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The uncontrolled hyperglycemia that characterizes diabetes mellitus (DM) causes several complications in the organism. DM is among the major causes of deaths, and the limited efficacy of current treatments push the search for novel drug candidates, also among natural compounds. We focused our attention on caffeoylmalic acid, a phenolic derivative extracted from Urtica dioica, a plant investigated for its potential against type 2 DM. This compound was tested for its antidiabetic activity in vitro through a glucose uptake assay, in vivo in a mouse DM model and through molecular docking towards α-amylase and α-glucosidase. The effects on glucose blood level, liver enzymes, insulin and creatinine levels as well as on lipid and blood parameters, considered biochemical markers of diabetes, were also evaluated. The results showed an antidiabetic activity in vitro and in vivo, as the compound stimulates glucose absorbtion and reduces blood glucose levels. Moreover, it ameliorates lipid profile, liver and blood parameters, with moderate effect on insulin secretion. Taken together, these findings pave the way for the compounds from this class of caffeoylmalic acid as potential antidiabetic compounds., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Citrulline malate (CM) is purported to be an ergogenic aid during various types of exercise performance. However, the effects of CM on repeated sprint performance (RSP) are under-explored. In a placebo-controlled, double-blind, counterbalanced cross-over design, male university-level team sport athletes (n = 13) performed two familiarization trials, after which CM or placebo (PLA) (8 × 1 g tablets each day) were taken on the 2 days prior to, and with breakfast on the morning of, each main experimental trial. The main experimental trials employed a RSP protocol consisting of 10 repetitions of 40 m maximal shuttle run test (MST) with a 30 s interval between the start of each sprint. Sprint times and heart rate were recorded throughout the MST, and blood lactate concentrations were measured before, immediately after, and 5 min after completing the MST. CM resulted in better RSP compared to PLA, as indicated by a lower sprint performance decrement (S dec : CM, 4.68% ± 1.82% vs. PLA, 6.10% ± 1.83%; p = 0.03; ES = 0.77), which was possibly influenced by the fastest sprint time being faster in CM (CM, 8.16 ± 0.34 s vs. PLA, 8.29 ± 0.39 s; p = 0.011; ES = 0.34). There were no differences between CM and PLA in average sprint time (p = 0.54), slowest sprint time (p = 0.48), blood lactate concentrations (p = 0.73) or heart rate (p = 0.18), nor was there a condition × time interaction effect across the 10 sprints (p = 0.166). Three days of CM supplementation (8 g daily) attenuated the sprint performance decrement during short-duration high-intensity exercise in the form of running RSP in male university-level team sport athletes., (© 2024 The Authors. European Journal of Sport Science published by Wiley‐VCH GmbH on behalf of European College of Sport Science.)

Objective: Metabolic reprogramming is an important coordinator of tumor development and resistance to therapy, such as the tendency of tumor cells to utilize glycolytic energy rather than oxidative phosphorylation, even under conditions of sufficient oxygen. Therefore, targeting metabolic enzymes is an effective strategy to overcome therapeutic resistance., Materials and Methods: We explored the differential expression and growth-promoting function of MDH2 by immunohistochemistry and immunoblotting experiments in lung cancer patients and lung cancer cells. Pentose phosphate pathway-related phenotypes (including ROS levels, NADPH levels, and DNA synthesis) were detected intracellularly, and the interaction of malate and proteinase 6PGD was detected in vitro. In vivo experiments using implanted xenograft mouse models to explore the growth inhibitory effect and pro-chemotherapeutic function of dimethyl malate (DMM) on lung cancer., Results: We found that the expression of malate dehydrogenase (MDH2) in the tricarboxylic acid cycle (TCA cycle) was increased in lung cancer. Biological function enrichment analysis revealed that MDH2 not only promoted oxidative phosphorylation, but also promoted the pentose phosphate pathway (PPP pathway). Mechanistically, it was found that malate, the substrate of MDH2, can bind to the PPP pathway metabolic enzyme 6PGD, inhibit its activity, reduce the generation of NADPH, and block DNA synthesis. More importantly, DMM can improve the sensitivity of lung cancer to the clinical drug cisplatin., Conclusion: We have identified malate as a natural inhibitor of 6PGD, which will provide new leads for the development of 6PGD inhibitors. In addition, the metabolic enzyme MDH2 and the metabolite malate may provide a backup option for cells to inhibit their own carcinogenesis, as the accumulated malate targets 6PGD to block the PPP pathway and inhibit cell cycle progression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: To evaluate the effectiveness of topical sialogogue spray containing 1% malic acid in the treatment of xerostomia in patients with type 2 diabetes mellitus., Material and Methods: A randomized double-blind controlled clinical trial was conducted on 52 patients with type 2 diabetes mellitus suffering from xerostomia. Patients were assigned equally to test group who received a topical sialogogue spray containing 1% malic acid and control group who received a placebo spray. Both groups were instructed to use the spray on demand for 2 weeks. The Summated Xerostomia Inventory-Dutch Version questionnaire (SXI-D) and the unstimulated salivary flow rate were evaluated for all patients at baseline, 2 and 4 weeks after malic acid/placebo application., Results: At 2 week's follow-up, the unstimulated salivary flow rate increased significantly from 0.059 ± 0.024 to 0.191 ± 0.064 and from 0.055 ± 0.026 to 0.078 ± 0.032 for test and control groups, respectively, with a statistically significant difference favoring the test group. SXI-D scores showed a significant decrease from 10.73 ± 2.22 to 8.38 ± 2.28 in the test group (p < 0.05), while in the control group it decreased from 10.62 ± 1.75 to 10.23 ± 1.48 (p > 0.05)., Conclusion: A sialogogue spray containing 1% malic acid increased the unstimulated salivary flow rate in patients with type 2 diabetes mellitus suffering from xerostomia., (© 2022 Wiley Periodicals LLC.)

Global soil acidification is increasing, enlarging aluminum (Al) availability in soils, leading to reductions in plant growth. This study investigates the effect of Al stress on the leaf growth zones of Rye (Secale cereale, cv Beira). Kinematic analysis showed that the effect of Al on leaf growth rates was mainly due to a reduced cell production rate in the meristem. Transcriptomic analysis identified 2272 significantly (log2fold > |0.5| FDR < 0.05) differentially expressed genes (DEGs) for Al stress. There was a downregulation in several DEGs associated with photosynthetic processes and an upregulation in genes for heat/light response, and H 2 O 2 production in all leaf zones. DEGs associated with heavy metals and malate transport were increased, particularly, in the meristem. To determine the putative function of these processes in Al tolerance, we performed biochemical analyses comparing the tolerant Beira with an Al sensitive variant RioDeva. Beira showed improved sugar metabolism and redox homeostasis, specifically in the meristem compared to RioDeva. Similarly, a significant increase in malate and citrate production, which are known to aid in Al detoxification in plants, was found in Beira. This suggests that Al tolerance in Rye is linked to its ability for Al exclusion from the leaf meristem., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The clinical significance of Pseudomonas aeruginosa infections and the tolerance of this opportunistic pathogen to antibiotic therapy makes the development of novel antimicrobial strategies an urgent need. We previously found that D,L-malic acid potentiates the activity of ciprofloxacin against P. aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium by increasing metabolic activity and tricarboxylic acid cycle activity. This suggested a potential new strategy to improve antibiotic therapy in P. aeruginosa infections. Considering the importance of the microenvironment on microbial antibiotic susceptibility, the present study aims to further investigate the effect of D,L-malate on ciprofloxacin activity against P. aeruginosa in physiologically relevant infection models, aiming to mimic the infection environment more closely. We used Caenorhabditis elegans nematodes, Galleria mellonella larvae, and a 3-D lung epithelial cell model to assess the effect of D,L-malate on ciprofloxacin activity against P. aeruginosa . D,L-malate was able to significantly enhance ciprofloxacin activity against P. aeruginosa in both G. mellonella larvae and the 3-D lung epithelial cell model. In addition, ciprofloxacin combined with D,L-malate significantly improved the survival of infected 3-D cells compared to ciprofloxacin alone. No significant effect of D,L-malate on ciprofloxacin activity against P. aeruginosa in C. elegans nematodes was observed. Overall, these data indicate that the outcome of the experiment is influenced by the model system used which emphasizes the importance of using models that reflect the in vivo environment as closely as possible. Nevertheless, this study confirms the potential of D,L-malate to enhance ciprofloxacin activity against P. aeru ginosa-associated infections., Competing Interests: The authors declare no conflict of interest.

