Expression Of Amino Acid Transporters Research Articles

Increased excitatory amino acid transporter 2 levels in basolateral amygdala astrocytes mediate chronic stress-induced anxiety-like behavior

Abstract The conventional perception of astrocytes as mere supportive cells within the brain has recently been called into question by empirical evidence, which has revealed their active involvement in regulating brain function and encoding behaviors associated with emotions. Specifically, astrocytes in the basolateral amygdala (BLA) have been found to play a role in the modulation of anxiety-like behaviors triggered by chronic stress. Nevertheless, the precise molecular mechanisms by which BLA astrocytes regulate chronic stress–induced anxiety-like behaviors remain to be fully elucidated. In this study, we found that in a mouse model of anxiety triggered by unpredictable chronic mild stress (UCMS), the expression of excitatory amino acid transporter 2 (EAAT2) was upregulated in the BLA. Interestingly, our findings indicate that the targeted knockdown of EAAT2 specifically within the BLA astrocytes was able to rescue the anxiety-like behavior in mice subjected to stress. Furthermore, we found that the overexpression of EAAT2 in the BLA, whether achieved through intracranial administration of EAAT2 agonists or through injection of EAAT2-overexpressing viruses with GfaABC1D promoters, evoked anxiety-like behavior in mice. Our single-nucleus RNA sequencing analysis further confirmed that chronic stress induced an upregulation of EAAT2 specifically in astrocytes in the BLA. Moreover, through in vivo calcium signal recordings, we found that the frequency of calcium activity in the BLA of mice subjected to chronic stress was higher compared with normal mice. After knocking down the expression of EAAT2 in the BLA, the frequency of calcium activity was not significantly increased, and anxiety-like behavior was obviously mitigated. Additionally, administration of an EAAT2 inhibitor in the BLA yielded a notable reduction in anxiety level among mice subjected to stress. These results suggest that BLA astrocytic EAAT2 plays a role in in the regulation of UCMS-induced anxiety-like behavior by impacting the activity of local glutamatergic neurons, and targeting EAAT2 in the BLA holds therapeutic promise for addressing anxiety disorders.

Effect of L-Type Amino Acid Transporter 1 Expression on Clinicopathological Features and Prognosis of Non-Hodgkin's Lymphoma

To detect the expression of L-type amino acid transporter 1 (LAT1) in non-Hodgkin's lymphoma (NHL) tissues, and analyze its effect on clinicopathological characteristics and prognosis of patients. A total of 92 NHL patients who were treated in our hospital from January 2017 to April 2019 were collected. The expression of LAT1 in NHL tissue was detected by immunohistochemistry and compared between patients with different pathological features (including sex, Ann Arbor stage, extranodal infiltration, Ki-67). The risk factors affecting mortality were analyzed using univariate and multivariate Cox proportional hazards regression. Receiver operating characteristic (ROC) curve was used to detect the predictive value of percentage of LAT1-positive cells in NHL tissue for patient mortality, and analyzing the effect of percentage of LAT1-positive cells on survival rate. LAT1 was positively expressed in NHL tissue. The high expression rate of LAT1 in Ann Arbor stage III and IV groups were higher than that in Ann Arbor stage I group, that in extranodal infiltration group was higher than non-extranodal infiltration group, and that in Ki-67 positive expression group was higher than Ki-67 negative expression group (all P < 0.05). The remission rate after 3 courses of treatment in high-LAT1 expression group was 70.7%, which was lower than 91.2% in low-LAT1 expression group (P < 0.05). Ann Arbor stage III and IV, extranodal invasion, Ki-67 positive expression and increased expression of LAT1 (LAT1-positive cell percentage score ≥2) were risk factors for mortality. The cut-off value of percentage of LAT1-positive cells for predicting NHL death was 45.6%, and the area under the ROC curve was 0.905 (95%CI: 0.897-0.924). The 3-year survival rate of high-LAT1 level group (the percentage of LAT1-positive cells≥45.6%) was 50.00%, which was lower than 78.26% of low-LAT1 level group (P < 0.05). The expression level of LAT1 in NHL tissue increases, which affects Ann Arbor stage and extranodal infiltration of patients. LAT1 is a risk factor for death.

