

PERBANDINGAN AKTIVITAS ENZIM ASPARTAT AMINOTRANSFERASE ANTARA SERUM YANG HEMOLISIS DAN SERUM NORMAL DENGAN METODE KINETIK-IFCC

Aspartic aminotransferase (AST) is a mitochondrial enzyme that catalyzes the back and forth transfer of amino groups from aspartic acid to ?-oxaloacetic acid to form glutamic acid and oxaloacetic acid. AST is contained in erythrocytes so that if there is hemolysis, a ruptured erythrocyte will cause an increase in the activity of the AST enzyme that comes out of the erythrocyte. This study aims to determine the effect of hemolysis in serum on AST enzyme activity. This research method is experimental by comparing normal serum with hemolytic serum. The research sample involved 30 employees of the Manding Sumenep Public Health Center who would be examined for the AST enzyme activity. The results showed that the AST enzyme activity in normal serum had an average of 24.367 IU / L, while the average hemolytic serum was 39.3 IU / L. Based on the results of these data, it shows that there is an increase in AST enzyme activity in hemolytic serum compared to normal serum.

Metabolic clearance of oxaloacetate and mitochondrial complex II respiration: Divergent control in skeletal muscle and brown adipose tissue

At low inner mitochondrial membrane potential (ΔΨ) oxaloacetate (OAA) accumulates in the organelles concurrently with decreased complex II-energized respiration. This is consistent with ΔΨ-dependent OAA inhibition of succinate dehydrogenase. To assess the metabolic importance of this process, we tested the hypothesis that perturbing metabolic clearance of OAA in complex II-energized mitochondria would alter O2 flux and, further, that this would occur in both ΔΨ and tissue-dependent fashion. We carried out respiratory and metabolite studies in skeletal muscle and interscapular brown adipose tissue (IBAT) directed at the effect of OAA transamination to aspartate (catalyzed by the mitochondrial form of glutamic-oxaloacetic transaminase, Got2) on complex II-energized respiration. Addition of low amounts of glutamate to succinate-energized mitochondria at low ΔΨ increased complex II (succinate)-energized respiration in muscle but had little effect in IBAT mitochondria. The transaminase inhibitor, aminooxyacetic acid, increased OAA concentrations and impaired succinate-energized respiration in muscle but not IBAT mitochondria at low but not high ΔΨ. Immunoblotting revealed that Got2 expression was far greater in muscle than IBAT mitochondria. Because we incidentally observed metabolism of OAA to pyruvate in IBAT mitochondria, more so than in muscle mitochondria, we also examined the expression of mitochondrial oxaloacetate decarboxylase (ODX). ODX was detected only in IBAT mitochondria. In summary, at low but not high ΔΨ, mitochondrial transamination clears OAA preventing loss of complex II respiration: a process far more active in muscle than IBAT mitochondria. We also provide evidence that OAA decarboxylation clears OAA to pyruvate in IBAT mitochondria.

Studies on the mechanism of energy metabolism via AMPK/PGC-1α signaling pathway induced by compatibility of Ligusticum chuanxiong Hort and Gastrodia.

AMP-activated protein kinase (AMPK) regulates overall energy consumption and energy intake through cytokines. Ligusticum striatum DC (CX) combined with Gastrodia elata Blume (TM) has been used for migraine treatment for millennia. When used alone in clinical practice, CX causes symptoms of thirst, irritability, and yellow urine and has influenced the levels of cytokines such as AMP that activate the AMPK pathway of energy metabolism. However, relationships between this compatibility prescription, integral biological energy metabolism, and the AMPK pathway remain unclear. Studies were performed by treating normal rats with physiological saline, CX extract, CX coupled TM extract, and TM extracts separately for 4 weeks. Food intake, water intake, urine output, stool output, and body weight were monitored once a week by the metabolic cage method. Values of FBG, BUN, TP, TC and TG in blood samples were detected approaching the whole blood automatic detector from 1 to 4 weeks. Na+-K+-ATPase, Ca2+-Mg2+-ATPase, cAMP, and cGMP activity were determined by the enzyme-linked immunosorbent assay (ELISA); the biological samples that were obtained at 1, 2, 3, and 4 weeks after drug administration were tested by GC-TOF-MS. Then real-time PCR and Western Blot were applied to detect changes in expression of some substances involved in energy metabolism. The results demonstrated that administering CX alone increased energy input, mobility, and respiratory exchange ratio, accelerated energy consumption, and caused inflammatory infiltration in the liver. CX coupled with TM led to lower energy metabolism and liver damage in comparison with CX used alone. Moreover, CX-treated rats harbored higher levels of differential metabolites (including pyrophosphate, oxaloacetic acid, and galactinol). Glycerophospholipid metabolism and the citrate cycle are closely related to the differential metabolites above. In addition, CX-induced unbalanced energy metabolism depends on cAMP activation mediated by the AMPK/PGC-1α pathway in rats. Our findings suggest that CX-induced energy metabolism imbalance was corrected after coupling with TM by mediating the AMPK/PGC-1α pathway.

