Comprehensive Analysis of the Mechanism of Anoikis in Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC), ranking as the second-leading cause of global mortality among malignancies, poses a substantial burden on public health worldwide. Anoikis, a type of programmed cell death, serves as a barrier against the dissemination of cancer cells to distant organs, thereby constraining the progression of cancer. Nevertheless, the mechanism of genes related to anoikis in HCC is yet to be elucidated. Methods: This paper's data (TCGA-HCC) were retrieved from the database of the Cancer Genome Atlas (TCGA). Differential gene expression with prognostic implications for anoikis was identified by performing both the univariate Cox and differential expression analyses. Through unsupervised cluster analysis, we clustered the samples according to these DEGs. By employing the least absolute shrinkage and selection operator Cox regression analysis (CRA), a clinical predictive gene signature was generated from the DEGs. The Cell-Type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) algorithm was used to determine the proportions of immune cell types. The external validation data (GSE76427) were procured from Gene Expression Omnibus (GEO) to verify the performance of the clinical prognosis gene signature. Western blotting and immunohistochemistry (IHC) analysis confirmed the expression of risk genes. Results: In total, 23 prognostic DEGs were identified. Based on these 23 DEGs, the samples were categorized into four distinct subgroups (clusters 1, 2, 3, and 4). In addition, a clinical predictive gene signature was constructed utilizing ETV4, PBK, and SLC2A1. The gene signature efficiently distinguished individuals into two risk groups, specifically low and high, demonstrating markedly higher survival rates in the former group. Significant correlations were observed between the expression of these risk genes and a variety of immune cells. Moreover, the outcomes from the validation cohort analysis aligned consistently with those obtained from the training cohort analysis. The results of Western blotting and IHC showed that ETV4, PBK, and SLC2A1 were upregulated in HCC samples. Conclusion: The outcomes of this paper underscore the effectiveness of the clinical prognostic gene signature, established utilizing anoikis-related genes, in accurately stratifying patients. This signature holds promise in advancing the development of personalized therapy for HCC.

miR-573 suppresses pancreatic cancer cell proliferation, migration, and invasion through targeting TSPAN1.

PURPOSE: To explore whether miR-573 can suppress pancreatic cancer cell proliferation, migration, and invasion by targeting TSPAN1. METHODS: The expression of miR-573 and TSPAN1 in pancreatic cancer tissues and cells lines was analyzed using RT-qPCR. The human pancreatic cancer cell line PANC­1 was transfected with miR-573 mimic, pcDNA3.1-TSPAN1, or genOFFTM st-h-TSPAN1. The effects of miR-573 and TSPAN1 on cell proliferation, colony formation, migration, and invasion were analyzed by CCK­8, colony formation, transwell migration, and invasion assay, respectively. Target genes of miR-573 were screened using bioinformatics tools and confirmed by dual-luciferase reporter assay and real-time PCR. The effects of miR-573 in vivo were observed using tumor xenografts. RESULTS: We found that miR-573 is downregulated and TSPAN1 is upregulated in pancreatic cancer tissues and cells lines. Function assays demonstrated that overexpression of miR-573 inhibited cell proliferation, colony formation, migration, and invasion of pancreatic cancer cells, as well as suppressing tumor growth in vivo. Target genes of miR-573 were predicted using bioinformatics tools and confirmed by dual-luciferase reporter assay and RT-qPCR or western blotting. Downregulation of TSPAN1 also inhibited cell proliferation, colony formation, migration, and invasion of pancreatic cancer cells. Furthermore, overexpression of TSPAN1 attenuated miR-573-induced inhibition of pancreatic cancer cell proliferation and migration. CONCLUSION: Our findings indicated that miR-573 suppresses pancreatic cancer cell proliferation, migration, and invasion through targeting TSPAN1. TSPAN1 targeted by miR-573 might be a potential therapeutic target for clinical treatment of pancreatic cancer.

