Search

Best1 and Best2 are two members of the bestrophin family of anion channels critically involved in the prevention of retinal degeneration and maintenance of intraocular pressure, respectively. Here, we solved glutamate- and γ-aminobutyric acid (GABA)-bound Best2 structures, which delineate an intracellular glutamate binding site and an extracellular GABA binding site on Best2, respectively, identified extracellular GABA as a permeable activator of Best2, and elucidated the co-regulation of Best2 by glutamate, GABA and glutamine synthetase in vivo. We further identified multiple small molecules as activators of the bestrophin channels. Extensive analyses were carried out for a potent activator, 4-aminobenzoic acid (PABA): PABA-bound Best1 and Best2 structures are solved and illustrate the same binding site as in GABA-bound Best2; PABA treatment rescues the functional deficiency of patient-derived Best1 mutations. Together, our results demonstrate the mechanism and potential of multiple small molecule candidates as clinically applicable drugs for bestrophin-associated diseases/conditions. Bestrophin anion channels are critical for the health of eyes. Here, authors identify multiple bestrophin activators including glutamate and γ-aminobutyric acid. [ABSTRACT FROM AUTHOR]

Ammonia nitrogen is a common environmental factor in aquaculture systems. High levels of ammonia nitrogen threaten the health of shrimp. This study aimed to investigate ammonia nitrogen stress in the hepatopancreas of Penaeus vannamei by integrating physiological, metabolome, and transcriptome analysis. The study revealed that exposure to ammonia nitrogen resulted in injury to the hepatopancreas, deformation disorder of the lumen, blurred boundaries, and increased vacuoles. After ammonia nitrogen stress, the activities of ammonia-metabolizing enzymes (glutamate dehydrogenase and glutamine synthetase) increased significantly, while the activity of transglutaminase decreased significantly. In addition, antioxidant enzymes (glutathione peroxidase, catalase, and superoxide dismutase) were significantly decreased. Additionally, the activity of energy metabolizing enzyme (hexokinase) was increased, and immune enzyme (alkaline phosphatase) activity was decreased. The accumulation of metabolites, such as d-myo-inositol, hexadecenoic acid, aspartate, and ornithine in the metabolite groups, showed significant differences under ammonia stress. Transcriptome analysis suggested that the KEGG pathways were significantly enriched in energy and primary metabolism, metabolism of cofactors and vitamins, and signaling pathways. The comprehensive analysis of metabolome and transcriptome displayed significant enrichment pathways, including TCA cycle, purine metabolism, pyrimidine metabolism, and folic acid biosynthesis under ammonia nitrogen stress. Simultaneously, six genes were found to respond to ammonia stress, including inositol oxygenase-like, histone h1-β, postembryonic, blood cell protein-glutamine-glutamyltransferase-like, receptor tyrosine protein phosphatase T-like, trichohyalin-like, and thyroxine 5-deiodinase-like. The results showed that ammonia nitrogen stress caused hepatopancreas tissue damage, changed in enzyme activities, metabolic process, and related gene expression in P. vannamei. [ABSTRACT FROM AUTHOR]

ve: Increasing tree productivity by genetic engineering methods is one of the main trends of forest biotechnology. A promising strategy for this is to improve the use efficiency of nitrogen, which is the main limiting factor of plant growth. For this purpose, the GS1 gene from Scots pine was transferred to downy birch (Betula pubescens) plants. This gene encodes the cytosolic form of glutamine synthetase, the main enzyme of nitrogen metabolism in plants. Methods: To assess the effects of insertion of this gene, the birch plant metabolome was analyzed using GC-MS and HPLC-MS. Results and Discussion: GC-MS analysis found 197 metabolites in birch extracts, but the metabolomes of two transgenic clones showed no statistically significant differences from the control. Using the S-plot based on the OPLS-DA model, 32 metabolite markers affecting the separation of control and transgenic birch plants were detected; 22 of them were identified. Three metabolites among them were nitrogen-containing, including γ-aminobutyric acid, the immediate precursor of which is glutamine. HPLC-MS analysis found 48 metabolites, but transgenic plants did not differ from the control. GC-MS, however, showed a decrease in the content of two phenolic compounds in transgenic plants, which is characteristic of improved nitrogen supply. Conclusions: The study shows that modification of nitrogen metabolism in birch plants does not significantly affect the biochemical composition of tree shoots. [ABSTRACT FROM AUTHOR]

Escherichia coli K12 and Lelliottia amnigena PTJIIT1005 bacteria were isolated from the polluted Yamuna River (Delhi, India) site, which can remediate nitrate from groundwater media under anaerobic conditions. BV-BRC (Bacterial and Viral Bioinformatics Resource Center) information system, RAST, and PGAP servers were used to annotate the nitrogen metabolism genes from the genome sequence of these microbes. Here we compared the strains L. amnigena PTJIIT1005 with E. coli K12 in the context of nitrogen metabolism genes. Sequence alignment, similarity percentage, and phylogenetic analysis were done to find similarities between the genes. Common nitrogen genes of these strains, like respiratory nitrate reductase, nitrite reductase, nitric oxide reductase, glutamine synthetase, and hydroxylamine reductase, have found good sequence similarity (83–94%) with each other. The PATRIC tool identified N-operons, and the nitrate reductase gene clusters were also determined as per literature survey. Protein–protein interaction network was constructed using STRING 12.0 database and Cytoscape v 3.10.0 software plug-in Network analyzer. On the basis of network topological parameters NarG, NarZ, NarY, NarH, NarI, NarV, NirB, NirD, NapA, and NapB are the key genes in network of E. coli K12 strain. Nar, NirB, NirD, NasA, NasB, NasC, NasD, NasE, and GlnA are the key genes in network of L. amnigena PTJIIT1005. Among these, NarG and NirB are the superhub genes because of having highest Betweenness centrality (BC) and node degree. The functional enrichment analysis was determined using PANTHER GENE ONTOLOGY and DAVID software exhibited their role in nitrogen metabolism pathway and nitrate assimilation. Further, SWISS-MODEL was used to predict the 3D protein structure of these enzymes, and after, these structures were validated by Ramachandran plot using the PROCHECK tool. The Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) method was used to determine the N-genes expression level in both strains. This study showed that E. coli K12 and L. amnigena PTJIIT1005 have common nitrogen metabolism genes involved in the same functional role, like the denitrification pathway. Additionally, operon arrangement study and PPI network revealed that E. coli K12 has only a denitrification pathway, while L. amnigena PTJIIT1005 has both an assimilation and denitrification pathway. PCR successfully amplified selected N-metabolizing genes, and the expression level of N-genes was high in strain L. amnigena PTJIIT1005. Our previous experimental study exhibited a better nitrate remediation rate in L. amnigena PTJIIT1005 over E. coli K12. This study confirmed the presence of assimilation and denitrification process through amplified N-metabolizing genes and showed high expression of N-genes in L. amnigena PTJIIT1005, which favor the evidence of better nitrate remediation in L. amnigena PTJIIT1005 over E. coli K12. [ABSTRACT FROM AUTHOR]

The mevalonate pathway produces essential lipid metabolites such as cholesterol. Although this pathway is negatively regulated by metabolic intermediates, little is known of the metabolites that positively regulate its activity. We found that the amino acid glutamine is required to activate the mevalonate pathway. Glutamine starvation inhibited cholesterol synthesis and blocked transcription of the mevalonate pathway—even in the presence of glutamine derivatives such as ammonia and α-ketoglutarate. We pinpointed this glutamine-dependent effect to a loss in the ER-to-Golgi trafficking of SCAP that licenses the activation of SREBP2, the major transcriptional regulator of cholesterol synthesis. Both enforced Golgi-to-ER retro-translocation and the expression of a nuclear SREBP2 rescued mevalonate pathway activity during glutamine starvation. In a cell model of impaired mitochondrial respiration in which glutamine uptake is enhanced, SREBP2 activation and cellular cholesterol were increased. Thus, the mevalonate pathway senses and is activated by glutamine at a previously uncharacterized step, and the modulation of glutamine synthesis may be a strategy to regulate cholesterol levels in pathophysiological conditions. Synopsis: Whether cholesterol biosynthesis can be adjusted to the availability of input carbon sources remains unclear. This study demonstrates that glutamine is directly sensed by and required for activation of the mevalonate pathway, coupling precursor availability to cellular lipid formation. Glutamine starvation inhibits cholesterol synthesis in human cells. Glutamine, but not its derivatives nor glucose, maintains expression of the rate-limiting lipid biosynthesis enzyme HMGCR. Glutamine synthesis from ammonia and glutamate sustains HMGCR in a cell-type-specific manner. Glutamine is required for the ER-to-Golgi trafficking of the SCAP-SREBP2 complex. Chronic mitochondrial dysfunction increases glutamine uptake and cholesterol levels in murine fibroblasts. The amino acid glutamine is directly sensed by the mevalonate pathway and acts as an anabolic switch coupling precursor availability to lipid biosynthesis. [ABSTRACT FROM AUTHOR]

