Recent Advances in Metabolic Engineering for the Biosynthesis of Phosphoenol Pyruvate-Oxaloacetate-Pyruvate-Derived Amino Acids.

The phosphoenol pyruvate-oxaloacetate-pyruvate-derived amino acids (POP-AAs) comprise native intermediates in cellular metabolism, within which the phosphoenol pyruvate-oxaloacetate-pyruvate (POP) node is the switch point among the major metabolic pathways existing in most living organisms. POP-AAs have widespread applications in the nutrition, food, and pharmaceutical industries. These amino acids have been predominantly produced in Escherichia coli and Corynebacterium glutamicum through microbial fermentation. With the rapid increase in market requirements, along with the global food shortage situation, the industrial production capacity of these two bacteria has encountered two bottlenecks: low product conversion efficiency and high cost of raw materials. Aiming to push forward the update and upgrade of engineered strains with higher yield and productivity, this paper presents a comprehensive summarization of the fundamental strategy of metabolic engineering techniques around phosphoenol pyruvate-oxaloacetate-pyruvate node for POP-AA production, including L-tryptophan, L-tyrosine, L-phenylalanine, L-valine, L-lysine, L-threonine, and L-isoleucine. Novel heterologous routes and regulation methods regarding the carbon flux redistribution in the POP node and the formation of amino acids should be taken into consideration to improve POP-AA production to approach maximum theoretical values. Furthermore, an outlook for future strategies of low-cost feedstock and energy utilization for developing amino acid overproducers is proposed.

Precision Medicine for Blood Glutamate Grabbing in Ischemic Stroke.

Glutamate grabbers, such as glutamate oxaloacetate transaminase (GOT), have been proposed to prevent excitotoxicity secondary to high glutamate levels in stroke patients. However, the efficacy of blood glutamate grabbing by GOT could be dependent on the extent and severity of the disruption of the blood-brain barrier (BBB). Our purpose was to analyze the relationship between GOT and glutamate concentration with the patient's functional status differentially according to BBB serum markers (soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) and leukoaraiosis based on neuroimaging). This retrospective observational study includes 906 ischemic stroke patients. We studied the presence of leukoaraiosis and the serum levels of glutamate, GOT, and sTWEAK in blood samples. Functional outcome was assessed using the modified Rankin Scale (mRS) at 3 months. A significant negative correlation between GOT and glutamate levels at admission was shown in those patients with sTWEAK levels > 2900 pg/mL (Pearson's correlation coefficient: -0.249; p < 0.0001). This correlation was also observed in patients with and without leukoaraiosis (Pearson's correlation coefficients: -0.299; p < 0.001 vs. -0.116; p = 0.024). The logistic regression model confirmed the association of higher levels of GOT with lower odds of poor outcome at 3 months when sTWEAK levels were >2900 pg/mL (OR: 0.41; CI 95%: 0.28-0.68; p < 0.0001) or with leukoaraiosis (OR: 0.75; CI 95%: 0.69-0.82; p < 0.0001). GOT levels are associated with glutamate levels and functional outcomes at 3 months, but only in those patients with leukoaraiosis and elevated sTWEAK levels. Consequently, therapies targeting glutamate grabbing might be more effective in patients with BBB dysfunction.

Effect of the mitochondrial transaminase (GOT2) on membrane potential-sensitive respiration in mitochondria of differentiated C2C12 muscle cells.

We previously showed that the transaminase inhibitor, aminooxyacetic acid, reduced respiration energized at complex II (succinate dehydrogenase, SDH) in mitochondria isolated from mouse hindlimb muscle. The effect required a reduction in membrane potential with resultant accumulation of oxaloacetate (OAA), a potent inhibitor of SDH. To specifically assess the effect of the mitochondrial transaminase, glutamic oxaloacetic transaminase (GOT2) on complex II respiration, and to determine the effect in intact cells as well as isolated mitochondria, we performed respiratory and metabolic studies in wildtype (WT) and CRISPR-generated GOT2 knockdown (KD) C2C12 myocytes. Intact cell respiration by GOT2KD cells versus WT was reduced by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to lower potential. In mitochondria of C2C12 KD cells, respiration at low potential generated by 1 µM FCCP and energized at complex II by 10 mM succinate + 0.5 mM glutamate (but not by complex I substrates) was reduced versus WT mitochondria. Although we could not detect OAA, metabolite data suggested that OAA inhibition of SDH may have contributed to the FCCP effect. C2C12 mitochondria differed from skeletal muscle mitochondria in that the effect of FCCP on complex II respiration was not evident with ADP addition. We also observed that C2C12 cells, unlike skeletal muscle, expressed glutamate dehydrogenase, which competes with GOT2 for glutamate metabolism. In summary, GOT2 KD reduced C2C12 respiration in intact cells at low potential. From differential substrate effects, this occurred largely at complex II. Moreover, C2C12 versus muscle mitochondria differ in complex II sensitivity to ADP and differ markedly in expression of glutamate dehydrogenase.NEW & NOTEWORTHY Impairment of the mitochondrial transaminase, GOT2, reduces complex II (succinate dehydrogenase, SDH)-energized respiration in C2C12 myocytes. This occurs only at low inner membrane potential and is consistent with inhibition of SDH. Incidentally, we observed that C2C12 mitochondria compared with muscle tissue mitochondria differ in sensitivity of complex II respiration to ADP and in the expression of glutamate dehydrogenase.

