Enzymes Glutamate Dehydrogenase Research Articles

Detection of Urea in Human Blood using Graphene-Based Amperometric Biosensor

Urea being an essential clinical analyte, its early and easy detection would be helpful in preventing various diseases. In this study, a graphene-based screen-printed enzymatic biosensor was fabricated for the blood urea detection by amperometric method. The electrode surface was modified with urease and glutamate dehydrogenase (Urs-GLDH) enzymes for sensitive and selective detection of urea with a wide detection range. The diffusion coefficient and the electron transfer rate constant of the screen-printed graphene electrode (SPGE) was found to be 8.53 × 10−7 cm2 s−1 and 7.58 × 10−5 cm s−1, respectively. The proposed graphene-based enzymatic urea biosensor can detect urea ranging from 2.5 to 30 mM with a sensitivity of 10.59 μA·mM−1·cm−2. The reliability of this enzymatic biosensor was confirmed by measuring urea in human blood and the results showed excellent agreement with the clinical testing method. Hence, the graphene-based biosensor described herein could provide rapid, sensitive, and selective detection of urea with low-cost and simple fabrication. In conclusion, this urea detection approach could be a promising method for developing feasible low-cost clinical diagnostic systems.

Dietary protein load affects the energy and nitrogen balance requiring liver glutamate dehydrogenase to maintain physical activity

Provision of amino acids to the liver is instrumental for gluconeogenesis while it requires safe disposal of the amino group. The mitochondrial enzyme glutamate dehydrogenase (GDH) is central for hepatic ammonia detoxification by deaminating excessive amino acids towards ureagenesis and preventing hyperammonemia. The present study investigated the early adaptive responses to changes in dietary protein intake in control mice and liver-specific GDH knockout mice (Hep-Glud1-/-). Mice were fed chow diets with a wide coverage of protein contents; i.e. suboptimal 10%, standard 20%, over optimal 30%, and high 45% protein diets; switched every 4 days. Metabolic adaptations of the mice were assessed in calorimetric chambers before tissue collection and analyses. Hep-Glud1-/- mice exhibited impaired alanine induced gluconeogenesis and constitutive hyperammonemia. The expression and activity of GDH in liver lysates were not significantly changed by the different diets. However, applying an in situ redox-sensitive assay on cryopreserved tissue sections revealed higher hepatic GDH activity in mice fed the high-protein diets. On the same section series, immunohistochemistry provided corresponding mapping of the GDH expression. Cosinor analysis from calorimetric chambers showed that the circadian rhythm of food intake and energy expenditure was altered in Hep-Glud1-/- mice. In control mice, energy expenditure shifted from carbohydrate to amino acid oxidation when diet was switched to high protein content. This shift was impaired in Hep-Glud1-/- mice and consequently the spontaneous physical activity was markedly reduced in GDH knockout mice. These data highlight the central role of liver GDH in the energy balance adaptation to dietary proteins.

Elevated CO2 concentration enhance carbon and nitrogen metabolism and biomass accumulation of Ormosiahosiei

Elevated CO2 concentrations may inhibit photosynthesis due to nitrogen deficiency, but legumes may be able to overcome this limitation and continue to grow. Our study confirms this conjecture well. First, we placed the two-year-old potted saplings of Ormosia hosiei (O. hosiei) (a leguminous tree species) in the open-top chamber (OTC) with three CO2 concentrations of 400 (CK), 600 (E1), and 800 μmol·mol−1 (E2) to simulate the elevated CO2 concentration environment. After 146 days, the light saturation point (LSP), light compensation point (LCP), apparent quantum efficiency (AQE), and dark respiration rate (Rd) of O. hosiei were increased under increasing CO2 concentration and obtain the maximum ribulose diphosphate (RuBP) carboxylation rate (Vc max) and RuBP regenerated photosynthetic electron transfer rate (Jmax) were also significantly increased under E2 treatment (P < 0.05). This results in a significant increase of the maximum assimilation rate (Amax) under elevated CO2 concentrations. Sucrose phosphate synthase (SPS) activity in sucrose metabolism increased in the leaves, more soluble sugars, starches, and sucrose was produced, but sucrose content only in leaves increased at E2, and more carbon flows to the roots. The activity of the NH4+ assimilating enzymes glutamine synthetase (GS), glutamate synthetase (GOGAT), and glutamate dehydrogenase (GDH) in the leaves of O. hosiei increases under elevated CO2 concentrations to promote nitrogen synthesis that reduces the content of ammonium nitrogen and increases the content of nitrate nitrogen. In addition, under E1 conditions, sucrose synthase (SS), direction of synthesis activity was highest and sucrose invertase (INV) activity was lowest, this means that the balance of C and N metabolism is maintained. While under E2 conditions SS activity decreased and INV activity increased, this increased C/N and nitrogen use efficiency. So, the elevated CO2 concentration promotes the accumulation of O. hosiei biomass, especially in the aboveground part, but did not have a significant effect on the accumulation of root biomass. This means that O. hosiei is able to cope under the elevated CO2 concentration without showing photosynthetic adaptation during the experimental period.

