Glutaminase Activity Research Articles

A novel method of Francisella infection of epithelial cells using HeLa cells expressing fc gamma receptor

BackgroundFrancisella tularensis, the causative agent of tularemia, is a facultative intracellular bacterium. Although the life cycle of this bacterium inside phagocytic cells (e.g., macrophages, neutrophils) has been well analyzed, the difficulty of gene silencing and editing genes in phagocytic cells makes it difficult to analyze host factors important for the infection. On the other hand, epithelial cell lines, such as HeLa, have been established as cell lines that are easy to perform gene editing. However, the infection efficiency of Francisella into these epithelial cells is extremely low.MethodsIn order to facilitate the molecular biological analysis of Francisella infection using epithelial cells, we constructed an efficient infection model of F. tularensis subsp. novicida (F. novicida) in HeLa cells expressing mouse FcγRII (HeLa-FcγRII), and the system was applied to evaluate the role of host GLS1 on Francisella infection.ResultsAs a result of colony forming unit count, HeLa-FcγRII cells uptake F. novicida in a serum-dependent manner and demonstrated an approximately 100-fold increase in intracellular bacterial infection compared to parental HeLa cells. Furthermore, taking advantage of the gene silencing capability of HeLa-FcγRII cells, we developed GLS1, a gene encoding glutaminase, knockdown cells using lentiviral sh RNA vector and assessed the impact of GLS1 on F. novicida infection. LDH assay revealed that GLS1-knockdown HeLa-FcγRII cells exhibited increased cytotoxicity during infection with F. novicida compared with control HeLa-FcγRII cells. Furthermore, the cell death was inhibited by the addition of ammonia, the metabolite produced through glutaminase activity. These results suggest that ammonia plays an important role in the proliferation of F. novicida.ConclusionsIn this report, we proposed a new cell-based infection system for Francisella infection using HeLa-FcγRII cells and demonstrated its effectiveness. This system has the potential to accelerate cell-based infection assays, such as large-scale genetic screening, and to provide new insights into Francisella infection in epithelial cells, which has been difficult to analyze in phagocytic cells.

Cucurbita Pepo L. Seed Oil Modulates Dyslipidemia and Neuronal Dysfunction in Tramadol-Induced Toxicity in Wistar Albino Rats.

Objective: The modulating effects of Cucurbita pepo seed oil (CPSO) on dyslipidemia and neuronal dysfunction in tramadol toxicity were studied. Methods: Fifty-six albino rats were divided into seven groups of eight rats each after a 2-week acclimatization period. All animals had unrestricted access to water and feed, and treatments were administered orally once daily for 42 days. Glutamate dehydrogenase and glutaminase activities were assessed using brain homogenate, while lipid profiles were analyzed in serum samples. Results: Tramadol toxicity was evidenced by significant (P < 0.05) increases in brain glutamate dehydrogenase along with significant (P < 0.05) decreases in the activities of glutaminase in the group administered only tramadol. Also, serum levels of total cholesterol, LDL-C and triglycerides also increased significantly (P < 0.05) following administration of tramadol with decreased level of HDL-C (P < 0.05). However, treatment with CPSO significantly restored the activities and levels of the altered biochemical parameters in a dose-dependent manner. The results of the biochemical investigation using the lipid profile and the enzymes of glutamate metabolism were corroborated by the results obtained from the histopathological examination of the brain. Conclusion: The results of this study therefore suggest that tramadol-induced dyslipidemia and neuronal dysfunction be managed and prevented by the administration of Cucurbita pepo seed oil.

Characterization of Mycobacterium smegmatis Glutaminase-Free Asparaginase (MSMEG_3173).

l-asparaginase is an enzyme catalyzing the hydrolysis of l-asparagine into l-aspartate and ammonia, which is of great therapeutic importance in tumor treatment. However, commercially available enzymes are associated with adverse effects, and searching for a new l-asparaginase with better pharmaceutical properties was the aim of this work. The coding sequence for Mycobacterium smegmatisl-asparaginase (MsA) was cloned and expressed. The recombinant protein showed high activity toward l-asparagine, whereas none was detected for l-glutamine. The enzymatic properties (K m = 1.403 ± 0.24 mM and k cat = 708.1 ± 25.05 s-1) indicate that the enzyme would be functional within the expected blood l-asparagine concentration, with good activity, as shown by k cat. The pH and temperature profiles suggest its use as a biopharmaceutical in humans. Molecular dynamics analysis of the MsA model reveals the formation of a hydrogen bond network involving catalytic residues with l-asparagine. However, the same is not observed with l-glutamine, mainly due to steric hindrance. Additionally, the structural feature of residue 119 being a serine rather than a proline has significant implications. These findings help explain the low glutaminase activity observed in MsA, like what is described for the Wolinella succinogenes enzyme. This establishes mycobacterial asparaginases as key scaffolds to develop biopharmaceuticals against acute lymphocytic leukemia.