Abstract 7052: p53 underpins a dependence on oxidative phosphorylation in glycolysis-competent colorectal cancer

Abstract The use of mitochondrial inhibitors to target oxidative phosphorylation (OXPHOS) in cancer treatment presents a challenge due to dose-limiting toxicities. Moreover, while glycolysis-deficient cancers are vulnerable to OXPHOS inhibition in preclinical models, the full extent of phenotypical and mechanistic consequences of inhibiting OXPHOS in cancers capable of glycolysis is not yet well understood. Our results presented here offer promising insights into potential therapeutic gains from combining p53 restoration strategies with OXPHOS inhibitors, even when applied to glycolysis-competent CRC cells. Treatment with mitochondrial complex I inhibitors did not cause energy stress in CRC cells capable of glycolysis. It does, however, induce DNA replication stress, apparent through an observed cell cycle arrest at the S phase, enrichment of the G2/M DNA-damage checkpoint regulation pathway, and replication fork slowdown. Intriguingly, CRC cells harboring wildtype p53 exhibited more severe replication stress than those carrying mutant p53. Furthermore, siRNA knockdown of p53 attenuates replication stress and reduces cell cycle arrest, underlining the important role of p53 in CRC cell responses to OXPHOS inhibition. Our targeted metabolomics analysis reveals that OXPHOS inhibition results in reductions in the purine nucleotides, adenine monophosphate (AMP) and guanine monophosphate (GMP), as well as the pyrimidine nucleotide, uridine monophosphate (UMP), in CRC cells, regardless of their p53 mutational status. By supplementing cell culture mediums with the purine nucleobases adenine and guanine, and the pyrimidine nucleoside uridine, we observed a partial reversal of the replication fork slowdown and reductions in cell viability. This suggests nucleotide deficiencies are involved in the induction of DNA replication stress caused by OXPHOS inhibition. The nucleotide deficiencies were associated with a decrease in the nucleobase precursor aspartate. By adding aspartate at a supraphysiological concentration, to overcome the low expression of the excitatory amino acid transporter 1 required for cellular import of aspartate, we were able to restore the levels of nucleotides. Collectively, our findings suggest broader potential cancer treatment paradigms via OXPHOS targeting, extending beyond glycolysis-deficient cancers. Our data uncovers that CRC cells, which commonly exhibit the glycolytic phenotype, are susceptible to OXPHOS inhibition, with those carrying wildtype p53 showing heightened sensitivity. Therefore, p53 status could serve as a biomarker for predicting CRC responses to OXPHOS inhibitors. Moreover, our findings suggest that combined strategies of restoring p53 function, using small molecules such as APR-246, might enable reduced dosage of OXPHOS inhibitors in CRC treatment, thereby mitigating their dose-limiting toxicities. Citation Format: Xiao Hong Zhao, Man Man Han, Qian Qian Yan, Yi Meng Yue, Kai Hong Ye, Yuan Yuan Zhang, Liu Teng, Liang Xu, Xiao Jing Shi, Ting La, Yu Chen Feng, Ran Xu, Vinod K. Narayana, David P. De Souza, Tao Liu, Mark Baker, Rick F. Thorne, Xu Dong Zhang, Song Chen, Lei Jin. p53 underpins a dependence on oxidative phosphorylation in glycolysis-competent colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7052.

Abstract 388: Identifying synthetic lethalities in GCN2 KO tumors using an in vivo targeted CRISPRi screen