Oxaloacetate as new inducer for osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

BACKGROUND: Mitochondrial organelles play a crucial role in cellular metabolism so different cell types exhibit diverse metabolic and energy demands. Therefore, alternations in the intracellular distribution, quantity, function, and structure of mitochondria are required for stem cell differentiation. Finding an effective inducer capable of modulating mitochondrial activity is critical for the differentiation of specific stem cells into osteo-like cells for addressing issues related to osteogenic disorders. This study aimed to investigate the effect of oxaloacetate (OAA) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro. METHODS AND RESULTS: First, the most favorable OAA concentration was measured through MTT assay and subsequently confirmed using acridine orange staining. Human ADSCs were cultured in osteogenic medium supplemented with OAA and analyzed on days 7 and 14 of differentiation. Various assays including alkaline phosphatase assay (ALP), cellular calcium content assay, mineralized matrix staining with alizarin red, catalase (CAT) and superoxide dismutase (SOD) activity, and real-time RT-PCR analysis of three bone-specific markers (ALP, osteocalcin, and collagen type I) were conducted to characterize the differentiated cells. Following viability assessment, OAA at a concentration of 1 µM was considered the optimal dosage for further studies. The results of osteogenic differentiation assays showed that OAA at a concentration of 1 × 10- 6 M significantly increased ALP enzyme activity, mineralization, CAT and SOD activity and the expression of bone-specific genes in differentiated cells compared to control groups in vitro. CONCLUSIONS: In conclusion, the fundings from this study suggest that OAA possesses favorable properties that make it a potential candidate for application in medical bone regeneration.

Tartrate fermentation with H2 production by a new member of Sporomusaceae enriched from rice paddy soil.

In rice paddies, soil and plant-derived organic matter are degraded anaerobically to methane (CH4), a powerful greenhouse gas. The highest rate of methane emission occurs during the reproductive stage of the plant when mostly dicarboxylic acids are exudated by the roots. The emission of methane at this stage depends largely on the cooperative interaction between dicarboxylic acid-fermenting bacteria and methanogenic archaea in the rhizosphere. The fermentation of tartrate, one of the major acids exudated, has been scarcely explored in rice paddy soils. In this work, we characterized an anaerobic consortium from rice paddy soil composed of four bacterial strains, whose principal member (LT8) can ferment tartrate, producing H2 and acetate. Tartrate fermentation was accelerated by co-inoculation with a hydrogenotrophic methanogen. The assembled genome of LT8 possesses a Na+-dependent oxaloacetate decarboxylase and shows that this bacterium likely invests part of the H2 produced to reduce NAD(P)+ to assimilate C from tartrate. The phylogenetic analysis of the 16S rRNA gene, the genome-based classification as well as the average amino acid identity (AAI) indicated that LT8 belongs to a new genus within the Sporomusaceae family. LT8 shares a few common features with its closest relatives, for which tartrate degradation has not been described. LT8 is limited to a few environments but is more common in rice paddy soils, where it might contribute to methane emissions from root exudates.IMPORTANCEThis is the first report of the metabolic characterization of a new anaerobic bacterium able to degrade tartrate, a compound frequently associated with plants, but rare as a microbial metabolite. Tartrate fermentation by this bacterium can be coupled to methanogenesis in the rice rhizosphere where tartrate is mainly produced at the reproductive stage of the plant, when the maximum methane rate emission occurs. The interaction between secondary fermentative bacteria, such as LT8, and methanogens could represent a fundamental step in exploring mitigation strategies for methane emissions from rice fields. Possible strategies could include controlling the activity of these secondary fermentative bacteria or selecting plants whose exudates are more difficult to ferment.