The latest advancements in cellular bioenergetics have revealed the potential of transferring chemical energy to biological energy for therapeutic applications. Despite efforts, a three-dimensional (3D) scaffold that can induce long-term bioenergetic effects and facilitate tissue regeneration remains a big challenge. Herein, the cellular energetic metabolism promotion ability of l-malate, an important intermediate of the tricarboxylic acid (TCA) cycle, was proved, and a series of bioenergetic porous scaffolds were fabricated by synthesizing poly(diol l-malate) (PDoM) prepolymers via a facial one-pot polycondensation of l-malic acid and aliphatic diols, followed by scaffold fabrication and thermal-cross-linking. The degradation products of the developed PDoM scaffolds can regulate the metabolic microenvironment by entering mitochondria and participating in the TCA cycle to elevate intracellular adenosine triphosphate (ATP) levels, thus promoting the cellular biosynthesis, including the production of collagen type I (Col1a1), fibronectin 1 (Fn1), and actin alpha 2 (Acta2/α-Sma). The porous PDoM scaffold was demonstrated to support the growth of the cocultured mesenchymal stem cells (MSCs) and promote their secretion of bioactive molecules [such as vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), and basic fibroblast growth factor (bFGF)], and this stem cells-laden scaffold architecture was proved to accelerate wound healing in a critical full-thickness skin defect model on rats.

Abstract: Gills, JL, Spliker, B, Glenn, JM, Szymanski, D, Romer, B, Lu, H-C, and Gray, M. Acute citrulline-malate supplementation increases total work in short lower-body isokinetic tasks for recreationally active females during menstruation. J Strength Cond Res 37(6): 1225-1230, 2023-Citrulline-malate (CM) exhibits acute ergogenic benefits through nitric oxide production (NO) and augmentation of vasodilatory properties. Nitric oxide is upregulated by estrogen and may influence CM's ergogenic efficacy in women. Therefore, the objective of this study was to evaluate the acute effects of CM supplementation on lower-body isokinetic performance in recreationally active women. Nineteen women (23.5 ± 3.1 years; 164.8 ± 7.0 cm; 61.9 ± 27.4 kg; 28.8 ± 8.1% body fat) completed 2 randomized, double-blind, crossover trials consuming CM (8 g CM + 12 g dextrose) or placebo (12 g dextrose). For testing trials, subjects were in the menstruating portion of the follicular phase of their menstrual cycle. Subjects performed a 5-repetition isokinetic leg extension protocol (5RP) followed by a 50-repetition isokinetic leg extension protocol (50RP), 60 minutes after supplement consumption. Repeated measures analysis of variance analysis showed that CM significantly increased total work completed, relative total work, and total work during maximum repetition compared with placebo ( p < 0.05); but no significant performance differences existed between trials for peak torque production ( p = 0.14) for the 5RP. No significant differences were identified between trials for peak torque production ( p = 0.69 ) or total work ( p = 0.33) completed during the 50RP. CM increased total work completed during the 5RP, but provided no ergogenic benefit during the 50RP in recreationally active menstruating women. CM amplifies power-based resistance exercise performance in women during the follicular phase of the menstrual cycle, potentially because of depressed estrogen levels. Additional research is needed to identify timing efficacy of CM to increase sport performance during each phase of the menstrual cycle., (Copyright © 2023 National Strength and Conditioning Association.)

The type strain of the species Alicyclobacillus acidoterrestris (DSM 3922) and the strain CB1 (accession number: KP144333) were studied in this research to assess the effects of three weak acids (malic, citric, and ascorbic acids), pH (3 or 4), and spore status (spores, and activated spores). Acids were used to prepare 7 different blends, and the blends used to reduce the pH of Malt Extract broth to 3 and 4; then, media were inoculated with spores or activated spores, stored at 45 °C (optimal temperature for A. acidoterrestris growth), and analyzed immediately and after 2 and 7 days. Data were preliminary standardized as increase/reduction of microbial population, compared to the initial concentration, and modelled through two different statistical approaches (multifactorial ANOVA, and multiple regression). Finally, a binary code (0-no growth or reduction of viable count; 1-growth) was used to perform a multiple regression analysis on the growth probability of A. acidoterrestris. Generally, ascorbic acid was the most effective compound, but other acids (e.g., malic acid) could contribute to increase the inactivation ratio; concerning spore status, the highest sensitivity of activated spores suggests that acids probably act during the outgrowing phase. Finally, the two strains showed different trends at pH 3.0, being the type strain the most resistant one., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Nearly half of the world's population is at risk of being infected by Plasmodium falciparum, the pathogen of malaria. Increasing resistance to common antimalarial drugs has encouraged investigations to find compounds with different scaffolds. Extracts of Artocarpus altilis leaves have previously been reported to exhibit in vitro antimalarial activity against P. falciparum and in vivo activity against P. berghei. Despite these initial promising results, the active compound from A. altilis is yet to be identified. Here, we have identified 2-geranyl-2', 4', 3, 4-tetrahydroxy-dihydrochalcone (1) from A. altilis leaves as the active constituent of its antimalarial activity. Since natural chalcones have been reported to inhibit food vacuole and mitochondrial electron transport chain (ETC), the morphological changes in food vacuole and biochemical inhibition of ETC enzymes of (1) were investigated. In the presence of (1), intraerythrocytic asexual development was impaired, and according to the TEM analysis, this clearly affected the ultrastructure of food vacuoles. Amongst the ETC enzymes, (1) inhibited the mitochondrial malate: quinone oxidoreductase (PfMQO), and no inhibition could be observed on dihydroorotate dehydrogenase (DHODH) as well as bc 1 complex activities. Our study suggests that (1) has a dual mechanism of action affecting the food vacuole and inhibition of PfMQO-related pathways in mitochondria., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest in this study., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Malate regulates blood pressure via nitric oxide production in salt-sensitive rats, a genetic model of hypertension. This study investigated the possible contributions of malate to blood pressure regulation and renal haemodynamics in normotensive rats. Malate (0.1, 0.3 and 1 μg/kg, iv) was injected into rats or L-nitro-arginine methyl ester (L-NAME)-treated rats and mean arterial blood pressure (MABP), cortical blood flow (CBF), and medullary blood flow (MBF), was measured. The clearance study involved infusion of malate at 0.1 μg/kg/h into rats, and MABP, CBF, MBF, glomerular filtration rate (GFR), urine volume (UV) and sodium output (U Na V) were determined. Mechanistic studies to evaluate the role of renal sodium channels involved the treatment with malate (600 mg/kg, po), amiloride (2.5 mg/kg, po) or hydrochlorothiazide (HCTZ) (10 mg/kg, po), and UV and U Na V were determined. Malate elicited significant peak reductions in MABP (124 ± 6.5 vs 105 ± 3.1 mmHg) at 0.1 μg/kg), CBF (231 ± 18.5 vs 205 ± 10.9 PU). L-NAME did not reverse the effect of malate on MABP but tended to blunt the effect on CBF (40%) and MBF (87%) at 0.3 μg/kg. Infusion of malate reduced MABP, CBF, and MBF in a time-dependent manner (p<0.05). Malate exerted a three-fold decrease in GFR in a time-related fashion (p<0.05) as well as increased UV. U Na V increased by 86% in malate-treated-amiloride rats (p<0.05). These data indicate that malate modulates blood pressure and exerts vascular and tubular effects on renal function that may involve epithelial sodium channels (E Na C)., Competing Interests: Declaration of competing Interest The authors have declared that there are no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objective: To evaluate an effect of ethylmethylhydroxypyridine succinate and ethylmethylhydroxypyridine malate on changes in mitochondrial function under experimental focal cerebral ischemia., Material and Methods: Focal cerebral ischemia was modeled in Wistar rats by thermocoagulation of the middle cerebral artery. Ethylmethylhydroxypyridine succinate («Mexidol») and ethylmethylhydroxypyridine malate («Ethoxidol») were injected into the tail vein 30 minutes after ischemia simulation and then for 3 days at doses of 50 mg/kg, 100 mg/kg and 150 mg/kg. After 72 hours, changes in neurological deficits, aerobic and anaerobic respiration activity, the concentration of mitochondrial hydrogen peroxide and apoptosis-inducing factor, as well as the activity of succinate dehydrogenase and cytochrome c oxidase in brain tissue supernatants were assessed., Results: The course administration of ethylmethylhydroxypyridine succinate and ethylmethylhydroxypyridine malate dose-dependently contributed to a decrease in the concentration of mitochondrial hydrogen peroxide and apoptosis-inducing factor in the brain tissue. The restoration of mitochondrial energy function was also shown with the use of ethylmethylhydroxypyridine succinate in all studied doses, while the administration of ethylmethylhydroxypyridine malate led to the restoration of mitochondrial-dependent energy production only at higher doses (100 mg/kg and 150 mg/kg)., Conclusion: The effect of malic acid and succinic acid salts of ethylmethylhydroxypyridine on the change in the redox and apoptosis-regulating function of mitochondria does not depend on the type of anion, whereas the change in the energy function of mitochondria is associated with the salt residue included in the drug structure and its dosage.