Gypenoside XVII Reduces Synaptic Glutamate Release and Protects against Excitotoxic Injury in Rats.

Excitotoxicity is a common pathological process in neurological diseases caused by excess glutamate. The purpose of this study was to evaluate the effect of gypenoside XVII (GP-17), a gypenoside monomer, on the glutamatergic system. In vitro, in rat cortical nerve terminals (synaptosomes), GP-17 dose-dependently decreased glutamate release with an IC50 value of 16 μM. The removal of extracellular Ca2+ or blockade of N-and P/Q-type Ca2+ channels and protein kinase A (PKA) abolished the inhibitory effect of GP-17 on glutamate release from cortical synaptosomes. GP-17 also significantly reduced the phosphorylation of PKA, SNAP-25, and synapsin I in cortical synaptosomes. In an in vivo rat model of glutamate excitotoxicity induced by kainic acid (KA), GP-17 pretreatment significantly prevented seizures and rescued neuronal cell injury and glutamate elevation in the cortex. GP-17 pretreatment decreased the expression levels of sodium-coupled neutral amino acid transporter 1, glutamate synthesis enzyme glutaminase and vesicular glutamate transporter 1 but increased the expression level of glutamate metabolism enzyme glutamate dehydrogenase in the cortex of KA-treated rats. In addition, the KA-induced alterations in the N-methyl-D-aspartate receptor subunits GluN2A and GluN2B in the cortex were prevented by GP-17 pretreatment. GP-17 also prevented the KA-induced decrease in cerebral blood flow and arginase II expression. These results suggest that (i) GP-17, through the suppression of N- and P/Q-type Ca2+ channels and consequent PKA-mediated SNAP-25 and synapsin I phosphorylation, reduces glutamate exocytosis from cortical synaptosomes; and (ii) GP-17 has a neuroprotective effect on KA-induced glutamate excitotoxicity in rats through regulating synaptic glutamate release and cerebral blood flow.

Purification and characterization of glutamate dehydrogenase from rainbow trout (Oncorhynchus mykiss) liver and molecular docking studies.

Glutamate dehydrogenase (GDH) participates in the energy metabolism of proteins and the synthesis of metabolites important for the organism. In this study, GDH enzyme was purified from the liver of rainbow trout (Oncorhynchus mykiss) by 2',5'-ADP Sepharose 4B affinity chromatography in one step. As a result of this purification process, GDH enzyme was purified 171-fold with 5.83U/mg protein-specific activity. The characterization experiments presented that the storage stability of the purified GDH enzyme was determined as -80°C; optimum temperature 40°C; it was determined that the optimum ionic strength was 100mM phosphate buffer and the optimum pH was 8.00. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and PAGE studies showed that the natural molar mass of the purified GDH enzyme was 346.74kDa, and the molar mass of its subunits was 53.71kDa. Km and Vmax values for substrates and coenzymes of GDH enzyme purified from rainbow trout liver were calculated, and the lowest Km value was found in NAD+ (1.86mM) and the highest Vmax value in NH4 + (1.79U/mL). The effects of some metal ions, vitamins, and solvents on the activity of the purified GDH enzyme were investigated and also IC50 values and inhibition types. The metal ion with the lowest IC50 value is Ag+ (8.65 ± 1.68μM), and the vitamin is B6 (0.77 ± 0.04mM). The binding affinities of inhibitors were investigated with molecular docking, based on the conformational state of GDH.