Premium-tasty japonica rice, distinguished by its unique flavor and enriched nutritional value, has attracted significant attention across Asia. Nitrogen metabolism plays a crucial role in determining the growth, development, and quality of rice grains. This study investigates the effects of nitrogen application rates on the enzymatic activities related to nitrogen metabolism, nitrogen metabolites, and grain protein content in the premium-tasty japonica rice variety, Suxiangjing 100. Using a range of nitrogen application rates (0, 80, 160, 240, 320, and 400 kg per hectare), comprehensive analyses were conducted across various developmental stages. Results indicate that as nitrogen application increased, there were distinct patterns in the enzyme activities and metabolite concentrations related to nitrogen metabolism in the leaves of premium-tasty japonica rice. The activities of nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT) peaked at moderate nitrogen applications, while excessive nitrogen led to a decrease in their activities. The activity of Glutamate Dehydrogenase (GDH) generally increased, but showed a decline after the filling stage. With increasing nitrogen application, the concentrations of nitrate nitrogen (NO3–N), ammonium nitrogen (NH4–N), free amino acids (FAA), and soluble proteins (SP) initially rose but declined at excessive application rates. The grain protein content increased, with levels of prolamin, glutelin, and globulin peaking at nitrogen applications of 240 kg/ha and 320 kg/ha, while changes in albumin levels were minimal. This study not only provides novel insights into the role of nitrogen in shaping the growth and quality of premium-tasty japonica rice, but also suggests refined nitrogen management strategies for sustainable agricultural practices. [ABSTRACT FROM AUTHOR]

The molecular mechanisms linking obstructive sleep apnea syndrome (OSA) to obesity and the development of metabolic diseases are still poorly understood. The role of hypoxia (a characteristic feature of OSA) in excessive fat accumulation has been proposed. The present study investigated the possible effects of hypoxia (4% oxygen) on de novo lipogenesis by tracking the major carbon sources in differentiating 3T3-L1 adipocytes. Gas-permeable cultuware was employed to cultivate 3T3-L1 adipocytes in hypoxia (4%) for 7 or 14 days of differentiation. We investigated the contribution of glutamine, glucose or acetate using 13C or 14C labelled carbons to the newly synthesized lipid pool, changes in intracellular lipid content after inhibiting citrate- or acetate-dependent pathways and gene expression of involved key enzymes. The results demonstrate that, in differentiating adipocytes, hypoxia decreased the synthesis of lipids from glucose (44.1 ± 8.8 to 27.5 ± 3.0 pmol/mg of protein, p < 0.01) and partially decreased the contribution of glutamine metabolized through the reverse tricarboxylic acid cycle (4.6% ± 0.2–4.2% ± 0.1%, p < 0.01). Conversely, the contribution of acetate, a citrate- and mitochondria-independent source of carbons, increased upon hypoxia (356.5 ± 71.4 to 649.8 ± 117.5 pmol/mg of protein, p < 0.01). Further, inhibiting the citrate- or acetate-dependent pathways decreased the intracellular lipid content by 58% and 73%, respectively (p < 0.01) showing the importance of de novo lipogenesis in hypoxia-exposed adipocytes. Altogether, hypoxia modified the utilization of carbon sources, leading to alterations in de novo lipogenesis in differentiating adipocytes and increased intracellular lipid content. [ABSTRACT FROM AUTHOR]

Boundary cap cells are a population of multipotent stem cells that have great potential for the use in the treatment of damaged nervous system. We studied the patterns of distribution of the gap junction protein connexin-43 (Cx43) in boundary cap cells of the ventral root of the spinal cord of rat embryos (E12-E20; n=40). It was found that Cx43 is expressed in ventral boundary cap cells at all stages of its existence during embryogenesis. At the early stages of prenatal development, the cytoplasmic distribution of Cx43 in the boundary cap cells predominates; at the later stages, Cx43-immunopositive punctate structures are identified. These puncta represent gap junction plaques between the cells. It can be assumed that during the early embryogenesis, Cx43 regulates the main histogenetic processes in boundary cap cells and only in the later stages of prenatal development, Cx43-mediated communications are formed between boundary cap cells. [ABSTRACT FROM AUTHOR]

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth. [ABSTRACT FROM AUTHOR]

Background: Polyaspartic Acid-Calcium (PASP-Ca) is a versatile and eco-friendly amino acid complex, primarily recognized for bolstering nitrogen use efficiency and crop productivity. However, the core significance of this complex remains enigmatic in potato crop. We hypothesized that simultaneous application of PASP-Ca with potato genotypes characterized by substantial root systems and high genetic potentials for nitrogen-use efficiency (NUE) would best address this knowledge gap. Methods: The synergistic effect of various PASP-Ca treatments on morph physiological, N-related, and enzymatic parameters coupled with their transcript levels (shoot and root) in four potato genotypes having contrasting NUEs under low and high N supplies in aeroponics. Results: PASP-Ca markedly boosted plant growth, yield components, and photosynthetic efficiency, with pronounced effects observed in nitrogen-efficient genotypes, especially Qingshu-9, emphasizing the importance of genotype selection in optimizing nitrogen utilization. Moreover, PASP-Ca treatments, particularly LN-P100 significantly enhances root system architecture (RSA), contributing to expanded root dimensions and improved nutrient acquisition capacity, especially under nitrogen-deficient conditions. Carbohydrate metabolism in potato tubers benefits from PASP-Ca treatment, leading to increased starch content, thereby impacting tuber quality. Among the N-assimilating enzymes, a large genotypic variation was observed for glutamine synthetase (GS), which may be considered a potential trait for improving NUE. Molecular analysis further elucidated the underlying mechanisms, demonstrating the upregulation of essential genes involved in nitrogen metabolism. Conclusions: The potential efficacy of PASP-Ca synergist as a novel accelerant for enhancing potato crop growth, biomass production, and nitrogen utilization efficiency, all coalescing seamlessly with the ethos of sustainable agricultural practices. [ABSTRACT FROM AUTHOR]

Nitrogen (N) is a macronutrient essential for plant growth and development; insufficient N availability has an extensive impact on the overall plant metabolism and productivity. Polyamines, short aliphatic amino-containing compounds naturally produced by living cells, have been widely reported to be beneficial for plant performance under several unfavorable conditions. In this work, the role of polyamine spermine (Spm) as a priming agent for wheat growing under N deficit was investigated. Wheat seeds were primed with 0.1 mM Spm for 3 h, germinated for 48 h, and then grown in N-sufficient or N-deficient media for 8 d. Spm treatment anticipated seed germination and, though priming with Spm did not result in any stimulatory effect on plants developed under an N-sufficient medium, under N limitation many of the studied traits improved compared with control plants. Under N deficit, wheat seedlings originated from Spm-primed seeds showed increased root length, developed the third leaf earlier, and maintained higher total N and protein contents and higher nitrate reductase (NR) and glutamine synthetase (GS) activities in their leaves, compared to control plants. Likewise, total amino acids increased in the roots of primed seedlings, and carbohydrates enhanced both in roots and leaves. Our findings indicate that seed priming with Spm promotes wheat germination and early seedling growth under N limitation, mainly by promoting root elongation and N allocation to the aerial part. [ABSTRACT FROM AUTHOR]

Leaves experience near-constant light fluctuations daily. Past studies have identified many limiting factors of slow photosynthetic induction when leaves transition from low light to high light. However, the contribution of photorespiration in influencing photosynthesis during transient light conditions is largely unknown. This study employs dynamic measurements of gas exchange and metabolic responses to examine the contribution of photorespiration in constraining net rates of carbon assimilation during light induction. This work indicates that photorespiratory glycine accumulation during the early light induction contributes 5–7% to the additional carbon fixed relative to the low light conditions. Mutants with large glycine pools under photorespiratory conditions (5-formyl THF cycloligase and hydroxypyruvate reductase 1) showed a transient spike in net CO2 assimilation during light induction, with glycine buildup accounting for 22–36% of the extra carbon assimilated. Interestingly, levels of many C3 cycle intermediates remained relatively constant in both mutants and wild-type throughout the light induction period where glycine accumulated, indicating that recycling of carbon into the C3 cycle via photorespiration is not needed to maintain C3 cycle activity under transient conditions. Furthermore, our data show that oxygen transient experiments can be used as a proxy to identify the photorespiratory component of light-induced photosynthetic changes. [ABSTRACT FROM AUTHOR]

The retina is light-sensitive neuronal tissue in the back of the eye. The phospholipid composition of the retina is unique and highly enriched in polyunsaturated fatty acids, including docosahexaenoic fatty acid (DHA). While it is generally accepted that a high DHA content is important for vision, surprisingly little is known about the mechanisms of DHA enrichment in the retina. Furthermore, the biological processes controlled by DHA in the eye remain poorly defined as well. Here, we combined genetic manipulations with lipidomic analysis in mice to demonstrate that acyl-CoA synthetase 6 (Acsl6) serves as a regulator of the unique composition of retinal membranes. Inactivation of Acsl6 reduced the levels of DHA-containing phospholipids, led to progressive loss of light-sensitive rod photoreceptor neurons, attenuated the light responses of these cells, and evoked distinct transcriptional response in the retina involving the Srebf1/2 (sterol regulatory element binding transcription factors 1/2) pathway. This study identifies one of the major enzymes responsible for DHA enrichment in the retinal membranes and introduces a model allowing an evaluation of rod functioning and pathology caused by impaired DHA incorporation/retention in the retina. Acyl-CoA synthetase 6 (Acsl6)-facilitated enrichment of docosahexaenoic acid (DHA) in the retina is essential for rod photoreceptor function, viability and control of basal activity of the Srebf1/2 pathway. [ABSTRACT FROM AUTHOR]