Poly (β-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries. In this study, electrolytic stimulation assisted PMLA production was developed. Firstly, it was found that the pentavalent nitrogen source (i.e., NO 3 - ) was more suitable for PMLA production. Secondly, a usual single-chamber bioelectric-fermentation system (BES) cannot improve PMLA production, which can only promote cell growth. Then, a new single-chamber BES with an external circulation was developed, where the PMLA metabolism was further intensified. Finally, the integration of NO 3 - addition and electrolytic stimulation mode (c) showed a positive synergy on the PMLA production. Compared to the case without NO 3 - addition and electrolytic stimulation, the PMLA production was increased by 22.9 % using the integrated process. Moreover, compared to the case without the electrolytic stimulation mode (c), it was revealed that the different genes involved in 12 metabolic subsystems using the integrated process, where 31 and 177 genes were up-regulated and down-regulated, respectively. The up-regulated genes were mainly participated in melanin metabolic process, catalase activity, and oxidoreductase activity. Hence, the integration of electrolytic stimulation represents a novel approach to improve PMLA production., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Myocardial dysfunction played a vital role in organ damage after sepsis. Fluid resuscitation was the essential treatment in which Lactate Ringer's solution (LR) was commonly used. Since LR easily led to hyperlactatemia, its resuscitation effect was limited. Malate Ringer's solution (MR) was a new resuscitation crystal liquid. Whether MR had a protective effect on myocardial injury in sepsis and the relevant mechanism need to be studied., Methods: The cecal ligation and puncture (CLP) inducing septic model and lipopolysaccharide (LPS) stimulating cardiomyocytes were used, and the cardiac function, the morphology and function of mitochondria were observed. The protective mechanism of MR on myocardial injury was explored by proteomics. Then the effects of TPP@PAMAM-MR, which consisted of the mitochondria- targeting polymer embodied malic acid, was further observed., Results: Compared with LR, MR resuscitation significantly prolonged survival time, improved the cardiac function, alleviated the damages of liver, kidney and lung following sepsis in rats. The proteomics of myocardial tissue showed that differently expressed proteins between MR and LR infusion involved oxidative phosphorylation, apoptosis. Further study found that MR decreased ROS, improved the mitochondrial morphology and function, and ultimately enhanced mitochondrial respiration and promoted ATP production. Moreover, MR infusion decreased the expression of apoptosis-related proteins and increased the expression of anti-apoptotic proteins. TPP@PAMAM@MA was a polymer formed by wrapping L-malic acid with poly amido amine (PAMAM) modified triphenylphosphine material. TPP@PAMAM-MR (TPP-MR), which was synthesized by replacing the L-malic acid of MR with TPP@PAMAM@MA, was more efficient in targeting myocardial mitochondria and was superior to MR in protecting the sepsis-inducing myocardial injury., Conclusion: MR was suitable for protecting myocardial injury after sepsis. The mechanism was related to MR improving the function and morphology of cardiomyocyte mitochondria and inhibiting cardiomyocyte apoptosis. The protective effect of TPP-MR was superior to MR., (© 2022. The Author(s).)

Aluminum (Al) stress in acidic soils has severe negative effects on crop productivity. In this study, the alleviating effect and related mechanism of malate on Al stress in quinoa (Chenopodium quinoa) seedlings were investigated. The findings indicated that malate alleviated the growth inhibition of quinoa seedlings under Al stress, maintained the enzymatic and nonenzymatic antioxidant systems, and aided resistance to the damage caused by excessive reactive oxygen species (ROS). Under Al stress, malate significantly increased the contents of chlorophyll and carotenoids in quinoa shoots by 103.8% and 240.7%, and significantly increased the ratios of glutathione (GSH)/oxidized glutathione (GSSG), and ascorbate (AsA)/dehydroascorbate (DHA) in roots by 59.9% and 699.2%, respectively. However, malate significantly decreased the superoxide radical (O 2 • - ), hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA) and Al contents in quinoa roots under Al stress by 32.7%, 60.9%, 63.1% and 49%, respectively. Moreover, the CqMADS family and the Al stress-responsive gene families (CqSTOP, CqALMT, and CqWRKY) were identified from the quinoa genome. Comprehensive expression profiling identified CqMADS68 as being involved in malate-mediated Al resistance. Transient overexpression of CqMADS68 increased Al tolerance in quinoa seedlings. More importantly, we found that CqMADS68 regulated the expression of CqSTOP6, CqALMT6 and CqWRKY88 and further demonstrated the interaction of CqMADS68 with CqSTOP6, CqALMT6 and CqWRKY88 by bimolecular fluorescence complementation (BIFC) experiments. Moreover, transient overexpression and physiological and biochemical analyses demonstrated that CqSTOP6, CqALMT6 and CqWRKY88 could also improve Al tolerance by maintaining the antioxidant capacity of quinoa seedlings. Taken together, these findings reveal that CqMADS68, CqSTOP6, CqALMT6 and CqWRKY88 may be important contributors to the Al tolerance regulatory network in quinoa, providing new insights into Al stress resistance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Polymalic acid (PMA) is a water-soluble polyester produced by Aureobasidium pullulans. In this study, the physiological response of A. pullulans after the addition of vegetable oils was investigated. Soybean oil (SBO) is pivotal for shortening fermentation time and achieving high PMA titer. With the addition of 1% (w/v) SBO, the titer and productivity of PMA was, respectively, increased by 34.2% and 80%. SBO acted as a chemical stimulatory agent rather than a carbon source, the enhancement on PMA production was attributed to the component of fatty acid. SBO induced the dimorphism (yeast-like cells and mycelia) of A. pullulans, in vitro enzyme activities indicated that the TCA oxidative branch for malic acid synthesis might be strengthened, which could generate more ATP for PMA synthesis, and the assay of intracellular energy supply validated this deduction. This study provided a new sight for recognizing the regulatory behavior of SBO in A. pullulans., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca 2+ ) entry through Ca v 1 channels triggered Ca 2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca 2+ -dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