Synergistic regulation of hydrogen sulfide and nitric oxide on biochemical components, exopolysaccharides, and nitrogen metabolism in nickel stressed rice field cyanobacteria.

The present study examined the regulatory mechanism of hydrogen sulfide (H2S) and nitric oxide (NO) in nickel (Ni) stressed cyanobacteria viz., Nostoc muscorum and Anabaena sp. by analyzing growth, photosynthetic pigments, biochemical components (protein and carbohydrate), exopolysaccharides (EPS), inorganic nitrogen content, and activity of enzymes comprised in nitrogen metabolism and Ni accumulation. The 1µM Ni substantially diminished growth by 18% and 22% in N. muscorum and Anabaena sp. respectively, along with declining the pigment contents (Chl a/Car ratio and phycobiliproteins), and biochemical components. It also exerted negative impacts on inorganic uptake of nitrate and nitrite contents; nitrate reductase and nitrite reductase; and ammonium assimilating enzymes (glutamine synthetase, glutamate synthase, and glutamate dehydrogenase exhibited a reverse trend) activities. Nonetheless, the adverse impact of Ni can be mitigated through the exogenous supplementation of NaHS [sodium hydrosulfide (8µM); H2S donor] and SNP [sodium nitroprusside (10µM); NO donor] which showed substantial improvement on growth, pigments, nitrogen metabolism, and EPS layer and noticeably occurred as a consequence of a substantial reduction in Ni accumulation content which minimized the toxicity effects. The accumulation of Ni on both the cyanobacterial cell surface (EPS layer) are confirmed by the SEM-EDX analysis. Further, the addition of NO scavenger (PTIO; 20µM) and inhibitor of NO (L-NAME; 100µM); and H2S scavenger (HT; 20µM) and H2S inhibitor (PAG; 50µM) reversed the positive responses of H2S and NO and damages were more prominent under Ni stress thereby, suggesting the downstream signaling of H2S on NO-mediated alleviation. Thus, this study concludes the crosstalk mechanism of H2S and NO in the mitigation of Ni-induced toxicity in rice field cyanobacteria.

Incidence of Clostridium difficile in Patients with Antibiotic Associated Diarrhea

Background: This study was performed to determine the magnitude of Clostridioides difficile infection (CDI) in a tertiary care hospital in patients with antibiotic associated diarrhea (AAD) and to study the risk factors associated with this disease.&#x0D; Methods: A descriptive study was conducted in the department of Microbiology in a tertiary care hospital during December 2019 to May 2021. Stool samples were collected from patients with signs and symptoms of AAD who had been consuming antibiotic or anticancer drugs durng six weeks before the sampling. The samples were subjected to C. difficile glutamate dehydrogenase (GDH) enzyme and CD toxin A &amp; B detection by Enzyme Linked Fluorescent Assay (ELFA). Patient’s demographic features and clinical details were noted and statistically correlated with the test results&#x0D; Results: Among the total 70 samples tested 20 (28%) were positive for GDH alone and 12 (17%) were positive for both GDH and CD toxin A and B. Fluoroquinolones was a significant risk factor in the study. Sepsis and colitis was found to have significant association with C.difficile infection in our study. The crude mortality rate was 17%.&#x0D; Conclusion: Prompt and precise diagnosis and knowledge about the risk factors of CDI helps in effective management and prevention of CDI.

Duration of Zearalenone Exposure Has Implications on Health Parameters of Lactating Cows.