The function of proteins depends on their correct structure and proper dynamics. Understanding the dynamics of target proteins facilitates drug design and development. However, dynamic information is often hidden in the spatial structure of proteins. It is important but difficult to identify the specific residues that play a decisive role in protein dynamics. Here, we report that a critical glycine residue (Gly463) dominates the motion of threonyl-tRNA synthetase (ThrRS) and the sensitivity of the enzyme to antibiotics. Obafluorin (OB), a natural antibiotic, is a novel covalent inhibitor of ThrRS. The binding of OB induces a large conformational change in ThrRS. Through five crystal structures, biochemical and biophysical analyses, and computational simulations, we found that Gly463 plays an important role in the dynamics of ThrRS. Mutating this flexible residue into more rigid residues did not damage the enzyme's three-dimensional structure but significantly improved the thermal stability of the enzyme and suppressed its ability to change conformation. These mutations cause resistance of ThrRS to antibiotics that are conformationally selective, such as OB and borrelidin. This work not only elucidates the molecular mechanism of the self-resistance of OB-producing Pseudomonas fluorescens but also emphasizes the importance of backbone kinetics for aminoacyl-tRNA synthetase-targeting drug development. Crystal structures and molecular dynamics simulations reveal that a critical glycine residue dominates the motion of threonyl-tRNA synthetase and the sensitivity of the enzyme to conformation selective inhibitors. [ABSTRACT FROM AUTHOR]

The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuA•AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5′-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuA•AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuA•AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels. AMP-forming acetyl-CoA synthetase (AcsA) is an essential enzyme in all domains of life. Here, the authors provide mechanistic insights into regulation of AcsA by lysine acetylation, uncovering a regulatory phosphotransacetylase activity. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) is often diagnosed late, leading to incomplete tumor removal, drug resistance and reduced chemotherapy efficacy. Curcumin has the potential for anti-cancer activity through various therapeutic properties and can improve the efficacy of chemotherapy. We aimed to investigate the synergistic effect of a combination of curcumin and gemcitabine against CCA, targeting the LAT2/glutamine pathway. This combination synergistically suppressed proliferation in gemcitabine-resistant CCA cells (KKU-213BGemR). It also resulted in a remarkable degree of CCA cell apoptosis and cell cycle arrest, characterized by a high proportion of cells in the S and G2/M phases. Knockdown of SLC7A8 decreased the expressions of glutaminase and glutamine synthetase, resulting in inhibited cell proliferation and sensitized CCA cells to gemcitabine treatment. Moreover, in vivo experiments showed that a combination curcumin and gemcitabine significantly reduced tumor size, tumor growth rate and LAT2 expression in a gemcitabine-resistant CCA xenograft mouse model. Suppression of tumor progression in an orthotopic CCA hamster model provided strong support for clinical application. In conclusion, curcumin synergistically enhances gemcitabine efficacy against gemcitabine-resistant CCA by induction of apoptosis, partly via inhibiting LAT2/glutamine pathway. This approach may be an alternative strategy for the treatment of gemcitabine-resistant in CCA patients. [ABSTRACT FROM AUTHOR]

This document discusses the modulation of γ-aminobutyric acid type A receptors (GABAA) by the endocannabinoid (eCB) system. The exact mechanism of this connection is still under debate, but experimental studies have shown that endocannabinoids enhance GABAA activity under conditions of low GABA concentrations and reduce GABAA activity at high GABA levels. The involvement of cannabinoid receptors in the regulation of GABAergic neurotransmission suggests that the eCB system may have anxiolytic effects through the modulation of GABAergic receptors. Additionally, the inhibition of glutamine synthetase (GS) influences GABAA receptor regulation, decreasing benzodiazepine binding. The expression of astrocytic GS is regulated by mitogen-activated protein kinases (MAPKs), with p38 upregulating and ERK1/2 downregulating GS. Further studies are needed to determine the exact mechanisms of these interactions. [Extracted from the article]

The elucidation of disease pathogenesis can be achieved by analyzing the low-abundance phosphopeptides in organisms. Herein, we developed a novel and easy-to-prepare polymer-coated nanomaterial. By improving the hydrophilicity and spatial conformation of the material, we effectively enhanced the adsorption of phosphopeptides and demonstrated excellent enrichment properties. The material was able to successfully enrich the phosphopeptides in only 1 min. Meanwhile, the material has high selectivity (1:2000), good loading capacity (100 μg/mg), excellent sensitivity (0.5 fmol), and great acid and alkali resistance. In addition, the material was applied to real samples, and 70 phosphopeptides were enriched from the serum of Parkinson's disease (PD) patients and 67 phosphopeptides were enriched from the serum of normal controls. Sequences Logo showed that PD is probably associated with threonine, glutamate, serine, and glutamine. Finally, gene ontology (GO) analysis was performed on phosphopeptides enriched in PD patients' serum. The results showed that PD patients expressed abnormal expression of the cholesterol metabolic process and cell–matrix adhesion in the biological process (BP), endoplasmic reticulum and lipoprotein in the cellular component (CC), and heparin-binding, lipid-binding, and receptor-binding in the molecular function (MF) as compared with normal individuals. All the experiments indicate that the nanomaterials have great potential in proteomics studies. [ABSTRACT FROM AUTHOR]

Nuclear factor erythroid 2-related factor 2 (NRF2) hyperactivation has been established as an oncogenic driver in a variety of human cancers, including non-small cell lung cancer (NSCLC). However, despite massive efforts, no specific therapy is currently available to target NRF2 hyperactivation. Here, we identify peptidylprolyl isomerase A (PPIA) is required for NRF2 protein stability. Ablation of PPIA promotes NRF2 protein degradation and blocks NRF2-driven growth in NSCLC cells. Mechanistically, PPIA physically binds to NRF2 and blocks the access of ubiquitin/Kelch Like ECH Associated Protein 1 (KEAP1) to NRF2, thus preventing ubiquitin-mediated degradation. Our X-ray co-crystal structure reveals that PPIA directly interacts with a NRF2 interdomain linker via a trans-proline 174-harboring hydrophobic sequence. We further demonstrate that an FDA-approved drug, cyclosporin A (CsA), impairs the interaction of NRF2 with PPIA, inducing NRF2 ubiquitination and degradation. Interestingly, CsA interrupts glutamine metabolism mediated by the NRF2/KLF5/SLC1A5 pathway, consequently suppressing the growth of NRF2-hyperactivated NSCLC cells. CsA and a glutaminase inhibitor combination therapy significantly retard tumor progression in NSCLC patient-derived xenograft (PDX) models with NRF2 hyperactivation. Our study demonstrates that targeting NRF2 protein stability is an actionable therapeutic approach to treat NRF2-hyperactivated NSCLC. Despite being an established oncogenic driver of non-small cell lung cancer (NSCLC), therapies targeting NRF2 hyperactivation are lacking. Here, the authors identify peptidylprolyl isomerase A (PPIA) as a mediator of NRF2 stability and demonstrate the efficacy of targeting this interaction with cyclosporin A in preclinical models of NSCLC. [ABSTRACT FROM AUTHOR]

Despite global warming, the response of rice yield to long-term warming in cool regions and its physiological mechanisms remain unknown. This study used the widely cultivated japonica rice Jiyang100 in Northeast China. Taking rice grown under natural temperatures as a control (CK), field warming treatments were conducted at the tillering-panicle initiation (T1), whole growth (T2), and grain-filling (T3) stages. The positive effects of T1, T2, and T3 on the total number of spikelets per hole increased the yield in both years, with average increases of 11.5%, 9.9% and 6.5% compared to CK, respectively. Warming treatments improved the stay-green traits, photosynthesis, sucrose synthesis, and nitrogen metabolism of rice. The yield was positively correlated with the relative chlorophyll content (SPAD), soluble sugar content, sucrose content, and the activities of sucrose phosphate synthase (SPS), nitrate reductase (NR), glutamine synthetase (GS), glutamine oxoglutarate aminotransferase (GOGAT) in flag leaves. In addition, SPAD had a positive correlation with soluble sugar content, soluble protein content, and the activities of NR, GS, GOGAT, glutamate dehydrogenase (GDH), but a negative correlation with acid invertase (AI) activity. The stay-green ability was positively correlated to the net photosynthetic rate (Pn), soluble sugar content and soluble protein content. The coupling interactions of stay-green traits, nitrogen and carbon metabolism increased the yield potential and yield supply capacity, increased yield under long-term warming conditions in the cool regions. Under gradual warming, the physiological response of rice in cool regions promotes plant growth and development, thereby increasing yield. [ABSTRACT FROM AUTHOR]

