Enzyme Glutamine Synthetase Research Articles

Glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase inhibition in the presence of pro-inflammatory cytokines contribute to the metabolic imbalance of diabetic retinopathy

Diabetic retinopathy (DR) is the leading cause of vision impairment in working age adults. In addition to hyperglycemia, retinal inflammation is an important driving factor for DR development. Although DR is clinically described as diabetes-induced damage to the retinal blood vessels, several studies have reported that metabolic dysregulation occurs in the retina prior to the development of microvascular damage. The two most commonly affected metabolic pathways in diabetic conditions are glycolysis and the glutamate pathway. We investigated the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutamine synthetase (GS) in an in-vitro model of DR incorporating high glucose and pro-inflammatory cytokines. We found that GAPDH and GS enzyme activity were not significantly affected in hyperglycemic conditions or after exposure to cytokines alone, but were significantly decreased in the DR model. This confirmed that pro-inflammatory cytokines IL-1β and TNFα enhance the hyperglycemic metabolic deficit. We further investigated metabolite and amino acid levels after specific pharmacological inhibition of GAPDH or GS in the absence/presence of pro-inflammatory cytokines. The results indicate that GAPDH inhibition increased glucose and addition of cytokines increased lactate and ATP levels and reduced glutamate levels. GS inhibition did not alter retinal metabolite levels but the addition of cytokines increased ATP levels and caused glutamate accumulation in Müller cells. We conclude that it is the action of pro-inflammatory cytokines concomitantly with the inhibition of the glycolytic or GS mediated glutamate recycling that contribute to metabolic dysregulation in DR. Therefore, in the absence of good glycemic control, therapeutic interventions aimed at regulating inflammation may prevent the onset of early metabolic imbalance in DR.

Small loci of astroglial glutamine synthetase deficiency in the postnatal brain cause epileptic seizures and impaired functional connectivity.

The astroglial enzyme glutamine synthetase (GS) is deficient in small loci in the brain in adult patients with different types of focal epilepsy; however, the role of this deficiency in the pathogenesis of epilepsy has been difficult to assess due to a lack of sufficiently sensitive and specific animal models. The aim of this study was to develop an in vivo approach for precise and specific deletions of the GS gene in the postnatal brain. We stereotaxically injected various adeno-associated virus (AAV)-Cre recombinase constructs into the hippocampal formation and neocortex in 22-70-week-old GSflox/flox mice to knock out the GS gene in a specific and focal manner. The mice were subjected to seizure threshold determination, continuous video-electroencephalographic recordings, advanced in vivo neuroimaging, and immunocytochemistry for GS. The construct AAV8-glial fibrillary acidic protein-green fluorescent protein-Cre eliminated GS in >99% of astrocytes in the injection center with a gradual return to full GS expression toward the periphery. Such focal GS deletion reduced seizure threshold, caused spontaneous recurrent seizures, and diminished functional connectivity. These results suggest that small loci of GS deficiency in the postnatal brain are sufficient to cause epilepsy and impaired functional connectivity. Additionally, given the high specificity and precise spatial resolution of our GS knockdown approach, we anticipate that this model will be extremely useful for rigorous in vivo and ex vivo studies of astroglial GS function at the brain-region and single-cell levels.

L‐glutamate inhibits blue mould caused by Penicillium expansum in apple fruits by altering the primary nitrogen and carbon metabolisms

SummaryAlthough several studies have shown the ability of l‐glutamate (glutamate) to mitigate the stress imposed by pathogens, the underlying mechanism is still in its infancy. To gain further knowledge, this study focussed on the effect of glutamate on primary nitrogen and carbon metabolisms during apple‐Penicillium expansum interaction. The obtained result showed that glutamate could effectively restrict blue mould rot development in apples, but had no direct impact on fungal growth in vitro. The application of glutamate increased the level of nitrogen in apples, resulting in a disrupted balance of carbon and nitrogen. Consistently, the key enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) involved in the GS/GOGAT cycle, and the deaminating activity of glutamate dehydrogenase (GDH), forming 2‐oxoglutarate and ammonium from glutamate, were promptly stimulated by glutamate. Interestingly, glutamate led to considerable consumption of the tricarboxylic acid (TCA) cycle intermediates, such as isocitric acid and citric acid, accompanied by the enhancement of malate dehydrogenase and succinate dehydrogenase activities. Collectively, exogenous application of glutamate might confer blue mould resistance in apples, at least in part, by redirecting host’s primary nitrogen and carbon metabolisms, such as the activation of the GS/GOGAT cycle, deaminating activity of GDH and the TCA cycle.

