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.

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.

Biochemical, structural, and kinetic characterization of PPi -dependent phosphoenolpyruvate carboxykinase from Propionibacterium freudenreichii.

Phosphoenolpyruvate carboxykinases (PEPCK) are a well-studied family of enzymes responsible for the regulation of TCA cycle flux, where they catalyze the interconversion of oxaloacetic acid (OAA) and phosphoenolpyruvate (PEP) using a phosphoryl donor/acceptor. These enzymes have typically been divided into two nucleotide-dependent classes, those that use ATP and those that use GTP. In the 1960's and early 1970's, a group of papers detailed biochemical properties of an enzyme named phosphoenolpyruvate carboxytransphosphorylase (later identified as a third PEPCK) from Propionibacterium freudenreichii (PPi -PfPEPCK), which instead of using a nucleotide, utilized PPi to catalyze the same interconversion of OAA and PEP. The presented work expands upon the initial biochemical experiments for PPi -PfPEPCK and interprets these data considering both the current understanding of nucleotide-dependent PEPCKs and is supplemented with a new crystal structure of PPi -PfPEPCK in complex with malate at a putative allosteric site. Most interesting, the data are consistent with PPi -PfPEPCK being a Fe2+ activated enzyme in contrast with the Mn2+ activated nucleotide-dependent enzymes which in part results in some unique kinetic properties for the enzyme when compared to the more widely distributed GTP- and ATP-dependent enzymes.

Immunodetection of Pyruvate Carboxylase Expression in Human Astrocytomas, Glioblastomas, Oligodendrogliomas, and Meningiomas.

Pyruvate carboxylase (PC) is an enzyme catalyzing the carboxylation of pyruvate to oxaloacetate. The enzymatic generation of oxaloacetate, an intermediate of the Krebs cycle, could provide the cancer cells with the additional anaplerotic capacity and promote their anabolic metabolism. Recent studies revealed that several types of cancer cells express PC. The gained anaplerotic capability of cells mediated by PC correlates with their expedited growth, higher aggressiveness, and increased metastatic potential. By immunohistochemical staining and immunoblotting analysis, we investigated PC expression among samples of different types of human brain tumors. Our results show that PC is expressed by the cells in glioblastoma, astrocytoma, oligodendroglioma, and meningioma tumors. The presence of PC in these tumors suppose that PC could support the anabolic metabolism of their cellular constituents by its anaplerotic capability.

Structural Comparison of hMDH2 Complexed with Natural Substrates and Cofactors: The Importance of Phosphate Binding for Active Conformation and Catalysis.

Malate dehydrogenase (MDH), which catalyzes a reversible conversion of L-malate to oxaloacetate, plays essential roles in common metabolic processes, such as the tricarboxylic acid cycle, the oxaloacetate-malate shuttle, and the glyoxylate cycle. MDH2 has lately been recognized as a promising anticancer target; however, the structural information for the human homologue with natural ligands is very limited. In this study, various complex structures of hMDH2, with its substrates and/or cofactors, were solved by X-ray crystallography, which could offer knowledge about the molecular and enzymatic mechanism of this enzyme and be utilized to design novel inhibitors. The structural comparison suggests that phosphate binds to the substrate binding site and brings the conformational change of the active loop to a closed state, which can secure the substate and cofactor to facilitate enzymatic activity.

[Activation of the adenosine A2A receptor at the acute stage of moderate traumatic brain injury enhances the neuroprotective effects of oxaloacetate].