Abstract The integrated stress response (ISR) is an intracellular signaling pathway that fosters cell adaptation to diverse stresses. The ISR is mediated by a family of four eIF2 kinases that each sense distinct cellular stresses. Activation of the eIF2 kinases results in reprogrammed gene expression through translational and transcriptional mechanisms that help prevent stress damage. Previously, we reported that prostate cancer (PCa) cells rely on GCN2 eIF2 kinase for maintenance of amino acid (AA) homeostasis to sustain growth (Cordova et al., 2022 eLife 11:e81083). GCN2 inhibition through pharmacological or genetic means results in decreased expression of AA transporters, depletion of intracellular AAs, and reduced growth of PCa cells in culture and mouse xenograft models. Although loss of GCN2 results in a significant reduction in PCa cell growth, minimal cell death is observed. We hypothesize that loss of GCN2 in PCa cells results in vulnerabilities that can be exploited and targeted to induce cell death. To optimize GCN2-targeted therapies for PCa, we utilized a targeted CRISPR-interference (CRISPRi) screen in castration-resistant 22Rv1 cells with functional wild type (WT) GCN2 or those with it deleted (GCN2 KO). The CRISPRi screen was performed in cells grown in culture or grown as tumor xenografts in mice and featured a guide library targeting genes that encode druggable targets, including kinases, phosphatases, and metabolic enzymes. While reduced expression of 152 genes was synthetically lethal with loss of GCN2 in the in vitro screen, more genes (202) were synthetically lethal with loss of GCN2 in vivo. Of note, reduced expression of many metabolic enzymes was synthetically lethal with GCN2 ablation only in the in vivo screen, potentially due to the abundance of nutrients in standard culture media. A group of six metabolism-related genes, identified as hits in the in vivo screen and targets of FDA-approved drugs, were selected for follow-up studies. One of these metabolic enzymes was GART, a trifunctional enzyme that carries out 3 of the 10 enzymatic steps in de novo purine biosynthesis. Based on our findings showing that GCN2 maintains AA levels to support nucleotide pools in PCa cells, we initially focused on this critical enzyme. Inhibition of GART using antifolates, including the FDA-approved Pemetrexed, resulted in minimal cell death of parental and GCN2 KO PCa cells cultured in standard culture media. However, when cultured in reduced nutrient media that better mimicked reduced nutrient availability in vivo, GART inhibition resulted in selective death of GCN2 KO cells. Our CRISPRi screening strategy uncovers new vulnerabilities in GCN2-inhibited PCa cells and reveals a general strategy to identify actionable targets with the potential to combine with GCN2 inhibitors for the treatment of PCa. Citation Format: Noah R. Sommers, Ricardo A. Cordova, Angela J. Klunk, Ronald C. Wek, Kirk A. Staschke. Identifying synthetic lethalities in GCN2 KO tumors using an in vivo targeted CRISPRi screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 388.

Investigation of the Effects of a Novel NOX2 Inhibitor, GLX7013170, against Glutamate Excitotoxicity and Diabetes Insults in the Retina.

Glutamate excitotoxicity and oxidative stress represent two major pathological mechanisms implicated in retinal disorders. In Diabetic Retinopathy (DR), oxidative stress is correlated to NADPH oxidase (NOX), a major source of Reactive Oxygen Species (ROS), and glutamate metabolism impairments. This study investigated the role of NOX2 and the novel NOX2 inhibitor, GLX7013170, in two models of a) retinal AMPA excitotoxicity [AMPA+GLX7013170 (10-4 M, intravitreally)] and b) early-stage DR paradigm (ESDR), GLX7013170: 14-day therapeutic treatment (topically, 20 μL/eye, 10 mg/mL (300 × 10-4 M), once daily) post-streptozotocin (STZ)-induced DR. Immunohistochemical studies for neuronal markers, nitrotyrosine, micro/macroglia, and real-time PCR, Western blot, and glutamate colorimetric assays were conducted. Diabetes increased NOX2 expression in the retina. NOX2 inhibition limited the loss of NOS-positive amacrine cells and the overactivation of micro/macroglia in both models. In the diabetic retina, GLX7013170 had no effect on retinal ganglion cell axons, but reduced oxidative damage, increased Bcl-2, reduced glutamate levels, and partially restored excitatory amino acid transporter (EAAT1) expression. These results suggest that NOX2 in diabetes is part of the triad, oxidative stress, NOX, and glutamate excitotoxicity, key players in the induction of DR. GLX7013170 is efficacious as a neuroprotective/anti-inflammatory agent and a potential therapeutic in retinal diseases, including ESDR.

Effects of the kinetic pattern of dietary glucose release on nitrogen utilization, the portal amino acid profile, and nutrient transporter expression in intestinal enterocytes in piglets