Aim: Idiopathic non-cirrhotic portal hypertension (INCPH) is a vascular disorder of uncertain origin. Diagnosis can be challenging on liver biopsy. Despite diverse histomorphologic findings documented in literature, studies on the frequency of these findings are lacking. This study aims to assess both the histomorphologic features and the immunoexpression patterns of CD34 and glutamine synthetase (GS) in liver biopsies and searched for their contribution to the pathologic diagnosis of INCPH. Materials and methods: Hematoxylin–eosin, CD34, and GS-stained liver needle biopsy sections of 16 patients clinically diagnosed with INCPH were retrospectively analyzed. Histologic findings such as portal vein narrowing, obliteration, or loss were grouped as major findings, while portal vein herniation, hypervascularized portal tracts, and periportal abnormal vessels were grouped as minor findings, and their frequency were evaluated. Periportal endothelial CD34 stained areas were measured via ocular micrometer. The distribution of GS immunoexpression was evaluated. Eighteen healthy liver donor biopsies were evaluated as controls. Results: In INCPH cases, 58% of portal tracts showed major findings, compared to 15% in the control group (p < 0.001). Minor findings were observed in 16% of INCPH cases and 7% of controls (p = 0.014). The number of portal tracts with histologic findings is significantly higher in INCPH than in control liver biopsies. Abnormal portal tract distribution, like being close to each other, was seen in 75% of INCPH cases but not in controls (p < 0.001). Nodular regenerative hyperplasia (NRH) was present in 31% of cases. Periportal CD34 expression was higher in INCPH, and affected areas were larger than in controls (p < 0.001). Irregular GS staining, i.e. GS staining with patchy distribution in zone 3, and/or periportal and zone 2 hepatocytes, was found in 62% of INCPH cases, while controls showed the usual pattern (p < 0.001). Conclusion: In the biopsy diagnosis of INCPH, in addition to the presence of major histologic findings and the amount of portal tracts displaying these features, the expression of endothelial CD34 in periportal areas, and irregular hepatocellular GS expression can also be considered as supporting feature. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains unknown. Here, we report that glucose-deprived ECs upregulate the GNG enzyme PCK2 and rely on a PCK2-dependent truncated GNG, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways, which can play key roles in redox homeostasis and phospholipid synthesis, respectively. Unexpectedly, however, even in normal glucose conditions, and independent of its enzymatic activity, PCK2 silencing perturbs proteostasis, beyond its traditional GNG role. Indeed, PCK2-silenced ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of protein aggregates in lysosomes and EC demise. Ultimately, loss of PCK2 in ECs impaired vessel sprouting. This study identifies a role for PCK2 in proteostasis beyond GNG. This study reports a unexpected role in endothelial cells (ECs) for PCK2 in proteostasis, independent of its gluconeogenic activity. Perturbation of this role likely contributed to the angiogenic defects observed in ECs with reduced PCK2 levels. [ABSTRACT FROM AUTHOR]

The gene products of PRS1-PRS5 in Saccharomyces cerevisiae are responsible for the production of PRPP (5-phospho-D-ribosyl-α-1-pyrophosphate). However, it has been demonstrated that they are also involved in the cell wall integrity (CWI) signalling pathway as shown by protein–protein interactions (PPIs) with, for example Slt2, the MAP kinase of the CWI pathway. The following databases: SGD, BioGRID and Hit Predict, which collate PPIs from various research papers, have been scrutinized for evidence of PPIs between Prs1-Prs5 and components of the CWI pathway. The level of certainty in PPIs was verified by interaction scores available in the Hit Predict database revealing that well-documented interactions correspond with higher interaction scores and can be graded as high confidence interactions based on a score > 0.28, an annotation score ≥ 0.5 and a method-based high confidence score level of ≥ 0.485. Each of the Prs1-Prs5 polypeptides shows some degree of interaction with the CWI pathway. However, Prs5 has a vital role in the expression of FKS2 and Rlm1, previously only documented by reporter assay studies. This report emphasizes the importance of investigating interactions using more than one approach since every method has its limitations and the use of different methods, as described herein, provides complementary experimental and statistical data, thereby corroborating PPIs. Since the experimental data described so far are consistent with a link between PRPP synthetase and the CWI pathway, our aim was to demonstrate that these data are also supported by high-throughput bioinformatic analyses promoting our hypothesis that two of the five PRS-encoding genes contain information required for the maintenance of CWI by combining data from our targeted approach with relevant, unbiased data from high-throughput analyses. [ABSTRACT FROM AUTHOR]

Background: Frailty is a multifaceted geriatric syndrome characterized by an increased vulnerability to stressful events. metabolomics studies are valuable tool for better understanding the underlying mechanisms of pathologic conditions. This review aimed to elucidate the metabolomics profile of frailty. Method: This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) 2020 statement. A comprehensive search was conducted across multiple databases. Initially, 5027 results were retrieved, and after removing duplicates, 1838 unique studies were subjected to screening. Subsequently, 248 studies underwent full-text screening, with 21 studies ultimately included in the analysis. Data extraction was performed meticulously by two authors, and the quality of the selected studies was assessed using the Critical Appraisal Skills Program (CASP) checklist. Results: The findings revealed that certain Branched-chain amino acids (BCAAs) levels were lower in frail subjects compared to robust subjects, while levels of glutamate and glutamine were higher in frail individuals. Moreover, sphingomyelins and phosphatidylcholines (PC) displayed a decreasing trend as frailty advanced. Additionally, other metabolic derivatives, such as carnitine, exhibited significant associations with frailty. These metabolites were primarily interconnected through biochemical pathways related to the tricarboxylic acid and urea cycles. Notably, frailty was associated with a decrease in metabolic derivatives, including carnitine. Conclusion: This study underscores the intricate relationship between essential metabolites, including amino acids and lipids, and their varying levels in frail individuals compared to their robust counterparts. It provides a comprehensive panel of metabolites, shedding light on their potential associations with frailty and expanding our understanding of this complex syndrome. [ABSTRACT FROM AUTHOR]

Pyruvate, the end product of glycolysis, is an important metabolite for the tricarboxylic acid (TCA) cycle and lipid metabolism. Pyruvate is converted to acetyl-CoA, which enters the TCA cycle for citrate formation. Citrate is the precursor for isocitrate that undergoes oxidative decarboxylation reaction forming α-ketoglutarate (α-KG) coupled with NADP+ reduction catalyzed by the enzyme isocitrate dehydrogenase (IDH). In cyanobacteria, α-KG donates its carbon skeleton to the glutamine synthetase-glutamine oxoglutarate aminotransferase (GS-GOGAT) pathway, thereby linking carbon and nitrogen metabolisms. In this study, the effect of pyruvate on IDH activity, its protein content, and its mRNA expression level were analyzed. Amongst the concentrations studied (100 µM, 250 µM, and 500 µM), in 250 µM externally supplied pyruvate, IDH registered the highest activity as well as maximum protein and mRNA contents when compared to the control. At this concentration, all biochemical parameters of nitrogen and carbon metabolisms- heterocyst frequency, nitrogenase, and glutamine synthetase activities, photosynthetic pigments, OEC (oxygen evolving complex) activity, carbohydrate content, proteins, and total biomass were also found to be highest. However, at an exposure to 500 µM pyruvate, there was a reduction in all the above-mentioned parameters indicating that although pyruvate is a key metabolite in the cellular metabolism, it tends to become toxic beyond this concentration. The compromised state of the cells was also evident from the micrographs developed from SEM (Scanning electron microscope) and TEM (Transmission electron microscope) studies under 500 µM pyruvate exposure. [ABSTRACT FROM AUTHOR]

Background: NLRP3 inflammasome activation is significantly associated with sepsis-induced acute kidney injury (S-AKI). Cytosolic DNA derived from damaged mitochondria has been reported to activate NLRP3 inflammasome via upregulating the cyclic GMP–AMP synthase (cGAS)—the stimulator of interferon genes (STING) axis in nucleus pulposus cell and cardiomyocytes. However, the regulatory effect of mitochondria DNA (mtDNA)–cGAS–STING axis on the NLRP3 inflammasome in S-AKI remains unclear. Methods: In the current study, we established an in vivo model of S-AKI by intraperitoneally injecting male C57BL/6 J mice with lipopolysaccharide (LPS). Next, selective cGAS inhibitor RU.521, and STING agonist DMXAA were intraperitoneally injected in the mice; then, blood urea nitrogen (BUN), serum creatinine (CRE), urinary kidney injury molecular-1 (KIM-1), pathological changes, and infiltrated neutrophils were detected to assess kidney injury. We also performed western blot and immunofluorescence assays to evaluate STING, cGAS, TBK-1, p-TBK-1, IRF3, p-IRF3, NF-kB, p-NF-kB, NLRP3, cleaved caspase-1, caspase-1, GSDMD-N, and GSDMD expression levels in kidney tissues. IL-18 and IL-1β in renal tissue were identified by ELISA. In vitro, we treated HK-2 cells with LPS to establish a cell model of S-AKI. Furthermore, ethidium bromide (EtBr) was administered to deplete mitochondria DNA (mtDNA). LPS-induced cytotoxicity was evaluated by LDH release assay. Protein expression of cGAS, STING, and NLRP3 in was quantified by western blot. Cytosolic mtDNA was detected by immunofluorescence and q-PCR. Released IL-1β and IL-18 in HK-2 supernatants were detected by ELISA. Results: LPS injection induced S-AKI in mice, as evidenced by neutrophil infiltration, tubular vacuolation, and increased levels of serum creatinine (CRE), blood urea nitrogen (BUN), and urinary KIM-1. In addition, LPS activated the cGAS–STING axis and NLRP3 inflammasome in vivo, illustrated by increased phosphorylation levels of TBK-1, IRF3, and NF-kB protein, increased ratio of cleaved caspase-1 to caspase-1 and GSDMD-N to GSDMD, and increased IL-1β and IL-18 levels. Moreover, the cGAS inhibitor RU.521 effectively attenuated NLRP3 inflammasome and S-AKI; however, these effects were abolished by treatment with the STING agonist DMXAA. Furthermore, cytosolic release of mtDNA and activation of the cGAS–STING–NLRP3 axis were observed in LPS-treated HK-2 cells. Inhibiting mtDNA replication by Ethidium Bromide (EtBr) treatment reduced cytosolic mtDNA accumulation and downregulated the cGAS–STING–NLRP3 axis, ameliorating the cytotoxicity induced by LPS. Conclusion: This study demonstrated that the cGAS–STING axis was triggered by cytosolic mtDNA and participated in the development of S-AKI by activating NLRP3 inflammasome. Reducing cytosolic mtDNA accumulation or inhibiting the cGAS–STING axis may be potential therapeutic targets for S-AKI. [ABSTRACT FROM AUTHOR]