Bacillus subtilis YisK possesses oxaloacetate decarboxylase activity and exhibits Mbl-dependent localization.

YisK is an uncharacterized protein in Bacillus subtilis previously shown to interact genetically with the elongasome protein Mbl. YisK overexpression leads to cell widening and lysis, phenotypes that are dependent on mbl and suppressed by mbl mutations. In the present work, we characterize YisK's localization, structure, and enzymatic activity. We show that YisK localizes as puncta that depend on Mbl. YisK belongs to the fumarylacetoacetate hydrolase (FAH) superfamily, and crystal structures revealed close structural similarity to two oxaloacetate (OAA) decarboxylases: human mitochondrial FAHD1 and Corynebacterium glutamicum Cg1458. We demonstrate that YisK can also catalyze the decarboxylation of OAA (K m = 134 µM, K cat = 31 min-1). A catalytic dead variant (YisK E148A, E150A) retains wild-type localization and still widens cells following overexpression, indicating these activities are not dependent on YisK catalysis. Conversely, a non-localizing variant (YisK E30A) retains wild-type enzymatic activity in vitro but localizes diffusely and no longer widens cells following overexpression. Together, these results suggest that YisK may be subject to spatial regulation that depends on the cell envelope synthesis machinery. IMPORTANCE The elongasome is a multiprotein complex that guides lengthwise growth in some bacteria. We previously showed that, in B. subtilis, overexpression of an uncharacterized putative enzyme (YisK) perturbed function of the actin-like elongasome protein Mbl. Here, we show that YisK exhibits Mbl-dependent localization. Through biochemical and structural characterization, we demonstrate that, like its mitochondrial homolog FAHD1, YisK can catalyze the decarboxylation of the oxaloacetate to pyruvate and CO2. YisK is the first example of an enzyme implicated in central carbon metabolism with subcellular localization that depends on Mbl.

Effect of biosal®, deltamethrin and lambda-cyhalothrin on the activity of GOT, GPT and total protein contents in two fodder pests Hermolaus modestus and Hermolaus ocimumi

Abstract The assessment of the comparative effect of biosal (phytopesticide), deltamethrin, and lambda-cyhalothrin (pyrethroids) were made against two fodder pests, Hermolaus modestus and Hermolaus ocimumi by filter paper impregnation method. The activity of total protein contents, GPT (glutamic-pyruvic transaminase) and GOT (glutamic oxaloacetate transaminase) were affected in Hermolaus modestus and Hermolaus ocimumi against biosal, deltamethrin, and lambda cyhalothrin. The activity of total protein contents in H. modestus was 31.053%, 4.607%, and 24.575%, against biosal, deltamethrin, and lambda-cyhalothrin, respectively. The activity of total protein contents was observed as 24.202%, 15.25%, and 56.036% against deltamethrin, lambda-cyhalothrin, and biosal, respectively in H. ocimumi. The activity of GOT was observed as 98.675% for biosal 33.95% for deltamethrin and 83.619% for lambda-cyhalothrin in H. modestus. The GOT activity was estimated in H. ocimumi as 78.831%, 47.645%, and 71.287% against biosal, deltamethrin, and lambda-cyhalothrin, respectively. The efficacy of GPT enzyme against biosal, deltamethrin, and lambda-cyhalothrin was calculated as 89.26%, 73.07%, and 47.58%, respectively in H. modestus. The H. ocimumi showed GPT activity as 77.58% for biosal, 68.84% for deltamethrin, and 52.67% for lambda-cyhalothrin, respectively.

Resumo A avaliação do efeito comparativo do biosal (fitopesticida), deltametrina e lambda-cialotrina (piretróides) foi feita contra duas pragas forrageiras, Hermolaus modestus e Hermolaus ocimumi, pelo método de impregnação com papel de filtro. A atividade do conteúdo de proteína total, GPT (transaminase glutâmico-pirúvica) e GOT (oxaloacetato transaminase glutâmico) foram afetados em Hermolaus modestus e Hermolaus ocimumi contra biosal, deltametrina e lambda cialotrina. A atividade do conteúdo de proteína total em H. modestus foi 31.053%, 4.607% e 24.575%, contra biosal, deltametrina e lambda-cialotrina, respectivamente. A atividade do conteúdo de proteína total foi observada como 24.202%, 15.25% e 56,036% contra deltametrina, lambda-cialotrina e biosal, respectivamente em H. ocimumi. A atividade do GOT foi observada em 98.675% para o biosal, 33,95% para a deltametrina e 83.619% para a lambda-cialotrina em H. modestus. A atividade do GOT foi estimada em H. ocimumi como 78.831%, 47.645% e 71.287% contra biosal, deltametrina e lambda-cialotrina, respectivamente. A eficácia da enzima GPT contra biosal, deltametrina e lambda-cialotrina foi calculada como 89.26%, 73.07% e 47.58%, respectivamente em H. modestus. A H. ocimumi apresentou atividade GPT de 77.58% para biosal, 68.84% para deltametrina e 52.67% para lambda-cialotrina, respectivamente.