Expression, characterization and cytotoxicity of recombinant l-asparaginase II from Salmonella paratyphi cloned in Escherichia coli

L-asparaginase is a remarkable antineoplastic enzyme used in medicine for the treatment of acute lymphoblastic leukemia (ALL) as well as in food industries. In this work, the L-asparaginase-II gene from Salmonella paratyphi was codon-optimized, cloned, and expressed in E. coli as a His-tag fusion protein. Then, using a two-step chromatographic procedure it was purified to homogeneity as confirmed by SDS-PAGE, which also showed its monomeric molecular weight to be 37 kDa. This recombinant L-asparaginase II from Salmonella paratyphi (recSalA) was optimally active at pH 7.0 and 40 °C temperature. It was highly specific for L-asparagine as a substrate, while its glutaminase activity was low. The specific activity was found to be 197 U/mg and the kinetics elements Km, Vmax, and kcat were determined to be 21 mM, 28 μM/min, and 39.6 S−1, respectively. Thermal stability was assessed using a spectrofluorometer and showed Tm value of 45 °C. The in-vitro effects of recombinant asparaginase on three different human cancerous cell lines (MCF7, A549 and Hep-2) by MTT assay showed remarkable anti-proliferative activity. Moreover, recSalA exhibited significant morphological changes in cancer cells and IC50 values ranged from 28 to 45.5 μg/ml for tested cell lines. To investigate the binding mechanism of SalA, both substrates L-asparagine and l-glutamine were docked with the protein and the binding energy was calculated to be −4.2 kcal mol−1 and − 4.4 kcal mol−1, respectively. In summary, recSalA has significant efficacy as an anticancer agent with potential implications in oncology while its in-vivo validation needs further investigation.

CB-839 induces reversible dormancy in lung tumor-cells

Glutaminase inhibitors are currently being explored as potential treatments for cancer. This study aimed to elucidate the molecular mechanisms underlying the effects of CB-839 on lung tumor cell lines compared to non-tumor cell lines. Viability assays based on NADPH-dependent dehydrogenases activity, ATP energy production, or mitochondrial reductase activity were used to determine that CB-839 caused significant tumor cell specific inhibition of cellular functions. Clonogenic survival assay revealed a dose dependent reduction in clonogenic survival of various lung tumor cells presenting estimated IC50 values between 10 and 90 nM, while no effect on non-tumor cells was observed. CB-839 led to a 20% reduction in glutaminase (GLS1, a mitochondrial enzyme that catalyzes the conversion of glutamine to glutamate) activity, and a dose-dependent reduced glutamine consumption in tumor cells and had no effect on non-tumor cells. Cell cycle analysis showed the CB-839 did not lead to cell cycle arrest. Apoptosis and necrosis assays revealed an only slight increase in apoptosis in tumor cells. Furthermore, a trypan blue exclusion assay revealed about 40% growth reduction in tumor cells at 0.1–1 μM CB-839 treatment. Surprisingly, treated cells resumed normal growth when re-plated in a drug-free medium, demonstrating reversibility. In hypoxic conditions, CB-839's effect on clonogenic survival was amplified in a dose dependent manner consistent with increased role of GLS1 for energy production under hypoxic conditions. In conclusion, these results suggest CB-839 efficacy is linked to temporary and reversible reduction in glutamine utilization suggesting induction of dormancy.

Role of L-glutamine in the regulation of rat Sertoli cell proliferation by FSH.

FSH leads to glutamine dependence, which is required for mTORC1 activation and in consequence Sertoli cell proliferation. The spermatogenic capacity of adult individuals depends on, among other factors, the number of Sertoli cells (SCs) that result from the proliferative waves during development. FSH upregulates SC proliferation at least partly, through the activation of the PI3K/Akt/mTORC1 pathway, among other mechanisms. It is widely known that mTORC1 is a sensor of amino acids. Among amino acids, glutamine acquires relevance since it might contribute to cell cycle progression through the modulation of mTORC1 activity. It has not been studied yet whether glutamine intervenes in FSH-mediated regulation of SC proliferation and cell cycle progression, or if FSH has any effect on glutamine metabolism. Eight-day-old rat SCs were incubated in culture media without glutamine or with glutamine in the absence or presence of a glutamine transporter inhibitor or a glutaminase activity inhibitor under basal conditions or stimulated with FSH. The results obtained show that FSH does not promote SC proliferation and mTORC1 activation in the absence of glutamine. Also, FSH modulates glutamine metabolism increasing glutaminase isoform 2 and reducing glutamine synthetaseexpression. FSH did not promote SC proliferation and mTORC1 activation when glutaminase activity was inhibited. The results suggest that glutamine or its metabolites might cooperate with FSH in the upregulation of SC proliferation through mTORC1. In addition, as FSH modulates glutamine metabolism through the induction of glutaminase isoform 2, the hormonal control of glutamine metabolism might be part of the intricate signaling network triggered by FSH, which is crucial to establish the population of mature SCs that supports the reproductive function.