The liver has multiple regeneration modes, including hepatocellular hypertrophy and self-renewal of hepatocytes. When hepatocyte proliferation is impaired, hepatic progenitor cells may proliferate through ductular reaction (DR), differentiate into hepatocytes, and contribute to fibrosis. However, the three-dimensional spatial relationship between DR and regenerating hepatocytes and dynamic changes in DR associated with fibrosis remain poorly understood. Here, we performed three-dimensional (3D) imaging of cleared 42 liver explants with chronic and acute liver diseases and 4 normal livers to visualize DR. In chronic hepatic liver diseases, such as viral hepatitis, steatohepatitis, autoimmune hepatitis, and cryptogenic cirrhosis, the total length and number of branches of DR showed a significant positive correlation. We studied the spatial relationship between DR and GS-expressing cells using glutamine synthetase (GS) and cytokeratin 19 (CK19) as markers of liver regeneration and DR, respectively. The percentage of CK19-positive cells that co-expressed GS was less than 10% in chronic liver diseases. In contrast, nearly one-third of CK19-positive cells co-expressed GS in acute liver diseases, and chronic cholestatic liver diseases, including primary biliary cholangitis and primary sclerosing cholangitis, showed no co-expression. We also found that DR was longer and had more branching in livers with progressive fibrosis compared to those with regressive fibrosis. Our results suggest that DR displays varying degrees of spatial complexity and contribution to liver regeneration. DR may serve as hepatobiliary junctions that maintain continuity between hepatocytes and bile ducts rather than hepatocyte regeneration in chronic liver diseases. [ABSTRACT FROM AUTHOR]

Glutamine synthetase (GS) is vital in maintaining ammonia and glutamate (Glu) homeostasis in living organisms. However, the natural enzyme relies on adenosine triphosphate (ATP) to activate Glu, resulting in impaired GS function during ATP-deficient neurotoxic events. To date, no reports demonstrate using artificial nanostructures to mimic GS function. In this study, we synthesize aggregation-induced emission active polyP-Mn nanosheets (STPE-PMNSs) based on end-labeled polyphosphate (polyP), exhibiting remarkable GS-like activity independent of ATP presence. Further investigation reveals polyP in STPE-PMNSs serves as phosphate source to activate Glu at low ATP levels. This self-feeding mechanism offers a significant advantage in regulating Glu homeostasis at reduced ATP levels in nerve cells during excitotoxic conditions. STPE-PMNSs can effectively promote the conversion of Glu to glutamine (Gln) in excitatory neurotoxic human neuroblastoma cells (SH-SY5Y) and alleviate Glu-induced neurotoxicity. Additionally, the fluorescence signal of nanosheets enables precise monitoring of the subcellular distribution of STPE-PMNSs. More importantly, the intracellular fluorescence signal is enhanced in a conversion-responsive manner, allowing real-time tracking of reaction progression. This study presents a self-sustaining strategy to address GS functional impairment caused by ATP deficiency in nerve cells during neurotoxic events. Furthermore, it offers a fresh perspective on the potential biological applications of polyP-based nanostructures. Glutamine synthetase (GS) relies on Adenosine triphosphate (ATP) to activate glutamate (Glu) and are vital for maintaining ammonia and Glu homeostasis, but GS function is impaired during ATP-deficient neurotoxic events. Here the authors report polyphosphate-manganese nanosheets having GS-like activity independent of ATP to promote the conversion of Glu to glutamine in excitatory neurotoxic cells. [ABSTRACT FROM AUTHOR]

The pathophysiology of Fronto Temporal Dementia (FTD) remains poorly understood, specifically the role of astroglia. Our aim was to explore the hypothesis of astrocytic alterations as a component for FTD pathophysiology. We performed an in-depth tri-dimensional (3-D) anatomical and morphometric study of glial fibrillary acidic protein (GFAP)-positive and glutamine synthetase (GS)-positive astrocytes in the human entorhinal cortex (EC) of FTD patients. The studies at this level in the different types of human dementia are scarce. We observed a prominent astrocyte atrophy of GFAP-positive astrocytes and co-expressing GFAP/GS astrocytes, characterised by a decrease in area and volume, whilst minor changes in GS-positive astrocytes in FTD compared to non-dementia controls (ND) samples. This study evidences the importance of astrocyte atrophy and dysfunction in human EC. We hypothesise that FTD is not only a neuropathological disease, but also a gliopathological disease having a major relevance in the understanding the astrocyte role in FTD pathological processes and development. [ABSTRACT FROM AUTHOR]

Key message: The current status of molecular regulation of nitrogen assimilation and recent advances made in gymnosperms are reviewed. Gymnosperms are a heterogeneous and ancient group of seed plants that includes conifers, ginkgos, cycads and gnetophytes. Molecular studies on extant gymnosperms have been constrained by some discouraging features for experimental research such as their long life cycles, large sizes, complex megagenomes and abundant phenolic compounds in their woody tissues. However, the development of high-throughput sequencing and refined multiomics technologies in the last few years has allowed to explore the molecular basis of essential processes in this ancient lineage of plants. Nitrogen is one of the main limiting factors determining vascular development and biomass production in woody plants. Therefore, nitrogen uptake, metabolism, storage and recycling are essential processes for fundamental gymnosperm biology. Here, recent progress in the molecular regulation of nitrogen assimilation in gymnosperms is reviewed and some future perspectives on this topic are outlined. [ABSTRACT FROM AUTHOR]

Metabolic syndrome combines major risk factors for cardiovascular disease, making deeper insight into its pathogenesis important. We here explore the mechanistic basis of metabolic syndrome by recruiting an essential patient cohort and performing extensive gene expression profiling. The mitochondrial fatty acid metabolism enzyme acyl-CoA synthetase medium-chain family member 3 (ACSM3) was identified to be significantly lower expressed in the peripheral blood of metabolic syndrome patients. In line, hepatic ACSM3 expression was decreased in mice with metabolic syndrome. Furthermore, Acsm3 knockout mice showed glucose and lipid metabolic abnormalities, and hepatic accumulation of the ACSM3 fatty acid substrate lauric acid. Acsm3 depletion markedly decreased mitochondrial function and stimulated signaling via the p38 MAPK pathway cascade. Consistently, Acsm3 knockout mouse exhibited abnormal mitochondrial morphology, decreased ATP contents, and enhanced ROS levels in their livers. Mechanistically, Acsm3 deficiency, and lauric acid accumulation activated nuclear receptor Hnf4α-p38 MAPK signaling. In line, the p38 inhibitor Adezmapimod effectively rescued the Acsm3 depletion phenotype. Together, these findings show that disease-associated loss of ACSM3 facilitates mitochondrial dysfunction via a lauric acid-HNF4a-p38 MAPK axis, suggesting a novel therapeutic vulnerability in systemic metabolic dysfunction. Synopsis: Metabolic syndrome constitutes major risk factors for cardiovascular disease but its molecular drivers remain unclear. This study establishes loss of acyl-coenzyme A synthetase (ACSM3), a mitochondrial lipid metabolism enzyme in the liver, as a cause for metabolic syndrome and related deleterious signaling. ACSM3 expression is strongly decreased in metabolic syndrome patients. Systemic or liver-specific Acsm3 depletion in mice results in metabolic syndrome and hepatic accumulation of its substrate lauric acid (C12:0). Acsm3 depletion impairs mitochondrial morphology and function. Acsm3 loss-induced mitochondrial dysfunction is caused by lauric acid-dependent activation of nuclear receptor HNF4A and p38 MAPK signaling. p38 MAPK inhibition rescues mitochondrial dysregulation and metabolic syndrome in Acsm3 knockout mice. Metabolic health in mice and patients depends on acyl-CoA synthetase ACSM3, which safeguards hepatic lipid turnover and mitochondrial fitness. [ABSTRACT FROM AUTHOR]