Effect of biosal®, deltamethrin and lambda-cyhalothrin on the activity of GOT, GPT and total protein contents in two fodder pests Hermolaus modestus and Hermolaus ocimumi / Efeito do biosal®, deltametrina e lambda-cialotrina na atividade do GOT, GPT e conteúdo de proteína total em duas pragas forrageiras Hermolaus modestus e Hermolaus ocimumi

Abstract The assessment of the comparative effect of biosal (phytopesticide), deltamethrin, and lambda-cyhalothrin (pyrethroids) were made against two fodder pests, Hermolaus modestus and Hermolaus ocimumi by filter paper impregnation method. The activity of total protein contents, GPT (glutamic-pyruvic transaminase) and GOT (glutamic oxaloacetate transaminase) were affected in Hermolaus modestus and Hermolaus ocimumi against biosal, deltamethrin, and lambda cyhalothrin. The activity of total protein contents in H. modestus was 31.053%, 4.607%, and 24.575%, against biosal, deltamethrin, and lambda-cyhalothrin, respectively. The activity of total protein contents was observed as 24.202%, 15.25%, and 56.036% against deltamethrin, lambda-cyhalothrin, and biosal, respectively in H. ocimumi. The activity of GOT was observed as 98.675% for biosal 33.95% for deltamethrin and 83.619% for lambda-cyhalothrin in H. modestus. The GOT activity was estimated in H. ocimumi as 78.831%, 47.645%, and 71.287% against biosal, deltamethrin, and lambda-cyhalothrin, respectively. The efficacy of GPT enzyme against biosal, deltamethrin, and lambda-cyhalothrin was calculated as 89.26%, 73.07%, and 47.58%, respectively in H. modestus. The H. ocimumi showed GPT activity as 77.58% for biosal, 68.84% for deltamethrin, and 52.67% for lambda-cyhalothrin, respectively.

Resumo A avaliação do efeito comparativo do biosal (fitopesticida), deltametrina e lambda-cialotrina (piretróides) foi feita contra duas pragas forrageiras, Hermolaus modestus e Hermolaus ocimumi, pelo método de impregnação com papel de filtro. A atividade do conteúdo de proteína total, GPT (transaminase glutâmico-pirúvica) e GOT (oxaloacetato transaminase glutâmico) foram afetados em Hermolaus modestus e Hermolaus ocimumi contra biosal, deltametrina e lambda cialotrina. A atividade do conteúdo de proteína total em H. modestus foi 31.053%, 4.607% e 24.575%, contra biosal, deltametrina e lambda-cialotrina, respectivamente. A atividade do conteúdo de proteína total foi observada como 24.202%, 15.25% e 56,036% contra deltametrina, lambda-cialotrina e biosal, respectivamente em H. ocimumi. A atividade do GOT foi observada em 98.675% para o biosal, 33,95% para a deltametrina e 83.619% para a lambda-cialotrina em H. modestus. A atividade do GOT foi estimada em H. ocimumi como 78.831%, 47.645% e 71.287% contra biosal, deltametrina e lambda-cialotrina, respectivamente. A eficácia da enzima GPT contra biosal, deltametrina e lambda-cialotrina foi calculada como 89.26%, 73.07% e 47.58%, respectivamente em H. modestus. A H. ocimumi apresentou atividade GPT de 77.58% para biosal, 68.84% para deltametrina e 52.67% para lambda-cialotrina, respectivamente.

Oxaloacetate as a Holy Grail Adjunctive Treatment in Gliomas: A Revisit to Metabolic Pathway.