Leptin stimulates synaptogenesis in hippocampal neurons via KLF4 and SOCS3 inhibition of STAT3 signaling.

Normal development of neuronal connections in the hippocampus requires neurotrophic signals, including the cytokine leptin. During neonatal development, leptin induces formation and maturation of dendritic spines, the main sites of glutamatergic synapses in the hippocampal neurons. However, the molecular mechanisms for leptin-induced synaptogenesis are not entirely understood. In this study, we reveal two novel targets of leptin in developing hippocampal neurons and address their role in synaptogenesis. First target is Kruppel-Like Factor 4 (KLF4), which we identified using a genome-wide target analysis strategy. We show that leptin upregulates KLF4 in hippocampal neurons and that leptin signaling is important for KLF4 expression in vivo. Furthermore, KLF4 is required for leptin-induced synaptogenesis, as shKLF4 blocks and upregulation of KLF4 phenocopies it. We go on to show that KLF4 requires its signal transducer and activator of transcription 3 (STAT3) binding site and thus potentially blocks STAT3 activity to induce synaptogenesis. Second, we show that leptin increases the expression of suppressor of cytokine signaling 3 (SOCS3), another well-known inhibitor of STAT3, in developing hippocampal neurons. SOCS3 is also required for leptin-induced synaptogenesis and sufficient to stimulate it alone. Finally, we show that constitutively active STAT3 blocks the effects of leptin on spine formation, while the targeted knockdown of STAT3 is sufficient to induce it. Overall, our data demonstrate that leptin increases the expression of both KLF4 and SOCS3, inhibiting the activity of STAT3 in the hippocampal neurons and resulting in the enhancement of glutamatergic synaptogenesis during neonatal development.

Synthesis of 3'-O-Alkyl Homologues and a Biotin Probe of Isorhamnetin and Evaluation of Cytotoxic Efficacy on Cancer Cells.

Isorhamnetin is a natural flavonoid which shows a variety of biological activities such as antioxidant, anti-inflammatory and antitumor. In order to identify the cellular binding protein of isorhamnetin as potential anti-cancer target, we first synthesized 3'-O-substituted quercetin as isorhamnetin homologues and evaluated the growth inhibitory activity of these derivatives on breast, colon and prostate cancer cell lines. The preliminary results showed that the 3'-O modification did not affect the cytotoxic activity of the scaffold. Analysis of the co-crystal structure and the docking pose of isorhamnetin with reported binding protein of isorhamnetin or quercetin indicated the 3'-O-substitution groups located outside of the binding pocket, which is in accordance with activity of 3'-O derivatives. Then a biotin conjugate of isorhamnetin with a tetraethylene glycol (PEG)4 linker at the 3' position was synthesized and the resulting probe retained the anti-proliferative activity on cancer cell lines, while the cellular fluorescence analysis showed the distribution of probe inside the cells which indicated the probe had limited cell permeability. Finally, pull down assay both in situ inside cells and in the cell lysates indicated the isorhamnetin biotin probe was capable of protein labeling in cell lysates. These findings provide the isorhamnetin 3'-O-biotin probe as a tool to reveal the target proteins of isorhamnetin.

AAIT: A novel prognostic model for HIV-negative patients with cryptococcal meningoencephalitis New Scoring Model for Non-HIV Patients with CM.