BackgroundPromoting the synchronization of glucose and amino acid release in the digestive tract of pigs could effectively improve dietary nitrogen utilization. The rational allocation of dietary starch sources and the exploration of appropriate dietary glucose release kinetics may promote the dynamic balance of dietary glucose and amino acid supplies. However, research on the effects of diets with different glucose release kinetic profiles on amino acid absorption and portal amino acid appearance in piglets is limited. This study aimed to investigate the effects of the kinetic pattern of dietary glucose release on nitrogen utilization, the portal amino acid profile, and nutrient transporter expression in intestinal enterocytes in piglets.MethodsSixty-four barrows (15.00 ± 1.12 kg) were randomly allotted to 4 groups and fed diets formulated with starch from corn, corn/barley, corn/sorghum, or corn/cassava combinations (diets were coded A, B, C, or D respectively). Protein retention, the concentrations of portal amino acid and glucose, and the relative expression of amino acid and glucose transporter mRNAs were investigated. In vitro digestion was used to compare the dietary glucose release profiles.ResultsFour piglet diets with different glucose release kinetics were constructed by adjusting starch sources. The in vivo appearance dynamics of portal glucose were consistent with those of in vitro dietary glucose release kinetics. Total nitrogen excretion was reduced in the piglets in group B, while apparent nitrogen digestibility and nitrogen retention increased (P < 0.05). Regardless of the time (2 h or 4 h after morning feeding), the portal total free amino acids content and contents of some individual amino acids (Thr, Glu, Gly, Ala, and Ile) of the piglets in group B were significantly higher than those in groups A, C, and D (P < 0.05). Cluster analysis showed that different glucose release kinetic patterns resulted in different portal amino acid patterns in piglets, which decreased gradually with the extension of feeding time. The portal His/Phe, Pro/Glu, Leu/Val, Lys/Met, Tyr/Ile and Ala/Gly appeared higher similarity among the diet treatments. In the anterior jejunum, the glucose transporter SGLT1 was significantly positively correlated with the amino acid transporters B0AT1, EAAC1, and CAT1.ConclusionsRational allocation of starch resources could regulate dietary glucose release kinetics. In the present study, group B (corn/barley) diet exhibited a better glucose release kinetic pattern than the other groups, which could affect the portal amino acid contents and patterns by regulating the expression of amino acid transporters in the small intestine, thereby promoting nitrogen deposition in the body, and improving the utilization efficiency of dietary nitrogen.

Anesthetic Sevoflurane Induces Enlargement of Dendritic Spine Heads in Mouse Neurons via Tau-Dependent Mechanisms.

Sevoflurane induces neuronal dysfunction and cognitive impairment. However, the underlying mechanism remains largely to be determined. Tau, cyclophilin D, and dendritic spine contribute to cognitive function. But whether changes in dendritic spines are involved in the effects of sevoflurane and the potential association with tau and cyclophilin D is not clear. We harvested hippocampal neurons from wild-type mice, tau knockout mice, and cyclophilin D knockout mice. We treated these neurons with sevoflurane at day in vitro 7 and measured the diameter of dendritic spine head and the number of dendritic spines. Moreover, we determined the effects of sevoflurane on the expression of excitatory amino acid transporter 3 (EAAT3), extracellular glutamate levels, and miniature excitatory postsynaptic currents (mEPSCs). Finally, we used lithium, cyclosporine A, and overexpression of EAAT3 in the interaction studies. Sevoflurane-induced tau phosphgorylation increased the diameter of dendritic spine head and decreased the number of dendritic spines in neurons harvested from wild-type and cyclophilin D knockout mice, but not tau knockout mice. Sevoflurane decreased the expression of EAAT3, increased extracellular glutamate levels, and decreased the frequency of mEPSCs in the neurons. Overexpression of EAAT3 mitigated the effects of sevoflurane on dendritic spines. Lithium, but not cyclosporine A, attenuated the effects of sevoflurane on dendritic spines. Lithium also inhibited the effects of sevoflurane on EAAT3 expression and mEPSCs. These data suggest that sevoflurane induces a tau phosphorylation-dependent demtrimental effect on dendritic spine via decreasing EAAT3 expression and increasing extracellular glutamate levels, leading to neuronal dysfunction.

Shifts in microbiota and gene expression of nutrient transporters, mucin and interleukins in the gut of fast-growing and slow-growing chickens infected by Salmonella Enteritidis.

Slow-growing breeds are more resistant to Salmonella infection compared to fast-growing broilers. However, it is unclear whether that is associated with innate resistance or rather rely on differences in Salmonella-induced gut responses. We investigated the microbial composition and gene expression of nutrient transporters, mucin, and interleukin in the gut of a fast-growing (Cobb500) and a slow-growing naked neck (NN) chicken breeds challenged with Salmonella Enteritidis. Hatchlings were inoculated at two days of age using sterile broth (sham) or Salmonella Enteritidis (SE) and distributed according to a completely randomized design into four treatments: Cobb-sham; Cobb-SE; NN-sham; and NN-SE. Cecal SE counting and microbial composition by 16S rRNA sequencing were determined at 24-, 96-, and 168-hours post-inoculation (hpi). Gene expression of amino acid (Asct1) and peptide transporters (PepT1), glucose transporters (Sglt1, Glut2 and Glut5) and mucin (Muc2) in the jejunum and expression of interleukins (IL1 beta, IL8, IL17 and IL22) in the cecum was assessed by qPCR at 24 and 168 hpi. NN birds were colonized by SE just as Cobb birds but showed innate upregulation of Muc2, IL8 and IL17 in comparison to Cobb. While nutrient transporter mRNA expression was impaired in SE-challenged Cobb birds, the opposite was observed in NN. There were no differences in microbial diversity at different sampling times for Cobb-SE, whereas the other groups had higher diversity and lower dominance at 24 hpi compared with 96 hpi and 168 hpi. NN birds apparently develop earlier gut microbial stability, have higher basal level of mucin gene expression as well as differential nutrient transporter and interleukin gene expression in the presence of SE which might mitigate the effects of SE infection compared to Cobb birds.