India experiences a significant amount of morbidity and mortality due to gliomas particularly glioblastoma multiforme (GBM), which ranks among the worst cancers. Oxaloacetate (OAA) is a human keto acid that is central to cellular metabolism; it has been recognized by the US FDA for use in GBM patients, triggering a review to revisit the cellular mechanism of its therapeutic action. Various cellular and molecular studies have proposed that instead of fueling the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), gliomas prefer to use glycolysis (the Warburg effect) to fuel macromolecules for the synthesis of nucleotides, fatty acids, and amino acids for the accelerated mitosis. A study found that oxaloacetate (OAA) inhibits human lactate dehydrogenase A (LDHA) in cancer cells, reversing the Warburg effect. Studies revealed that OAA supplementation reduced Warburg glycolysis, improved neuronal cell bioenergetics, and triggered brain mitochondrial biogenesis, thereby enhancing the efficacy of standard treatment. Similarly, OAA has been found in preclinical investigations to be able to decrease tumor development and survival rates by blocking the conversion of glutamine to alpha-ketoglutarate (alpha-KG) in the TCA cycle and lowering nicotinamide adenine dinucleotide phosphate (NADPH) levels. OAA is a safe adjuvant that has the potential to be an effective therapy in gliomas when combined with temozolomide (TMZ) chemotherapy and routine surgery.

Aspartic Acid in Health and Disease.

Aspartic acid exists in L- and D-isoforms (L-Asp and D-Asp). Most L-Asp is synthesized by mitochondrial aspartate aminotransferase from oxaloacetate and glutamate acquired by glutamine deamidation, particularly in the liver and tumor cells, and transamination of branched-chain amino acids (BCAAs), particularly in muscles. The main source of D-Asp is the racemization of L-Asp. L-Asp transported via aspartate-glutamate carrier to the cytosol is used in protein and nucleotide synthesis, gluconeogenesis, urea, and purine-nucleotide cycles, and neurotransmission and via the malate-aspartate shuttle maintains NADH delivery to mitochondria and redox balance. L-Asp released from neurons connects with the glutamate-glutamine cycle and ensures glycolysis and ammonia detoxification in astrocytes. D-Asp has a role in brain development and hypothalamus regulation. The hereditary disorders in L-Asp metabolism include citrullinemia, asparagine synthetase deficiency, Canavan disease, and dicarboxylic aminoaciduria. L-Asp plays a role in the pathogenesis of psychiatric and neurologic disorders and alterations in BCAA levels in diabetes and hyperammonemia. Further research is needed to examine the targeting of L-Asp metabolism as a strategy to fight cancer, the use of L-Asp as a dietary supplement, and the risks of increased L-Asp consumption. The role of D-Asp in the brain warrants studies on its therapeutic potential in psychiatric and neurologic disorders.

High preoperative blood oxaloacetate and 2-aminoadipic acid levels are associated with postoperative delayed neurocognitive recovery.

Introduction: This study aimed to identify preoperative blood biomarkers related to development of delayed neurocognitive recovery (dNCR) following surgery. Methods: A total of 67 patients (≥65 years old) who underwent head and neck tumor resection under general anesthesia were assessed using the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA). Preoperative serum metabolomics were determined using widely targeted metabolomics technology. Results: Of the 67 patients, 25 developed dNCR and were matched to 25 randomly selected patients from the remaining 42 without dNCR. Differential metabolites were selected using the criteria of variable importance in projection > 1.0 in orthogonal partial least squares discrimination analysis, false discovery rate <0.05, and fold-change >1.2 or <0.83 to minimize false positives. Preoperative serum levels of oxaloacetate (OR: 1.054, 95% CI: 1.027-1.095, P = 0.001) and 2-aminoadipic acid (2-AAA) (OR: 1.181, 95% CI: 1.087-1.334, P = 0.001) were associated with postoperative dNCR after adjusting for anesthesia duration, education, and age. Areas under the curve for oxaloacetate and 2-AAA were 0.86 (sensitivity: 0.84, specificity: 0.88) and 0.86 (sensitivity: 0.84, specificity: 0.84), respectively. High levels of preoperative oxaloacetate and 2-AAA also were associated with postoperative decreased MoCA (ß: 0.022, 95% CI: 0.005-0.04, P = 0.013 for oxaloacetate; ß: 0.077, 95%CI: 0.016-0.137, P = 0.014 for 2-AAA) and MMSE (ß: 0.024, 95% CI: 0.009-0.039, P = 0.002 for oxaloacetate; ß: 0.083, 95% CI: 0.032-0.135, P = 0.002 for 2-AAA) scores after adjusting for age, education level, and operation time. Conclusion: High preoperative blood levels of oxaloacetate and 2-AAA were associated with increased risk of postoperative dNCR. Clinical trial registration: https://classic.clinicaltrials.gov/ct2/show/NCT05105451, identifier NCT05105451.