Alterations in Primary Carbon Metabolism in Cucumber Infected with Pseudomonas syringae pv lachrymans: Local and Systemic Responses.

The reconfiguration of the primary metabolism is essential in plant–pathogen interactions. We compared the local metabolic responses of cucumber leaves inoculated with Pseudomonas syringae pv lachrymans (Psl) with those in non-inoculated systemic leaves, by examining the changes in the nicotinamide adenine dinucleotides pools, the concentration of soluble carbohydrates and activities/gene expression of carbohydrate metabolism-related enzymes, the expression of photosynthesis-related genes, and the tricarboxylic acid cycle-linked metabolite contents and enzyme activities. In the infected leaves, Psl induced a metabolic signature with an altered [NAD(P)H]/[NAD(P)+] ratio; decreased glucose and sucrose contents, along with a changed invertase gene expression; and increased glucose turnover and accumulation of raffinose, trehalose, and myo-inositol. The accumulation of oxaloacetic and malic acids, enhanced activities, and gene expression of fumarase and l-malate dehydrogenase, as well as the increased respiration rate in the infected leaves, indicated that Psl induced the tricarboxylic acid cycle. The changes in gene expression of ribulose-l,5-bis-phosphate carboxylase/oxygenase large unit, phosphoenolpyruvate carboxylase and chloroplast glyceraldehyde-3-phosphate dehydrogenase were compatible with a net photosynthesis decline described earlier. Psl triggered metabolic changes common to the infected and non-infected leaves, the dynamics of which differed quantitatively (e.g., malic acid content and metabolism, glucose-6-phosphate accumulation, and glucose-6-phosphate dehydrogenase activity) and those specifically related to the local or systemic response (e.g., changes in the sugar content and turnover). Therefore, metabolic changes in the systemic leaves may be part of the global effects of local infection on the whole-plant metabolism and also represent a specific acclimation response contributing to balancing growth and defense.

Oxaloacetate acid ameliorates paraquat-induced acute lung injury by alleviating oxidative stress and mitochondrial dysfunction.

Acute lung injury (ALI) is the primary cause of death among patients with acute paraquat (PQ) poisoning, whereby peroxidative damage is an important mechanism underlying PQ-induced lung injury. There is a lack of effective interventional drugs for patients with PQ poisoning. Oxaloacetic acid (OAA) participates in multiple in vivo metabolic processes, whereby it facilitates the clearance of reactive oxygen species (ROS) and improves mitochondrial function. The study aimed to assess the protective effects of OAA on PQ-induced ALI and elucidate the underlying molecular mechanism. Our data demonstrated that OAA treatment significantly alleviated PQ-induced ALI and improved the survival rate of PQ-poisoned mice, and also alleviated PQ-induced cellular oxidative stress and mitochondrial dysfunction. OAA-mediated alleviation of PQ-induced mitochondrial dysfunction depends on the following mechanisms which may explain the above findings: 1) OAA effectively cleared intracellular ROS, inhibited ROS accumulation, and mitochondrial depolarization; 2) OAA inhibited the downregulation of L-OPA1 and MFN2 caused by PQ and promoted a dynamic balance of mitochondrial fusion and fission, and 3) the expression of PGC-1α, TFAM, COX2, and COX4I1, increased significantly following OAA intervention which improved mitochondrial respiratory functions and promoted its biogenesis and energy metabolism in damaged cells. In conclusion, OAA effectively cleared ROS and improved mitochondrial dysfunction, thereby significantly improving ALI caused by PQ poisoning and the animal survival rate. Therefore, OAA may be a potential drug for the treatment of PQ poisoning.