A primary metabolite malate is secreted from guard cells in response to the phytohormone abscisic acid (ABA) and elevated CO2. The secreted malate subsequently facilitates stomatal closure in plants. Here, we investigated the molecular mechanism of malate-induced stomatal closure using inhibitors and ABA signaling component mutants of Arabidopsis thaliana. Malate-induced stomatal closure was impaired by a protein kinase inhibitor, K252a, and also by the disruption of a receptor-like kinase GHR1, which mediates activation of calcium ion (Ca2+) channel by reactive oxygen species (ROS) in guard cells. Malate induced ROS production in guard cells while the malate-induced stomatal closure was impaired by a peroxidase inhibitor, salicylhydroxamic acid, but not by the disruption of Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases, RBOHD and RBOHF. The malate-induced stomatal closure was impaired by Ca2+ channel blockers, verapamil, and niflumic acid. These results demonstrate that the malate signaling is mediated by GHR1 and ROS in Arabidopsis guard cells., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Copper stress in the presence of exogenous methyl jasmonate and Serratia plymuthica in a complete trifactorial design with copper (0, 50 µM), methyl jasmonate (0, 1, 10 µM) and Serratia plymuthica (without and with inoculation) was studied on the physiological parameters of Phaseolus coccineus. Copper application reduced biomass and allantoin content, but increased chlorophyll and carotenoids contents as well as catalase and peroxidases activities. Jasmonate did not modify biomass and organic acids levels under copper treatment, but additional inoculation elevated biomass and content of tartrate, malate and succinate. Jasmonate used alone or in combination with bacteria increased superoxide dismutase activity in copper application. With copper, allantoin content elevated at lower jasmonate concentration, but with additional inoculation - at higher jasmonate concentration. Under copper stress, inoculation resulted in higher accumulation of tartrate, malate and citrate contents in roots, which corresponded with lower allantoin concentration in roots. Combined with copper, inoculation reduced catalase and guaiacol peroxidase activities, whereas organic acids content was higher. Under metal stress, with bacteria, jasmonate reduced phenolics content, elevated superoxide dismutase and guaiacol peroxidase activities. The data indicate that jasmonate and S. plymuthica affected most physiological parameters of P. coccineus grown with copper and revealed some effect on biomass., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Propionic acid (PA) predominantly accumulates in tissues and biological fluids of patients affected by propionic acidemia that may manifest chronic renal failure along development. High urinary excretion of maleic acid (MA) has also been described. Considering that the underlying mechanisms of renal dysfunction in this disorder are poorly known, the present work investigated the effects of PA and MA (1-5 mM) on mitochondrial functions and cellular viability in rat kidney and cultured human embryonic kidney (HEK-293) cells. Mitochondrial membrane potential (∆ψm), NAD(P)H content, swelling and ATP production were measured in rat kidney mitochondrial preparations supported by glutamate or glutamate plus malate, in the presence or absence of Ca 2+ . MTT reduction and propidium iodide (PI) incorporation were also determined in intact renal cells pre-incubated with MA or PA for 24 h. MA decreased Δψm and NAD(P)H content and induced swelling in Ca 2+ -loaded mitochondria either respiring with glutamate or glutamate plus malate. Noteworthy, these alterations were fully prevented by cyclosporin A plus ADP, suggesting the involvement of mitochondrial permeability transition (mPT). MA also markedly inhibited ATP synthesis in kidney mitochondria using the same substrates, implying a strong bioenergetics impairment. In contrast, PA only caused milder changes in these parameters. Finally, MA decreased MTT reduction and increased PI incorporation in intact HEK-293 cells, indicating a possible association between mitochondrial dysfunction and cell death in an intact cell system. It is therefore presumed that the MA-induced disruption of mitochondrial functions involving mPT pore opening may be involved in the chronic renal failure occurring in propionic acidemia., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

As a nitric oxide (NO) enhancer, citrulline malate (CM) has recently been touted as a potential ergogenic aid to both resistance and high-intensity exercise performance, as well as the recovery of muscular performance. The mechanism has been associated with enhanced blood flow to active musculature, however, it might be more far-reaching as either ammonia homeostasis could be improved, or ATP production could be increased via greater availability of malate. Moreover, CM might improve muscle recovery via increased nutrient delivery and/or removal of waste products. To date, a single acute 8 g dose of CM on either resistance exercise performance or cycling has been the most common approach, which has produced equivocal results. This makes the effectiveness of CM to improve exercise performance difficult to determine. Reasons for the disparity in conclusions seem to be due to methodological discrepancies such as the testing protocols and the associated test-retest reliability, dosing strategy (i.e., amount and timing), and the recent discovery of quality control issues with some manufacturers stated (i.e., citrulline:malate ratios). Further exploration of the optimal dose is therefore required including quantification of the bioavailability of NO, citrulline, and malate following ingestion of a range of CM doses. Similarly, further well-controlled studies using highly repeatable exercise protocols with a large aerobic component are required to assess the mechanisms associated with this supplement appropriately. Until such studies are completed, the efficacy of CM supplementation to improve exercise performance remains ambiguous., (© 2021. The Author(s).)

This study investigated the effects of combinations of acetic or malic acid and various solutes (salt, glucose, glycine, or sucrose) on the survival of Escherichia coli O157:H7 in laboratory broth. Additionally, the effectiveness of combining organic acids and various concentrations of salt (0-18%) or sucrose (0-100%) with different water activity values against E. coli O157:H7 were evaluated. For treatment of 1% malic acid, the addition of 3% salt showed synergistic effect. Whereas, when 3% salt, glucose, glycine, or sucrose was added to 1% acetic acid, the solutes antagonized the action of the acid against E. coli O157:H7. Acetic, lactic, or propionic acid combined with salt at either 7 or 9% or sucrose at 60, 80, or 100% resulted in the highest resistance of E. coli O157:H7. From a result of evaluating the membrane fatty acid (MFA) composition of cells, salt or sucrose significantly increased levels of saturated fatty acids (SFAs) or SFAs and cyclopropane fatty acids, respectively. From the results of this study, the addition of solutes and organic compounds may increase the tolerance of E. coli O157:H7 to acetic, lactic, and propionic acid treatments and that the salt or sucrose significantly affects cell MFA composition., (Copyright © 2021. Published by Elsevier Ltd.)

Particulate matters (PMs) increase oxidative stress and inflammatory response in different tissues. PMs disrupt the formation of primary cilia in various skin cells, including keratinocytes and melanocytes. In this study, we found that 2-isopropylmalic acid (2-IPMA) promoted primary ciliogenesis and restored the PM2.5-induced dysgenesis of primary cilia in dermal fibroblasts. Moreover, 2-IPMA inhibited the generation of excessive reactive oxygen species and the activation of stress kinase in PM2.5-treated dermal fibroblasts. Further, 2-IPMA inhibited the production of pro-inflammatory cytokines, including IL-6 and TNF-α, which were upregulated by PM2.5. However, the inhibition of primary ciliogenesis by IFT88 depletion reversed the downregulated cytokines by 2-IPMA. Moreover, we found that PM2.5 treatment increased the MMP-1 expression in dermal fibroblasts and a human 3-D-skin model. The reduced MMP-1 expression by 2-IPMA was further reversed by IFT88 depletion in PM2.5-treated dermal fibroblasts. These findings suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in dermal fibroblasts.

Citrulline malate (CitMal) is a dietary supplement that is suggested to enhance strength training performance. However, there is conflicting evidence on this matter. Thus, the purpose of this meta-analysis was to determine whether supplementing with CitMal prior to strength training could increase the total number of repetitions performed before reaching voluntary muscular failure. A systematic search was conducted wherein the inclusion criteria were double-blind, placebo-controlled studies in healthy participants that examined the effect of CitMal on repetitions to failure during upper body and lower body resistance exercises. The Hedges's g standardized mean differences (SMD) between the placebo and CitMal trials were calculated and used in a random effect model. Two separate subanalyses were performed for upper body and lower body exercises. Eight studies, including 137 participants who consisted of strength-trained men (n = 101) and women (n = 26) in addition to untrained men (n = 9), fulfilled the inclusion criteria. Across the studies, 14 single-joint and multijoint exercises were performed with an average of 51 ± 23 total repetitions during 5 ± 3 sets per exercise at ∼70% of one-repetition maximum. Supplementing with 6-8 g of CitMal 40-60 min before exercise increased repetitions by 3 ± 5 (6.4 ± 7.9%) compared with placebo (p = .022) with a small SMD (0.196). The subanalysis for the lower body resulted in a tendency for an effect of the supplement (8.1 ± 8.4%, SMD: 0.27, p = .051) with no significant effect for the upper body (5.7 ± 8.4%, SMD: 0.16, p = .131). The current analysis observed a small ergogenic effect of CitMal compared with placebo. Acute CitMal supplementation may, therefore, delay fatigue and enhance muscle endurance during high-intensity strength training.

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis 1-3 . Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate 4,5 . Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy) 6-8 .

