Search Results (568)

Background/Objective: Prolonged ethanol use can induce behavioral and physiological alterations and impair the liver’s capacity to metabolize ethanol, leading to hepatocyte injury. The spectrum of this disease ranges from fat accumulation to severe liver damage, including alcohol-related hepatitis and cirrhosis. MC-100093, a beta-lactam non-antibiotic compound, is known to modulate glutamate transporter-1 (GLT-1) and reduce ethanol drinking. Therefore, this study examined the effects of two optimal doses of MC-100093 on ethanol consumption and, consequently, ethanol-induced hepatic injury in both male and female alcohol-preferring (P) rats. Methods: Animals were divided into four groups and exposed to ethanol for six weeks. In week six, rats received intraperitoneal injections of saline, 100 mg/kg MC-100093, or 150 mg/kg MC-100093 for five consecutive days. Liver tissue samples were analyzed to assess GLT-1 and the cystine/glutamate antiporter (xCT), triglyceride levels, collagen type-1 (COL-1) deposition, and inflammatory cytokines. Results: MC-100093 effectively attenuated ethanol-induced liver damage by decreasing ethanol intake and restoring GLT-1, xCT, and COL-1 levels in the livers of both male and female P rats. Moreover, MC-100093 reversed ethanol-induced inflammation and reduced hepatic triglyceride levels. Conclusions: Our data show that MC-100093 effectively mitigates ethanol-induced liver injury. Full article

The cerebellum, long studied as the principal center for motor coordination, is now recognized as a participant in epileptogenic network dynamics through the pathway running from the cerebellar cortex to the deep cerebellar nuclei and on to thalamic and cortical targets. Purkinje cells are the sole inhibitory output of the cerebellar cortex, and Bergmann glia, a radial astrocyte population apposed to Purkinje cell dendrites and parallel-fiber synapses, sustain the perisynaptic environment on which Purkinje cell firing depends through glutamate clearance by GLAST/EAAT1, potassium buffering by Kir4.1, and calcium-dependent signaling. In cortex and hippocampus, loss of the equivalent functions in protoplasmic astrocytes lowers seizure threshold and produces spontaneous seizures, but no comparable experiment has been performed in Bergmann glia. Molecular genetics has identified numerous epilepsy-associated genes, of which only SLC1A3, KCNJ10 and AQP4 have cell-type-resolved expression in Bergmann glia; the remainder act on cerebellar circuits through neurons. In human epilepsy, cerebellar atrophy with Purkinje cell loss and Bergmann gliosis tracks cumulative seizure burden and antiseizure medication exposure rather than a primary cerebellar lesion and is therefore best read as reactive. Reactive astrogliosis is nonetheless accompanied by reduced glutamate transport and potassium buffering in sclerotic hippocampus, and whether cerebellar reactive gliosis carries the same homeostatic cost has not been measured. This review sets out the circuit-level, genetic and neuropathological evidence bearing on that question and specifies the experiments that would answer it. Full article

Background/Objectives. VHHs (Nanobodies^®) are camelid single-domain antibody fragments with broad applicability in central nervous system (CNS) research, including blood–brain barrier transport, modulation of pathological proteins and molecular detection. However, few VHHs have been developed for cell-type-specific targeting within the brain. Astrocytes play essential roles in CNS homeostasis and contribute to many neurological disorders, making them an appealing target for diagnostic and therapeutic approaches. Here, we report the development and validation of the first VHHs directed against the astrocyte-enriched glutamate transporters GLT-1 and GLAST-1. Methods. VHHs were generated from a camelid immune library using phage display. Target specificity and affinity were evaluated in cells overexpressing the mouse and human transporters, as well as in iPSC-derived astrocytes. Target engagement and astrocyte specificity was further assessed ex vivo on brain sections using immunohistochemistry and in dissociated mouse brain cell suspensions by flow cytometry. VHH in vivo binding was evaluated by stereotactic injections within the brain striatum. Results. One anti-GLT-1 VHH and four anti-GLAST-1 VHHs demonstrated high specificity and apparent nanomolar affinity when binding tocells overexpressing the mouse transporters. The anti-GLT-1 VHH was additionally cross-reactive with the human ortholog, as demonstrated in overexpressing cells and iPSC-derived astrocytes. All VHHs showed target engagement on mouse brain sections and astrocyte specificity in dissociated mouse brain cell suspensions. VHH direct brain delivery resulted in localized transporter-specific signal around the injection site, whereas the non-binding isotype control VHH produced no detectable signal. Conclusions. Here we present the first set of VHHs capable of selectively recognizing astrocyte-enriched glutamate transporters GLT-1 and GLAST-1 in vitro, ex vivo, and in vivo. These VHHs constitute promising tools for astrocyte detection in research applications and the cross-reactive anti-GLT-1 lead might hold potential for future diagnostic or therapeutic strategies, including cell-targeted payload delivery. Full article