Apparent ileal amino acid digestibility, gut morphometrics, and gene expression of peptide and amino acid transporters in broiler chickens fed low-crude-protein diets supplemented with crystalline amino acids with soybean meal, canola meal, or corn DDGS as protein feedstuffs.

We investigated the impact of using canola meal (CM) or corn distillers dried grain soluble (cDDGS) in place of soybean meal (SBM) in low-crude-protein diets supplemented with amino acids (AA) on AA digestibility, gut morphometrics, and AA transporter genes in broiler chicken. On day 0, 540 Cobb 500 male broilers were allocated to six diets in 36-floor pens. The positive control (PC) was a corn-SBM diet with adequate crude protein (CP). The CP level of negative control (NC) was decreased by 45 and 40 g kg-1 relative to PC for grower and finisher phases, respectively. The subsequent two diets had the same CP levels as NC but with cDDGS added at 50 or 125 g kg-1. The last two diets had the same CP as NC but with CM added at 50 or 100 g kg-1. Dietary CP reduction in corn-SBM diets increased (P < 0.05) the digestibility of Lys (88.5%), Met (90.7%), Thr (77.4%), Cys (80.7%), and Gly (84.7%). Increasing levels of cDDGS linearly decreased (P < 0.05) the digestibility of Asp, Cys, Glu, and Ser, whereas increasing CM level linearly decreased (P < 0.05) the digestibility of Cys, Pro, and Ser. The CP reduction in corn-SBM diets produced downward expression of peptide transporter1 and decreased (P < 0.05) absolute pancreas and ileum weight and length of jejunum and ileum. Partial replacement of SBM with alternative protein feedstuffs (cDDGS or CM) in low-CP diets had minimal effects on AA digestibility and mRNA levels of peptides and AA transporters. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The effect of reducing dietary energy on performance, intestinal morphology and intestinal peptide and amino acid transporters in broiler chicks

AbstractBackgroundPrevious research has investigated the impact of different factors such as gender and age of birds, protein and amino acid levels, protein origin and the physical form of the diet, on the mRNA expression of intestinal peptides and amino acid transporters. Nevertheless, there have been no attempts to examine the impact of dietary energy density on the mRNA abundance of intestinal amino acid and peptide transporters in humans or animals. This is the first reported case in both humans and animals.ObjectivesThis experiment aimed to evaluate the influence of two levels of metabolizable energy ([ME]; 2950 and 2850 kcal ME/kg diet) on performance and jejunal morphology and mRNA expression of amino acid (B0AT, b0,+AT, CAT1 and y+LAT1) and peptide (PepT1) transporters in broiler chickens during the starter period (0–10 days of age).MethodsTwo hundred and seventy day‐old male Ross 308 broiler chickens were randomly assigned to two dietary treatments with nine replicates and fifteen birds in each replicate. At 10 days of age, the outcome variables were measured, which included the assessment of the mRNA abundance of PepT1, b0,+AT, B0AT, y+LAT1 and CAT1 in the jejunal section using real‐time PCR.ResultsThe findings of this study revealed no significant impact of the dietary treatments on feed intake, body weight gain, feed conversion ratio and the jejunal mRNA abundance of PepT1, CAT1 and y+LAT1 (p &gt; 0.05). However, a reduction in dietary ME level from 2950 to 2850 kcal ME/kg diet resulted in a decrease in the mRNA level of B0AT and b0,+AT (p ≤ 0.05). The reduction in dietary energy level did not lead to any significant changes in villus width, villus height, crypt depth, villus surface area and villus height to crypt depth ratio (p &gt; 0.05).ConclusionThe results demonstrated that decreasing dietary energy density may alter the expression of amino acid transporters in the intestine while having no impact on the growth performance and morphometric structure of the intestine.

Differential amino acid transporter expression in adult Drosophila melanogaster tissues.