Gegen Qinlian Decoction reverses oxaliplatin resistance in colorectal cancer by inhibiting YTHDF1-regulated m6A modification of GLS1

BackgroundColorectal cancer (CRC) and its chemoresistance pose significant threats to human health. Gegen Qinlian Decoction (GQD) is frequently employed alongside chemotherapy drugs for the treatment of CRC and various intestinal disorders. Despite its widespread use, there is limited research investigating the mechanisms through which GQD reverses chemoresistance. PurposeThis study investigated the mechanism by which GQD reverses oxaliplatin (OXA) resistance in CRC. MethodsA YTH N6-methyladenosine RNA binding protein 1 (YTHDF1)-knockdown OXA-resistant cell line was constructed by lentivirus to clarify YTHDF1-mediated chemoresistance through the regulation of glutaminase 1 (GLS1). The efficacy of GQD in reversing OXA resistance in CRC in vitro was evaluated by Cell Counting Kit-8, western blotting, quantitative real-time polymerase chain reaction, and glutaminase activity assays. In vivo validation was performed by constructing tumor xenografts in nude mice with OXA-resistant cells. In addition, mouse feces were collected and a 16S rDNA assay was performed to assess the regulation of intestinal flora by GQD. ResultsOverexpression of YTHDF1 upregulated GLS1 expression and induced OXA-resistance in CRC. GQD induced apoptosis in LoVo/OXAR, increased OXA accumulation in LoVo/OXAR, inhibited expression of YTHDF1 and GLS1 when administered alone and in combination with OXA, and suppressed GLS1 activity to reverse drug resistance with good synergistic effects. GQD and OXA combination or GLS1 inhibitor alleviated OXA toxicity, reduced the volume of tumor xenografts in nude mice, inhibited YTHDF1 and GLS1 protein expression and GLS1 activity, adjusted the intestinal flora, and significantly reversed the increased Firmicutes/Bacteroidetes ratio. ConclusionGQD has shown superior efficacy in reversing OXA-resistance and increasing sensitivity. These findings indicate that the therapy combined with GQD has potential utility in the treatment of OXA-resistant CRC.

Norovirus NS1/2 protein increases glutaminolysis for efficient viral replication.

Viruses are obligate intracellular parasites that rely on host cell metabolism for successful replication. Thus, viruses rewire host cell pathways involved in central carbon metabolism to increase the availability of building blocks for successful propagation. However, the underlying mechanisms of virus-induced alterations to host metabolism are largely unknown. Noroviruses (NoVs) are highly prevalent pathogens that cause sporadic and epidemic viral gastroenteritis. In the present study, we uncovered several strain-specific and shared host cell metabolic requirements of three murine norovirus (MNV) strains, MNV-1, CR3, and CR6. While all three strains required glycolysis, glutaminolysis, and the pentose phosphate pathway for optimal infection of macrophages, only MNV-1 relied on host oxidative phosphorylation. Furthermore, the first metabolic flux analysis of NoV-infected cells revealed that both glycolysis and glutaminolysis are upregulated during MNV-1 infection of macrophages. Glutamine deprivation affected the viral lifecycle at the stage of genome replication, resulting in decreased non-structural and structural protein synthesis, viral assembly, and egress. Mechanistic studies further showed that MNV infection and overexpression of the non-structural protein NS1/2 increased the enzymatic activity of the rate-limiting enzyme glutaminase. In conclusion, the inaugural investigation of NoV-induced alterations to host glutaminolysis identified NS1/2 as the first viral molecule for RNA viruses that regulates glutaminolysis either directly or indirectly. This increases our fundamental understanding of virus-induced metabolic alterations and may lead to improvements in the cultivation of human NoVs.