Sepsis-induced disseminated intravascular coagulation (DIC) carries approximately 44% mortality and has no targeted mechanistic therapy. Stable isotope tracer data show whole-blood glutathione (GSH) synthesis falling by approximately 60% in sepsis despite paradoxically elevated cysteine flux, consistent with enzymatic failure at the glutamate-cysteine ligase (GCL) step rather than substrate shortage. We propose that GCL failure may unify endothelial coagulopathy and innate immune dysfunction in DIC, linked to the NLRP3/GSDMD pyroptotic coagulation pathway through both cytosolic synthesis and mitochondrial import failure. Cross-dataset bioinformatic analysis was conducted across four independent GEO datasets. Three whole-blood cohorts (n = 200; 144 sepsis, 56 controls; two platforms) were selected for this study (two adult cohorts and one pediatric cohort). All cohorts were strictly filtered to day 1 baseline samples to prevent pseudoreplication. Differential expression was evaluated via Mann–Whitney U with Benjamini–Hochberg correction, with xCell deconvolution resolving bulk signals into cell-type-specific components via Spearman’s correlation. Mitochondrial GSH transporter and receptor-mediated mitophagy genes were evaluated using the same pipeline as an independent evidential layer. A fourth dataset of isolated monocytes (GSE46955; n = 14 individuals, 22 samples) was analyzed to assess AFG3L2 and SLC25A39 co-transcription. Across cohorts, NLRP3 transcript abundance positively correlated with monocyte and neutrophil enrichment scores (e.g., rho = +0.633 and +0.537). Conversely, transcripts for GCLC (rho = −0.530) and SLC25A39 (rho = −0.400), the principal mitochondrial GSH importer, inversely correlated with these same myeloid population scores. Furthermore, the receptor-mediated mitophagy transcripts FUNDC1 and BNIP3L were significantly downregulated and inversely correlated with expanding myeloid fractions. These bulk transcriptomic patterns reveal a strong inverse relationship between the expression of inflammasome components and GSH synthesis, import, and clearance machinery, though further studies are required to confirm functional protein activation or enzymatic failure. Longitudinal analysis of isolated monocytes, utilizing a linear mixed-effects model to account for repeated within-patient sampling, demonstrated significant aggregate co-transcription between AFG3L2 and SLC25A39 (p = 0.012), indicating tight co-regulation of the importer and its protease. These findings, derived from sepsis-versus-control cohorts without DIC adjudication, suggest that the transcriptional suppression of GSH synthesis and mitochondrial import machinery correlates strongly with NLRP3 and GSDMD transcript upregulation in estimated myeloid populations. Extending this axis to a mechanistic role in DIC is a hypothesis supported by the independent literature, not a direct finding of the present dataset. Because this bottleneck appears enzymatic rather than substrate-dependent, N-acetylcysteine alone may be insufficient to restore homeostasis. We therefore hypothesize that combining intravenous glutathione with an Fe–S cluster-promoting agent, such as hinokitiol, could mechanistically address these findings. However, this remains strictly a preclinical hypothesis that requires extensive investigation to resolve existing safety concerns, particularly the potential for ionophore toxicity in humans. Full article