Cryptococcal meningitis (CM) is a common opportunistic infection in HIV-negative patients, with mortality rates as high as those in the HIV-negative population. This requires accurate initial clinical decision-making, warranting the development of a prognostic score. Two groups of patients were investigated separately to develop a novel prognostic model (AAIT) for HIV-negative patients with CM. A retrospective analysis of 201 HIV-negative patients with CM was conducted to develop the CM prognostic score. In addition, the CM cohort (n = 21) was recruited longitudinally to verify the new prognostic score. Meanwhile, the association between the prognostic score and 1-year mortality of CM was expounded. AAIT (age, albumin, combined bacterial infection, and total triiodothyronine) is a novel prognostic score based on age, albumin level, combined bacterial infection, and total triiodothyronine (TT3) level, which were significantly higher in nonsurvivors than in survivors (0.68 [-0.70 to 1.55] vs - 1.72 [-3.75 to -0.73], P < .00). Regarding the AAIT-predicted 1-year mortality, the area under the receiver operating characteristic curve (AUROC) value was 0.857, whereas it was 0.965 for the validation cohort. In the induction period, different treatment options did not seem to significantly improve the 1-year survival rate. AAIT is a straightforward and clear prognostic score that can add value to predict the outcomes in HIV-negative patients with CM. In addition, controlling infection and increasing the albumin and TT3 levels may help improve clinical outcomes in HIV-negative patients with CM. LAY ABSTRACT: AAIT (age, albumin, combined bacterial infection, and total triiodothyronine) is a straightforward and clear prognostic score that can add value to predict the outcomes HIV-negative patients with CM.

LncRNA MALAT1 Promotes Ulcerative Colitis by Upregulating lncRNA ANRIL.

BACKGROUND: LncRNA MALAT1 contributes to the inflammatory responses induced by lipopolysaccharides (LPS), which shares similar pathogenesis with ulcerative colitis (UC), indicating the potential involvement of MALAT1 in UC. METHODS: Expression of MALAT1 and lncRNA ANRIL in both UC patients and healthy controls was analyzed by RT-qPCR. ROC curve analysis was used to evaluate the diagnostic value of MALAT1 for UC. Cell transfections were performed to analyze the interactions between MALAT1 and ANRIL. Cell apoptosis was analyzed by cell apoptosis assay. RESULTS: In the present study, we found that MALAT1 was upregulated in colonic mucosa tissues of UC patients in comparison with healthy controls. Plasma levels of MALAT1 were also higher in UC patients than in healthy controls, and upregulation of plasma MALAT1 distinguished UC patients from healthy controls. ANRIL was also upregulated in colonic mucosa tissues of UC patients than in that of healthy controls. ANRIL and MALAT1 were significantly and positively correlated in UC patients but not in healthy controls. Normal colonic epithelial cells with ANRIL overexpression showed no significantly changed MALAT1 overexpression, while MALAT1 overexpression led to promoted ANRIL expression. MALAT1 and ANRIL overexpression led to promoted apoptosis of FHCs. CONCLUSION: MALAT1 promotes ulcerative colitis by upregulating ANRIL.

Optical Control of the GTP Affinity of K-Ras(G12C) by a Photoswitchable Inhibitor.

Photocontrol of protein activity is an emerging field in biomedicine. For optical control of a mutant small GTPase K-Ras(G12C), we developed small-molecule inhibitors with photoswitchable efficacy, where one configuration binds the target protein and exert different pharmacological effects upon light irradiation. The compound design was based on the structure feature of a previously identified allosteric pocket of K-Ras(G12C) and the chemical structure of covalent inhibitors, and resulted in the synthesis and characterization of two representative azobenzene-containing compounds. Nucleotide exchange assays demonstrated the different efficacy to control the GTP affinity by photoswitching of one potent compound PS-C2, which would be a useful tool to probe the conformation of mutational K-Ras. Our study demonstrated the feasibility of designing photoswitchable modulators from allosteric covalent inhibitor of small GTPases.

Effects of acute and chronic nicotine on catecholamine neurons of the nucleus of the solitary tract.