Kinetics characterization of a low immunogenic recombinant l-asparaginase from Phaseolus vulgaris with cytotoxic activity against leukemia cells

l-asparaginases play a crucial role in the treatment of acute lymphoblastic leukemia (ALL), a type of cancer that mostly affects children and teenagers. However, it is common for these molecules to cause adverse reactions during treatment. These downsides ignite the search for novel asparaginases to mitigate these problems. Thus, this work aimed to produce and characterize a recombinant asparaginase from Phaseolus vulgaris (Asp-P). In this study, Asp-P was expressed in Escherichia coli with high yields and optimum activity at 40 °C, pH 9.0. The enzyme Km and Vmax values were 7.05 mM and 1027 U/mg, respectively. Asp-P is specific for l-asparagine, showing no activity against l-glutamine and other amino acids. The enzyme showed a higher cytotoxic effect against Raji than K562 cell lines, but only at high concentrations. In silico analysis indicated that Asp-P has lower immunogenicity than a commercial enzyme. Asp-P induced biofilm formation by Candida sp. due to sublethal dose, showing an underexplored potential of asparaginases. The absence of glutaminase activity, lower immunogenicity and optimal activity similar to physiological temperature conditions are characteristics that indicate Asp-P as a potential new commercial enzyme in the treatment of ALL and its underexplored application in the treatment of other diseases.

Reduced adipocyte glutaminase activity promotes energy expenditure and metabolic health

Glutamine and glutamate are interconverted by several enzymes and alterations in this metabolic cycle are linked to cardiometabolic traits. Herein, we show that obesity-associated insulin resistance is characterized by decreased plasma and white adipose tissue glutamine-to-glutamate ratios. We couple these stoichiometric changes to perturbed fat cell glutaminase and glutamine synthase messenger RNA and protein abundance, which together promote glutaminolysis. In human white adipocytes, reductions in glutaminase activity promote aerobic glycolysis and mitochondrial oxidative capacity via increases in hypoxia-inducible factor 1α abundance, lactate levels and p38 mitogen-activated protein kinase signalling. Systemic glutaminase inhibition in male and female mice, or genetically in adipocytes of male mice, triggers the activation of thermogenic gene programs in inguinal adipocytes. Consequently, the knockout mice display higher energy expenditure and improved glucose tolerance compared to control littermates, even under high-fat diet conditions. Altogether, our findings highlight white adipocyte glutamine turnover as an important determinant of energy expenditure and metabolic health.

AMPK targets PDZD8 to trigger carbon source shift from glucose to glutamine

The shift of carbon utilization from primarily glucose to other nutrients is a fundamental metabolic adaptation to cope with decreased blood glucose levels and the consequent decline in glucose oxidation. AMP-activated protein kinase (AMPK) plays crucial roles in this metabolic adaptation. However, the underlying mechanism is not fully understood. Here, we show that PDZ domain containing 8 (PDZD8), which we identify as a new substrate of AMPK activated in low glucose, is required for the low glucose-promoted glutaminolysis. AMPK phosphorylates PDZD8 at threonine 527 (T527) and promotes the interaction of PDZD8 with and activation of glutaminase 1 (GLS1), a rate-limiting enzyme of glutaminolysis. In vivo, the AMPK-PDZD8-GLS1 axis is required for the enhancement of glutaminolysis as tested in the skeletal muscle tissues, which occurs earlier than the increase in fatty acid utilization during fasting. The enhanced glutaminolysis is also observed in macrophages in low glucose or under acute lipopolysaccharide (LPS) treatment. Consistent with a requirement of heightened glutaminolysis, the PDZD8-T527A mutation dampens the secretion of pro-inflammatory cytokines in macrophages in mice treated with LPS. Together, we have revealed an AMPK-PDZD8-GLS1 axis that promotes glutaminolysis ahead of increased fatty acid utilization under glucose shortage.

Sirtuin 5 alleviates apoptosis and autophagy stimulated by ammonium chloride in bovine mammary epithelial cells.

Ammonia (NH3) is an irritating and harmful gas that affects cell apoptosis and autophagy. Sirtuin 5 (SIRT5) has multiple enzymatic activities and regulates NH3-induced autophagy in tumor cells. In order to determine whether SIRT5 regulates NH3-induced bovine mammary epithelial cell apoptosis and autophagy, cells with SIRT5 overexpression or knockdown were generated and in addition, bovine mammary epithelial cells were treated with SIRT5 inhibitors. The results showed that SIRT5 overexpression reduced the content of NH3 and glutamate in cells by inhibiting glutaminase activity in glutamine metabolism, and reduced the ratio of ADP/ATP. The results in the SIRT5 knockdown and inhibitor groups were comparable, including increased content of NH3 and glutamate in cells by activating glutaminase activity, and an elevated ratio of ADP/ATP. It was further confirmed that SIRT5 inhibited the apoptosis and autophagy of bovine mammary epithelial cells through reverse transcription-quantitative PCR, western blot, flow cytometry with Annexin V FITC/PI staining and transmission electron microscopy. In addition, it was also found that the addition of LY294002 or Rapamycin inhibited the PI3K/Akt or mTOR kinase signal, decreasing the apoptosis and autophagy activities of bovine mammary epithelial cells induced by SIRT5-inhibited NH3. In summary, the PI3K/Akt/mTOR signal involved in NH3-induced cell autophagy and apoptosis relies on the regulation of SIRT5. This study provides a new theory for the use of NH3 to regulate bovine mammary epithelial cell apoptosis and autophagy, and provides guidance for improving the health and production performance of dairy cows.