Effects of the potassium application rate on lipid synthesis and eating quality of two rice cultivars

Lipid content has an important effect on rice eating quality, but the effects of fertilizer application rate on the lipid synthesis and eating quality of rice are not well understood. Potassium (K) has a strong influence on rice quality and the requirement for K fertilizer in rice is greater than for nitrogen (N) and phosphorus (P) fertilizers. To investigate the effects of K fertilizer on the lipid synthesis and eating quality of rice, we used Nanjing 9108 (NJ9108, japonica) and IR72 (indica) rice as experimental materials and four K levels: K0 (0 kg ha–1), K1 (90 kg ha–1), K2 (135 kg ha–1) and K3 (180 kg ha–1). The results showed that the lipid content, free fatty acid (FFA) content, unsaturated fatty acid (UFA) content, malonyl-CoA (MCA) content, phosphatidic acid (PA) content, lipid synthesis-related enzyme activities and eating quality first increased and then decreased with increasing K in both cultivars. The maximum values were obtained under K2. However, the saturated fatty acid (SFA) content showed the opposite trend. No significant differences were found in pyruvate (PYR) content among the K treatments. The protein and oxaloacetic acid (OAA) contents and phosphoenolpyruvate carboxylase (PEPCase) activity of NJ9108 first decreased and then increased with increasing K, and the minimum values were obtained under K2; while IR72 showed the opposite trend and the maximum values were obtained under K1. Overall, increasing K optimized the fatty acid components and increased the lipid content and eating quality of rice by enhancing lipid synthesis-related enzyme activities and regulating substrate competition for lipid and protein synthesis. The optimal K application rate for lipid synthesis, eating quality and grain yield was 135 kg ha–1 for both cultivars.

Nutritional Component Analyses in Different Varieties of Actinidia eriantha Kiwifruit by Transcriptomic and Metabolomic Approaches.

Actinidia eriantha is a unique germplasm resource for kiwifruit breeding. Genetic diversity and nutrient content need to be evaluated prior to breeding. In this study, we looked at the metabolites of three elite A. eriantha varieties (MM-11, MM-13 and MM-16) selected from natural individuals by using a UPLC-MS/MS-based metabolomics approach and transcriptome, with a total of 417 metabolites identified. The biosynthesis and metabolism of phenolic acid, flavonoids, sugars, organic acid and AsA in A. eriantha fruit were further analyzed. The phenolic compounds accounted for 32.37% of the total metabolites, including 48 phenolic acids, 60 flavonoids, 7 tannins and 20 lignans and coumarins. Correlation analysis of metabolites and transcripts showed PAL (DTZ79_15g06470), 4CL (DTZ79_26g05660 and DTZ79_29g0271), CAD (DTZ79_06g11810), COMT (DTZ79_14g02670) and FLS (DTZ79_23g14660) correlated with polyphenols. There are twenty-three metabolites belonging to sugars, the majority being sucrose, glucose arabinose and melibiose. The starch biosynthesis-related genes (AeglgC, AeglgA and AeGEB1) were expressed at lower levels compared with metabolism-related genes (AeamyA and AeamyB) in three mature fruits of three varieties, indicating that starch was converted to soluble sugar during fruit maturation, and the expression level of SUS (DTZ79_23g00730) and TPS (DTZ79_18g05470) was correlated with trehalose 6-phosphate. The main organic acids in A. eriantha fruit are citric acid, quinic acid, succinic acid and D-xylonic acid. Correlation analysis of metabolites and transcripts showed ACO (DTZ79_17g07470) was highly correlated with citric acid, CS (DTZ79_17g00890) with oxaloacetic acid, and MDH1 (DTZ79_23g14440) with malic acid. Based on the gene expression, the metabolism of AsA acid was primarily through the L-galactose pathway, and the expression level of GMP (DTZ79_24g08440) and MDHAR (DTZ79_27g01630) highly correlated with L-Ascorbic acid. Our study provides additional evidence for the correlation between the genes and metabolites involved in phenolic acid, flavonoids, sugars, organic acid and AsA synthesis and will help to accelerate the kiwifruit molecular breeding approaches.