Cognitive fatigability is the time-dependent destabilization of cognitive performance under sustained demand and is distinct from subjective fatigue. Neurophysiological effects may persist after demand. To link performance instability during demand with delayed recovery, this non-systematic, hypothesis-driven Review focuses on the extracellular milieu, which reflects and shapes neural activity. This Review operationally defines the interstitial ionic state as extracellular K⁺, Ca²⁺, Mg²⁺, pH, and extracellular space volume. Neurons, glia, and the vasculature generate this state; astrocytes couple neuromodulatory input to K⁺ and glutamate transport, metabolism, and water movement. Astrocyte involvement should be assessed through transport and extracellular variables rather than Ca²⁺ alone. This Review proposes that sustained cognitive demand constrains maintenance of this milieu during information processing and reconfiguration afterward. Delayed recovery may reflect incomplete reconfiguration of the milieu or incomplete recovery of neuronal and astrocytic responsiveness. Within the literature examined, no direct test of this integrated hypothesis under ordinary sustained cognitive demand was identified. The hypothesis predicts that recovery trajectories will outperform single post-demand measurements in forecasting renewed-demand performance and that recovery-phase manipulation of ion regulation or cellular responsiveness will alter fatigability. Brain fatigue may partly reflect reduced capacity to maintain and flexibly reconfigure the extracellular milieu across brain states. Full article

Background/objectives: Febuxostat (FBXT), a xanthine oxidase (XO) inhibitor, is a urate-lowering drug commonly used to treat gout. XO is also a promising therapeutic target for inflammatory bowel disease (IBD). This study aimed to improve the efficacy and safety of FBXT repurposed for the treatment of IBD. Methods: FBXT was conjugated with the acidic amino acids aspartic acid (Asp) and glutamic acid (Glu) via amide linkages to yield the colon-specific conjugates FBXT-Asp and FBXT-Glu. Results: The results revealed that amino acid conjugation decreased the distribution coefficient of FBXT from 1.451 to −1.217 for FBXT-Asp and −0.859 for FBXT-Glu. In parallel, conjugation retarded FBXT transport across the small intestinal wall, with a more pronounced effect observed with Glu conjugation than with Asp conjugation. The conjugates were converted to FBXT in cecal contents under a nitrogen atmosphere while remaining intact in pH 1.2 buffer and small intestinal contents. The cecal conversion percentages at 8 and 24 h were 7.1% and 22%, respectively, for FBXT-Asp and 17% and 54.8%, respectively, for FBXT-Glu. Consistent with these in vitro results, oral gavage of FBXT-Glu resulted in greater FBXT accumulation in the rat cecum than that observed with free FBXT. In rats with 2,4-dinitrobenzenesulfonic acid-induced colitis, oral gavage of FBXT-Glu at a dose equivalent to 20 mg of FBXT ameliorated colonic damage and inflammation. This effect was more potent than that of FBXT at the same dose or sulfasalazine (SSZ), a currently used anti-IBD drug. Additionally, FBXT-Glu did not produce detectable blood levels of FBXT, whereas FBXT was detected at concentrations of up to 6.7 μM at 2 h after oral gavage of FBXT at the corresponding dose. Conclusions: FBXT-Glu may serve as an anti-colitis agent with therapeutic and toxicological advantages over FBXT and could be a viable alternative to SSZ. Full article

Background/Objectives: High-protein (HP) diets may influence body composition, lipid metabolism, oxidative stress, and mitochondrial function, but their interaction with probiotics remains unclear. This study evaluated the effects of HP feeding, alone or combined with Lactobacillus acidophilus and Bifidobacterium lactis, on metabolic, redox, mitochondrial, and plasma amino acid parameters in Wistar rats. Methods: Thirty-two male Wistar rats were allocated to standard diet (SD), SD plus probiotics, HP diet, or HP plus probiotics for 10 weeks. Body weight gain, food and energy intake, visceral adiposity, plasma biochemical profile, adipose tissue histology, oxidative stress markers, mitochondrial electron transport chain activity, and plasma amino acids were assessed. Results: Food and energy intake did not differ among groups. HP-fed animals showed lower weight gain, reduced visceral fat, and lower total cholesterol and triglycerides. Probiotics modulated selected redox-related outcomes, including methylglyoxal and sulfhydryl levels, with tissue-specific responses. Complex I activity increased in adipose tissue and skeletal muscle, and plasma amino acid profiles were altered, particularly glutamate, leucine, aspartate, alanine, and methionine. Conclusions: HP feeding, with or without probiotics, modulated metabolic, redox, mitochondrial, and amino acid-related parameters in Wistar rats. These findings suggest that HP diets and probiotics may influence cardiometabolic adaptations, although tissue-specific responses and mechanisms require further investigation. Full article