Abstract PO2-15-11: Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models

Abstract Background: Glutamine is an important metabolic substrate in many aggressive tumors, with comparable importance to glucose metabolism. Utilizing human breast cancer mouse xenograft models, we studied the kinetics of the PET imaging agent, [5-11C] glutamine, a biochemical authentic substrate for glutamine metabolism, to further characterize the metabolism of glutamine and downstream labeled metabolites. Studies were performed with and without inhibition of the enzyme, glutaminase (GLS), the first step in glutamine catabolism that generates glutamate, and key target for therapy directed to glutamine-metabolizing cancers. Methods: The study used xenograft mouse models for two breast cancer cell lines, HCC1806, a highly glutaminolytic triple-negative cell line, and MCF-7, a hormone receptor positive line with only low levels of glutaminolysis. Mice were injected with 5-[11C]L-glutamine, and the contributions of individual metabolites to the total [11C] signal in blood and tumor tissue were estimated at 10, 20, and 30 minutes after injection by assaying the fractional contributions of [5-11C] glutamine and labeled metabolites, specifically [5-11C] glutamate, [11C] CO2 and all other metabolites with [11C], in both blood and tumor tissue. This generated estimated time activity curves for [5-11C] glutamine and downstream metabolites in both HCC1806 and MCF-7 mouse models with and without CB-839 treatment, an inhibitor of GLS. We also investigated a compartment model that describes these data, including model simulations that fit the model to match the measured curves. Results: We found that, out to 30 minutes post-injection, the vast majority of tumor radioactivity was in the form of either 5-11C] glutamine or [5-11C] glutamate, with smaller amounts of radioactivity in the form of more downstream metabolites, including [11C]CO2. Systemic administration of the glutaminase inhibitor alters the arterial input curve by reducing the systemic rate of conversion of [5-11C] glutamine to [5-11C] glutamate. However, in GLS active tumors, [5-11C] glutamate retained in the large cellular glutamate pool represents a greater fraction of total radioactivity than in the blood and leads to a total tumor time-activity curve that is only marginally different than the total tumor time-activity curve after GLS inhibition. Data from blood and tissue analysis were used to test a hypothetical kinetic model for [5-11C] glutamine, which provided estimates of glutamate and glutamate tumor tissue concentrations and tumor GLS activity that agree with direct measurements in the animal models. Conclusion: The study of [ 5-11C] glutamine in breast cancer models leads to kinetic insight that are in line with biochemical studies and direct measurement taken for tumor tissue obtained from the animal models. Retention of metabolized [5-11C] glutamine as [5-11C] glutamate in a large tissue pool in tumors with high GLS activity makes non-invasive inference of GLS activity challenging without additional ex vivo analysis, supporting the use of non-metabolized glutamine analogs such as such as [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) to infer in vivo glutamine metabolism. Citation Format: Christopher Hensley, Prashanth Padakanti, Hoon Choi, Christina Dulal, Hsiaojiu Lee, Austin Pantel, Rong Zhou, David Mankoff. Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-11.

Effects of Different Organic Fertilizer Substitutions for Chemical Nitrogen Fertilizer on Soil Fertility and Nitrogen Use Efficiency of Foxtail Millet