Organismal macronutrient intake modulates organ and tissue function. Dietary amino acids play essential roles in metabolic processes that support normal tissue growth, repair, and function. For example, in Drosophila melanogaster , protein-deficient diets lead to reduced overall organismal growth during larval development and severely decreased egg production in adult females. Multiple tissues, therefore, must sense and respond to dietary protein input. Amino acid transporter proteins facilitate the movement of amino acids across cellular membranes. Based on high-throughput expression studies, the Drosophila genome is predicted to encode 58 amino acid transporters. We have set out to determine if there are tissue-specific amino acid requirements for proper tissue function by first assessing the complement of amino acid transporters expressed in several adult tissues. Using RT-PCR to assess transcript levels, we find that most of the 24 amino acid transporters examined are expressed in the head, thorax, abdomen, gut, and ovary, while a subset shows differential transcript expression. This work will serve as the foundation for future studies addressing the impact of physiological factors, like nutrition, on amino acid sensing by individual tissues.

Comparative study of susceptibility to methylmercury cytotoxicity in cell types composing rat peripheral nerves: a higher susceptibility of dorsal root ganglion neurons.

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

Dietary methionine supplementation during the estrous cycle improves follicular development and estrogen synthesis in rats.

The follicle is an important unit for the synthesis of steroid hormones and the oocyte development and maturation in mammals. However, the effect of methionine supply on follicle development and its regulatory mechanism are still unclear. In the present study, we found that dietary methionine supplementation during the estrous cycle significantly increased the number of embryo implantation sites, as well as serum contents of a variety of amino acids and methionine metabolic enzymes in rats. Additionally, methionine supplementation markedly enhanced the expression of rat ovarian neutral amino acid transporters, DNA methyltransferases (DNMTs), and cystathionine gamma-lyase (CSE); meanwhile, it significantly increased the ovarian concentrations of the metabolite S-adenosylmethionine (SAM) and glutathione (GSH). In vitro data showed that methionine supply promotes rat follicle development through enhancing the expression of critical gene growth differentiation factor 9 and bone morphogenetic protein 15. Furthermore, methionine enhanced the relative protein and mRNA expression of critical genes related to estrogen synthesis, ultimately increasing estrogen synthesis in primary ovarian granulosa cells. Taken together, our results suggested that methionine promoted follicular growth and estrogen synthesis in rats during the estrus cycle, which improved embryo implantation during early pregnancy. These findings provided a potential nutritional strategy to improve the reproductive performance of animals.

Unraveling differential characteristics and mechanisms of nitrogen uptake in wheat cultivars with varied nitrogen use efficiency

Nitrogen uptake is crucial to wheat nitrogen use efficiency (NUE). The study's findings indicate that both high- and low-NUE cultivars exhibited highest nitrogen uptake efficiency (NupE) under 0.2 mM nitrogen. Under 2 mM nitrogen, their NupEs decrease significantly, while uptakes to NO3− were notably higher than that of NH4+. Strikingly, high-NUE cultivars exhibited a significantly higher NH4+ uptake rate than low NUE cultivars, resulting in a marked improvement in their ability to take up nitrogen. The NUEs of the cultivars with 5 mM nitrogen were almost half that of 2 mM nitrogen. NO3− uptake primarily occurred in the mature zone of roots, while NH4+ uptake took place in the root tip meristem and elongation zones. Notably, the NH4+ uptake in root tip meristematic zone of high-NUE cultivar was significantly higher than that of low NUE cultivar. Furthermore, the NO3− uptake of high-NUE cultivar in the root mature zone was significantly higher than that of low-NUE cultivar under 2 mM nitrogen. These findings were consistent with the significantly higher expression levels of TaAMT in root tip and of TaNRT in root mature area of high-NUE cultivar compared to low-NUE cultivar, respectively, enabling efficient absorption of NO3− and NH4+ and transport of NO3− to shoot. The high-NUE cultivars showed elevated expression of amino acid transporters further promoting nitrogen uptake, and conversion of nitrogen into ureides and amino acids further facilitated inorganic nitrogen uptake by roots. The differential findings offer valuable insights into novel variety breeding of high NUE in the future.

Increased glucose metabolism and impaired glutamate transport in human astrocytes are potential early triggers of abnormal extracellular glutamate accumulation in hiPSC-derived models of Alzheimer's disease.