Glutamate dehydrogenase 1 (GLUD1) is an important enzyme in glutamine metabolism. Previously, we found GLUD1 was down-regulated in tumor tissues of hepatocellular carcinoma (HCC) patients by proteomics study. To explore its role in the progression of HCC, the expressional level of GLUD1 was firstly examined and presented as that both the protein and mRNA levels were down-regulated in tumor tissues compared to the normal liver tissues. GLUD1 overexpression significantly inhibited HCC cells proliferation, migration, invasion and tumor growth both in vitro and in vivo, while GLUD1 knocking-down promoted HCC progression. Metabolomics study of GLUD1 overexpressing and control HCC cells showed that 129 differentially expressed metabolites were identified, which mainly included amino acids, bases, and phospholipids. Moreover, metabolites in mitochondrial oxidative phosphorylation system (OXPHOS) were differentially expressed in GLUD1 overexpressing cells. Mechanistic studies showed that GLUD1 overexpression enhanced mitochondrial respiration activity and reactive oxygen species (ROS) production. Excessive ROS lead to mitochondrial apoptosis that was characterized by increased expression levels of p53, Cytochrome C, Bax, Caspase 3 and decreased expression level of Bcl-2. Furthermore, we found that the p38/JNK MAPK pathway was activated in GLUD1 overexpressing cells. N-acetylcysteine (NAC) treatment eliminated cellular ROS and blocked p38/JNK MAPK pathway activation, as well as cell apoptosis induced by GLUD1 overexpression. Taken together, our findings suggest that GLUD1 inhibits HCC progression through regulating cellular metabolism and oxidative stress state, and provide that ROS generation and p38/JNK MAPK pathway activation as promising methods for HCC treatment. [ABSTRACT FROM AUTHOR]

Cytidine triphosphate (CTP), as a substance involved in the metabolism of phospholipids, proteins and nucleic acids, has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline. In this study, we established an in vitro six-enzyme cascade system to generate CTP. To avoid thermodynamic bottlenecks, we employed a circuitous and two-stage reaction strategy. Using cytidine as the key substrate, the final product CTP is obtained via the deamination and uridine phosphorylation pathways, relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate. Several extremophilic microbial-derived deaminases were screened and characterized, and a suitable cytidine deaminase was selected to match the first-stage reaction conditions. In addition, directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition, along with a 1.7-fold increase in activity over the wild type. After optimizing the reaction conditions, we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM, and the yield of CTP exceeded 82% within 10.0 h. [ABSTRACT FROM AUTHOR]

Insect cuticular lipids, especially epicuticular hydrocarbons (CHC), have a significant role in insect ecology and interactions with other organisms, including fungi. The CHC composition of a specific insect species may influence the outcome of the interaction with a specific fungal strain. Some insects, such as Piezodorus guildinii, have low susceptibility towards fungal infections seemingly due to their CHC composition. The entomopathogenic fungus Beauveria bassiana can assimilate CHC and incorporate them as building blocks via cytochrome P450 monooxygenases (CYPs). However, little is known about other enzymes that promote the degradation/assimilation of these cuticular components. In this study, we performed a transcriptomic analysis to evaluate the in vitro response of two virulence-contrasting B. bassiana strains when grown on three different P. guildinii CHC sources. We found a different expression profile of virulence-related genes, as well as different GO and KEGG parameters enriched at 4 days post-inoculation, which could help account for the intrinsic virulence and for an alkane-priming virulence enhancement effect. The hypovirulent strain predominantly showed higher expression of cuticle penetration genes, including chitinases, proteases, and CYPs, with GO term categories of "heme binding," "monooxygenase activity," and "peroxisome" pathways enriched. The hypervirulent strain showed higher expression of cell wall remodeling and cell cycle genes, and cuticle adhesion and a distinct set of CYPs, with GO categories of "DNA-binding transcription factor activity" and KEGG pathways corresponding to "meiosis-yeast" and "cell cycle" enriched. These results suggest a delay and alternate routes in pathogenicity-related metabolism in the hypovirulent strain in comparison with the hypervirulent strain. Key points: •Transcriptomics of two B. bassiana strains grown in P. guildinii cuticular components •Virulence-related genes correlated with virulence enhancement towards P. guildinii •Differentially expressed genes, GOs and KEGGs showed different metabolic timelines associated with virulence [ABSTRACT FROM AUTHOR]

The cyanobacterium Synechococcus elongatus PCC 7942 accumulates alarmone guanosine tetraphosphate (ppGpp) under stress conditions, such as darkness. A previous study observed that artificial ppGpp accumulation under photosynthetic conditions led to the downregulation of genes involved in the nitrogen assimilation system, which is activated by the global nitrogen regulator NtcA, suggesting that ppGpp regulates NtcA activity. However, the details of this mechanism have not been elucidated. Here, we investigate the metabolic responses associated with ppGpp accumulation by heterologous expression of the ppGpp synthetase RelQ. The pool size of 2-oxoglutarate (2-OG), which activates NtcA, is significantly decreased upon ppGpp accumulation. De novo 13C-labeled CO2 assimilation into the Calvin-Benson-Bassham cycle and glycolytic intermediates continues irrespective of ppGpp accumulation, whereas the labeling of 2-OG is significantly decreased under ppGpp accumulation. The low 2-OG levels in the RelQ overexpression cells could be because of the inhibition of metabolic enzymes, including aconitase, which are responsible for 2-OG biosynthesis. We propose a metabolic rearrangement by ppGpp accumulation, which negatively regulates 2-OG levels to maintain carbon and nitrogen balance. Elevated levels of alarmone guanosine tetraphosphate (ppGpp) in the cyanobacterium Synechococcus elongatus reduce de novo synthesis of 2-oxoglutarate, potentially by inhibiting metabolic enzymes like aconitase. [ABSTRACT FROM AUTHOR]

GABA and glutamate are the most abundant neurotransmitters in the CNS and play a pivotal part in synaptic stability/plasticity. Glutamate and GABA homeostasis is important for healthy aging and reducing the risk of various neurological diseases, while long-term imbalance can contribute to the development of neurodegenerative disorders, including Alzheimer's disease (AD). Normalization of the homeostasis has been discussed as a promising strategy for prevention and/or treatment of AD, however, data on the changes in the GABAergic and glutamatergic systems with age, as well as on the dynamics of AD development, are limited. It is not clear whether imbalance of the excitatory/inhibitory systems is the cause or the consequence of the disease development. Here we analyzed the age-related alterations of the levels of glutamate, GABA, as well as enzymes that synthesize them (glutaminase, glutamine synthetase, GABA-T, and GAD67), transporters (GLAST, GLT-1, and GAT1), and relevant receptors (GluA1, NMDAR1, NMDA2B, and GABAAr1) in the whole hippocampus of the Wistar rats and of the senescence-accelerated OXYS rats, a model of the most common (> 95%) sporadic AD. Our results suggest that there is a decline in glutamate and GABA signaling with age in hippocampus of the both rat strains. However, we have not identified significant changes or compensatory enhancements in this system in the hippocampus of OXYS rats during the development of neurodegenerative processes that are characteristic of AD. [ABSTRACT FROM AUTHOR]

Background: Diverse microbes, such as bacteria, are of immense worth to mangrove ecosystems due to their adaptability to salinity and variable environmental characteristics conditions. Pyridine and its derivatives compose most heterocyclic aromatic compounds largely produced by human activities that lead to environmental pollution. Bacteria have a crucial role in the nutrient cycling of carbon and nitrogen etc., to understand their functional involvement with environmental factors or ecosystem functioning as well as the species invasion and domestic or agriculture pyridine degradation pollution activities that threaten the mangrove ecosystem. Methods: This work established the genetic-based molecular degradation of organic compounds in the mangrove ecosystem, which ultimately makes the availability of nutrients. As well as the effects of various abiotic factors on pyridine degradation to discover the pyridine degradation and the removal of ammonia nitrogen and the proposed nitrogen metabolism pathway. Results: The novel bacterial strain NM1-A2 was isolated from mangrove sediments and, after 16S rRNA gene sequence analysis identified as Bacillus aryabhattai. NM1-A2 completely degraded pyridine within a 100 h incubation period at a temperature of 35 °C, an initial pH of 7.0, glucose and a pyridine concentration of 500 mg/L. The pseudo-first-order kinetics model described the pyridine biodegradation profile of NM1-A2 well. Interestingly, within 96 h the strain achieved almost complete pyridine degradation with a total organic carbon (TOC) removal of 87.9% ± 0.19% (from 377.52 ± 6.9 mg/L to 45.65 ± 0.14 mg/L). Within 96 h, the pyridine ring in the total nitrogen (TN) fraction at the maximum concentration of 55.31 ± 0.17 mg/L, furtherly 51.3% ± 2.39% of (TN) converted into Ammonia nitrogen (NH4+-N). Furthermore, NM1-A2 exhibited its pyridine biodegradation activity decreased by only 4% after three consecutive cycles (48 h each). Moreover, NM1-A2 possessed nrt-ABCD nitrate transport family and gltABCD operons that participate in the activities of glutamine and glutamate synthetase in NH4+ conversion in the nitrogen cycle. Furthermore, the nitrogen metabolism genes (nrtA, nrtB, nrtC, nirB, nirD, gltB, gltD, glnA) exhibited expression in NM1-A2. Conclusion: This research offers a potential treatment strategy for pyridine degradation in the mangrove ecosystem. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetase (aaRS) is a core component for genetic code expansion (GCE), a powerful technique that enables the incorporation of noncanonical amino acids (ncAAs) into a protein. The aaRS with polyspecificity can be exploited in incorporating additional ncAAs into a protein without the evolution of new, orthogonal aaRS/tRNA pair, which hence provides a useful tool for probing the enzyme mechanism or expanding protein function. A variant (N346A/C348A) of pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) exhibited a wide substrate scope of accepting over 40 phenylalanine derivatives. However, for most of the substrates, the incorporation efficiency was low. Here, a MbPylRS (N311A/C313A) variant was constructed that showed higher ncAA incorporation efficiency than its homologous MmPylRS (N346A/C348A). Next, N-terminal of MbPylRS (N311A/C313A) was engineered by a greedy combination of single variants identified previously, resulting in an IPE (N311A/C313A/V31I/T56P/A100E) variant with significantly improved activity against various ncAAs. Activity of IPE was then tested toward 43 novel ncAAs, and 16 of them were identified to be accepted by the variant. The variant hence could incorporate nearly 60 ncAAs in total into proteins. With the utility of this variant, eight various ncAAs were then incorporated into a lanthanide-dependent alcohol dehydrogenase PedH. Incorporation of phenyllactic acid improved the catalytic efficiency of PedH toward methanol by 1.8-fold, indicating the role of modifying protein main chain in enzyme engineering. Incorporation of O-tert-Butyl-L-tyrosine modified the enantioselectivity of PedH by influencing the interactions between substrate and protein. Enzymatic characterization and molecular dynamics simulations revealed the mechanism of ncAAs affecting PedH catalysis. This study provides a PylRS variant with high activity and substrate promiscuity, which increases the utility of GCE in enzyme mechanism illustration and engineering. [ABSTRACT FROM AUTHOR]