The purpose of the present study was to investigate the effect of glutamate scavenger oxaloacetate (OA) combined with CGS21680, an adenosine A2A receptor (A2AR) agonist, on acute traumatic brain injury (TBI), and to elucidate the underlying mechanisms. C57BL/6J mice were subjected to moderate-level TBI by controlled cortical impact, and then were treated with OA, CGS21680, or OA combined with CGS21680 at acute stage of TBI. At 24 h post TBI, neurological severity score, brain water content, glutamate concentration in cerebrospinal fluid (CSF), mRNA and protein levels of IL-1ß and TNF-α, mRNA level and activity of glutamate oxaloacetate aminotransferase (GOT), and ATP level of brain tissue were detected. The results showed that neurological deficit, brain water content, glutamate concentration in CSF, and the inflammatory cytokine IL-1ß and TNF-α production were exacerbated in CGS21680 treated mice. Administrating OA suppressed the rise of both glutamate concentration in CSF and brain water content, and elevated the ATP level of cerebral tissue. More interestingly, neurological deficit, brain edema, glutamate concentration, IL-1ß and TNF-α levels were ameliorated significantly in mice treated with OA combined with CGS21680. The combined treatment exhibited better therapeutic effects than single OA treatment. We also observed that GOT activity was enhanced in single CGS21680 treatment group, and both the GOT mRNA level and GOT activity were up-regulated in early-stage combined treatment group. These results suggest that A2AR can improve the efficiency of GOT and potentiate the ability of OA to metabolize glutamate. This may be the mechanism that A2AR activation in combination group augmented the neuroprotective effect of OA rather than aggravated the brain damages. Taken together, the present study provides a new insight for the clinical treatment of TBI with A2AR agonists and OA.

Oxaloacetate Treatment For Mental And Physical Fatigue In Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Long-COVID fatigue patients: a non-randomized controlled clinical trial.

BACKGROUND: There is no approved pharmaceutical intervention for Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS). Fatigue in these patients can last for decades. Long COVID may continue to ME/CFS, and currently, it is estimated that up to 20 million Americans have significant symptoms after COVID, and the most common symptom is fatigue. Anhydrous Enol-Oxaloacetate, (AEO) a nutritional supplement, has been anecdotally reported to relieve physical and mental fatigue and is dimished in ME/CFS patients. Here, we examine the use of higher dosage AEO as a medical food to relieve pathological fatigue. METHODS: ME/CFS and Long-COVID patients were enrolled in an open label dose escalating "Proof of Concept" non-randomized controlled clinical trial with 500 mg AEO capsules. Control was provided by a historical ME/CFS fatigue trial and supporting meta-analysis study, which showed average improvement with oral placebo using the Chalder Scale of 5.9% improvement from baseline. At baseline, 73.7% of the ME/CFS patients were women, average age was 47 and length of ME/CFS from diagnosis was 8.9 years. The Long-COVID patients were a random group that responded to social media advertising (Face Book) with symptoms for at least 6 months. ME/CFS patients were given separate doses of 500 mg BID (N = 23), 1,000 mg BID (N = 29) and 1000 mg TID (N = 24) AEO for six weeks. Long COVID patients were given 500 mg AEO BID (N = 22) and 1000 mg AEO (N = 21), again over a six-week period. The main outcome measure was to compare baseline scoring with results at 6 weeks with the Chalder Fatigue Score (Likert Scoring) versus historical placebo. The hypothesis being tested was formulated prior to data collection. RESULTS: 76 ME/CFS patients (73.7% women, median age of 47) showed an average reduction in fatigue at 6 weeks as measured by the "Chalder Fatigue Questionnaire" of 22.5% to 27.9% from baseline (P < 0.005) (Likert scoring). Both physical and mental fatigue were significantly improved over baseline and historical placebo. Fatigue amelioration in ME/CFS patients increased in a dose dependent manner from 21.7% for 500 mg BID to 27.6% for 1000 mg Oxaloacetate BID to 33.3% for 1000 mg TID. Long COVID patients' fatigue was significantly reduced by up to 46.8% in 6-weeks. CONCLUSIONS: Significant reductions in physical and metal fatigue for ME/CFS and Long-COVID patients were seen after 6 weeks of treatment. As there has been little progress in providing fatigue relief for the millions of ME/CFS and Long COVID patients, anhydrous enol oxaloacetate may bridge this important medical need. Further study of oxaloacetate supplementation for the treatment of ME/CFS and Long COVID is warranted. Trial Registration https://clinicaltrials.gov/ct2/show/NCT04592354 Registered October 19, 2020. 1,000 mg BID Normalized Fatigue Data for Baseline, 2-weeks and 6-weeks evaluated by 3 Validated Fatigue Scoring Questionnaires.