Glutamate recycling between neurons and astrocytes is essential to maintain neurotransmitter homeostasis. Disturbances in glutamate homeostasis, resulting in excitotoxicity and neuronal death, have been described as a potential mechanism in Alzheimer's disease (AD) pathophysiology. However, glutamate neurotransmitter metabolism in different human brain cells, particularly astrocytes, has been poorly investigated at the early stages of AD. We sought to investigate glucose and glutamate metabolism in AD by employing human induced pluripotent stem cell (hiPSC)-derived astrocytes and neurons carrying mutations in the amyloid precursor protein (APP) or presenilin-1 (PSEN-1) gene as found in familial types of AD (fAD). Methods such as live-cell bioenergetics and metabolic mapping using [13C]-enriched substrates were used to examine metabolism in the early stages of AD. Our results revealed greater glycolysis and glucose oxidative metabolism in astrocytes and neurons with APP or PSEN-1 mutations, accompanied by an elevated glutamate synthesis compared to control WT cells. Astrocytes with APP or PSEN-1 mutations exhibited reduced expression of the excitatory amino acid transporter 2 (EAAT2), and glutamine uptake increased in mutated neurons, with enhanced glutamate release specifically in neurons with a PSEN-1 mutation. These results demonstrate a hypermetabolic phenotype in astrocytes with fAD mutations possibly linked to toxic glutamate accumulation. Our findings further identify metabolic imbalances that may occur in the early phases of AD pathophysiology.

Ultrasmall epibiont Nanosynbacter lyticus strain TM7x and host bacteria transcriptional activity after initial host parasitism

ABSTRACT Background Oral Saccharibacteria Nanosynbacter lyticus strain TM7× lives as an ultrasmall epibiont on the surface of its host, Schaalia odontolytica strain XH001. Establishing this interaction is a poorly understood multi-step process. The recovery phase marks a shift in the TM7×/host interaction, switching from the early killing phase, with extensive host cell death, to a stable symbiosis phase where the host and epibiont can grow together. Results Transcriptomes of TM7× and host, XH001, were captured during the recovery phase and compared to uninfected host and the early host/epibiont interaction (initial encounter). XH001 showed increased expression for rhamnose cell wall components and for the precursor to peptidoglycan while TM7× showed increases in the peptidoglycan pathway. Transporter expression was generally increased for both organisms during recovery compared to the initial encounter, though, XH001 showed lower amino acid transporter expression. Consistent with host parasitism, XH001 showed increased expression of various stress-related genes during recovery while TM7× showed reduced stress. TM7× displayed higher expression of type IV pili, consistent with increased attachment to new hosts. Conclusion As TM7× is a member of the broadly distributed Candidate Phyla Radiation with small genomes lacking numerous biosynthetic pathways, this study provides further insights into how these epibionts interact and modulate their host bacteria.

Molecular insights into disease-associated glutamate transporter (EAAT1 / SLC1A3) variants using in silico and in vitro approaches

Glutamate is an essential excitatory neurotransmitter and an intermediate for energy metabolism. Depending on the tumor site, cancer cells have increased or decreased expression of excitatory amino acid transporter 1 or 2 (EAAT1/2, SLC1A3/2) to regulate glutamate uptake for the benefit of tumor growth. Thus, EAAT1/2 may be an attractive target for therapeutic intervention in oncology. Genetic variation of EAAT1 has been associated with rare cases of episodic ataxia, but the occurrence and functional contribution of EAAT1 mutants in other diseases, such as cancer, is poorly understood. Here, 105 unique somatic EAAT1 mutations were identified in cancer patients from the Genomic Data Commons dataset. Using EAAT1 crystal structures and in silico studies, eight mutations were selected based on their close proximity to the orthosteric or allosteric ligand binding sites and the predicted change in ligand binding affinity. In vitro functional assessment in a live-cell, impedance-based phenotypic assay demonstrated that these mutants differentially affect L-glutamate and L-aspartate transport, as well as the inhibitory potency of an orthosteric (TFB-TBOA) and allosteric (UCPH-101) inhibitor. Moreover, two episodic ataxia-related mutants displayed functional responses that were in line with literature, which confirmed the validity of our assay. Of note, ataxia-related mutant M128R displayed inhibitor-induced functional responses never described before. Finally, molecular dynamics (MD) simulations were performed to gain mechanistic insights into the observed functional effects. Taken together, the results in this work demonstrate 1) the suitability of the label-free phenotypic method to assess functional variation of EAAT1 mutants and 2) the opportunity and challenges of using in silico techniques to rationalize the in vitro phenotype of disease-relevant mutants.