Late adolescence mortality in mice with brain-specific deletion of the volume-regulated anion channel subunit LRRC8A.

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated the brain-wide conditional LRRC8A knock-out mice (LRRC8A bKO) using NestinCre-driven Lrrc8aflox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase. Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.

Role of oxylipin on Luffa seedlings exposed to NaCl and UV-B stresses: An insight into mechanism

Nowadays, among several abiotic stresses, salt, especially NaCl and UV-B are of major concern. They lead to deleterious effects on plant growth and ultimately affect crop productivity. The present study was planned to find out some ameliorative solution against these stresses. Here, the modulatory action of two oxylipins, namely, methyl jasmonate (MeJA) and 12-Oxo-phytodienoic acid (OPDA) on growth, photosynthetic performance, nitrate/ammonia assimilating enzymes, and nutritive values of Luffa Mill. seedlings grown under NaCl (20 and 40 mM) and/or enhanced UV-B stresses (ambient: 8.2 kJ m−2 d−1 + additional: 2.2 kJ m−2 s−1) were analyzed. Both the stresses when given alone, negatively affected the fresh mass, root/shoot ratio, leaf area, photosynthetic pigments content, photosynthetic oxygen yield and, chlorophyll a fluorescence kinetic parameter. This decline was further aggravated upon combined exposure to the stressors. However, supplementation of MeJA/OPDA effectively counteracted the negative impact on important growth-regulating processes. The activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) enzymes, as well as the contents of inorganic nitrogen, protein, and carbohydrate, were increased with the supplementation of MeJA/OPDA. The increase in the Na+ and Cl‾ contents due to NaCl or/and UV-B was depreciated by MeJA or OPDA. Ameliorating behaviour of MeJA or OPDA is correlated with improved photosynthetic activity and nitrogen metabolism. These findings, point out that supplementation of MeJA/OPDA, particularly OPDA more favourably regulated the growth-promoting activities, which can be linked with the mitigation of NaCl and UV-B stress.

Dysembryoplastic neuroepithelial tumors: A single-institutional series with special reference to glutamine synthetase expression

Dysembryoplastic neuroepithelial tumors (DNT) is a benign (World Health Organisation, WHO, grade I) glioneuronal tumor and it represent one of the most frequent neoplasm in patient affected by seizures. The epileptic neuronal activity can be determined by abnormal synchronization, excessive glutamate excitation and\\or inadequate GABA inhibition. Increasing evidence suggests that the astrocytes might be involved in this process even if neurons play a relevant role. In particular astrocytes promote the clearance of glutamate, a potent excitatory neurotransmitter of the central nervous system. Indeed, elevated concentrations of extracellular glutamate may determine iper-excitability and seizures as well as other neurological disorders. So, astrocytes, converting glutamate into glutamine via the enzyme glutamine synthetase (GS), could play a protective anti-seizures role. In the present study, we analyzed the immunohistochemical expression of GS in 20 DNTs specimens documenting a constant immunoistochemical expression of GS in astrocytes of the lesional tissue and of the cerebral cortex.

Quercetin and catechin assuage redox imbalance and neurochemical dysfunction in rotenone-induced neurotoxicity: A comparative in vivo experiment supported by in silico study