There is a limited research focus on evaluating the detrimental effects of prolonged zearalenone (ZEN) intake on dairy cows' health under controlled conditions. This experiment was conducted to evaluate whether the length of exposure to a ZEN-contaminated total mixed ration (TMR) at a level of 9.45 mg per day can negatively influence animal health parameters, such as milk composition, rumen and fecal fermentation, and the chewing activity of lactating dairy cows. For this experiment, we used 18 lactating Simmental cows that were fed a diet of 60% forage and 40% concentrate (on dry matter basis) for 26 consecutive days. The first 4 days were for adaptation prior to the first sampling day (day 0). The sampling events took place on day 0 (baseline) without ZEN, followed by day 1, day 7, day 14, and day 21 (with toxin). Dry matter intake (DMI) and ruminating chews per minute increased on the third week of ZEN inclusion; meanwhile, ruminating, eating, and drinking times were not affected. Most milk composition variables were also unaffected. Rumen fluid osmolality increased on day 21 and total short-chain fatty acids (SCFA) of ruminal fluid decreased on day 7. Fecal SCFA increased on day 21 and the acetate-to-propionate ratio increased from day 1 onwards, showing the influence of toxin intake. Animal health parameters, like heart rate, respiratory rate, and body temperature, were negatively influenced by ZEN intake, all increasing consistently on days 4 and 6, 9 and 12, and 16 and 18, respectively. The liver enzyme glutamate dehydrogenase decreased in response to ZEN intake on day 7. A total daily ZEN intake at the level of 9.45 mg did not show detrimental effects on DMI. Nevertheless, certain health parameters were negatively affected, including body temperature, respiratory rate, and heart rate, starting from the 7th day of ZEN intake, with additional signs of possible loss of water balance on the last sampling day.

Unravelling the interplay of different traits and parameters related to nitrogen use efficiency in wheat for climate‐resilient agriculture

AbstractEnhancing Nitrogen Use Efficiency (NUE) is extremely important towards mitigating climate change, especially in wheat where the NUE is less than 50%. Hence, optimizing grain yield under reduced application of nitrogenous fertilizer is a significant challenge. To address this challenge, a comprehensive study was conducted to investigate various agronomic traits and morphological, biochemical and molecular parameters related to NUE. This study explored their interrelationships and effects on grain yield, providing novel insights that were not previously reported. A set of 278 diverse wheat genotypes were assessed, encompassing eight NUE‐related field traits. All traits' values were reduced under stressed N (ranging from 7.5% to 77.5%) except Nitrogen Utilization Efficiency (NUtE) and NUE. Data analysis showed a significant positive correlation between grain yield and all other NUE‐related traits (r2 value ranged from .23 to 1.00), highlighting their relevance in comprehending the biological NUE of wheat plants. Principal component analysis (PCA) also revealed that N at head and N at harvest were more connected with gain yield, NUE and biomass under the optimum N condition, but less connected with gain yield and NUE under the stressed N condition. To complement the field data, representative genotypes were further subjected to a hydroponics experiment under absolute N control to study the different morphological parameters, photosynthetic pigments and the performance of essential N‐ and C‐metabolizing enzymes at the seedling stage. N stress had a detrimental impact on the majority of the parameters (−0.84% to −79.8%). Nitrite reductase (NiR), glutamate dehydrogenase (GDH) and isocitrate dehydrogenase (ICDH) enzymes as well as root length (RL), root fresh weight (RFW) and CS transcript, were positively affected by 5.9%–35.6%. The correlation analysis highlighted the substantial influence of four key N‐metabolizing enzymes, namely nitrate reductase (NR), glutamine synthetase (GS), glutamate oxo‐glutarate aminotransferase (GOGAT), and GDH on grain yield. Additionally, this study highlighted the direct and indirect associations between seedling parameters and field traits, where shoot and root length were found to be most significant for N acquisition, especially under N stress. In conclusion, these findings offer valuable insights into the intricate network of traits and parameters influencing wheat grain yield under varying N regimes.

Identification of candidate genes and residues for improving nitrogen use efficiency in the N-sensitive medicinal plant Panax notoginseng