Conventional fertilizer management can destroy the structure of soil. Replacing chemical fertilizers with organic fertilizers can improve soil quality and nitrogen use efficiency. We aimed to study the effects of organic fertilizer substitutions for chemical nitrogen fertilizer on soil fertility and nitrogen use efficiency in order to clarify the effectiveness of the available nutrient management measures in improving soil quality and increasing foxtail millet yield. A field experiment was carried out over two consecutive years, and a total of six treatments were set up: no fertilizer (CK), chemical nitrogen fertilizer alone (N), the substitution of 25% of chemical nitrogen fertilizer with bio-organic fertilizer (N25A1), the substitution of 25% of chemical nitrogen fertilizer with fermented mealworm manure (N25B1), the substitution of 50% of chemical nitrogen fertilizer with bio-organic fertilizer (N50A2), and the substitution of 50% of chemical nitrogen fertilizer with fermented mealworm manure (N50B2). The results of this study show the following: (1) Compared with chemical nitrogen fertilizer, the substitution of organic fertilizer for nitrogen fertilizer reduced the bulk density and solid phase of the soil, and it increased the total porosity, water content, liquid phase, and gas phase of the soil. (2) Compared with nitrogen fertilizer, the use of an organic fertilizer increased the contents of nitrate nitrogen, ammonium nitrogen, and total nitrogen in the soil by 13.59~52.56%, 4.47~18.27%, and 4.40~12.09%, respectively. The content of alkaline nitrogen increased by 1.70~32.37%, and the contents of soil available potassium, available phosphorus, and organic matter also increased. (3) The activities of sucrase, urease, glutaminase, and asparaginase were improved by replacing chemical nitrogen fertilizer with organic fertilizer. The N25 treatments performed better than the N50 treatments, and fermented mealworm manure performed better than biological organic fertilizer. (4) A moderate application of organic fertilizer (N25) can increase the grain yield, ear weight, grain weight, and 1000-grain weight of foxtail millet, whereas excessive application of organic fertilizer (N50) can reduce foxtail millet yield. (5) Replacing chemical nitrogen fertilizer with organic fertilizer can improve the agronomic use efficiency, physiological efficiency, biased productivity, harvest index, and apparent use efficiency of nitrogen fertilizer. In this study, the substitution of 25% of chemical nitrogen fertilizer with fermented mealworm manure was the best combination for restoring crop productivity and soil quality.

Effect of bioremediation or organic treatments of soil contaminated with some heavy metals on the biological and L- glutaminase activity

For the purpose of identify the ability of bioremediation or organic treatments of soil to reduce the harmful effect of heavy metals including Zn, pb and cd on the biological activity such as soil respiration, the total number of fungi and the activity of the L-glutaminase enzyme have been studied. A laboratory incubation experiment was applied in the Soil Microbiology Laboratory in the Department of Soil Sciences and Water Resources in the College of Agriculture– Basrah University. The soil of the surface layer (0-30 cm) from Qurna district, north of Basrah province was contaminated with the critical levels of the heavy metals cd, Pb, Zn and were placed in an incubator at 30°C for 14 days. The biological and organic treatments process was applied by inoculating soil contaminated with Aspergillus niger or humic acid (40 L ha-1) or sterilized cow manure (4%), incubated at 25°C for 30 days, then some biological parameters were estimated at the periods of 10, 20 and 30 days. Results have been showed that an inhibitory effect of the studied heavy metals on the biological parameters, and the Cd has been recorded the highest percentage of inhibition of total number of fungi (30.67%), the amount of CO2 released (13.93%) and the activity of the L-glutaminase enzyme (45.33%) compared to the control treatment. The ability of the treatments materials to remove heavy metals were (89.02%) for A. niger compared to humic acid (63.48%) and cow manure (48.71%). It was reflected in an increase in the number of fungi, which recorded 152, 167.75, 174.5 cfu g-1 when treated with A. niger, cow manure and humic acid, respectively compared to the control treatment which was (65.92 cfu g-1). The biological and organic treatments also increased the level of liberated CO2 from the polluted soil to 268.62, 253.86, 303.59 mg CO2 g-1, respectively compared to the control treatment. The activity of the L-glutaminase enzyme has increased due to application of treatments. It was 57.02, 48.97, 70.41 µg NH4+ g-1 2hr.-1 for inoculated with A. niger or treated with cow manure and humic acid, respectively, while the control treatment was 37.19 µg NH4+ g-12hr.-1.

Long-Term Cropping Management Practices Affect the Biochemical Properties of an Alabama Ultisol

Interest in improving the long-term sustainability of agricultural production systems has focused on identifying management practices that promote soil health. No tillage, cover cropping, and amending soils with broiler (Gallus gallus domesticus L.) litter are commonly adopted conservation practices that have been shown to improve soil fertility and crop yield. However, the overall influence of these conservation practices on soil health in the southeastern US are not well understood. Thus, a study was conducted to evaluate the influence of tillage, broiler litter (BL) applications, and cropping systems on soil biochemical properties. Soils were collected from field research plots under long-term management (&gt;than 25 years of tillage, 15 years of broiler litter application, and 15 years of cropping system). Soil microbial biomass, C, N, and P, amidohydrolases, and dissolved organic matter (DOM) were evaluated as indicators of soil health. Adopting tillage and BL into the agricultural management system modified the biochemical parameters of the soils evaluated. Most of these modifications occurred in the 0–5 cm depth. Higher microbial biomass carbon (MBC; 85%) and nitrogen (MBN; 10%) and enzyme activities of asparaginase (65%) and glutaminase (70%) were observed in the 0–5 cm depth under no tillage (NT) compared to conventional tillage (CT), indicating greater biological activities were established in these soil ecosystems. Broiler litter applications increased microbial biomass N and activities of asparaginase and glutaminase in both soil depths. In addition, microbial biomass phosphorus (MBP) was increased following BL application in the 0–5 cm depth. The results suggest that long-term management of NT and BL additions can improve the health of eroded southeastern US soils by altering the soil biochemical parameters.