Antibacterial mechanism of polysaccharides from the leaves of Lindera aggregata (Sims) Kosterm. by metabolomics based on HPLC/MS

Lindera aggregata (Sims) Kosterm. is a traditional Chinese herb, which has been proven to have excellent antibacterial activity. In this work, we firstly extracted the polysaccharides from the leaves of Lindera aggregata (Sims) Kosterm. (LLPs), and explored their antibacterial activity and related mechanisms. The experimental results show that LLPs are a good antibacterial agent, which can damage the cell structure of bacteria and lead to the leakage of intracellular lysates. Compared with Escherichia coli (E. coli), LLPs showed better inhibitory activity against Staphylococcus aureus (S. aureus). Furthermore, the administration of LLPs not only led to the upregulation of the levels of fructose-1,6-bisphosphate (F-1,6-P) and citric acid in the glycolysis and tricarboxylic acid cycle pathways in bacteria, but also resulted in the down-regulation of the levels of oxaloacetate (OAA) and 1,3-diphosphoglycerate (1,3-BPG). This study confirmed that LLPs have good antibacterial activity, and broaden the application of the leaves of Lindera aggregata (Sims) Kosterm. in the antibacterial field. It provides ideas for exploring the antibacterial mechanism of active ingredients of Chinese herbal medicine through metabolomics.

Optimum flux rerouting for efficient production of naringenin from acetate in engineered Escherichia coli

BackgroundMicrobial production of naringenin has received much attention owing to its pharmaceutical applicability and potential as a key molecular scaffold for various flavonoids. In the microbial fermentation, a cheap and abundant feedstock is required to achieve an economically feasible bioprocess. From this perspective, utilizing acetate for naringenin production could be an effective strategy, with the advantages of both low-cost and abundant feedstock. For the efficient production of naringenin using acetate, identification of the appropriate regulatory node of carbon flux in the biosynthesis of naringenin from acetate would be important. While acetyl-CoA is a key precursor for naringenin production, carbon flux between the TCA cycle and anaplerosis is effectively regulated at the isocitrate node through glyoxylate shunt in acetate metabolism. Accordingly, appropriate rerouting of TCA cycle intermediates from anaplerosis into naringenin biosynthesis via acetyl-CoA replenishment would be required.ResultsThis study identified the isocitrate and oxaloacetate (OAA) nodes as key regulatory nodes for the naringenin production using acetate. Precise rerouting at the OAA node for enhanced acetyl-CoA was conducted, avoiding extensive loss of OAA by fine-tuning the expression of pckA (encoding phosphoenolpyruvate carboxykinase) with flux redistribution between naringenin biosynthesis and cell growth at the isocitrate node. Consequently, the flux-optimized strain exhibited a significant increase in naringenin production, a 27.2-fold increase (with a 38.3-fold increase of naringenin yield on acetate) over that by the unoptimized strain, producing 97.02 mg/L naringenin with 21.02 mg naringenin/g acetate, which is a competitive result against those in previous studies on conventional substrates, such as glucose.ConclusionsCollectively, we demonstrated efficient flux rerouting for maximum naringenin production from acetate in E. coli. This study was the first attempt of naringenin production from acetate and suggested the potential of biosynthesis of various flavonoids derived from naringenin using acetate.

Soil drought decreases oil synthesis and increases protein synthesis in cottonseed kernel during the flowering and boll formation of cotton