6-methoxydihydroavicine, the alkaloid extracted from Macleaya cordata (Willd.) R. Br. (Papaveraceae), triggers RIPK1/Caspase-dependent cell death in pancreatic cancer cells through the disruption of oxaloacetic acid metabolism and accumulation of reactive oxygen species.

BACKGROUND: Many extracts and purified alkaloids of M. cordata (Papaveraceae family) have been reported to display promising anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis in many cancer types. However, no evidence currently exists for anti-pancreatic cancer activity of alkaloids extracted from M. cordata, including a novel alkaloid named 6­methoxy dihydrosphingosine (6-Methoxydihydroavicine, 6-ME) derived from M. cordata fruits. PURPOSE: The aim of this study was to investigate the anti-tumor effects of 6-ME on PC cells and the underlying mechanism. METHODS: CCK-8, RTCA, and colony-formation assays were used to analyze PC cell growth. Cell death ratios, changes in MMP and ROS levels were measured by flow cytometry within corresponding detection kits. A Seahorse XFe96 was employed to examine the effects of 6-ME on cellular bioenergetics. Western blot and q-RT-PCR were conducted to detect changes in target molecules. RESULTS: 6-ME effectively reduced the growth of PC cells and promoted PCD by activating RIPK1, caspases, and GSDME. Specifically, 6-ME treatment caused a disruption of OAA metabolism and increased ROS production, thereby affecting mitochondrial homeostasis and reducing aerobic glycolysis. These responses resulted in mitophagy and RIPK1-mediated cell death. CONCLUSION: 6-ME exhibited specific anti-tumor effects through interrupting OAA metabolic homeostasis to trigger ROS/RIPK1-dependent cell death and mitochondrial dysfunction, suggesting that 6-ME could be considered as a highly promising compound for PC intervention.

The metabolic importance of the overlooked asparaginase II pathway.

The asparaginase II pathway consists of an asparagine transaminase [l-asparagine + α-keto acid ⇆ α-ketosuccinamate + l-amino acid] coupled to ω-amidase [α-ketosuccinamate + H2O → oxaloacetate + NH4+]. The net reaction is: l-asparagine + α-keto acid + H2O → oxaloacetate + l-amino acid + NH4+. Thus, in the presence of a suitable α-keto acid substrate, the asparaginase II pathway generates anaplerotic oxaloacetate at the expense of readily dispensable asparagine. Several studies have shown that the asparaginase II pathway is important in photorespiration in plants. However, since its discovery in rat tissues in the 1950s, this pathway has been almost completely ignored as a conduit for asparagine metabolism in mammals. Several mammalian transaminases can catalyze transamination of asparagine, one of which - alanine-glyoxylate aminotransferase type 1 (AGT1) - is important in glyoxylate metabolism. Glyoxylate is a precursor of oxalate which, in the form of its calcium salt, is a major contributor to the formation of kidney stones. Thus, transamination of glyoxylate with asparagine may be physiologically important for the removal of potentially toxic glyoxylate. Asparaginase has been the mainstay treatment for certain childhood leukemias. We suggest that an inhibitor of ω-amidase may potentiate the therapeutic benefits of asparaginase treatment.

Unveiling a key role of oxaloacetate-glutamate interaction in regulation of respiration and ROS generation in nonsynaptic brain mitochondria using a kinetic model.