We have demonstrated that chemotaxis to l-malate facilitated motility of Ralstonia pseudosolanacearum MAFF 106611, a causative agent of bacterial wilt, to plant roots. Here, we evaluated the assumption that the disruption of chemotaxis to l-malate leads to inhibition of plant infection by R. pseudosolanacearum MAFF 106611. Chemotactic assays revealed that chemotaxis to l-malate was completely or partially inhibited in the presence of l-, d-, and dl-malate, respectively. Moreover, l-malate served as a carbon and energy source for R. pseudosolanacearum MAFF 106611, while d-malate inhibited the growth of this bacterium. In the sand-soak inoculation virulence assay for tomato plants, the addition of l-, d-, and dl-malate to sand suppressed the plant infection. We concluded that supplementation of l- and dl-malate suppresses tomato plant infection with R. pseudosolanacearum MAFF 106611 by disrupting its chemotaxis to l-malate, while d-malate suppresses it by both the disruption of l-malate chemotaxis and inhibition of growth., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Pancreatic cancer remains intractable owing to the lack of effective therapy for unresectable cases. Activating mutations of K-ras are frequently found in pancreatic cancers, but these have not yet been targeted by cancer therapies. The Keap1-Nrf2 system plays a crucial role in mediating the oxidative stress response, which also contributes to cancer progression. Nrf2 activation reprograms the metabolic profile to promote the proliferation of cancer cells. A recent report suggested that K-ras - and Nrf2-active lung cancer cells are sensitive to glutamine depletion. This finding led to the recognition of glutaminase inhibitors as novel anticancer agents. In the current study, we used murine pancreatic cancer tissues driven by mutant K-ras and p53 to establish cell lines expressing constitutively activated Nrf2. Genetic or pharmacological Nrf2 activation in cells via Keap1 deletion or Nrf2 activation sensitized cells to glutaminase inhibition. This phenomenon was confirmed to be dependent on K-ras activation in human pancreatic cancer cell lines harboring mutant K-ras , i.e., Panc-1 and MiaPaCa-2 in response to DEM pretreatment. This phenomenon was not observed in BxPC3 cells harboring wildtype K-ras . These results indicate the possibility of employing Nrf2 activation and glutaminase inhibition as novel therapeutic interventions for K-ras mutant pancreatic cancers.

Reduced expression of the plasma membrane citrate transporter INDY (acronym I'm Not Dead, Yet) extends life span in lower organisms. Deletion of the mammalian Indy (mIndy) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target.

Citrulline-malate (CM) purportedly increases exercise performance through increased nitric oxide production. The effects of CM on muscular strength performance are well-documented; however, the benefits of CM on aerobic and anaerobic biking performance are not well researched. Therefore, the present investigation examined the acute CM supplementation effects on aerobic and anaerobic cycling performance in recreationally active males. Methods : 28 recreationally active males (20.9 ± 2.8 years) completed randomized, double-blind, crossover trials consuming CM (12g dextrose + 8g CM) or a placebo (12g dextrose). Participants performed an aerobic cycling protocol (time-to-exhaustion [TTE]), followed by a subsequent 30-second Wingate cycling test, 60-minutes after supplement consumption. Results : Dependent t -tests showed no significant differences ( p  > 0.05) for TTE (PLA: 315.4 s ± 137.7 s; CM: 314.1 s ± 107.1 s) and Total Work Completed (TWC) (PLA: 74.7 ± 34.1 kilojoules (kJ); CM: 74.1 ± 26.4 kJ) during the aerobic cycling protocol. Dependent t -tests also showed no significant differences ( p  > 0.05) for mean watts (PLA: 586.1 ± 87.7 Watts (W); CM: 588.0 ± 93.0 W), peak watts (PLA: 773.0 ± 136.7 W; CM: 786.7 ± 133.0 W), and fatigue index (PLA: 12.9 ± 6.4 FI; CM: 14.3 ± 7.2 FI) during the Wingate protocol. Repeated-measures ANOVA results indicated a significant effect between each 5 s interval ( p  < 0.001), but no differences were observed between trials ( p  > 0.05). Conclusion : Acute CM supplementation in recreationally active males provides no ergogenic benefit in aerobic cycling performance followed by an anaerobic cycling test.

Development of low-cost and eco-friendly approaches to fight bacterial pathogens is especially needed in aquaculture. We previously showed that exogenous malate reprograms zebrafish's metabolome to potentiate zebrafish survival against Vibrio alginolyticus infection. However, the underlying mechanism is unknown. Here, we use GC-MS based metabolomics to identify the malate-triggered metabolic shift. An activated TCA cycle and elevated taurine are identified as the key metabolic pathways and the most crucial biomarker of the reprogrammed metabolome, respectively. Taurine elevation is attributed to the activated TCA cycle, which is further supported by the increased expression of genes in the metabolic pathway of taurine biosynthesis from the isocitrate of the TCA cycle to taurine. Exogenous taurine increases the survival of zebrafish against V. alginolyticus infection as malate did. Moreover, exogenous taurine and malate regulate the expression of innate immunity genes and promote the generation of reactive oxygen species and nitrogen oxide in a similar way. The two metabolites can alleviate the excessive immune response to bacterial challenge, which protects fish from bacterial infection. These results indicate that malate enhances the survival of zebrafish to V. alginolyticus infection via taurine. Thus, our study highlights a metabolic approach to enhance a host's ability to fight bacterial infection.

This study was conducted to evaluate the antibacterial effect of 460-470 nm light-emitting diodes illumination (460/470 LED) combined with various antimicrobials at inactivating Escherichia coli O157:H7 and identify the antibacterial mechanisms. When carvacrol, thymol, citric acid, malic acid, citrus fruit extract, 3% NaCl, or 5% NaCl was combined with 460/470 LED, there was a higher reduction in E. coli O157:H7 compared to 460/470 LED treatment or antimicrobials alone at 4 °C. Particularly, a marked synergistic effect (>8.74 log 10 CFU/ml) was observed when 460/470 LED was combined with carvacrol, malic acid, citrus fruit extract, or 3% NaCl. Levels of intracellular ROS and lipid peroxidation of E. coli O157:H7 were higher in the combination of 460/470 LED and antimicrobials compared to individual treatments. Moreover, the combination treatment increased depolarization of the cell membrane leading to membrane damage as well as the loss of DNA integrity. Thus, adding antimicrobial treatment to 460/470 LED could improve its efficacy against pathogenic bacteria such as E. coli O157:H7., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study aimed to examine the influence of two prebiotics, fructo-oligosaccharides (FOS) and xylo-oligosaccharides (XOS), on probiotic properties (resistance to low pH and bile salt, hydrophobicity and auto-aggregation), metabolites production, and antimicrobial activity of probiotic Lactiplantibacillus (L. pentosus S42 and L. plantarum S61) and Levilactobacillus (L. brevis S27) strains isolated from fermented olive. The results demonstrated the ability of strains to ferment XOS more than FOS as a sole carbon source, resulting in pH reduction. The prebiotics (FOS and XOS) significantly increased (p < 0.05) their survival in gastro-intestinal conditions (low pH and 0.3% of bile salts), as well as their hydrophobicity, auto-aggregation and production of proteins, compared to glucose (control). The major organic acids produced by Lactiplantibacillus and Levilactobacillus strains, were oxalic, malic and lactic acids from FOS, XOS and glucose, respectively. No antimicrobial activity was observed from cell-free supernatant (CFS) of Lactiplantibacillus and Levilactobacillus strains obtained from FOS. In the presence of XOS the organic acids, produced by Lactiplantibacillus and Levilactobacillus strains, were more diverse with high contents, and exhibited higher antifungal and antibacterial activities, more than that of FOS and glucose. The combination of L. plantarum S61 and XOS demonstrated the highest inhibition zones ranges of 20.7-22.2 mm against pathogenic bacteria and 29.2-30 mm against yeasts. This combination can be used in production of antifungal preservatives and pharmaceuticals, against pathogenic and spoilage yeasts.