BackgroundNitrogen (N) metabolism-related key genes and conserved amino acid sites in key enzymes play a crucial role in improving N use efficiency (NUE) under N stress. However, it is not clearly known about the molecular mechanism of N deficiency-induced improvement of NUE in the N-sensitive rhizomatous medicinal plant Panax notoginseng (Burk.) F. H. Chen. To explore the potential regulatory mechanism, the transcriptome and proteome were analyzed and the three-dimensional (3D) information and molecular docking models of key genes were compared in the roots of P. notoginseng grown under N regimes.ResultsTotal N uptake and the proportion of N distribution to roots were significantly reduced, but the NUE, N use efficiency in biomass production (NUEb), the recovery of N fertilizer (RNF) and the proportion of N distribution to shoot were increased in the N0-treated (without N addition) plants. The expression of N uptake- and transport-related genes NPF1.2, NRT2.4, NPF8.1, NPF4.6, AVP, proteins AMT and NRT2 were obviously up-regulated in the N0-grown plants. Meanwhile, the expression of CIPK23, PLC2, NLP6, TCP20, and BT1 related to the nitrate signal-sensing and transduction were up-regulated under the N0 condition. Glutamine synthetase (GS) activity was decreased in the N-deficient plants, while the activity of glutamate dehydrogenase (GDH) increased. The expression of genes GS1-1 and GDH1, and proteins GDH1 and GDH2 were up-regulated in the N0-grown plants, there was a significantly positive correlation between the expression of protein GDH1 and of gene GDH1. Glu192, Glu199 and Glu400 in PnGS1 and PnGDH1were the key amino acid residues that affect the NUE and lead to the differences in GDH enzyme activity. The 3D structure, docking model, and residues of Solanum tuberosum and P. notoginseng was similar.ConclusionsN deficiency might promote the expression of key genes for N uptake (genes NPF8.1, NPF4.6, AMT, AVP and NRT2), transport (NPF1.2 and NRT2.4), assimilation (proteins GS1 and GDH1), signaling and transduction (genes CIPK23, PLC2, NLP6, TCP20, and BT1) to enhance NUE in the rhizomatous species. N deficiency might induce Glu192, Glu199 and Glu400 to improve the biological activity of GS1 and GDH, this has been hypothesized to be the main reason for the enhanced ability of N assimilation in N-deficient rhizomatous species. The key genes and residues involved in improving NUE provide excellent candidates for the breeding of medicinal plants.

Glutamate dehydrogenase in "Liverworld"-A study in selected species to explore a key enzyme of plant primary metabolism in Marchantiophyta.

In plants, glutamate dehydrogenase (GDH) is an ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate in glutamate. It contributes to both the amino acid homeostasis and the management of intracellular ammonium, and it is regarded as a key player at the junction of carbon and nitrogen assimilation pathways. To date, information about the GDH of terrestrial plants refers to a very few species only. We focused on selected species belonging to the division Marchantiophyta, providing the first panoramic overview of biochemical and functional features of GDH in liverworts. Native electrophoretic analyses showed an isoenzymatic profile less complex than what was reported for Arabidposis thaliana and other angiosperms: the presence of a single isoform corresponding to an α-homohexamer, differently prone to thermal inactivation on a species- and organ-basis, was found. Sequence analysis conducted on amino acid sequences confirmed a high similarity of GDH in modern liverworts with the GDH2 protein of A. thaliana, strengthening the hypothesis that the duplication event that gave origin to GDH1-homolog gene from GDH2 occurred after the evolutionary bifurcation that separated bryophytes and tracheophytes. Experiments conducted on Marchantia polymorpha and Calypogeia fissa grown in vitro and compared to A. thaliana demonstrated through in gel activity detection and monodimensional Western Blot that the aminating activity of GDH resulted in strongly enhanced responses to ammonium excess in liverworts as well, even if at a different extent compared to Arabidopsis and other vascular species. The comparative analysis by bi-dimensional Western Blot suggested that the regulation of the enzyme could be, at least partially, untied from the protein post-translational pattern. Finally, immuno-electron microscopy revealed that the GDH enzyme localizes at the subcellular level in both mitochondria and chloroplasts of parenchyma and is specifically associated to the endomembrane system in liverworts.

Detection of Clostridium difficile among diarrheic children using cultural and polymerase chain reaction technique

Introduction: Clostridium difficile is the most common cause of antibiotic-associated diarrhea and colitis. Several methods are available for the detection of C. difficile in stool samples. This study aimed to use glutamate dehydrogenase (GDH), toxin detection, culture and polymerase chain reaction (PCR) techniques for the diagnosis of this pathogen. Methodology: A total of 300 stool samples were collected from children with hospital acquired diarrhea (HA-D), community acquired diarrhea (CA-D), and hospitalized non-diarrheic children as control with ages ranging from 6 months to 6 years (mean 3.7 ± 1.7). Each stool sample was divided into two parts; one part was tested for the enzyme GDH, toxin A and B and then cultured on selective media; and the other part for direct DNA extraction. Results: From a total of 300 stool samples, 9 (3.0%) were positive for C. difficile by the PCR technique, 7 (7%) samples of which were from HA-D cases and 2 (2.0%) from CA-D cases; the control group samples were negative. The enzyme GDH was detected in 12 (12%) samples and toxins A and B in 8 (8%) samples from HA-D cases compared to 5 (5%) and 2 (2%), respectively from CA-D cases. Both GDH and the toxins were negative in control samples. Only 19 (19.0%) samples from HA-D cases gave suspected growth and all of these were negative by PCR. Conclusions: Based on the results of this study, we conclude that the PCR technique is the only reliable method for the diagnosis of this pathogen.