BackgroundQuercetin and catechin are structurally-related compounds that are capable of preventing or attenuating oxidative damage and mitochondrial dysfunction. Rotenone is a naturally occurring compound that has been used to model neurotoxicity characterized by Parkinson disease-like symptoms. PurposeThis study investigated the comparative property of quercetin and catechin, to ameliorate oxidative damage and neurochemical dysfunction in hippocampal and cortical regions of brain of rats intoxicated with rotenone in vivo, supported by in silico study. MethodMale Wistar rats were subcutaneously administered rotenone for 10 days followed by post-treatment with catechin or quercetin (5, 10 and 20 mg/kg) for 3 days. Markers of oxidative stress and neurochemical dysfunction were biochemically estimated in the hippocampus and cortex of the brain of rats while in silico study was carried out to evaluate the molecular interaction of some neurochemical enzymes with the compounds. ResultQuercetin and catechin ameliorated the reduction in complex 1 and Na+K+ATPase activities while attenuating the elevated lactate dehydrogenase activity in the brain of rotenone-intoxicated rats. Disturbances in acetylcholine, dopamine and glutamate metabolism as well as the evoked oxidative stress in rotenone-intoxicated rats were mitigated by quercetin and catechin. The in silico study revealed the key molecular interaction between the flavonoids and targeted enzymes (tyrosine hydroxylase, monoamine oxidase, glutamine synthetase and Na+K+ATPase) to be by hydrogen bond and hydrophobic interaction. ConclusionCatechin and quercetin significantly assuaged cortical and hippocampal redox stress and attenuated neurochemical dysfunction caused by rotenone toxicity. Furthermore, this study highlighted that the neuroprotective potentials of the flavonoids is a function of their structural patterns, and quercetin elicited superior activity over catechin due to its structural advantages.

Astroglial Glutamine Synthetase and the Pathogenesis of Mesial Temporal Lobe Epilepsy.

The enzyme glutamine synthetase (GS), also referred to as glutamate ammonia ligase, is abundant in astrocytes and catalyzes the conversion of ammonia and glutamate to glutamine. Deficiency or dysfunction of astrocytic GS in discrete brain regions have been associated with several types of epilepsy, including medically-intractable mesial temporal lobe epilepsy (MTLE), neocortical epilepsies, and glioblastoma-associated epilepsy. Moreover, experimental inhibition or deletion of GS in the entorhinal-hippocampal territory of laboratory animals causes an MTLE-like syndrome characterized by spontaneous, recurrent hippocampal-onset seizures, loss of hippocampal neurons, and in some cases comorbid depressive-like features. The goal of this review is to summarize and discuss the possible roles of astroglial GS in the pathogenesis of epilepsy.

Metabolic Responses to Contrasting Levels of N Fertilization during Ear Leaf Transition from Assimilation to Remobilization at Grain Filling in Maize

MAIZE grain yield is shaped by source-sink relationships during grain filling. Such relations are driven by complex metabolic changes, responsive to N availability and are not fully understood. Here, the impact of limited and sufficient N fertilization on the metabolic interconversions in the ear leaf of a new maize field-grown hybrid (Tzi8 × Mo17) during its critical transition from assimilation to remobilization were chronologically investigated at 0, 5, 10, 15, and 20 days after pollination. N-deficient plants produced 43% less grain yield and 13% less biomass however, they had 51% higher root dry weight and higher Root/Shoot ratio at anthesis. The low N-induced reduction in yield and biomass accumulation was associated with earlier chlorophyll degradation and overall decrease in leaf chlorophyll, total soluble proteins, carbon (C: sucrose & total soluble sugars), and N assimilates (leaf N, nitrate, ammonia & amino acids). In contrast, N-deficient plants accumulated 18% more starch and 24 % flavonoids than N-sufficient plants and such responses were driven by low N-induced sink limitation. N-deficient plants also had significantly higher activities of N remobilizing (asparaginase & protease) but lower activities of N assimilating (nitrate reductase & glutamine synthetase) enzymes compared to N-sufficient plants. Glutamate and aspartate followed by branched amino acids dominated the amino acids pool under both N conditions. Altogether, the accumulation of starch and flavonoids and the induction of N remobilizing enzymes represent low N-specific responses whereas the rest of responses depict the common metabolic interconversions between adequate and limited N-induced responses during maize grain filling.

Deletion of Glutamine Synthetase Gene Disrupts the Survivability and Infectivity of Leishmania donovani.