To control Pseudomonas and Shewanella as important psychrotrophic spoilage bacteria in fish meat, we used ethanolic extracts of oregano (Origanum vulgare subsp. vulgare) and nettle (Urtica dioica), with phytochemical characterisation of the extracts and their bioactive compounds. Liquid chromatography coupled with photodiode array detection and electrospray ionisation-mass spectrometry was used for qualitative compositional determination of the extracts. Four main compounds were identified in the oregano extract, with rosmarinic acid the most abundant, followed by three glycosylated phenolics, one of which is reported for the first time in O. vulgare: 4'-O-β-d-glucopyranosyl-3',4'-dihydroxybenzyl-4-hydroxybenzoate. Six main compounds were identified in the nettle extract, as caffeoylmalic acid and five flavonoid glycosides. These oregano and nettle ethanolic extracts showed in-vitro antimicrobial activities against selected Pseudomonas and Shewanella strains in broth and fish meat homogenate when evaluated at two inoculum concentrations. The antimicrobial activities were more pronounced for the nettle extract at the lower inoculum concentration, and for both the Shewanella strains. Growth inhibition in the fish meat homogenate was evaluated at 3.13 mg/mL and 1.56 mg/mL at 5 °C. Again, the nettle extract showed greater antimicrobial activity, which was seen as the lowest maximum growth rate, followed by the oregano extract, which was inhibitory only at 3.13 mg/mL. Finally, the extracts were applied to fish meat that was then stored at 5 °C for 9 days. Evaluation here was for the counts of the mesophilic, psychrotrophic, Pseudomonas and H 2 S producers. These confirmed the better antimicrobial effects of the nettle extract, especially against the H 2 S-producing bacteria, which included Shewanella. Both of the extracts were rich in glycosides of flavonoids and phenolic acids. The enzymatic activities of the Pseudomonas and Shewanella spoilage bacteria and their actions on the phenolic glycosides from natural sources will be further investigated., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We evaluated the antioxidant and skin-lightening activities of 4-hydroxycinnamoyl-malate (4-HCM) in vitro ; the material is a water-soluble component of Citrus junos callus. 4-HCM was waterextracted from callus grown on MS medium containing picloram to enhance growth. The antioxidant activity of 4-HCM was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzoline-6-sulfonate) (ABTS) free-radical-scavenging assays. 4-HCM-mediated inhibition of extracellular matrix protein glycation was assessed using the elastin and collagen glycation assays. Inhibition of skin pigmentation was evaluated by measuring anti-tyrosinase activity and melanogenesis in melanoma cells. 4-HCM was then incorporated into elastic nanoliposomes (ENLs) and delivered topically to enhance percutaneous absorption. At 5 mM, the free-radical-scavenging activity of 4-HCM was 54.2±0.631% in the ABTS assay, comparable to that of 1 mM ascorbic acid (46.5± 0.17%). The IC 50 value for inhibition of advanced glycation end-product formation from elastin was 1.25±0.23 mM; collagen glycation was completely inhibited when the 4-HCM level was >5 mM. At 0.5 mM, the material afforded 49.2±3.09% inhibition of tyrosinase activity and, at 10 mM, reduced the melanocyte melanin content by 78% without significant cellular toxicity. Moreover, 4-HCM-loaded ENLs exhibited 569% enhanced permeation of 4-HCM across the human epidermis, which may afford skin-lightening if included in cosmetic formulations.

2-Isopropylmalic acid (2-IPMA) and 3-isopropylmalic acid (3-IPMA), recently discovered in wines, were simultaneously quantified in forty wines by UHPLC-MS/MS triple quadrupole. Principal component analysis displayed that red wines were more correlated with high amounts of 2-IPMA (average content 31.60 mg/L); white wines were mostly characterized by low levels of both organic acids. No correlation of theirs levels to other wine features (wine ageing or alcoholic content) were found. 2-IPMA and 3-IPMA showed MICs values of 4096 mg/L and MBCs values of 8192 mg/L or higher against several food borne pathogens. In association, an interesting lower MIC and MBC values (2048 mg/L and 4096 mg/L respectively) were observed against Y. enterocolitica. Interestingly, 3-IPMA showed a mild antioxidant activity by DPPH assay (EC 50  = 3940 mg/L), higher than that of 2-IPMA (EC 50  > 4800 mg/L). No toxicity of these compounds against human colorectal and liver cells (TB assay) was observed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Promiscuous activities of enzymes may serve as starting points for the evolution of new functions. However, most experimental examples of promiscuity affording an observable phenotype necessitate the artificial overexpression of the target enzyme. Here, we show that 3-isopropylmalate dehydrogenase (IPMDH), an enzyme involved in leucine biosynthesis, has a secondary activity on d-malate, which is sufficient for d-malate assimilation under physiological conditions where the enzyme is upregulated. In vitro, the turnover constant (k cat ) of IPMDH for d-malate is about 30-fold lower than the k cat for 3-isopropylmalate, yet sufficiently high to support the growth on d-malate. From an evolutionary perspective, our results highlight the possibility of phenotype emergence triggered by arbitrary changes in environmental conditions and prior to any mutational event., (© 2020 Federation of European Biochemical Societies.)

Gonzalez, AM and Trexler, ET. Effects of citrulline supplementation on exercise performance in humans: A review of the current literature. J Strength Cond Res 34(5): 1480-1495, 2020-L-citrulline, a nonessential amino acid found primarily in watermelon, has recently garnered much attention for its potential to augment L-arginine bioavailability, nitric oxide production, and exercise performance. Over the past decade, L-citrulline has received considerable scientific attention examining potentially ergogenic properties for both aerobic and anaerobic exercise performance. Thus, the purpose of this article is to summarize the theoretical rationale behind L-citrulline supplementation and to comprehensively review the available scientific evidence assessing the potential ergogenic value of L-citrulline supplementation on vascular function and exercise performance in humans. In addition, research that has investigated the potential synergistic effects of L-citrulline with other dietary ingredients (e.g., arginine, antioxidants, nitrates, and branched-chain amino acids) is reviewed. Oral L-citrulline and citrulline malate supplementation have shown to increase plasma citrulline and arginine concentrations, along with total nitrate and nitrite concentrations. Although blood flow enhancement is a proposed mechanism for the ergogenic potential of L-citrulline, evidence supporting acute improvements in vasodilation and skeletal muscle tissue perfusion after supplementation is scarce and inconsistent. Nevertheless, several studies have reported that L-citrulline supplementation can enhance exercise performance and recovery. Given the positive effects observed from some investigations, future studies should continue to investigate the effects of both acute and chronic supplementation with L-citrulline and citrulline malate on markers of blood flow and exercise performance and should seek to elucidate the mechanism underlying such effects.

Objectives: To investigate the impact of gustatory stimulants of salivary secretion (GSSS) on Sjögren's syndrome patients' self-perception of xerostomia, oral health-related quality of life (OHRQoL) and salivary secretion., Methods: A total of 110 Sjögren's syndrome patients were randomly allocated to be treated with either a malic acid lozenge or a citric acid mouthwash and then crossed over. Before and after the interventions, the Xerostomia Inventory 5 (SXI-5-PL) and the Oral Health Impact Profile (OHIP-14-PT) questionnaires (both in the Portuguese language) were administered to patients. Unstimulated, mechanical and gustatory-stimulated salivary flows were determined. Repeated measures and between-subject analyses were performed. Statistical significance was set at 5%., Results: After the intervention and within each group, both GSSS elicited a reduction in the SXI-5-PL and OHIP-14-PT scores and an increase in salivary output, significant in the malic acid lozenge group. The malic acid treatment resulted in a greater effect size and percentage improvement than citric acid mouthwash. The malic acid lozenge also produced a significant greater salivary output than the citric acid rising solution., Conclusions: In Sjögren's syndrome patients, lozenges containing malic acid increased saliva production and xerostomia relief, resulting in improved quality of life., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: Athletes are increasingly exploring ways to enhance their physical performance. Increasing blood flow to the working tissues through endothelium-dependent vasodilation is one factor athletes use to realize these results. Sports supplements such as pre-workouts tout this benefit; however, many have not been tested under laboratory conditions to examine the effects of commonly used supplements on vasodilation. Two popular supplements are Nitrosigine® and citrulline malate (CM). Thus, the purpose of this experiment was to determine the effects of Nitrosigine and CM on vasodilation using ultrasound and flow mediated dilation (FMD)., Methods: Healthy, normotensive, and physically active male (n = 16) and female (n = 8) young adults participated in the present investigation. We utilized a randomized, double-blind, within-subjects design where participants reported for three trials, each preceded by a 7-day washout period. Baseline FMD measurement was obtained for each visit, followed by consumption of one clinical dose CM (8 g), Nitrosigine (1.5 g), or dextrose placebo (8 g). Following a 60-min digestion period, FMD was repeated. Supplementation order was randomized controlling for potential order effects., Results: Repeated measures ANOVA yielded a significant supplement (3) x time (2) effect (p < .001), such that Nitrosigine and CM yielded a greater improvement in FMD response than placebo. After supplementation, Nitrosigine and CM increased FMD by 31 and 34%, respectively, compared to a decrease of 2% during the placebo trial. After allometric scaling of the FMD values, supplement x time effect remained significant (p = .001) and changes were similar to non-scaled results. Nitrosigine (23%) and CM (25%) generated significantly greater allometric scaled FMD values when compared to the placebo trial (0.60%)., Discussion: Both Nitrisigine and CM increased endothelial-dependent vasodilation as measured by a change in FMD. Increased vasodilation leads to an increase in skeletal muscle blood flow resulting in potential improvements in exercise performance.