Roles of malate and aspartate in gluconeogenesis in various physiological and pathological states.

Gluconeogenesis, a pathway for glucose synthesis from non-carbohydrate substances, begins with the synthesis of oxaloacetate (OA) from pyruvate and intermediates of citric acid cycle in hepatocyte mitochondria. The traditional view is that OA does not cross the mitochondrial membrane and must be shuttled to the cytosol, where most enzymes involved in gluconeogenesis are compartmentalized, in the form of malate. Thus, the possibility of transporting OA in the form of aspartate has been ignored. In the article is shown that malate supply to the cytosol increases only when fatty acid oxidation in the liver is activated, such as during starvation or untreated diabetes. Alternatively, aspartate synthesized from OA by mitochondrial aspartate aminotransferase (AST) is transported to the cytosol in exchange for glutamate via the aspartate-glutamate carrier 2 (AGC2). If the main substrate for gluconeogenesis is an amino acid, aspartate is converted to OA via urea cycle, therefore, ammonia detoxification and gluconeogenesis are simultaneously activated. If the main substrate is lactate, OA is synthesized by cytosolic AST, glutamate is transported to the mitochondria through AGC2, and nitrogen is not lost. It is concluded that, compared to malate, aspartate is a more suitable form of OA transport from the mitochondria for gluconeogenesis.

Oxaloacetate regulates complex II respiration in brown fat: dependence on UCP1 expression.

We previously found that skeletal muscle mitochondria incubated at low membrane potential (ΔΨ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low, accumulate oxaloacetate (OAA) in amounts sufficient to inhibit complex II respiration. We proposed a mechanism wherein low ΔΨ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance, we carried out studies of mice with inherently different levels of IBAT mitochondrial inner membrane potential. Isolated complex II (succinate)-energized IBAT mitochondria from obesity-resistant 129SVE mice compared with obesity-prone C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower ΔΨ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower ΔΨ, exhibited much lower respiration, twofold greater OAA concentrations, much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared with the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater ΔΨ, ROS, and NADH, but equal or greater O2 flux compared with WT mitochondria. GDP addition, compared with no GDP, increased ΔΨ and complex II respiration in wild-type (WT) mice associated with much less OAA. Respiration on complex I substrates followed the more classical dynamics of greater respiration at lower ΔΨ. These findings support the abovementioned mechanism for OAA- and ΔΨ-dependent complex II respiration and support its physiological relevance.NEW & NOTEWORTHY We examined mitochondrial respiration initiated at mitochondrial complex II in mice with varying degrees of brown adipose tissue UCP1 expression. We show that, by affecting inner membrane potential, UCP1 expression determines reverse electron transport from complex II to complex I and, consequently, the NADH/NAD+ ratio. Accordingly, this regulates the level of oxaloacetate accumulation and the extent of oxaloacetate inhibition of complex II.

Prebiotic Synthesis of Aspartate Using Life's Metabolism as a Guide.

A protometabolic approach to the origins of life assumes that the conserved biochemistry of metabolism has direct continuity with prebiotic chemistry. One of the most important amino acids in modern biology is aspartic acid, serving as a nodal metabolite for the synthesis of many other essential biomolecules. Aspartate's prebiotic synthesis is complicated by the instability of its precursor, oxaloacetate. In this paper, we show that the use of the biologically relevant cofactor pyridoxamine, supported by metal ion catalysis, is sufficiently fast to offset oxaloacetate's degradation. Cu2+-catalysed transamination of oxaloacetate by pyridoxamine achieves around a 5% yield within 1 h, and can operate across a broad range of pH, temperature, and pressure. In addition, the synthesis of the downstream product ß-alanine may also take place in the same reaction system at very low yields, directly mimicking an archaeal synthesis route. Amino group transfer supported by pyridoxal is shown to take place from aspartate to alanine, but the reverse reaction (alanine to aspartate) shows a poor yield. Overall, our results show that the nodal metabolite aspartate and related amino acids can indeed be synthesised via protometabolic pathways that foreshadow modern metabolism in the presence of the simple cofactor pyridoxamine and metal ions.

Oxaloacetate enhances and accelerates regeneration in young mice by promoting proliferation and mineralization.