Background: Type 1 Diabetes Mellitus (T1DM) is preceded by a prolonged asymptomatic autoimmune phase during which islet autoantibodies represent the earliest detectable biomarkers of disease. Although Radioligand Binding Assays (RBAs) remain the reference method for autoantibody detection, their use is limited by the requirement of radioactive reagents, specialized facilities and high operational costs, highlighting the need for sensitive, non-radiometric assays suitable for large-scale screening. Objective and Methods: In this study, we developed a bridge Fluorophore-Linked Immunosorbent Assay (b-FLIA) for the simultaneous detection of glutamic acid decarboxylase autoantibodies (GADA) and zinc transporter 8 autoantibodies (ZnT8A) using a recombinant ZnT8/GAD65 chimeric antigen expressed in a baculovirus–insect cell system. Assay performance was evaluated using sera from healthy controls and diabetic patients previously characterized by RBA. Results: The b-FLIA achieved an area under the receiver operating characteristic curve of 0.9867, with 90.5% sensitivity and 100% specificity for the detection of ZnT8A and/or GADA. Furthermore, the assay showed an almost perfect agreement with RBA (κ = 0.912) and displayed excellent analytical precision (intra-assay CV 1.34%, inter-assay CV 2.88%). Conclusions: These findings demonstrate that the combination of a baculovirus-expressed ZnT8/GAD65 chimeric antigen with a bridge fluorescence immunoassay constitutes a practical and sensitive platform for multiplex detection of diabetes-associated autoantibodies, with potential application in routine laboratories and future population-based screening programs. Full article

Glial mechanisms regulate neuronal excitability through multiple mechanisms, including the control of extracellular inhibitory signaling. One such mechanism is the Glu/GABA exchange process, in which glutamate uptake is coupled to GABA release, with GABA being synthesized from the polyamine putrescine, placing putrescine at a central position in metabolic pathways that may influence epileptiform activity. Here, we examined how pharmacological manipulation of key enzymes of putrescine metabolism affects seizure-like activity in the low-[Mg²⁺] in vitro model of frontotemporal epilepsy, complemented by in vivo recordings in the non-convulsive absence epilepsy model Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. Increasing putrescine availability by inhibiting spermidine synthase with trans-4-methylcyclohexylamine significantly reduced both the duration and appearance of seizure-like events. Strikingly, simultaneous inhibition of monoamine oxidase B (MAO-B) and diamine oxidase (DAO) with deprenyl and aminoguanidine almost completely abolished seizure-like events in vitro and markedly suppressed spike–wave discharges in WAG/Rij rats. This effect was largely reversed by blockade of GAT-2/3 transporters with SNAP-5114, suggesting the presence and significant anti-epileptic potential of a MAO-B- and DAO-independent putrescine–GABA synthesis pathway. Together, these findings indicate that the anticonvulsant effects of putrescine metabolism arise from the coordinated engagement of multiple parallel pathways rather than from a single dominant enzymatic route. Putrescine thus appears to function as a metabolic hub whose increased availability can be channeled into several anticonvulsant processes, suggesting that therapeutic strategies enhancing putrescine-dependent inhibitory pathways at the network level may offer promising avenues for the modulation of epileptiform activity. Full article