We studied the effects of three organic acids (citric acid, tartaric acid and malic acid) on the biomass, photosynthetic pigment content and photosynthetic parameters of Salix variegata under Cd stress and observed the ultrastructure of mesophyll cells in each treatment. Cd stress significantly reduced photosynthesis by reducing the content of pigments and disrupting chloroplast structure, which consequently decreased the biomass. However, respective addition of three organic acids greatly increased the biomass of S. variegata under Cd stress. Among them, the effect of malic acid or tartaric acid on shoot and total biomass accumulation was greater than that of citric acid. The alleviation of biomass probably related with the photosynthetic process. Results revealed that treatment with each organic acid enhanced the net photosynthesis rate under Cd stress. Malic acid promoted plant growth and biomass by increasing the chlorophyll content and mitigating damage to the photosynthetic apparatus resulting from Cd stress. Tartaric acid had little impact on the photosynthetic pigment content, but it was important in mitigating the ultrastructural damage of plants caused by Cd. Addition of citric acid significantly increased the carotenoid as well as the number and volume of chloroplasts in mesophyll cells, while the mitigation of structural damage in the photosynthetic apparatus was weaker than that in tartaric acid or malic acid treatment. It is concluded that application of tartaric acid or malic acid is effective in increasing the growth potential of S. variegata under Cd stress and thus can be a promising approach for the phytoremediation of Cd-contaminated soil., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The remediation effect of organic acids in heavy metal contaminated soil was widely studied. However, the comprehensive evaluation of organic acids on micro-ecological environment in heavy metal contaminated soil was less known. Herein, this experiment was conducted to investigate the impact of malic acid, citric acid and oxalic acid on soil fertility, cadmium (Cd) speciation and ecotoxicity in contaminated soil. Especially, to evaluate the ecotoxicity of Cd, high-throughput sequencing was used to investigate the soil bacterial community structure and diversity after incubation with organic acids. The results showed that obvious changes in soil pH were not observed. Whereas, the contents of available phosphorus (Olsen-P) and alkali hydrolysable nitrogen (Alkeline-N) evidently increased with a significant difference. Furthermore, compared to control, the proportion of acetic acid-extractable Cd increased by 3.06-6.63%, 6.11-9.43% and 1.91-6.22% respectively in the groups amended with malic acid, citric acid and oxalic acid, which indicated that citric acid did better in improving the availability of Cd than malic acid and oxalic acid. In terms of biological properties, citric acid did best in bacteria count increase, enzyme activities and bacterial community structure improvement. Accordingly, these results provided a better understanding for the influence of organic acids on the micro-ecological environment in Cd contaminated soil, based on physicochemical and biological analysis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: dl-Malic acid (dl-M) is used widely in cosmetic formulations as a pH-adjuster or as a preservative. dl-M is used as an exfoliator in the form of α-hydroxy acids. However, the role of dl-M in skin diseases (including atopic dermatitis (AD)) has not been studied deeply. We wished to reveal the effect of dl-M on AD induced by 2,4-dinitrochlorobenzene (DNCB) in Balb/c mice. Methods: The thickness and immune-cell infiltration into the dermis and epidermis were evaluated. Moreover, serum levels of cytokines, as well as expression of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) in tissue were measured in AD mice. We also studied the effect of dl-M on inflammatory mediators in a human keratinocyte (HaCaT) cell line. Results: The dl-M (high) group improved skin condition compared with the DNCB-treated group. The dl-M (high) group inhibited phosphorylation of MAPK and NF-κB in skin tissue. dl-M reduced serum levels of interleukin-4 and IgE. Finally, dl-M decreased the expression of thymus and activation-regulated chemokine, monocyte chemoattractant protein-1 and intercellular cell adhesion molecule induced by interferon-gamma/tumor necrosis factor-α in HaCaT cells. Discussion: These results suggest that dl-M can improve the skin conditions of AD mice.

Trexler, ET, Keith, DS, Schwartz, TA, Ryan, ED, Stoner, L, Persky, AM, and Smith-Ryan, AE. Effects of citrulline malate and beetroot juice supplementation on blood flow, energy metabolism, and performance during maximum effort leg extension exercise. J Strength Cond Res 33(9): 2321-2329, 2019-Citrulline malate (CitMal) and beetroot juice (BEET) are increasingly popular ergogenic aids, but few studies have rigorously investigated their effects on resistance exercise performance and underlying mechanisms. The current randomized, double-blind, crossover study evaluated the effects of CitMal and BEET supplementation on blood flow, metabolic efficiency, and performance during maximal isokinetic leg extension exercise. After familiarization, 27 recreationally active men (age: 22 ± 4 years) completed 3 visits in which subjects ingested a treatment beverage (CitMal [8 g], BEET [400-mg nitrate], or placebo [PLA]), followed by a 2-hour rest period, warm-up, and 5 sets of 30 concentric leg extensions. Before and after exercise, ultrasound was used to measure diameter (aDIAM) and blood flow (aBF) of the superficial femoral artery, along with cross-sectional area and echo intensity of the vastus lateralis. Plasma analytes (lactate, nitrate/nitrite [NOx], and urea nitrogen [BUN]) were also assessed at these times, and indirect calorimetry was used to measure energy expenditure and respiratory exchange ratio before and during exercise. Resting NOx values were higher in BEET (233.2 ± 1.1 μmol·L) compared with CitMal (15.3 ± 1.1, p < 0.0001) and PLA (13.4 ± 1.1, p < 0.0001). Postexercise NOx values, adjusted for resting differences, were higher in BEET (86.3 ± 1.2 μmol·L) than CitMal (21.3 ± 1.1, p < 0.0001) and PLA (18.1 ± 1.1, p < 0.0001). No other variables were affected by treatment (all p > 0.05). While BEET increased NOx, neither treatment was found to enhance performance, blood flow, metabolic efficiency, nor the hormonal response to leg extension exercise.

Farney, TM, Bliss, MV, Hearon, CM, and Salazar, DA. The effect of citrulline malate supplementation on muscle fatigue among healthy participants. J Strength Cond Res 33(9): 2464-2470, 2019-The focus of the investigation was to examine the effects of citrulline malate (CM) on muscular fatigue in healthy, recreationally trained participants. Twelve participants (men = 6 and women = 6) (24.1 ± 3.9 years) visited the laboratory on 3 separate days, all separated by 1 week. Each visit consisted of consuming 1 of 3 treatments: placebo, CM (8 g), and control in which no drink mixture was consumed. For each day of testing, participants consumed assigned treatment and performed 1 high-intensity exercise trial consisting of squats, lunge jumps, squat jumps, and lateral jumps. Participants performed the exercises in the listed order, which was designated as 1 round. Each participant performed 3 rounds, with the work-to-rest ratio being 20 seconds of work and 30 seconds of rest. A 1-minute rest was given between rounds. A pre/post-exercise isokinetic leg extension test was performed to measure for peak power, peak torque, and rate of fatigue. In addition, blood lactate was obtained pre/post-exercise. There were no treatment or interaction effects (p > 0.05) for peak torque, peak power, rate of fatigue, or blood lactate accumulation. However, there was a statistical significant decrease from pre/post-exercise for peak torque (p = 0.003), peak power (p = 0.003), and rate of fatigue (p = 0.001). In addition, lactate accumulation did increase significantly from pre/post-exercise (p = 0.0001). Finally, neither total work nor final heart rate was statistically significant between the treatments (p > 0.05). Citrulline malate was not effective in improving performance or alleviating fatigue after a high-intensity exercise session.