Successful anti-tumor effects with two novel bifunctional chemotherapeutic compounds that combine a LAT1 substrate with cytotoxic moieties in aggressive T-cell lymphomas

T-cell lymphomas are aggressive neoplasms characterized by poor responses to current chemotherapeutic agents. Expression of the l-type amino acid transporter 1 (LAT 1, SLC7A5) allows for the expansion of healthy T-cell counterparts, and upregulation of LAT1 has been reported in precursor T-cell acute leukemia. Therefore, the expression of LAT1 was evaluated in a cohort of cutaneous and peripheral T-cell lymphomas. The findings demonstrated that LAT1 is upregulated in aggressive variants and absent in low-grade or indolent disease such as mycosis fungoides. In addition, upregulated LAT1 expression was seen in a large proportion of aggressive peripheral T-cell lymphomas, including peripheral T-cell lymphoma not otherwise specific (PTCL-NOS) and angioimmunoblastic T-cell lymphoma (AITL). The anti-tumor effects of two novel non-cleavable and bifunctional compounds, QBS10072S and QBS10096S, that combine a potent cytotoxic chemotherapeutic domain (tertiary N-bis(2-chloroethyl)amine) with the structural features of a selective LAT1 substrate (aromatic β-amino acid) were tested in vitro and in vivo in T-cell lymphoma cell lines. The findings demonstrated decreased survival of T-cell lymphoma lines with both compounds. Overall, the results demonstrate that LAT1 is a valuable biomarker for aggressive T-cell lymphoma counterparts and QBS10072S and QBS10096S are successful therapeutic options for these aggressive diseases.

Compensatory Processes in Striatal Neurons Expressing the Tyrosine Hydroxylase Gene in Transgenic Mice in a Model of Parkinson's Disease.

The mammalian striatum is known to contain non-dopaminergic neurons that express dopamine (DA)-synthesizing enzymes and produce DA, responsible for the regulation of motor function. This study assessed the expression of DA-synthesizing enzymes in striatal neurons and their role in DA synthesis in transgenic mice expressing the green fluorescent protein (GFP) gene under the tyrosine hydroxylase (TH) gene promoter in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease (PD). We showed that, in Parkinsonian animals, the number of neurons expressing the TH gene increased by 1.9 times compared with the control (0.9% NaCl), which indicates a compensatory response to the DAergic denervation of the striatum. This assumption is supported by a 2.5-fold increase in the expression of genes for TH and transcription factor Nurr1 and a 1.45-fold increase in the expression of the large amino acid transporter 1 gene. It is noteworthy that, in Parkinsonian mice, in contrast to the controls, DA-synthesizing enzymes were found not only in nerve fibers but also in neuronal cell bodies. Indeed, TH or TH and aromatic L-amino acid decarboxylase (AADC) were detected in GFP-positive neurons, and AADC was detected in GFP-negative neurons. These neurons were shown to synthesize DA, and this synthesis is compensatorily increased in Parkinsonian mice. The above data open the prospect of improving the treatment of PD by maintaining DA homeostasis in the striatum.

Overexpression of the LAT1 in primary human trophoblast cells increases the uptake of essential amino acids and activates mTOR signaling.

The System L amino acid transporter, particularly the isoform Large Neutral Amino Acid Transporter Small Subunit 1 (LAT1) encoded bySLC7A5, is believed to mediate the transfer of essential amino acids in the human placenta. Placental System L amino acid transporter expression and activity is decreased in pregnancies complicated by IUGR and increased in fetal overgrowth. However, it remains unknown if changes in the expression of LAT1 are mechanistically linked to System L amino acid transport activity. Here we combined overexpression approaches with protein analysis and functional studies in cultured primary human trophoblast (PHT) cells to test the hypothesis that SLC7A5overexpression increases the uptake of essential amino acids and activates mTOR signaling in PHT cells. Overexpression of SLC7A5 resulted in a marked increase in protein expression of LAT1 in the PHT cells microvillous plasma membrane and System L amino acid transporter activity. Moreover, mTOR signaling was activated, and System A amino acid transporter activity increased following SLC7A5overexpression, suggesting coordination of trophoblast amino transporter expression and activity to ensure balanced nutrient flux to the fetus. This is the first report showing that overexpression of LAT1 is sufficient to increase the uptake of essential amino acids in PHT cells, which activates mTOR, a master regulator of placental function. The decreased placental System L activity in human IUGR and the increased placental activity of this transporter system in some cases of fetal overgrowth may directly contribute to changes in fetal amino acid availability and altered fetal growth in these pregnancy complications.