Glutamine synthetase (GS) is one of the most important metabolic enzymes which catalyzes ligation of glutamate and ammonia to form glutamine. Previous studies from our lab had revealed significant differences in parasite and host GS enzyme which warranted us to further work on its relevance in parasite. To analyze glutamine synthetase function in Leishmania, we generated GS overexpressors and knockout mutants and evaluated their ability to grow in vitro in monocyte differentiated macrophage and in vivo by infections in BALB/c mice. GS knocked out strain showed significant growth retardation with delayed cell cycle progression and morphological alteration. Null mutants exhibited attenuated infectivity both in in vitro and in vivo experiments and the effect was reverted back when infected with GS complemented parasites. This indicated that the alterations in phenotype observed were indeed due to GS knockout. GS knockout also made the parasite increasingly sensitive to Miltefosine. Detailed investigation of mode of parasite death upon Miltefosine treatment by dual staining with Annexin-V conjugated FITC and propidium iodide, pointed towards apoptotic or necrotic mode of cell death. This is the first report to confirm that GS is essential for the survivability and infectivity of Leishmania donovani, and can be exploited as a potential drug-target.

Transcriptome Analysis for Fraxinus mandshurica Rupr. Seedlings from Different Carbon Sequestration Provenances in Response to Nitrogen Deficiency

To explore the molecular regulatory mechanism of high-carbon (C) sequestration Fraxinus mandshurica Rupr. (F. mandshurica) provenance and the expression profile of F. mandshurica during nitrogen (N) starvation, the foliage and roots of the annual Wuchang (WC) seedlings with greater C amount and Hailin (HL) seedlings with smaller C amount, which were grown in N-deficient nutrition and complete N, were used for RNA-seq and physiological determination, respectively. One thousand and fifty-seven differentially expressed genes (DEGs) between WC and HL and 8173 DEGs related to N deficiency were identified, respectively. The root of F. mandshurica responded to N deficiency more strongly than foliar. The target genes that responded to N deficiency in roots were mainly regulatory genes (transcription factors, hormones and protein kinases), and their response patterns were upregulated. The growth and N concentration in both WC and HL were reduced by the N deficiency, which might result from the decrease of the leaf Nitrate reductase (NR) and glutamine synthetase (GS) enzyme activity and ABA content, although the root-to-shoot ratio; lateral root number; lignin content; endogenous hormones content (GA, IAA and ZR); root GS and glutamate synthetase activity and transcriptional level of most of the regulatory genes were increased. The C sequestration capacity in WC was greater than that in HL, which related to the higher GS enzymes activity and transcriptional levels of regulatory genes and metabolic genes (terpenes, carbohydrates, and lipid energy). However, the C sequestration advantage of WC was significantly reduced by the N deficiency, which was due to the smaller response to N deficiency compared to HL.

Cover crops in the inter-row of a table grape vineyard managed with irrigation sensors: Effects on yield, quality and glutamine synthetase activity in leaves

Researchers, farmers and viticulturists have focused on the development and adoption of alternative management strategies that are more environmentally and economically sustainable. One strategy could be cover crops, which are often sowed as a mix of non-leguminous and leguminous species, with positive effects on nitrogen nutrition of the vines . Irrigation of cover cropped vineyards can be managed with the use of wireless soil sensors. Nitrogen (N) is the most important soil mineral nutrient for plant growth and glutamine synthetase (GS) is not only responsible for N assimilation, but is also involved in different responses to both biotic or abiotic stresses. Therefore, the aims of this this study were to determine the effects of two different soil management practices (cover crops and tillage) on the vegetative activity of Italia table grape, as well as on fruit yield and quality and on the GS enzyme activity at different phenological stages of the grapevine. White clover reduced the canopy of Italia vines but increased the GS activity; no effects were reported for yield and quality of harvested clusters.

Physiological response of ryegrass (Lolium spp.) grown at different temperatures to glufosinate ammonium application

Efficacy of glufosinate ammonium is influenced by temperature. However, several studies show variable results. The response of ryegrass (Lolium spp.) to glufosinate ammonium activity at different temperatures was investigated. Ryegrass was grown at 10/15, 15/20, 20/25, and 25/30 °C (night/day) temperature regimes. Ammonia accumulation, glutamine synthetase, glutamate dehydrogenase, and nitrate reductase activity was analysed on control plants (0 g a.i. ha−1) and treated plants (900 g a.i. ha−1) 24 hours after application (HAA). Photosynthesis studies were conducted on plants treated with 0 and 900 g a.i. ha−1 glufosinate ammonium dosage rates 24, 48, and 72 HAA using an infra-red gas analyser. An increase in glutamine synthetase enzyme activity was observed with increasing temperature after glufosinate ammonium application. In contrast, glutamine dehydrogenase enzyme activity decreased with increasing temperature in treated plants. Accumulation of ammonia was not the main cause of ryegrass mortality. Better control of ryegrass with glufosinate ammonium at cooler temperatures was mainly attributed to the rapid decline in glutamine synthetase activity 24 HAA.