The accumulation of some free amino acids and their metabolism were investigated in Dunaliella sp. cells adapted to three salt levels (1, 2, and 3 M NaCl) in the presence or absence of 5 mM Arginine (Arg) to assess the relationship between salinity tolerance and nitrogen metabolism. Salt-adapted cells showed a large decrease in density depending on salt concentration but accumulated high concentrations of free amino acids such as proline, Arg, and γ-aminobutyric acid (GABA). Salinity increased the activities of enzymes involved in nitrogen metabolism, including nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (NADH-GOGAT), as well as several amino acid metabolizing enzymes such as arginase, arginine decarboxylase (ADC), ornithine aminotransferase (OAT), glutamate dehydrogenase (GDH), and glutamate decarboxylase (GDC). Arg treatment improved cell density at suspensions of 1 and 2 M NaCl, which was associated with a high accumulation in glutamate and GABA and decreased Arg and proline. In contrast, Arg and GABA accumulated in Arg-treated cells at 3 M NaCl, without changes in proline and glutamate levels. Accumulation of GABA in Arg-treated cells occurred while GDC activity decreased or remained unchanged. Like amino acids, the activities of all enzymes involved in amino acid metabolization were differently changed by the Arg treatment. These results favor a determining role for nitrogen metabolism in tolerance of Dunaliella sp. cells to long-term salinity. Arg treatment balances the metabolism of carbon and nitrogen, perhaps by different modifying the activity of nitrogen and amino acid metabolizing enzymes and operating the biosynthesis of signaling molecules. [ABSTRACT FROM AUTHOR]

Sulforaphane (SFN) is a type of phytochemical found in many cruciferous vegetables that has been shown to positively benefit the control of Type 2 Diabetes Mellitus (T2DM). The search was done from 2000 until December 2022 using PubMed, Scopus, Web of Sciences, and Google Scholar databases. We included all in vitro, in vivo, and clinical trials. Sulforaphane has been demonstrated to activate the PI3K/AKT and AMP-activated protein kinase pathways and the glucose transporter type 4 to increase insulin production and reduce insulin resistance. Interestingly, SFN possesses protective effects against diabetes complications, such as diabetic-induced hepatic damage, vascular inflammation and endothelial dysfunction, nephropathy, and neuropathy via nuclear factor erythroid 2-related factor 2 activation that leads to the translation of several anti-oxidant enzymes and regulation glycolysis, pentose phosphate pathway, fatty acid metabolism, glutamine metabolism, and glutathione metabolism. Furthermore, multiple clinical trial studies emphasized the ameliorating effects of SFN on T2DM patients. This review provides sufficient evidence for further research and development of sulforaphane as a hypoglycemic drug. [ABSTRACT FROM AUTHOR]

Pathophysiology of sporadic Alzheimer's disease (SAD) and familial Alzheimer's disease (FAD) remains poorly known, including the exact role of neuroglia and specifically astroglia, in part because studies of astrocytes in human Alzheimer's disease (AD) brain samples are scarce. As far as we know, this is the first study of a 3-D immunohistochemical and microstructural analysis of glial fibrillary acidic protein (GFAP)- and glutamine synthetase (GS)-positive astrocytes performed in the entorhinal cortex (EC) of human SAD and FAD samples. In this study, we report prominent atrophic changes in GFAP and GS astrocytes in the EC of both SAD and FAD characterised by a decrease in area and volume when compared with non-demented control samples (ND). Furthermore, we did not find neither astrocytic loss nor astrocyte proliferation or hypertrophy (gliosis). In contrast with the astrogliosis classically accepted hypothesis, our results show a highly marked astrocyte atrophy that could have a major relevance in AD pathological processes being fundamental and key for AD mnesic and cognitive alterations equivalent in both SAD and FAD. [ABSTRACT FROM AUTHOR]

Obesity is a major health concern that poses significant risks for many other diseases, including diabetes, cardiovascular disease, and cancer. Prevalence of these diseases varies by biological sex. This study utilizes a mouse (C57BL/6J) model of obesity to analyze liver and fecal metabolic profiles at various time points of dietary exposure: 5, 9, and 12 months in control or high fat diet (HFD)-exposed mice. Our study discovered that the female HFD group has a more discernable perturbation and set of significant changes in metabolic profiles than the male HFD group. In the female mice, HFD fecal metabolites including pyruvate, aspartate, and glutamate were lower than control diet-exposed mice after both 9th and 12th month exposure time points, while lactate and alanine were significantly downregulated only at the 12th month. Perturbations of liver metabolic profiles were observed in both male and female HFD groups, compared to controls at the 12th month. Overall, the female HFD group showed higher lactate and glutathione levels compared to controls, while the male HFD group showed higher levels of glutamine and taurine compared to controls. These metabolite-based findings in both fecal and liver samples for a diet-induced effect of obesity may help guide future pioneering discoveries relating to the analysis and prevention of obesity in people, especially for females. [ABSTRACT FROM AUTHOR]

Uncoupling of biological nitrogen fixation from ammonia assimilation is a prerequisite step for engineering ammonia excretion and improvement of plant-associative nitrogen fixation. In this study, we have identified an amino acid substitution in glutamine synthetase, which provides temperature sensitive biosynthesis of glutamine, the intracellular metabolic signal of the nitrogen status. As a consequence, negative feedback regulation of genes and enzymes subject to nitrogen regulation, including nitrogenase is thermally controlled, enabling ammonia excretion in engineered Escherichia coli and the plant-associated diazotroph Klebsiella oxytoca at 23 °C, but not at 30 °C. We demonstrate that this temperature profile can be exploited to provide diurnal oscillation of ammonia excretion when variant bacteria are used to inoculate cereal crops. We provide evidence that diurnal temperature variation improves nitrogen donation to the plant because the inoculant bacteria have the ability to recover and proliferate at higher temperatures during the daytime. Engineering ammonium excretion diazotrophs suffers from severe penalties to the bacteria. Here, the authors utilize a thermo-sensitive glutamine synthetase-based regulatory switch that permits diurnal changes in diazotrophic lifestyle, coincident with seasonal temperatures for cereal cultivation. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a toxic substance that is uptake by plants from soils, Cd easily transfers into the food chain. Considering global food security, eco-friendly, cost-effective, and metal detoxification strategies are highly demandable for sustainable food crop production. The purpose of this study was to investigate how citric acid (CA) alleviates or tolerates Cd toxicity in Brassica using a proteome approach. In this study, the global proteome level was significantly altered under Cd toxicity with or without CA supplementation in Brassica. A total of 4947 proteins were identified using the gel-free proteome approach. Out of these, 476 proteins showed differential abundance between the treatment groups, wherein 316 were upregulated and 160 were downregulated. The gene ontology analysis reveals that differentially abundant proteins were involved in different biological processes including energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense, heavy metal detoxification, plant development, and cytoskeleton and cell wall structure in Brassica leaves. Interestingly, several candidate proteins such as superoxide dismutase (A0A078GZ68) l-ascorbate peroxidase 3 (A0A078HSG4), glutamine synthetase (A0A078HLB2), glutathione S-transferase DHAR1 (A0A078HPN8), glutamine synthetase (A0A078HLB2), cysteine synthase (A0A078GAD3), S-adenosylmethionine synthase 2 (A0A078JDL6), and thiosulfate/3-mercaptopyruvate sulfur transferase 2 (A0A078H905) were involved in antioxidant defense system and sulfur assimilation-involving Cd-detoxification process in Brassica. These findings provide new proteome insights into CA-mediated Cd-toxicity alleviation in Brassica, which might be useful to oilseed crop breeders for enhancing heavy metal tolerance in Brassica using the breeding program, with sustainable and smart Brassica production in a metal-toxic environment. [ABSTRACT FROM AUTHOR]

Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi-oligomeric machines. Here we describe a structural dissection of the archaeal Methanosarcina mazei (Mm) GS and its regulation. We show that Mm GS forms unstable dodecamers. Strikingly, we show this Mm GS oligomerization property is leveraged for a unique mode of regulation whereby labile Mm GS hexamers are stabilized by binding the nitrogen regulatory protein, GlnK1. Our GS-GlnK1 structure shows that GlnK1 functions as molecular glue to affix GS hexamers together, stabilizing formation of GS active-sites. These data, therefore, reveal the structural basis for a unique form of enzyme regulation by oligomer modulation. Glutamine synthetases (GS) play vital roles in cellular nitrogen assimilation and hence these enzymes, which form large oligomeric machines, are tightly regulated. Here the authors reveal the molecular mechanism behind a unique form of GS regulation involving oligomer modulation. [ABSTRACT FROM AUTHOR]

Intermittent (or bolus) feeding regimens in critically ill patients have been of increasing interest to clinicians and scientists. Changes in amino acid, fat and carbohydrate metabolites over time might yet deliver other benefits (e.g. modulation of the circadian rhythm and sleep, and impacts on ghrelin secretion, insulin resistance and autophagy). We set out to characterise these changes in metabolite concentration. The Intermittent versus Continuous Feeding in Critically Ill paitents study (NCT02358512) was an eight-centre single-blinded randomised controlled trial. Patients were randomised to received a continuous (control arm) or intermittent (6x/day, intervention arm) enteral feeding regimen. Blood samples were taken on trial days 1, 7 and 10 immediately before and 30 min after intermittent feeds, and at equivalent timepoints in the control arm. A pre-planned targeted metabolomic analysis was performend using Nuclear Resonance Spectroscopy. Five hundred and ninety four samples were analysed from 75 patients. A total of 24 amino acid-, 19 lipid based-, and 44 small molecule metabolite features. Across the main two axes of variation (40–60% and 6–8% of variance), no broad patterns distinguished between intermittent or continuous feeding arms, across intra-day sampling times or over the 10 days from initial ICU admission. Logfold decreases in abundance were seen in metabolites related to amino acids (Glutamine − 0.682; Alanine − 0.594), ketone body metabolism (Acetone − 0.64; 3-Hydroxybutyric Acid − 0.632; Acetonacetic Acid − 0.586), fatty acid (carnitine − 0.509) and carbohydrate metabolism (Maltose − 0.510; Citric Acid − 0.485). 2–3 Butanediol, a by-product of sugar-fermenting microbial metabolism also decreased (− 0.489). No correlation was seen with change in quadriceps muscle mass for any of the 20 metabolites varying with time (all p > 0.05). Increasing severity of organ failure was related to increasing ketone body metabolism (3 Hydroxybutyric Acid-1 and − 3; p = 0.056 and p = 0.014), carnitine deficiency (p = 0.002) and alanine abundancy (p − 0.005). A 6-times a day intermittent feeding regimen did not alter metabolite patterns across time compared to continuous feeding in critically ill patients, either within a 24 h period or across 10 days of intervention. Future research on intermittent feeding regimens should focus on clinical process benefits, or extended gut rest and fasting. [ABSTRACT FROM AUTHOR]

Evidence indicates that transcranial direct current stimulation (tDCS) provides therapeutic benefits in different situations, such as epilepsy, depression, inflammatory and neuropathic pain. Despite the increasing use of tDCS, its cellular and molecular basis remains unknown. Astrocytes display a close functional and structural relationship with neurons and have been identified as mediators of neuroprotection in tDCS. Considering the importance of hippocampal glutamatergic neurotransmission in nociceptive pathways, we decided to investigate short-term changes in the hippocampal astrocytes of rats subjected to tDCS, evaluating specific cellular markers (GFAP and S100B), as well as markers of astroglial activity; glutamate uptake, glutamine synthesis by glutamine synthetase (GS) and glutathione content. Data clearly show that a single session of tDCS increases the pain threshold elicited by mechanical and thermal stimuli, as evaluated by von Frey and hot plate tests, respectively. These changes involve inflammatory and astroglial neurochemical changes in the hippocampus, based on specific changes in cell markers, such as S100B and GS. Alterations in S100B were also observed in the cerebrospinal fluid of tDCS animals and, most importantly, specific functional changes (increased glutamate uptake and increased GS activity) were detected in hippocampal astrocytes. These findings contribute to a better understanding of tDCS as a therapeutic strategy for nervous disorders and reinforce the importance of astrocytes as therapeutic targets. Highlights: tDCS has many proposed therapeutic uses, but its mechanism of action remains unclear. There is some evidence to suggest the involvement of glial cells in the effect of tDCS. A single tDCS increases the pain threshold elicited by mechanical and thermal stimuli. This mechanism involves inflammatory and astroglial changes in the hippocampus. Increased glutamate uptake and GS activity confirm the astroglial functionality changes. [ABSTRACT FROM AUTHOR]

Symbiotic associations with Symbiodiniaceae have evolved independently across a diverse range of cnidarian taxa including reef-building corals, sea anemones, and jellyfish, yet the molecular mechanisms underlying their regulation and repeated evolution are still elusive. Here, we show that despite their independent evolution, cnidarian hosts use the same carbon-nitrogen negative feedback loop to control symbiont proliferation. Symbiont-derived photosynthates are used to assimilate nitrogenous waste via glutamine synthetase–glutamate synthase-mediated amino acid biosynthesis in a carbon-dependent manner, which regulates the availability of nitrogen to the symbionts. Using nutrient supplementation experiments, we show that the provision of additional carbohydrates significantly reduces symbiont density while ammonium promotes symbiont proliferation. High-resolution metabolic analysis confirmed that all hosts co-incorporated glucose-derived 13C and ammonium-derived 15N via glutamine synthetase–glutamate synthase-mediated amino acid biosynthesis. Our results reveal a general carbon-nitrogen negative feedback loop underlying these symbioses and provide a parsimonious explanation for their repeated evolution. This study found that cnidarian animals including corals, anemones and jellyfish share a common mechanism to regulate their association with symbiotic algae. Despite evolving independently, these animals all use the same carbon-nitrogen negative feedback loop for algae control, shedding light on the repeated evolution of these marine symbiotic relationships. [ABSTRACT FROM AUTHOR]

Tuberculosis caused by Mycobacterium tuberculosis (M.tb) has killed millions worldwide. Antibiotic resistance leads to the ineffectiveness of the current therapies. Aminoacyl tRNA synthetase (aaRS) class of proteins involved in protein synthesis are promising bacterial targets for developing new therapies. Here, we carried out a systematic comparative study on the aaRS sequences from M.tb and human. We listed important M.tb aaRS that could be explored as potential M.tb targets alongside the detailed conformational space analysis of methionyl-tRNA synthetase (MetRS) in apo- and substrate-bound form, which is among the proposed targets. Understanding the conformational dynamics is central to the mechanistic understanding of MetRS, as the substrate binding leads to the conformational changes causing the reaction to proceed. We performed the most complete simulation study of M.tb MetRS for 6 microseconds (2 systems × 3 runs × 1 microsecond) in the apo and substrate-bound states. Interestingly, we observed differential features, showing comparatively large dynamics for the holo simulations, whereas the apo structures became slightly compact with reduced solvent exposed area. In contrast, the ligand size decreased significantly in holo structures possibly to relax ligand conformation. Our findings correlate with experimental studies, thus validating our protocol. Adenosine monophosphate moiety of the substrate exhibited quite higher fluctuations than the methionine. His21 and Lys54 were found to be the important residues forming prominent hydrogen bond and salt-bridge interactions with the ligand. The ligand-protein affinity decreased during simulations as computed by MMGBSA analysis over the last 500 ns trajectories, which indicates the conformational changes upon ligand binding. These differential features could be further explored for designing new M.tb inhibitors. [ABSTRACT FROM AUTHOR]

Frogs evolved terrestrial development multiple times, necessitating mechanisms to avoid ammonia toxicity at early stages. Urea synthesis from ammonia is a key adaptation that reduces water dependence after metamorphosis. We tested for early expression and plasticity of enzymatic mechanisms of ammonia detoxification in three terrestrial-breeding frogs: foam-nest-dwelling larvae of Leptodactylus fragilis (Lf) and arboreal embryos of Hyalinobatrachium fleischmanni (Hf) and Agalychnis callidryas (Ac). Activity of two ornithine-urea cycle (OUC) enzymes, arginase and CPSase, and levels of their products urea and CP in tissues were high in Lf regardless of nest hydration, but reduced in experimental low- vs. high-ammonia environments. High OUC activity in wet and dry nests, comparable to that under experimental high ammonia, suggests terrestrial Lf larvae maintain high capacity for urea excretion regardless of their immediate risk of ammonia toxicity. This may aid survival through unpredictably long waiting periods before rain enables their transition to water. Moderate levels of urea and CP were present in Hf and Ac tissues and enzymatic activities were lower than in Lf. In both species, embryos in drying clutches can hatch and enter the water early, behaviorally avoiding ammonia toxicity. Moreover, glutamine synthetase was active in early stages of all three species, condensing ammonia and glutamate to glutamine as another mechanism of detoxification. Enzyme activity appeared highest in Lf, although substrate and product levels were higher in Ac and Lf. Our results reveal that multiple biochemical mechanisms of ammonia detoxification occur in early life stages of anuran lineages that evolved terrestrial development. [ABSTRACT FROM AUTHOR]