Changes in Plasma Pyruvate and TCA Cycle Metabolites upon Increased Hepatic Fatty Acid Oxidation and Ketogenesis in Male Wistar Rats.

Altered hepatic mitochondrial fatty acid β-oxidation and associated tricarboxylic acid (TCA) cycle activity contributes to lifestyle-related diseases, and circulating biomarkers reflecting these changes could have disease prognostic value. This study aimed to determine hepatic and systemic changes in TCA-cycle-related metabolites upon the selective pharmacologic enhancement of mitochondrial fatty acid β-oxidation in the liver, and to elucidate the mechanisms and potential markers of hepatic mitochondrial activity. Male Wistar rats were treated with 3-thia fatty acids (e.g., tetradecylthioacetic acid (TTA)), which target mitochondrial biogenesis, mitochondrial fatty acid β-oxidation, and ketogenesis predominantly in the liver. Hepatic and plasma concentrations of TCA cycle intermediates and anaplerotic substrates (LC-MS/MS), plasma ketones (colorimetric assay), and acylcarnitines (HPLC-MS/MS), along with associated TCA-cycle-related gene expression (qPCR) and enzyme activities, were determined. TTA-induced hepatic fatty acid β-oxidation resulted in an increased ratio of plasma ketone bodies/nonesterified fatty acid (NEFA), lower plasma malonyl-CoA levels, and a higher ratio of plasma acetylcarnitine/palmitoylcarnitine (C2/C16). These changes were associated with decreased hepatic and increased plasma pyruvate concentrations, and increased plasma concentrations of succinate, malate, and 2-hydroxyglutarate. Expression of several genes encoding TCA cycle enzymes and the malate-oxoglutarate carrier (Slc25a11), glutamate dehydrogenase (Gdh), and malic enzyme (Mdh1 and Mdh2) were significantly increased. In conclusion, the induction of hepatic mitochondrial fatty acid β-oxidation by 3-thia fatty acids lowered hepatic pyruvate while increasing plasma pyruvate, as well as succinate, malate, and 2-hydroxyglutarate.

Sage leaf rock rose water extract: a bio-solution for enhancing the growth and salt stress resistance of sorghum plants.

Sorghum bicolor, a versatile cereal grain, holds significant agronomic importance globally and plays a crucial role in addressing food insecurity. However, salinity, a major abiotic stress, poses a threat to food production by reducing soil fertility and hindering plant growth and yield. In this study, we investigated the potential of Cistus salviifolius water extract (CSE) in mitigating salt stress in sorghum plants. Salt stress severely impacted plant growth, biomass, and chlorophyll production, and reduced indole-3-acetic acid (IAA) levels, which negatively affected plant development. Salt stress also led to the buildup of reactive oxygen species (ROS), hence, resulting in oxidative harm to sorghum plants and also affecting their carbon and nitrogen metabolism. On the other hand, CSE treatments increased IAA and chlorophyll content which promoted growth under stress. Furthermore, this extract exhibited strong ROS scavenging capacity and safeguarded plants against oxidative stress by enhancing the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase) and increasing the production of osmolytes. Additionally, CSE treatments enhanced the activities of carbon/nitrogen enzymes (phosphoenolpyruvate carboxylase, malate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and glutamine synthase), promoting energy synthesis and crop growth. This led to a significant increase in sorghum growth in salted soil with the highest rise recorded for 5mg/L of CSE (an increase of 48.23% and 158.36% in length and weight compared to the salt control), which highlights this extract's potential as a biostimulant to enhance crop tolerance to salinity and contribute to sustainable agriculture.