Differential Expression of Mitogen Activated Protein Kinase (MAPK) and Stress-Related Genes in Rice Overexpressing MPK3 and MPK6 under Abiotic Stress

Plants being sessile have ability to adapt themselves in constantly changing and challenging environment by perceiving external/internal cues and transducing them down to the nucleus for appropriate cellular reorganization. Activation of mitogen-activated protein kinases (MAPKs) is a common reaction of plant cells towards such cues in development-related signal transduction pathways. The downstream events after the activation of MAPKs are largely unknown in plants. In the present study, we examined the effect of arsenic, salt and drought stress on growth parameters and transcript profiling of MAPKs, silicon transporters andstress responsivegenes in transgenic rice overexpressing OsMPK3 and OsMPK6 under the control of a dexamethasone (DEX)-inducible promoter, and possible role MAPKs in stress tolerance. Our results revealed that OsMPK3 and OsMPK6 overexpressing lines under arsenic stress and drought stress enhanced the transcript level of stress responsive genes, encoding enzymes superoxide dismutase, ascorbate peroxidase, glutamine synthetase and aldehyde oxidase. Upregulation in transcripts profiling of three silicon transporter genes OsLsi1, OsLsi2 and OsLsi6 showed improved stress tolerance ability of OsMPK3 and OsMPK6 overexpressing lines by effective absorption of silicon. The fundamental knowledge about the function of MPK3 and MPK6, important member of MAPKs family may also lead to an engineered plant tolerant to various abiotic and biotic stresses.

Chronic Valproic Acid Administration Increases Plasma, Liver, and Brain Ammonia Concentration and Suppresses Glutamine Synthetase Activity.

Asymptomatic valproic acid (VPA)-induced hyperammonemia in the absence of liver impairment is fairly common. However, the underlying mechanisms through which VPA causes elevation in plasma ammonia (NH4) remains under investigation. Male Sprague Dawley rats (n = 72) were randomly allocated to receive VPA 400 mg/kg, 200 mg/kg, or vehicle IP daily for either 8, 14, or 28 consecutive days. The behavioral effects of VPA were assessed. Plasma, liver, and prefrontal cortex (PFC), striatum (Str), and cerebellum (Cere) were collected 1 h post last injection and assayed for NH4 concentration and glutamine synthetase (GS) enzyme activity. Chronic VPA treatment caused attenuation of measured behavioral reflexes (p < 0.0001) and increase in plasma NH4 concentration (p < 0.0001). The liver and brain also showed significant increase in tissue NH4 concentrations (p < 0.0001 each) associated with significant reduction in GS activity (p < 0.0001 and p = 0.0003, respectively). Higher tissue NH4 concentrations correlated with reduced GS activity in the liver (r = −0.447, p = 0.0007) but not in the brain (r = −0.058, p = 0.4). Within the brain, even though NH4 concentrations increased in the PFC (p = 0.001), Str (p < 0.0001), and Cere (p = 0.01), GS activity was reduced only in the PFC (p < 0.001) and not in Str (p = 0.2) or Cere (p = 0.1). These results suggest that VPA-induced elevation in plasma NH4 concentration could be related, at least in part, to the suppression of GS activity in liver and brain tissues. However, even though GS is the primary mechanism in brain NH4 clearance, the suppression of brain GS does not seem to be the main factor in explaining the elevation in brain NH4 concentration. Further research is urgently needed to investigate brain NH4 dynamics under chronic VPA treatment and whether VPA clinical efficacy in treating seizure disorders and bipolar mania is impacted by its effect on GS activity or other NH4 metabolizing enzymes.

Targeting Glutamine Synthesis Inhibits Stem Cell Adipogenesis in Vitro.