Glutamate plays diverse roles in neuronal cells, affecting cell energetics and reactive oxygen species (ROS) generation. These roles are especially vital for neuronal cells, which deal with high amounts of glutamate as a neurotransmitter. Our analysis explored neuronal glutamate implication in cellular energy metabolism and ROS generation, using a kinetic model that simulates electron transport details in respiratory complexes, linked ROS generation and metabolic reactions. The analysis focused on the fact that glutamate attenuates complex II inhibition by oxaloacetate, stimulating the latter's transformation into aspartate. Such a mechanism of complex II activation by glutamate could cause almost complete reduction of ubiquinone and deficiency of oxidized form (Q), which closes the main stream of electron transport and opens a way to massive ROS generating transfer in complex III from semiquinone radicals to molecular oxygen. In this way, under low workload, glutamate triggers the respiratory chain (RC) into a different steady state characterized by high ROS generation rate. The observed stepwise dependence of ROS generation on glutamate concentration experimentally validated this prediction. However, glutamate's attenuation of oxaloacetate's inhibition accelerates electron transport under high workload. Glutamate-oxaloacetate interaction in complex II regulation underlies the observed effects of uncouplers and inhibitors and acceleration of Ca2+ uptake. Thus, this theoretical analysis uncovered the previously unknown roles of oxaloacetate as a regulator of ROS generation and glutamate as a modifier of this regulation. The model predicted that this mechanism of complex II activation by glutamate might be operative in situ and responsible for excitotoxicity. Spatial-time gradients of synthesized hydrogen peroxide concentration, calculated in the reaction-diffusion model with convection under a non-uniform local approximation of nervous tissue, have shown that overproduction of H2O2 in a cell causes excess of its level in neighbor cells.

Enzymatic activation of pyruvate kinase increases cytosolic oxaloacetate to inhibit the Warburg effect.

Pharmacological activation of the glycolytic enzyme PKM2 or expression of the constitutively active PKM1 isoform in cancer cells results in decreased lactate production, a phenomenon known as the PKM2 paradox in the Warburg effect. Here we show that oxaloacetate (OAA) is a competitive inhibitor of human lactate dehydrogenase A (LDHA) and that elevated PKM2 activity increases de novo synthesis of OAA through glutaminolysis, thereby inhibiting LDHA in cancer cells. We also show that replacement of human LDHA with rabbit LDHA, which is relatively resistant to OAA inhibition, eliminated the paradoxical correlation between the elevated PKM2 activity and the decreased lactate concentration in cancer cells treated with a PKM2 activator. Furthermore, rabbit LDHA-expressing tumours, compared to human LDHA-expressing tumours in mice, displayed resistance to the PKM2 activator. These findings describe a mechanistic explanation for the PKM2 paradox by showing that OAA accumulates and inhibits LDHA following PKM2 activation.

Blood glutamate scavengers increase pro-apoptotic signaling and reduce metastatic melanoma growth in-vivo.

Inhibition of extracellular glutamate (Glu) release decreases proliferation and invasion, induces apoptosis, and inhibits melanoma metastatic abilities. Previous studies have shown that Blood-glutamate scavenging (BGS), a novel treatment approach, has been found to be beneficial in attenuating glioblastoma progression by reducing brain Glu levels. Therefore, in this study we evaluated the ability of BGS treatment to inhibit brain metastatic melanoma progression in-vivo. RET melanoma cells were implanted in C56BL/6J mice to induce brain melanoma tumors followed by treatment with BGS or vehicle administered for fourteen days. Bioluminescent imaging was conducted to evaluate tumor growth, and plasma/CSF Glu levels were monitored throughout. Immunofluorescence staining of Ki67 and 53BP1 was used to analyze tumor cell proliferation and DNA double-strand breaks. In addition, we analyzed CD8, CD68, CD206, p-STAT1 and iNOS expression to evaluate alterations in tumor micro-environment and anti-tumor immune response due to treatment. Our results show that BGS treatment reduces CSF Glu concentration and consequently melanoma growth in-vivo by decreasing tumor cell proliferation and increasing pro-apoptotic signaling in C56BL/6J mice. Furthermore, BGS treatment supported CD8+ cell recruitment and CD68+ macrophage invasion. These findings suggest that BGS can be of potential therapeutic relevance in the treatment of metastatic melanoma.

Oxaloacetate treatment preserves motor function in SOD1G93A mice and normalizes select neuroinflammation-related parameters in the spinal cord.