Treatment of animals with fatty liver disease using a drug based on the seeds of Silybum marianum

Medicinal plants are a source of various therapeutic preparations. Therefore, the aim of our work was to prepare a liposomal drug from extract from the seeds of Silybum marianum (L.) Gaertn., adding tocopherol acetate, lecithin, squalene, and Tween 80. The drug was used on the laboratory animals (rats) intramuscularly to measure the efficacy of treatment of experimentally modeled toxic fatty liver disease. The fatty infiltration of the liver in the rats was caused by tetrachloromethane (ССl4). The efficacy of the liposomal drug based on the extract from S. marianum seeds was studied on 25 animals in which the liver pathology had been caused by 50% oil solution of ССl4, administered in the dose of 5 mL per kg of body weight. The diseased rats were divided into five groups, each consisting five animals. Animals of the four experimental groups – first, second, third, and fourth - received the drug intramuscularly in the doses of 0.05, 0.25, 0.50, and 1.50 mL/kg of body weight three times every two days, respectively. At the same time, the control rats received three-time intramuscular injection of physiological solution in the dose of 0.5 mL/kg of body weight. Treatment of the animals with fatty liver disease by injections of the drug based on the extract from S. marianum seeds normalized the general condition, significantly improved the functions and structure of the liver. Biochemical studies of blood serum of the sick animals after the treatment revealed increase in albumin content, which may suggest reduction of the protein-synthesizing function of the liver. The normalization of the bile-forming and bile-excreting functions of the liver, and also elimination of cholestasis were evidenced by reduced contents of bile acids and total bilirubin and increased total cholesterol in the blood serum of the rats. After treating the animals with the created drug, we saw decrease in the activity of the liver-indicator enzymes (aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, and glutamate dehydrogenase) in the blood serum, which is a sign of recovery of the structure of hepatocytes and elimination of cytolysis. Histological studies of the liver of the treated rats confirmed the positive effect of the liposomal drug on the organ’s structure. In the future studies, we plan to test this combination of agents in treatment of agricultural and domestic animals with liver pathologies.

Syndromic Panel Testing Among Patients With Infectious Diarrhea: The Challenge of Interpreting Clostridioides difficile Positivity on a Multiplex Molecular Panel.

Including Clostridioides difficile (CD) in gastrointestinal multiplex molecular panels (GIPCR) presents a diagnostic challenge. Incidental detection by polymerase chain reaction (PCR) without consideration of pretest probability (PTP) may inadvertently delay diagnoses of other treatable causes of diarrhea and lead to prescription of unnecessary antibiotics. We conducted a retrospective study to determine the frequency at which clinicians characterize PTP and disease severity in adult patients who test positive for CD by GIPCR. We organized subjects into cohorts based on the status of their CD PCR, glutamate dehydrogenase enzyme immunoassay (GDH), and toxin A/B detection, as well as by high, moderate, or low CD PTP. We used multivariable regression models to describe predictors of toxin positivity. We identified 483 patients with positive CD PCR targets. Only 22% were positive for both GDH and CD toxin. Among patients with a low PTP for CDI, 11% demonstrated a positive CD toxin result compared to 63% of patients with a high PTP. A low clinician PTP for CD infection (CDI) correlated with a negative CD toxin result compared to cases of moderate-to-high PTP for CDI (odds ratio, 0.19 [95% confidence interval, .10-.36]). Up to 64% of patients with negative GDH and CD toxin received CD treatment. Only receipt of prior antibiotics, fever, and a moderate-to-high clinician PTP were statistically significant predictors of toxin positivity. Patients with a positive CD PCR were likely to receive treatment regardless of PTP or CD toxin results. We recommend that CD positivity on GIPCR be interpreted with caution, particularly in the setting of a low PTP.

Is Clostridioides difficile toxins detection necessary when the glutamate dehydrogenase enzyme is detected?

Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. To define if GDH determination is redundant to that of toxins. Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.

Uncovering Actions of Type 3 Deiodinase in the Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD).