Glutamine is the most abundant amino acid in the body and has a metabolic role as a precursor for protein, amino sugar and nucleotide synthesis. After glucose, glutamine is the main source of energy in cells and has recently been shown to be an important carbon source for de novo lipogenesis. Glutamine is synthesized by the enzyme glutamine synthetase, a mitochondrial enzyme that is active during adipocyte differentiation suggesting a regulatory role in this process. The aim of our study was therefore to investigate whether glutamine status impacts on the differentiation of adipocytes and lipid droplet accumulation. Mouse mesenchymal stem cells (MSCs) were submitted to glutamine deprivation (i.e. glutamine-free adipogenic medium in conjunction with irreversible glutamine synthetase inhibitor, methionine sulfoximine - MSO) during differentiation and their response was compared with MSCs differentiated in glutamine-supplemented medium (5, 10 and 20 mM). Differentiated MSCs were assessed for lipid content using Oil Red O (ORO) staining and gene expression was analysed by qPCR. Intracellular glutamine levels were determined using a colorimetric assay, while extracellular glutamine was measured using liquid chromatography-mass spectrometry (LC-MS). Glutamine deprivation largely abolished adipogenic differentiation and lipid droplet formation. This was accompanied with a reduction in intracellular glutamine concentration, and downregulation of gene expression for classical adipogenic markers including PPARγ. Furthermore, glutamine restriction suppressed isocitrate dehydrogenase 1 (IDH1) gene expression, an enzyme which produces citrate for lipid synthesis. In contrast, glutamine supplementation promoted adipogenic differentiation in a dose-dependent manner. These results suggest that the glutamine pathway may have a previously over-looked role in adipogenesis. The underlying mechanism involved the glutamine-IDH1 pathway and could represent a potential therapeutic strategy to treat excessive lipid accumulation and thus obesity.

Enhanced Contextual Fear Memory and Elevated Astroglial Glutamate Synthase Activity in Hippocampal CA1 BChE shRNA Knockdown Mice.

Butyrylcholinesterase (BChE) efficiently hydrolyzes acetylcholine (ACh) at high concentrations when acetylcholinesterase (AChE) is substrate-inhibited. Recent studies have shown that BChE also has a function that is independent of ACh, but it has not been fully explored. Low BChE expression is accompanied with higher stress-induced aggression and ghrelin levels in stress models, and BChE knockout mice exhibit cognitive and memory impairments. However, the role of BChE in posttraumatic stress disorder (PTSD) remains unclear. In the present study, we investigated the role of BChE in contextual fear memory and its regulatory effect on the expression of factors related to the glutamate (Glu)-glutamine (Gln) cycle via knockdown studies. We used AAVs and lentiviruses to knockdown BChE expression in the mouse hippocampal CA1 region and C8D1A astrocytes. Our behavioral data from those mice injected with AAV-shBChE in the hippocampal CA1 region showed strengthened fear memory and increased dendritic spine density. Elevated Glu levels and glutamine synthetase (GS) enzyme activity were detected in contextual fear conditioned-BChE knockdown animals and astrocytes. We observed that an AAV-shBChE induced lowering of BChE expression in the hippocampus CA1 region enhanced contextual fear memory expression and promoted the astrocytic Glu-Gln cycle but did not elevate ACh-hydrolyzing activity. This study provides new insight into the regulatory role of BChE in cognition and suggests potential target for stress-related psychiatric disorder such as PTSD where patients experience fear after exposure to severe life-threatening traumatic events.

Evaluation of the bacteria formulation different inoculum densities on growth and development of Euphorbia pulcherrima

Abstract This research was carried out to examine the effects of different doses of bacterial formulation comprised of a mixture of Bacillus megaterium TV-91C, Pantoea agglomerans RK-92 and Kluyvera cryocrescens TV-113C strains on the plant development and bract quality of poinsettias (Euphorbia pulcherrima Willd. ex Klotzsch), which are one of the most important potted plants grown for their fleshy bracts of the horticultural sector. The study was carried out in a climate-controlled research greenhouse from 8 August 2018 to 15 January 2019 in Erzurum (Turkey). Rooted cuttings of poinsettia [E. pulcherrima Willd. ex Klotzsch cv. Christmas Feelings (CvF)] were used as plant materials in the study. Each of the solutions containing bacterial suspensions 52.5 (T1), 105 (T2), 210 (T3), 420 (T4) and 840 (T5) mL · L−1 of water was diluted 5 times with water and 200 mL · pot−1 was applied to the plant rhizosphere. This study revealed that positive changes incurred in plant height, main flower stem length, plant crown width, bract diameter, green leaf and bract leaf area, leaf total nitrogen content and nitrate reductase enzyme activity parameters in poinsettia plants with increasing doses of the bacterial formulation. This effect has reached the maximum level in the number of bracts, main flower stem length, bract diameter, nitrate reductase enzyme activity, plant fresh weight and maximal root length parameters with the T5 application which has the highest bacterial formulation concentration. The most efficient application of glutamine synthetase enzyme activity was determined as T4.