In cotton production, drought is particularly prominent in the flowering and boll formation stage, which significantly affects the cottonseed yield and quality. As the two main storage products in cottonseed kernel, the formation of oil and protein will be affected inescapably under soil drought. Still, the effects of soil drought on the synthesis of cottonseed oil and protein are poorly investigated. To this end, experiments were conducted using two cultivars with contrasting drought tolerance (Dexiamian 1 and Yuzaomian 9110) under three water levels [soil relative water content: control (75 ± 5)%, mild drought (60 ± 5)% and severe drought (45 ± 5)%] from 2017 to 2019. We examined the responses of metabolic activities in cottonseed kernels to drought. Otherwise, 13 CO 2 and 15 N-urea isotope labeling were applied to verify the trend of oil and protein accumulation under soil drought. Results showed that drought decreased cottonseed yield per plant due to the decreases of single-seed weight and cottonseed number. And drought decreased oil content (% dry weight −1 ) and increased protein content (% dry weight −1 ) in cottonseed kernel. According to the findings, drought decreased 13 C content and limited the flow of carbon into oil synthesis pathway; moreover, drought decreased the expression of GhPEPC1 and GhDGAT and activities of phosphoenolpyruvate carboxylase (PEPCase) and diacylglycerol acyltransferase (DGAT), which restrained oil accumulation. Increased protein content under drought was attributed to the increased translocation of plant nitrogen to cottonseed kernel, higher expression of GhGS and GhGOGAT , glutamine synthetase (GS) and glutamate synthase (GOGAT) activities. The Yuzaomian 9110 was more sensitive to drought stress with a higher loss of cottonseed yield and yield components than Dexiamian 1. This study provides insight into the physiological mechanisms of cottonseed oil and protein synthesis under drought and provides a theoretical basis for the utilization of cottonseed oil and protein in the future. Annotation: PEP, phosphoenolpyruvic acid; OAA, oxaloacetic acid; TCA, tricarboxylic acid cycle; Ace-CoA, acetyl coenzyme A; Mal-CoA, Malonyl mono-CoA; G3P, glyceraldehyde 3-phosphate; DAG, diacylglycerol; TAG, triacylglycerol; NADPH, nicotinamide adenine dinucleotide phosphate; PEPCase, Phosphoenolpyruvate carboxylase; ACCase, acetyl-CoA carboxylase; DGAT, diacylglycerol acyltransferase; GS, glutamine synthetase; GOGAT, glutamate synthase; AST, aspartate aminotransferase; ALT, alanine aminotransferase. • Cottonseed yield was significantly decreased under soil drought. • Drought stress significantly restrained 13 C into oil in cottonseed kernels, and enhanced 15 N into kernels. • The oil content was restrained under drought due to lower carbon substrates and enzyme activities. • The increase of nitrogen accumulation and protein synthesis enhanced kernel protein content under drought.

Assessment of plasma amino acids, purines, tricarboxylic acid cycle metabolites, and lipids levels in NSCLC patients based on LC-MS/MS quantification

Non-small cell lung cancer (NSCLC) is the most common type of malignant tumor of the lung with poor prognosis. Currently, there is still no effective strategy for diagnosing lung cancer from the perspective of multiple biomarkers containing both polar and nonpolar molecules. In order to explore the pathological changes of NSCLC at the endogenous molecule levels, and further establish the strategy for identifying and monitoring drug efficacy of NSCLC, targeted metabolomics and lipidomics studies were established with NSCLC patients. Polar metabolites including 21 amino acids, 7 purines, 6 tricarboxylic acid (TCA) cycle metabolites, and nonpolar lipids like phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), sphingomyelin (SM), and ceramide (Cer), diacylglycerol (DG), triacylglycerol (TG), were quantitatively determined based on LC-MS/MS, taking into account their metabolism were significantly concerned with the occurrence of lung cancer in previous study. As a result, 14 polar metabolites and 16 lipids were prominently altered in the plasma of NSCLC patients, among which, after multivariate statistical analysis, LPC 18:0 (sn-2), L-Phenylalanine (Phe), oxaloacetic acid (OAA) and xanthine (XA) were screened out as potential small molecules and lipid biomarkers for NSCLC. Furthermore, a new strategy for formulating equation of NSCLC identification was proposed and clinical utility was successfully evaluated through Kangai injection treatment to NSCLC patients. Taking together, this study investigated the pathological changes of NSCLC from the perspective of endogenous polar and nonpolar molecules, and shed a light on identification of NSCLC.

Spore Powder of Paecilomyces hepiali Shapes Gut Microbiota to Relieve Exercise-Induced Fatigue in Mice.