Amyotrophic lateral sclerosis (ALS) remains a devastating motor neuron disease with limited treatment options. Oxaloacetate treatment has a neuroprotective effect in rodent models of seizure and neurodegeneration. Therefore, we treated the ALS model superoxide dismutase 1 (SOD1) G93A mice with oxaloacetate and evaluated their neuromuscular function and lifespan. Treatment with oxaloacetate beginning in the presymptomatic stage significantly improved neuromuscular strength measured during the symptomatic stage in the injected mice compared to the non-treated group. Oxaloacetate treatment starting in the symptomatic stage significantly delayed limb paralysis compared with the non-treated group. For lifespan analysis, oxaloacetate treatment did not show a statistically significant positive effect, but the treatment did not shorten the lifespan. Mechanistically, SOD1G93A mice showed increased levels of tumor necrosis factor-α (TNFα) and peroxisome proliferative activated receptor gamma coactivator 1α (PGC-1α) mRNAs in the spinal cord. However, oxaloacetate treatment reverted these abnormal levels to that of wild-type mice. Similarly, the altered expression level of total NF-κB protein returned to that of wild-type mice with oxaloacetate treatment. These results suggest that the beneficial effects of oxaloacetate treatment in SOD1G93A mice may reflect the effects on neuroinflammation or bioenergetic stress.

Safety and target engagement profile of two oxaloacetate doses in Alzheimer's patients.

INTRODUCTION: Brain bioenergetics are defective in Alzheimer's disease (AD). Preclinical studies find oxaloacetate (OAA) enhances bioenergetics, but human safety and target engagement data are lacking. METHODS: We orally administered 500 or 1000 mg OAA, twice daily for 1 month, to AD participants (n = 15 each group) and monitored safety and tolerability. To assess brain metabolism engagement, we performed fluorodeoxyglucose positron emission tomography (FDG PET) and magnetic resonance spectroscopy before and after the intervention. We also assessed pharmacokinetics and cognitive performance. RESULTS: Both doses were safe and tolerated. Compared to the lower dose, the higher dose benefited FDG PET glucose uptake across multiple brain regions (P < .05), and the higher dose increased parietal and frontoparietal glutathione (P < .05). We did not demonstrate consistent blood level changes and cognitive scores did not improve. CONCLUSIONS: 1000 mg OAA, taken twice daily for 1 month, is safe in AD patients and engages brain energy metabolism.

Multicomponent Reaction of Aldehydes, Amines and Oxalacetate Analogues Leading to Biologically Attractive Pyrrole Derivatives.

Pyrrole is a very important pharmacophoric moiety. It has been widely incorporated into the skeleton of antitumor, anti-inflammatory, antibacterial, antioxidant and antifungal active substances. Access to this key heterocycle by diverse routes is particularly attractive in terms of chemistry, and also from the environmental point of view. The present minireview summarizes the reported methods for the preparation of highly substituted pyrrole derivatives based on the one-pot multicomponent reaction of aldehydes, primary amines, and oxalacetate analogues as well as their biology.

Heteroexpression and biochemical characterization of thermostable citrate synthase from the cyanobacteria Anabaena sp. PCC7120.

The present study recombinantly expressed a citrate synthase from cyanobacteria Anabaena sp. PCC7120 (AnCS) in Escherichia coli and characterized its enzymatic activity. The molecular mass of native AnCS was 88,533.1 Da containing two 44,162.7 Da subunits. Recombinant AnCS revealed the highest activity at pH 9.0 and 25 °C. AnCS displayed high thermal stability with a half-life time (t1/2) of approximately 6.5 h at 60 °C, which was more thermostable than most CS from general organisms, but less than those from hyperthermophilic bacteria. The Km values of oxaloacetate and acetyl-CoA were 138.50 and 18.15 µM respectively, suggesting a higher affinity to acetyl-CoA than oxaloacetate. Our inhibition assays showed that AnCS activity was not severely affected by most metal ions, but was strongly inhibited by Cu2+ and Zn2+. Treatments with ATP, ADP, AMP, NADH, and DTT depressed the AnCS activity. Overall, our results provide information on the enzymatic properties of AnCS, which contributes to the basic knowledge on CS selection for industrial utilizations.

ATP citrate lyase: A central metabolic enzyme in cancer.