DNA-Encoded Glutamine Synthetase Enzyme as Ammonia-Lowering Therapeutic for Hyperammonemia.

Hyperammonemia is a dangerous life-threatening metabolic complication characterized by markedly elevated ammonia levels that can lead to irreversible brain damage if not carefully monitored. Current pharmacological treatment strategies available for hyperammonemia patients are suboptimal and associated with major side effects. In this study, we focus on developing and evaluating the in vivo delivery of novel DNA-encoded glutamine synthetase (GS) enzymes for the treatment of hyperammonemia. Direct in vivo delivered DNA-encoded GS enzyme was evaluated in ammonium acetate-induced hyperammonemia and thioacetamide-induced acute liver injury (ALI) models in C57BL/6 mice. In ammonium acetate-induced hyperammonemia model, we achieved a 30.5% decrease in blood ammonia levels 15 min postadministration of ammonium acetate, with DNA-encoded GS-treated group. Significant increase in survival was observed in ALI model with the treated mice. A comparison of the secreted versus intracellular DNA-encoded GS enzyme demonstrated similar increases in survival in the ALI model, with 40% mortality in the secreted enzymes and 30% mortality in the intracellular enzymes, as compared with 90% mortality in the control group. Direct in vivo delivery of DNA-encoded GS demonstrated important ammonia-lowering potential. These results provide the initial steps toward development of delivered DNA as a potential new approach to ammonia-lowering therapeutics.

1745P SARS-CoV-2 infects metabolically-primed epithelial cells in lung cancer models

The novel coronavirus SARS-CoV-2 is the cause of the respiratory illness COVID-19—a global pandemic affecting over 4 million individuals worldwide. Viruses efficiently replicate by hijacking host cell machinery to obtain macromolecules and energy by similar mechanisms as cancer cells. Since viral infection is known to alter cellular nutrient requirements, this study explores the metabolites and metabolic pathways associated with SARS-CoV-2 infection. Bulk and single-cell seequencing data from cell lines and tumor samples were retrieved from publically available datasets. Transcriptional data were retrieved from publically available datasets of gefitinib- and erlotinib-resistant EGFR-mutant cell lines and Calu3 and A549 cells mock treated or infected with SARS-CoV-2. Single-cell RNAseq datasets of EGFR-mutant PC-9 mock and osimertinib treated were downloaded from GEO. 225 metabolites were profiled in CCLE cell lines using LC-MS. To identify metabolic features of cells able to be infected by SARS-CoV-2 via the ACE2 receptor, metabolites associated with ACE2 expression were investigated. ACE2 expression positively correlates with glutamine in upper aerodigestive tract cell lines. Consistent with this, ACE2 expression was examined against a list of 253 metabolism-associated genes and GLUL, which encodes an enzyme (glutamine synthetase) responsible for conversion of glutamate to glutamine, was significantly positively correlated in NSCLC, HNSCC, and SCLC cell lines and confirmed in human tumor datasets. Additionally, GLS, which encodes the enzyme (glutaminase) that catalyzes the opposing reaction, is negatively correlated with ACE2 expression. Further, we analyzed RNA sequencing data from NSCLC cell lines infected with SARS-CoV-2 for 24 hours and revealed that upon infection there is a down regulation of GLUL signifying a metabolic-shift away from glutamine as the cells undergo EMT. We show that SARS-CoV-2 targeting of ACE-2 expressing, metabolically-primed epithelial cells is advantageous to exploit the abundance of glutamine to synthesize nucleotides for rapid replication and viral spread.