Cell metabolism coordinates the biochemical reactions that produce carbon and ATP in order for the cell to proliferate, differentiate, and respond to environmental changes. Cell type determines metabolic demand, so proliferating skeletal progenitors and differentiated osteoblasts exhibit different levels of cell metabolism. Limb regeneration is an energetically demanding process that involves multiple types of tissues and cell functions over time. Dysregulation of cell metabolism in aged mice results in impaired regeneration, a defect that can be rescued in part by the administration of oxaloacetate (OAA). A better understanding of how cell metabolism regulates regeneration in general, and how these changes can be modulated to benefit potential regenerative strategies in the future is needed. Here we sought to better understand the effects of OAA on young mice and determine whether the same mechanism could be tapped to improve regeneration without an aged-defect. We also asked which dosing time periods were most impactful for promoting regenerative outcomes, and whether these effects were sustained after dosing was stopped. Consistent with our findings in aged mice we found that OAA enhanced regeneration by accelerating bone growth, even beyond control measures, by increasing trabecular thickness, decreasing trabecular spacing, and improving the patterning by decreasing the taper, making the regenerated bone more like an unamputated digit. Our data suggests that the decrease in spacing, an improvement over aged mice, may be due to a decrease in hypoxia-driven vasculature. Our findings suggest that OAA, and similar metabolites, may be a strong tool to promote regenerative strategies and investigate the mechanisms that link cell metabolism and regeneration.

Protective effect of glutamic-oxaloacetic transaminase on hippocampal neurons in Alzheimer's disease using model mice.

Alzheimer's disease (AD), a neurodegenerative disease affecting the elderly, frequently causes cognitive impairment and memory decline, and there are currently no effective therapeutic drugs available. Glutamate excitotoxicity is one of the pathogeneses of AD, and there is evidence that glutamic-oxaloacetic transaminase (GOT) can significantly reduce glutamate concentrations in the hippocampi of mice, but its role in APP/PS1 transgenic mice is unknown. We investigated the improvement of neurological function and related protein expression following subcutaneous injection of GOT in mice with AD. We performed immunohistochemical staining on the brain tissue of 3-, 6-, and 12-month-old mice and found that the content of the ß-amyloid protein Aß1-42 in the 6 months old GOT treatment group was significantly reduced. Meanwhile, the APP-GOT group outperformed the APP group in the water maze and spatial object recognition experiments. The number of neurons in the hippocampal CA1 area of the APP-GOT group increased when compared to the APP group according to Nissl staining. Electron microscopic examination of the hippocampal CA1 area demonstrated that the number of synapses in the APP-GOT group was more than that in the APP group, and the mitochondrial structure was relatively complete. Finally, the protein content of the hippocampus was detected. In comparison to the APP group, SIRT1 content increased in the APP-GOT group whereas Aß1-42 content decreased, and Ex527 could reverse this trend. These results suggest that GOT can significantly improve the cognitive function of mice in the early stage of AD, and the underlying mechanism may be through decreasing Aß1-42 and increasing SIRT1 expressions.

Vibrio cholerae Alkalizes Its Environment via Citrate Metabolism to Inhibit Enteric Growth In Vitro.

Vibrio cholerae is a Gram-negative pathogen, living in constant competition with other bacteria in marine environments and during human infection. One competitive advantage of V. cholerae is the ability to metabolize diverse carbon sources, such as chitin and citrate. We observed that when some V. cholerae strains were grown on a medium with citrate, the medium's chemical composition turned into a hostile alkaline environment for Gram-negative bacteria, such as Escherichia coli and Shigella flexneri. We found that although the ability to exclude competing bacteria was not contingent on exogenous citrate, V. cholerae C6706 citrate metabolism mutants ΔoadA-1, ΔcitE, and ΔcitF were not able to inhibit S. flexneri or E. coli growth. Lastly, we demonstrated that while the V. cholerae C6706-mediated increased medium pH was necessary for the enteric exclusion phenotype, secondary metabolites, such as bicarbonate (protonated to carbonate in the raised pH) from the metabolism of citrate, enhanced the ability to inhibit the growth of E. coli. These data provide a novel example of how V. cholerae outcompetes other Gram-negative bacteria. IMPORTANCE Vibrio cholerae must compete with other bacteria in order to cause disease. Here, we show that V. cholerae creates an alkaline environment, which is able to inhibit the growth of other enteric bacteria. We demonstrate that V. cholerae environmental alkalization is linked to the capacity of the bacteria to metabolize citrate. This behavior could potentially contribute to V. cholerae's ability to colonize the human intestine.