Babaodan overcomes cisplatin resistance in cholangiocarcinoma via inhibiting YAP1

Context Cholangiocarcinoma with highly heterogeneous, aggressive, and multidrug resistance has a poor prognosis. Although babaodan (BBD) combined with cisplatin improved non-small cell lung cancer efficacy, its impact on overcoming resistance in cholangiocarcinoma remains unexplored. Objective This study explored the role and mechanism of BBD on cisplatin resistance in cholangiocarcinoma cells (CCAs). Materials and methods Cisplatin-resistant CCAs were exposed to varying concentrations of cisplatin (25–400 μg/mL) or BBD (0.25–1.00 mg/mL) for 48 h. IC50 values, inhibition ratios, apoptosis levels, DNA damage, glutathione (GSH) levels, oxidized forms of GSH, total GSH content, and glutaminase relative activity were evaluated using the cell counting kit 8, flow cytometry, comet assay, and relevant assay kits. Results BBD-reduced the cisplatin IC50 in CCAs from 118.8 to 61.83 μg/mL, leading to increased inhibition rate, apoptosis, and DNA damage, and decreased expression of B-cell lymphoma-2, p-Yes-associated protein 1/Yes-associated protein 1, solute carrier family 1 member 5, activating transcription factor 4, and ERCC excision repair 1 in a dose-dependent manner with maximum reductions of 78.97%, 51.98%, 54.03%, 56.59%, and 63.22%, respectively; bcl2-associated X and gamma histone levels were increased by 0.43–115.77% and 22.15–53.39%. The impact of YAP1 knockdown on cisplatin-resistant CCAs resembled BBD. GSH, oxidized GSH species, total GSH content, and glutaminase activity in cisplatin-resistant CCAs with BBD treatment also decreased, while YAP1 overexpression countered BBD’s effects. Discussion and conclusion This study provides a scientific basis for BBD clinical application and provides a new direction for BBD biological mechanism research.

Ammoniagenic Action of Valproate without Signs of Hepatic Dysfunction in Rats: Possible Causes and Supporting Evidence.

(1) Background: Valproic acid (VPA) is one of the frequently prescribed antiepileptic drugs and is generally considered well tolerated. However, VPA neurologic adverse effects in the absence of liver failure are fairly common, suggesting that in the mechanism for the development of VPA-induced encephalopathy, much more is involved than merely the exposure to hyperammonemia (HA) caused by liver insufficiency to perform detoxification. Taking into account the importance of the relationship between an impaired brain energy metabolism and elevated ammonia production, and based on the ability of VPA to interfere with neuronal oxidative pathways, the current study intended to investigate a potential regional ammoniagenic effect of VPA on rats' brains by determining activities of the enzymes responsible for ammonia production and neutralization. (2) Methods: Rats received a single intraperitoneal injection of VPA (50, 100, 250, 500 mg/kg). Plasma, the neocortex, the cerebellum, and the hippocampus were collected at 30 min after injection. The levels of ammonia, urea, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured in blood plasma. The activities of glutaminase and glutamate dehydrogenase (GDH) in mitochondria and the activities of AMP deaminase (AMPD), adenosine deaminase (ADA), and glutamine synthetase (GS) in cytosolic fractions isolated from rat brain regions were measured. Ammonia, ALT, and AST values were determined in the mitochondrial and cytosolic fractions. (3) Results: Multi-dose VPA treatment did not significantly affect the plasma levels of ammonia and urea or the ALT and AST liver enzymes. Significant dose-independent increases in the accumulation of ammonia were found only in the cytosol from the cerebellum and there was a strong correlation between the ammonia level and the ADA activity in this brain structure. A significant decrease in the AMPD and AST activities was observed, while the ALT activity was unaffected. Only the highest VPA dose (500 mg/kg) was associated with significantly less activity of GS compared to the control in all studied brain structures. In the mitochondria of all studied brain structures, VPA caused a dose-independent increases in ammonia levels, a high concentration of which was strongly and positively correlated with the increased GDH and ALT activity, while glutaminase activity remained unchanged, and AST activity significantly decreased compared to the control in all studied brain structures. (4) Conclusions: This study highlights the rat brain region-specific ammoniagenic effects of VPA, which may manifest themselves in the absence of hyperammonemia. Further research should analyze how the responsiveness of the different brain regions may vary in VPA-treated animals that exhibit compromised energy metabolism, leading to increased ammoniagenesis.