ACLY links energy metabolism provided by catabolic pathways to biosynthesis. ACLY, which has been found to be overexpressed in many cancers, converts citrate into acetyl-CoA and OAA. The first of these molecules supports protein acetylation, in particular that of histone, and de novo lipid synthesis, and the last one sustains the production of aspartate (required for nucleotide and polyamine synthesis) and the regeneration of NADPH,H+(consumed in redox reaction and biosynthesis). ACLY transcription is promoted by SREBP1, its activity is stabilized by acetylation and promoted by AKT phosphorylation (stimulated by growth factors and glucose abundance). ACLY plays a pivotal role in cancer metabolism through the potential deprivation of cytosolic citrate, a process promoting glycolysis through the enhancement of the activities of PFK 1 and 2 with concomitant activation of oncogenic drivers such as PI3K/AKT which activate ACLY and the Warburg effect in a feed-back loop. Pending the development of specific inhibitors and tailored methods for identifying which specific metabolism is involved in the development of each tumor, ACLY could be targeted by inhibitors such as hydroxycitrate and bempedoic acid. The administration of citrate at high level mimics a strong inhibition of ACLY and could be tested to strengthen the effects of current therapies.

Metabolic response of vetiver grass (Chrysopogon zizanioides) to acid mine drainage.

Acid mine drainage (AMD) is a sulfuric discharge containing metals and particulates that can spread to nearby water sources, imposing toxicity and physical stress to living things. We have shown that vetiver grass (Chrysopogon zizanioides) is capable of tolerating and treating AMD-impacted water from the abandoned Tab-Simco mining site from southern Illinois, though little is known about its tolerance mechanisms. We conducted metabolomic analyses of vetiver shoots and roots after relatively short- and long-term periods of exposure to Tab-Simco AMD. The metabolic shift of vetiver shoots was dramatic with longer-term AMD exposure, including upregulation of amino acid and glutathione metabolism, cellular respiration and photosynthesis pathways, with downregulation of phosphorylated metabolites. Meanwhile, the roots demonstrated drastic downregulation of phospholipids and phosphorylated metabolites, cellular respiration, glyoxylate metabolism, and amino acid metabolism. Vetiver accumulated ornithine and oxaloacetate in the shoots, which could function for nitrogen storage and various intracellular functions, respectively. Organic acids and glutathione were secreted from the roots for rhizospheric metal-chelation, whereas phosphorylated metabolites were recycled for phosphorus. These findings reveal AMD-induced metabolic shifts in vetiver grass, which are seemingly unique in comparison to independent abiotic stresses reported previously.

In vitro nitazoxanide exposure affects energetic metabolism of Taenia crassiceps.

Nitazoxanide (NTZ) is a broad-spectrum drug used in intestinal infections, but still poorly explored in the treatment of parasitic tissular infections. This study aimed to evaluate the in vitro responses of the energetic metabolism of T. crassiceps cysticerci induced by NTZ. The organic acids of the tricarboxylic acid cycle, products derived from fatty acids oxidation and protein catabolism were analyzed. These acids were quantified after 24 h of in vitro exposure to different NTZ concentrations. A positive control group was performed with albendazole sulfoxide (ABZSO). The significant alterations in citrate, fumarate and malate concentrations showed the NTZ influence in the tricarboxylic acid (TCA) cycle. The non-detection of acetate confirmed that the main mode of action of NTZ is effective against T. crassiceps cysticerci. The statistical differences in fumarate, urea and beta-hydroxybutyrate concentrations showed the NTZ effect on protein catabolism and fatty acid oxidation. Therefore, the main energetic pathways such as the TCA cycle, protein catabolism and fatty acids oxidation were altered after in vitro NTZ exposure. In conclusion, NTZ induced a significant metabolic stress in the parasite indicating that it may be used as an alternative therapeutic choice for cysticercosis treatment. The use of metabolic approaches to establish comparisons between anti parasitic drugs mode of actions is proposed.

Biochemical characterization of phosphoenolpyruvate carboxykinases from Arabidopsis thaliana.

ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.