Effects of glutamate oxaloacetate transaminase on reactive oxygen species in Ganoderma lucidum.

Nitrogen metabolism can regulate mycelial growth and secondary metabolism in Ganoderma lucidum. As an important enzyme in intracellular amino acid metabolism, glutamate oxaloacetate transaminase (GOT) has many physiological functions in animals and plants, but its function in fungi has been less studied. In the present study, two GOT isoenzymes were found in G. lucidum; one is located in the mitochondria (GOT1), and the other is located in the cytoplasm (GOT2). The reactive oxygen species (ROS) level was increased in got1 silenced strains and was approximately 1.5-fold higher than that in the wild-type (WT) strain, while silencing got2 did not affect the ROS level. To explore how GOT affects ROS in G. lucidum, experiments related to the generation and elimination of intracellular ROS were conducted. First, compared with that in the WT strain, the glutamate content, one of the substrates of GOT, decreased when got1 or got2 was knocked down, and the glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH) content decreased by approximately 38.6%, 19.3%, and 40.1% in got1 silenced strains, got2 silenced strains, and got1/2 co-silenced strains respectively. Second, GOT also affects glucose metabolism. The pyruvate (PA), acetyl-CoA and α-ketoglutarate (α-KG) contents decreased in got1 and got2 silenced strains, and the transcription levels of most genes involved in the glycolytic pathway and the tricarboxylic acid cycle increased. The NADH content was increased in got1 silenced strains and got2 silenced strains, and the NAD+/NADH ratio was decreased, which might result in mitochondrial ROS production. Compared with the WT strain, the mitochondrial ROS level was approximately 1.5-fold higher in the got1 silenced strains. In addition, silencing of got1 or got2 resulted in a decrease in antioxidant enzymes, including superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase. Finally, ganoderic acid (GA) was increased by approximately 40% in got1 silenced strains compared with the WT strain, while silencing of got2 resulted in a 10% increase in GA biosynthesis. These findings provide new insights into the effect of GOT on ROS and secondary metabolism in fungi. KEY POINTS: • GOT plays important roles in ROS level in Ganoderma lucidum. • Silencing of got1 resulted in decrease in GSH content and antioxidant enzymes activities, but an increase in mitochondrial ROS level in G. lucidum. • Silencing of got1 and got2 resulted in an increase in ganoderic acid biosynthesis in G. lucidum.

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.

Clinical profile and biochemical abnormalities in Scrub Typhus: A cross-sectional study.

Introduction: Scrub Typhus (ST) is an acute febrile illness caused by obligate intracellular bacteria of the family Rickettsia. It is often unrecognized and neglected but prevalent in tropical regions of endemic areas. The tragedy behind this diagnostic dilemma is non-specific clinical signs and symptoms, limited awareness, unavailability of diagnostic facilities, and low index of suspicion among the physicians. To address the knowledge gap, we tried to find out a proper panel of laboratory investigations to diagnose the disease and predict its progression because of the uncertainty of the course of the disease in a tertiary care hospital in western Nepal. Methods: This is a hospital laboratory-based prospective study conducted at Gandaki Medical College- Teaching Hospital (GMC-TH) for a period of two years. Among 988 cases of acute febrile illness, 40 seropositive cases of ST were enrolled in the study. We excluded those who did not give consent for the participation, those who were under 17 years of age, and those who had preexisting liver dysfunctions and other co-morbidities and dual seropositive with other infectious etiologies. We used descriptive statistics to analyze the data in terms of demography, clinical features, and laboratory parameters. Results: Out of 988 febrile patients, we included 40 confirmed cases of ST aged between 17 and 70 years during the study-period. Maximum seropositive cases were from Tanahun district 14 (35%), with predominance among the women (70%). The cases were prevalent in the age group 30-60 years, 19 (47.5%), and in the month of October 15 (37.5%). The commonest complaints were fever in 40 (100%), headache in 20 (50%), eschar in 11 (27.5%). Laboratory parameters showed anemia in 22 (55%), hypoalbuminemia in 11 (27.5%), leukopenia in 5 (12.5%), leukocytosis in 9 (22.5%), thrombocytopenia in 13 (32.5%), raised transaminase levels, SGPT in 21 (52.5%) and SGOT in 14 (26%) ST patients. Conclusion: We found clinical and laboratory profiles in patients with ST were varied and nonspecific. However, knowledge of these findings may evoke the recognition of ST and give a clue to the progression of the disease.