Audiogenic kindling activates glutamatergic system in the hippocampus of rats with genetic predisposition to audiogenic seizures

Temporal lobe epilepsy (TLE) development is associated with dysregulation of glutamatergic transmission in the hippocampus; however, detailed molecular mechanisms of pathological changes are still poorly understood. In the present study, we performed the complex analysis of glutamatergic system in the hippocampus of Krushinsky-Molodkina (KM) rats genetically prone to audiogenic seizures (AGS). Daily AGS stimulations (audiogenic kindling) were used to reproduce the dynamics of TLE development. Naïve KM rats were used as a control. After 14 AGS, at the stage of developing TLE, KM rats demonstrated significant upregulation of extracellular signal-regulated kinases (ERK) 1 and 2, cAMP response element-binding protein (CREB), and c-Fos in the hippocampus indicating activation of the hippocampal cells. These changes were accompanied with an increase in glutaminase and vesicular glutamate transporter (VGLUT) 2 suggesting the activation of glutamate production and loading into the synaptic vesicles. After 21 AGS, when TLE was fully-established, alterations were similar but more pronounced, with higher activation of glutaminase, increase in glutamate production, upregulation of VGLUT1 and 2, and Fos-related antigen 1 (Fra-1) along with c-Fos. Analysis of glutamate receptors showed variable changes. Thus, after 14 AGS, simultaneous increase in metabotropic glutamate receptor mGluR1 and decrease in ionotropic N-methyl-D-aspartate (NMDA) receptors could reflect compensatory anti-epileptic mechanism, while further kindling progression induced upregulation of ionotropic receptors, probably, contributing to the hippocampal epileptization. However, we revealed practically no alterations in the expression of synaptic proteins. Altogether, obtained results suggested that overactivation of glutamate production in the hippocampus strongly contributed to TLE development in KM rats.

P003 Metabolic Reprogramming in Intestinal Fibrosis: Integrating Metabolomics, Single-Cell Transcriptomics and Isotope Tracing

Abstract Background This study aims to systematically elucidate the metabolic landscape of intestinal fibrosis in Crohn's disease, revealing the key reprogrammed metabolic pathways in the fibrotic microenvironment. Methods We collected normal and fibrosis tissues from 122 Crohn's disease patients undergoing surgery for fibrosis-induced intestinal obstruction, and established DSS/TNBS-induced murine intestinal fibrosis model. To measure metabolites level in normal and fibrotic tissues, untargeted metabolomics was performed. C13-labeled glutamine was used for metabolic tracing to delineate metabolic fluxes in vitro and in vivo. We performed integration analysis by merging metabolomics and metabolic tracing data with established single-cell transcriptomic profiles. Isolation and further validation of human intestinal stromal cells were carried out, and gene expression was assessed using qRT-PCR and immunofluorescence. Results Significant metabolic heterogeneity was observed between normal and fibrotic tissues, with differential metabolites enriched in glucose and amino acid metabolism. Notably, intermediates of glycolysis were significantly increased, while tricarboxylic acid (TCA) cycle intermediates were decreased in fibrotic regions (p&amp;lt;0.05). Amino acids were broadly downregulated in fibrosis, whereas glutamate and glutamine were highly enriched (p&amp;lt;0.05). Mechanistic insights into these changes were provided by isotope tracing, revealing enhanced glycolysis and weakened TCA flux attributed to transition from TCA intermediate α-KG to glutamate. Moreover, catabolism of glutamine to glutamate was enhanced. Glutamate from above pathways converted to proline, which is the ingredient of extracellular matrix (ECM). Single-cell transcriptomic analysis indicated a high enrichment of genes related to glutamine metabolism in stromal cells, with significant upregulation of enzymes such as GLS (Glutaminase) and PYCR1 (Pyrroline-5-carboxylate Reductase 1) involved in glutamine and proline synthesis. Immunofluorescence and qRT-PCR confirmed the upregulation of GLS and PYCR1. Further experiments using primary stromal cells and murine models demonstrated that inhibiting GLS activity reduced ECM synthesis, alleviating intestinal fibrosis. Conclusion In intestinal fibrosis, glycolysis and glutamine catabolism enhanced. Stromal cells utilized the reprogrammed metabolic pathway to synthesize glutamate, thereby promoting pathological ECM anabolism. Targeting the key enzyme GLS represent a potential therapeutic strategy for intestinal fibrosis.