Salt stress is frequently encountered by plants and is a major constraint on crop production. Wheat (Triticum aestivum L.) is a staple cereal crop that is important for human health and food security. However, the response mechanisms of roots from different wheat varieties to salt stress remain unclear. In this study, we analyzed the complex response mechanisms of the salt-tolerant variety Longjian 114 (LJ114) and the salt-sensitive variety Chinese Spring (CS) to salt stress using integrated transcriptomic and metabolomic analyses. The results showed that LJ114 maintained better growth and exhibited higher antioxidant enzyme activities and greater osmotic adjustment capacity under salt stress. Differentially expressed genes in LJ114 were predominantly enriched in flavonoid biosynthesis, alpha-linolenic acid metabolism, and zeatin biosynthesis under salt stress. Metabolomic profiling identified 1366 differential metabolites, with LJ114 specifically enriched in tyrosine metabolism, tryptophan metabolism, glycerophospholipid metabolism, and fatty acid metabolism under salt stress. Integrated analyses further highlighted ABC transporters and alanine, aspartate, and glutamate metabolism as hub pathways, with notably higher GAD expression and GABA accumulation under salt stress. These findings suggest new insights into the molecular mechanisms underlying wheat salt tolerance and propose potential candidate targets for enhancing crop salinity tolerance. Full article

During swine gestation, the placenta serves as the sole interface for nutrient, gas, and signaling molecule exchange between the dam and fetuses, and its function critically determines pregnancy outcomes. Branched-chain amino acids (BCAAs)—leucine, isoleucine, and valine—are substrates for protein synthesis, precursors for glutamate and glutathione, and signaling molecules that activate the mechanistic target of rapamycin (mTOR) pathway. This review synthesizes current evidence on BCAA transport, catabolism, and signaling in the porcine placenta, organized around four mechanistic axes: transporter-mediated transplacental transfer, local catabolism supporting energy production and antioxidant defense, mTORC1-dependent regulation of growth and differentiation, and modulation of endocrine and immune homeostasis. Both deficiency and excess of BCAAs compromise placental function, contributing to intrauterine growth restriction and metabolic programming abnormalities through a U-shaped dose–response relationship. Critical knowledge gaps remain, including the reliance on rodent and in vitro models, the absence of standardized supplementation protocols and dose–response data, unaddressed confounders (parity, litter size, environment), and the lack of formal power analyses or meta-analytic synthesis. We propose precision dietary strategies to optimize BCAA supplementation during gestation for improved reproductive performance in swine. Full article

Semen quality is a critical determinant of male fertility in poultry, yet the testicular metabolic and molecular mechanisms underlying seminal traits in geese remain poorly understood. This study aimed to characterize the testicular metabolomic and transcriptomic profiles of Zhedong white geese with divergent semen quality, and to identify key regulatory pathways and candidate genes influencing semen quality. Untargeted metabolomics and RNA sequencing were performed on testicular tissues from geese with high (H) and low (L) semen quality. Serum hormone measurements revealed significantly increased testosterone (T) and decreased follicle-stimulating hormone (FSH) levels in the H group compared with the L group (p < 0.05). Metabolomic analysis identified 207 differential metabolites (DEMs), which were significantly enriched in glycosylphosphatidylinositol (GPI)-anchor biosynthesis, the citrate (TCA) cycle, and alanine, aspartate, and glutamate metabolism. Integrated transcriptomic and metabolomic analyses, combined with KEGG Markup Language (KGML) network analysis, revealed a candidate regulatory network involving potential metabolites (arachidonic acid, pyruvic acid, fumaric acid, malic acid and beta-alanine), signaling pathways (the citrate (TCA) cycle, arginine biosynthesis, ABC transporters, glycine/serine/threonine metabolism, and efferocytosis), and candidate genes (PLCB1, PLCB2, NT5C1A, TAT, LOC106042968 and ABCG8). Notably, PLCB1, which was significantly up-regulated in the H group, emerged as a candidate gene of particular interest that may be implicated in the regulation of metabolic pathways underlying semen quality in ganders. These findings provide novel insights into the testicular metabolic and transcriptomic determinants of semen quality in geese, and implicate PLCB1 and associated pathways as potential targets for improving male fertility in poultry. Full article