To evaluate the role of D3 in the progression of MAFLD in an animal model. Male/adult Sprague Dawley rats (n = 20) were allocated to a control group (2.93 kcal/g) and high-fat diet group (4.3 kcal/g). Euthanasia took place on the 28th week. D3 activity and expression, Uncoupling Protein 2 (UCP2) and type 1 deiodinase (D1) expression, oxidative stress status, mitochondrial, Krebs cycle and endoplasmic reticulum homeostasis in liver tissue were measured. We observed an increase in D3 activity/expression (p < 0.001) related to increased thiobarbituric acid reactive substances (TBARS) and carbonyls and diminished reduced glutathione (GSH) in the MAFLD group (p < 0.05). There was a D3-dependent decrease in UCP2 expression (p = 0.01), mitochondrial capacity, respiratory activity with increased endoplasmic reticulum stress in the MAFLD group (p < 0.001). Surprisingly, in an environment with lower T3 levels due to high D3 activity, we observed an augmented alpha-ketoglutarate dehydrogenase (KGDH) and glutamate dehydrogenase (GDH) enzymes activity (p < 0.05). Induced D3, triggered by changes in the REDOX state, decreases T3 availability and hepatic mitochondrial capacity. The Krebs cycle enzymes were altered as well as endoplasmic reticulum stress. Taken together, these results shed new light on the role of D3 metabolism in MAFLD.

Hyperinsulinism/hyperammonemia syndrome caused by biallelic SLC25A36 mutation.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome has been known to be caused by dominant gain-of-function mutations in GLUD1, encoding the mitochondrial enzyme glutamate dehydrogenase. Pathogenic GLUD1 mutations enhance enzymatic activity by reducing its sensitivity to allosteric inhibition by GTP. Two recent independent studies showed that a similar HI/HA phenotype can be caused by biallelic mutations in SLC25A36, encoding pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine and guanine nucleotides across the inner mitochondrial membrane: one study reported a single case caused by a homozygous truncating mutation in SLC25A36 resulting in lack of expression of SLC25A36 in patients' fibroblasts. A second study described two siblings with a splice site mutation in SLC25A36, causing reduction of mitochondrial GTP content, putatively leading to hyperactivation of glutamate dehydrogenase. In an independent study, through combined linkage analysis and exome sequencing, we demonstrate in four individuals of two Bedouin Israeli related families the same disease-causing SLC25A36 (NM_018155.3) c.284 + 3A > T homozygous splice-site mutation found in the two siblings. We demonstrate that the mutation, while causing skipping of exon 3, does not abrogate expression of mRNA and protein of the mutant SLC25A36 in patients' blood and fibroblasts. Affected individuals had hyperinsulinism, hyperammonemia, borderline low birth weight, tonic-clonic seizures commencing around 6months of age, yet normal intellect and no significant other morbidities. Chronic constipation, hypothyroidism, and developmental delay previously described in a single patient were not found. We thus verify that biallelic SLC25A36 mutations indeed cause HI/HA syndrome and clearly delineate the disease phenotype.

Synergistic mitigation of nickel toxicity in pepper (Capsicum annuum) by nitric oxide and thiourea via regulation of nitrogen metabolism and subcellular nickel distribution.

Nickel (Ni) contamination hinders plant growth and yield. Nitric oxide (NO) and thiourea (Thi) aid plant recovery from heavy metal damage, but their combined effects on pepper (Capsicum annuum ) plant tolerance to Ni stress need more study. Sodium nitroprusside (0.1mM, SNP) and 400mgL-1 Thi, alone and combined, were studied for their impact on pepper growth under Ni toxicity. Ni stress reduces chlorophyll, PSII efficiency and leaf water and sugar content. However, SNP and Thi alleviate these effects by increasing leaf water, proline and sugar content. It also increased the activities of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase. Nickel stress lowered nitrogen assimilation enzymes (nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase and glutamate dehydrogenase) and protein content, but increased nitrate, ammonium and amino acid content. SNP and Thi enhanced nitrogen assimilation, increased protein content and improved pepper plant growth and physiological functions during Ni stress. The combined treatment reduced Ni accumulation, increased Ni in leaf cell walls and potentially in root vacuoles, and decreased Ni concentration in cell organelles. It effectively mitigated Ni toxicity to vital organelles, surpassing the effects of SNP or Thi use alone. This study provides valuable insights for addressing heavy metal contamination in agricultural soils and offers potential strategies for sustainable and eco-friendly farming practices.