Paecilomyces hepiali, a fungal strain isolated from natural Ophiocordyceps sinensis, contains similar pharmacologically active components, has been used widely as a substitute of O. sinensis in functional food and medicine. However, the components and anti-fatigue effects of P.hepiali spores and their mechanisms of action are largely unknown. Here, we compared the chemical composition in P.hepiali spore (HPS) and mycelium (HPM) by liquid chromatography with tandem mass spectrometry analysis. We found 85 metabolites with significant differences, and HPS contains more L-Malic acid, Oxalacetic acid, Fructose-1,6-bisphosphate, and L-Arginine than HPM. Then we evaluated their anti-fatigue effects and regulatory effects on the gut microbiota in mice. The forced swimming time (SW) was only significantly increased in HPS groups: the high and low dose of the HPS group was 101% and 72% longer than the control group, respectively. Both HPS and HPM treatment decreased lactic acid, blood urea nitrogen, creatine kinase while increased lactate dehydrogenase (LDH) levels in the blood. Moreover, mice treated with HPS and HPM showed less skeletal muscle fiber spacing and breakage. The relative abundance of Alistips, Eubacterium, Bacterium, Parasutterella, and Olsenella in the gut microbiota of the HPS group was higher than that in the HPM group through 16S rRNA gene sequencing analysis. These changes may be related to the regulation of nucleotide, amino acid, and carbohydrate metabolism. Correlation analysis between the gut microbiota and fatigue-related indicators suggested that Alistips, Clostridium, Akkermansia, Olsenella, and Lactobacillus were positively correlated with the SW and LDH content. Our findings demonstrated that HPS has beneficial anti-fatigue effects by regulating gut microbiota.

Plasma Metabolomics and Machine Learning-Driven Novel Diagnostic Signature for Non-Alcoholic Steatohepatitis.

We performed targeted metabolomics with machine learning (ML)-based interpretation to identify metabolites that distinguish the progression of nonalcoholic fatty liver disease (NAFLD) in a cohort. Plasma metabolomics analysis was conducted in healthy control subjects (n = 25) and patients with NAFL (n = 42) and nonalcoholic steatohepatitis (NASH, n = 19) by gas chromatography-tandem mass spectrometry (MS/MS) and liquid chromatography-MS/MS as well as RNA sequencing (RNA-seq) analyses on liver tissues from patients with varying stages of NAFLD (n = 12). The resulting metabolomic data were subjected to routine statistical and ML-based analyses and multi-omics interpretation with RNA-seq data. We found 6 metabolites that were significantly altered in NAFLD among 79 detected metabolites. Random-forest and multinomial logistic regression analyses showed that eight metabolites (glutamic acid, cis-aconitic acid, aspartic acid, isocitric acid, α-ketoglutaric acid, oxaloacetic acid, myristoleic acid, and tyrosine) could distinguish the three groups. Then, the recursive partitioning and regression tree algorithm selected three metabolites (glutamic acid, isocitric acid, and aspartic acid) from these eight metabolites. With these three metabolites, we formulated an equation, the MetaNASH score that distinguished NASH with excellent performance. In addition, metabolic map construction and correlation assays integrating metabolomics data into the transcriptome datasets of the liver showed correlations between the concentration of plasma metabolites and the expression of enzymes governing metabolism and specific alterations of these correlations in NASH. Therefore, these findings will be useful for evaluation of altered metabolism in NASH and understanding of pathophysiologic implications from metabolite profiles in relation to NAFLD progression.

Markov State Study of Electrostatic Channeling within the Tricarboxylic Acid Cycle Supercomplex.

The high efficiency of cascade reactions in supramolecular enzyme nanoassemblies, known as metabolons, has attracted substantial attention in various fields ranging from fundamental biochemistry and molecular biology to recent applications in biofuel cells, biosensors, and chemical synthesis. One reason for the high efficiency of metabolons is the structures formed by sequential enzymes that allow the direct transport of intermediates between consecutive active sites. The supercomplex of malate dehydrogenase (MDH) and citrate synthase (CS) is an ideal example of the controlled transport of intermediates via electrostatic channeling. Here, using a combination of molecular dynamics (MD) simulations and a Markov state model (MSM), we examined the transport process of the intermediate oxaloacetate (OAA) from MDH to CS. The MSM enables the identification of the dominant transport pathways of OAA from MDH to CS. Analysis of all pathways using a hub score approach reveals a small set of residues that control OAA transport. This set includes an arginine residue previously identified experimentally. MSM analysis of a mutated complex, where the identified arginine is replaced by alanine, led to a 2-fold decrease in transfer efficiency, also consistent with experimental results. This work provides a molecular-level understanding of the electrostatic channeling mechanism and will enable the further design of catalytic nanostructures utilizing electrostatic channeling.