Background: Studies have persuasively demonstrated that citrulline has a key role in the arginine-nitric oxide system, increasing nitric oxide bioavailability, an important mediator of peripheral vasodilation., Objective: To analyze the inter-individual post-exercise hypotension responsiveness following acute citrulline supplementation in hypertensives., Methods: Forty hypertensives were randomly assigned to one of the four experimental groups (control-placebo, control-citrulline, exercise-placebo, and exercise-citrulline). They ingested placebo or citrulline malate [CM] (6 grams). During the exercise session, individuals performed 40 minutes of walking/running on a treadmill at 60-70% of HR reserve. For the control session, the individuals remained seated at rest for 40 minutes. Office blood pressure (BP) was taken every 10 minutes until completing 60 minutes after the experimental session. The ambulatory BP device was programmed to take the readings every 20 minutes (awake time) and every 30 minutes (sleep time) over the course of 24 hours of monitoring. Statistical significance was defined as p < 0.05., Results: Unlike the other experimental groups, there were no "non-responders" in the exercise/citrulline (EC) for "awake" (systolic and diastolic BP) and "24 hours" (diastolic BP). The effect sizes were more consistent in the EC for systolic and diastolic ambulatorial BP response. The effects were "large" (> 0.8) for "awake", "asleep", and "24 hours" only in the EC for diastolic BP., Conclusion: CM supplementation can increase the post-exercise hypotensive effects in hypertensives. In addition, the prevalence of non-responders is lower when associated with aerobic exercise and CM supplementation.

Organic acids play an important role in metal uptake and trafficking in plants. Therefore, the role of exogenous citrate and malate on Cd tolerance was studied in the seedlings of Oryza sativa L. cv MTU 7029. Seedlings were exposed to Cd plus organic acids in hydroponics and monitored changes in Cd accumulation, expression of metal transporters, chlorophyll fluorescence, and antioxidants. It found that organic acid supplements decrease Cd accumulation in leaf because of up-regulation of tonoplast localized heavy metal ATPase (OsHMA3) which allows vacuolar sequestration of Cd in the root. Malic acid helped Cd exclusion in the root too. A shift in Cd speciation from sulphhydryl to the carboxylic group also noticed in the roots of plants exposed to organic acids. Treatment of organic acids was effective to prevent Cd inducible Fe deficiency via up-regulation of the iron-regulated transporter (OsIRT1), increase in ferric chelate reductase activity, and formation of Cd stabilized Fe 3+ - organic acid complex respectively. Also, exposure to organic acids increased the accumulation of antioxidants such as anthocyanin and glutathione (GSH) under Cd stress. Above changes assisted in upholding of photosynthetic electron transport and biomass productivity during the course of Cd treatment with organic acid supplements., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the effect of two organic acids [malic acid (MA) and citric acid (CA)], Lactobacillus plantarum (LP) and their mixtures on the fermentation of alfalfa silage. The wilted and chopped alfalfa was ensiled in vacuum-sealed plastic bags (200-230 g per bag) with following additives: 0.5% distilled water (control), 0.5% DL-MA, 0.5% CA, LP (1 · 10 6  cfu/g), 0.5% DL-MA plus LP (MA+LP) or 0.5% CA plus LP (CA+LP) (application rates based on fresh weight). Each treatment had four replicates and was stored at room temperature. After 60 d of ensiling, decreases in pH, non-protein N (NPN) and increases in lactic acid were observed in all additive-treated silages (p < 0.01). Compared with treatment LP, higher pH and lower lactic acid concentrations were observed after MA or CA addition (p < 0.05) and further application of MA or CA together with LP did not promote lactic acid fermentation in ensiled alfalfa. Furthermore, additives decreased the proportion of saturated fatty acids and increased the proportion of polyunsaturated fatty acids in ensiled alfalfa, with the highest level in CA-treated silages (p < 0.05). Although MA and CA were not as effective as LP in improving silage fermentation quality, treatments MA+LP and CA+LP were more effective in decreasing silage NPN, and CA was most effective in inhibiting biohydrogenation of fatty acids in ensiled alfalfa.

A green alga, Chlamydomonas reinhardtii, was used to verify whether a simple Biotic Ligand Model (BLM) could be used to predict carefully controlled short-term biouptake for the lanthanide, Nd. In the absence of ligands or competitors, Nd biouptake was well described by a Michaelis-Menten equation with an affinity constant, K Nd , of 10 6.8  M -1 and a maximum internalization flux of J max  = 1.70 × 10 -14  mol cm -2 s -1 . For bi-metal mixtures containing Nd and Ca, Mg, Sm or Eu, Nd uptake could also be well modelled by assigning experimentally determined affinity constants of K Ca  = 10 2.6  M -1 , K Mg  = 10 3.4  M -1 , K Sm  = 10 6.5  M -1 and K Eu  = 10 6.5  M -1 . The similar values of K m and J max for the three rare earth elements (REEs): Sm, Eu and Nd is consistent with them sharing a common metal uptake site. On the other hand, in the presence of the small organic ligands (citric or malic acid), neither, free or total Nd concentrations could be used to quantitatively predict Nd internalization fluxes. In other words, in order to predict biouptake by simple BLM determinations, it was necessary to consider that the Nd complexes were bioavailable. The data strongly suggest that risk evaluations of the REE will require a new paradigm and new tools for evaluating bioavailability., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Dozens of Gram negative pathogens use one or more type III secretion systems (T3SS) to disarm host defenses or occupy a beneficial niche during infection of a host organism. While the T3SS represents an attractive drug target and dozens of compounds with T3SS inhibitory activity have been identified, few T3SS inhibitors have been validated and mode of action determined. One issue is the lack of standardized orthogonal assays following high throughput screening. Using a training set of commercially available compounds previously shown to possess T3SS inhibitory activity, we demonstrate the utility of an experiment pipeline comprised of six distinct assays to assess the stages of type III secretion impacted: T3SS gene copy number, T3SS gene expression, T3SS basal body and needle assembly, secretion of cargo through the T3SS, and translocation of T3SS effector proteins into host cells. We used enteropathogenic Yersinia as the workhorse T3SS-expressing model organisms for this experimental pipeline, as Yersinia is sensitive to all T3SS inhibitors we tested, including those active against other T3SS-expressing pathogens. We find that this experimental pipeline is capable of rapidly distinguishing between T3SS inhibitors that interrupt the process of type III secretion at different points in T3SS assembly and function. For example, our data suggests that Compound 3, a malic diamide, blocks either activity of the assembled T3SS or alters the structure of the T3SS in a way that blocks T3SS cargo secretion but not antibody recognition of the T3SS needle. In contrast, our data predicts that Compound 4, a haloid-containing sulfonamidobenzamide, disrupts T3SS needle subunit secretion or assembly. Furthermore, we suggest that misregulation of copy number control of the pYV virulence plasmid, which encodes the Yersinia T3SS, should be considered as a possible mode of action for compounds with T3SS inhibitory activity against Yersinia .

Cell models of mitochondrial complex I (CI) deficiency display activation of glycolysis to compensate for the loss in mitochondrial ATP production. This adaptation can mask other relevant deficiency-induced aberrations in cell physiology. Here we investigated the viability, mitochondrial morphofunction, ROS levels and ATP homeostasis of primary skin fibroblasts from Leigh Syndrome (LS) patients with isolated CI deficiency. These cell lines harbored mutations in nuclear DNA (nDNA)-encoded CI genes (NDUFS7, NDUFS8, NDUFV1) and, to prevent glycolysis upregulation, were cultured in a pyruvate-free medium in which glucose was replaced by galactose. Following optimization of the cell culture protocol, LS fibroblasts died in the galactose medium, whereas control cells did not. LS cell death was dose-dependently inhibited by pyruvate, malate, oxaloacetate, α-ketoglutarate, aspartate, and exogenous NAD + (eNAD), but not by lactate, succinate, α-ketobutyrate, and uridine. Pyruvate and eNAD increased the cellular NAD + content in galactose-treated LS cells to a different extent and co-incubation studies revealed that pyruvate-induced rescue was not primarily mediated by NAD + . Functionally, in LS cells glucose-by-galactose replacement increased mitochondrial fragmentation and mass, depolarized the mitochondrial membrane potential (Δψ), increased H 2 DCFDA-oxidizing ROS levels, increased mitochondrial ATP generation, and reduced the total cellular ATP content. These aberrations were differentially rescued by pyruvate and eNAD, supporting the conclusion that these compounds rescue galactose-induced LS cell death via different mechanisms. These findings establish a cell-based strategy for intervention testing and enhance our understanding of CI deficiency pathophysiology.