Vesicular (synaptic) zinc is a neuromodulator that fine-tunes synaptic transmission and sensory processing across many brain areas, including the brainstem, hippocampus, amygdala, and neocortex. Throughout the central auditory system, synaptic zinc plays a crucial role in modulating neurotransmission as well as baseline and adaptive sound processing. However, the developmental changes in the protein expression levels and localization of the vesicular zinc transporter (ZnT3), which loads synaptic zinc into presynaptic vesicles, remain unknown—due in part to the lack of robust ZnT3 antibodies. To address this question, we used the recently developed and validated ZnT3-HA transgenic mouse line, in which the ZnT3 protein contains a C-terminal HA epitope tag. We performed immunohistochemical staining and confocal microscopy in the central auditory system across development to localize and quantify changes in ZnT3 expression and explore potential colocalization of ZnT3 with the vesicular glutamate and GABA transporters VGLUT1 and VGAT, respectively. We found that ZnT3 expression increased significantly between P7 and P14 in both the dorsal cochlear nucleus (DCN) and the auditory cortex (AC), reaching a stable overall expression level and layer distribution by P21. Both in the DCN and AC, ZnT3 was mainly colocalized with VGLUT1. We did not find any significant levels of ZnT3 expression in either the IC or the auditory thalamus. Together, these results highlight major developmental changes in zinc signaling that may affect synaptic transmission and plasticity, as well as normal and pathological sound processing. Full article

The BTBR T+ Itpr3tf/J (BTBR) strain is a widely used model of neurodevelopmental disorders, characterized by an altered neuroanatomy, including a full-penetrant agenesis of the corpus callosum. While the adult BTBR brain has been studied thoroughly, less is known about how its brain develops prenatally and about when neurodevelopmental trajectories begin to differ from neurotypical strains. Here, we conducted a comparative histological analysis of fetal brain development across multiple developmental stages in BTBR and C57BL/6J (B6) mice, focusing on neocortical and midline development. BTBR mice showed lower fetal weight but comparable somite counts (assessed at 12.5 days post coitum, dpc) compared to B6 controls, indicating similar developmental timing. Neocortical layering showed a reduced ventricular zone fraction in BTBR fetuses at 15.5 dpc, without a change in overall cortical thickness. Concurrently, midline cell populations immunoreactive for the glutamate aspartate transporter (GLAST) failed to undergo remodeling in subsequent stages, resulting in the failure to form a GLAST+ indusium griseum and the lack of callosal projections crossing the midline at 17.5 dpc, which instead develop normally in B6 fetuses. These findings offer structural correlates for the early neuropathology of the BTBR brain. Full article

High-altitude environments expose aquatic organisms to complex environmental challenges, including fluctuations in dissolved oxygen levels, water temperature, and other habitat conditions. The freshwater snail Cipangopaludina cathayensis has limited dispersal ability and is strongly associated with local habitat conditions, making it a suitable species for investigating biological responses to long-term environmental variation. In this study, high-altitude and low-altitude populations of Cipangopaludina cathayensis were compared using biochemical assays, shell elemental composition analysis, transcriptomics, and metabolomics to explore altitude-associated physiological and molecular responses. Environmental monitoring showed that the high-altitude habitat exhibited lower water temperatures, greater thermal variability, and significantly higher dissolved oxygen availability than the low-altitude habitat, indicating distinct environmental conditions between the two populations. The high-altitude population exhibited significantly higher superoxide dismutase (SOD) and pyruvate kinase (PK) activities than the low-altitude population, whereas malondialdehyde (MDA) content and total ATPase (T-ATPase) activity showed no significant differences between populations. Shell microstructure and elemental analyses revealed altitude-associated differences in biomineralization. Transcriptomic analysis identified differentially expressed genes involved in intracellular transport, including KIF10, KIF11, KIF13, KIF17, MYO3, and MYO7A, as well as apoptosis-associated genes, including CASP2, CASP3, and BCL-2. Integrated transcriptomic and metabolomic analyses further highlighted purine metabolism, D-amino acid metabolism, insulin resistance, and alanine, aspartate and glutamate metabolism as shared enriched pathways between the two populations. These findings indicate that Cipangopaludina cathayensis may cope with high-altitude environments through coordinated regulation of antioxidant defense, intracellular transport, apoptosis-associated pathways, and metabolic homeostasis. Full article