Nicotine is an addictive drug that has broad effects throughout the brain. One site of action is the nucleus of the solitary tract (NTS), where nicotine initiates a stress response and modulates cardiovascular and gastric function through nicotinic acetylcholine receptors (nAChRs). Catecholamine (CA) neurons in the NTS influence stress and gastric and cardiovascular reflexes, making them potential mediators of nicotine's effects; however nicotine's effect on these neurons is unknown. Here, we determined nicotine's actions on NTS-CA neurons by use of patch-clamp techniques in brain slices from transgenic mice expressing enhanced green fluorescent protein driven by the tyrosine hydroxylase promoter (TH-EGFP). Picospritzing nicotine both induced a direct inward current and increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in NTS-CA neurons, effects blocked by nonselective nAChR antagonists TMPH and MLA. The increase in sEPSC frequency was mimicked by nAChRα7 agonist AR-R17779 and blocked by nAChRα7 antagonist MG624. AR-R17779 also increased the firing of TH-EGFP neurons, an effect dependent on glutamate inputs, as it was blocked by the glutamate antagonist NBQX. In contrast, the nicotine-induced current was mimicked by nAChRα4ß2 agonist RJR2403 and blocked by nAChRα4ß2 antagonist DHßE. RJR2403 also increased the firing rate of TH-EGFP neurons independently of glutamate. Finally, both somatodendritic and sEPSC nicotine responses from NTS-CA neurons were larger in nicotine-dependent mice that had under gone spontaneous nicotine withdrawal. These results demonstrate that 1) nicotine activates NTS-CA neurons both directly, by inducing a direct current, and indirectly, by increasing glutamate inputs, and 2) NTS-CA nicotine responsiveness is altered during nicotine withdrawal.

Microbial Infections as a Trigger for Acute-on-Chronic Liver Failure: A Review.

ABSTRACT Microbial infection is an important cause of acute-on-chronic liver failure (ACLF), which is a syndrome that results in multiple organ dysfunction or failure and is accompanied by an increased short-term risk of mortality. Early detection and treatment of microbial infection can effectively reduce the mortality of patients with ACLF. However, antimicrobial resistance has recently increased due to the increased use of antimicrobial agents. Therefore, it is important to choose appropriate antibiotics and antifungal agents for early prevention or treatment of patients with microbial infection and ACLF to reduce the occurrence of drug resistance and to reduce patient mortality. This review summarizes the current status in the understanding of the epidemiology, pathogenesis, early diagnosis, treatment, and strategies for prevention of microbial infection in patients with ACLF.

Azobenzene-based small molecular photoswitches for protein modulation.

Molecular photoswitches are a class of chemical structures that can readily isomerize between distinct geometries upon irradiation with light. Molecular photoswitches are utilized to control protein structure and function with temporal and spatial precision. In this review, we summarize the recent progress in the development of azobenzene-based molecular photoswitches and their applications in the photocontrol of protein structure and function. For clarity of discussion, we divide the known photoswitchable proteins into different categories: protein motifs, ion channels, receptors, and enzymes. Basic approaches and considerations for the structure-guided design of photoswitchable ligands are discussed. The applications and limitations of current photoswitches are also discussed.

High glucose increases action potential firing of catecholamine neurons in the nucleus of the solitary tract by increasing spontaneous glutamate inputs.

Glucose is a crucial substrate essential for cell survival and function. Changes in glucose levels impact neuronal activity and glucose deprivation increases feeding. Several brain regions have been shown to respond to glucoprivation, including the nucleus of the solitary tract (NTS) in the brain stem. The NTS is the primary site in the brain that receives visceral afferent information from the gastrointestinal tract. The catecholaminergic (CA) subpopulation within the NTS modulates many homeostatic functions including cardiovascular reflexes, respiration, food intake, arousal, and stress. However, it is not known if they respond to changes in glucose. Here we determined whether NTS-CA neurons respond to changes in glucose concentration and the mechanism involved. We found that decreasing glucose concentrations from 5 mM to 2 mM to 1 mM, significantly decreased action potential firing in a cell-attached preparation, whereas increasing it back to 5 mM increased the firing rate. This effect was dependent on glutamate release from afferent terminals and required presynaptic 5-HT3Rs. Decreasing the glucose concentration also decreased both basal and 5-HT3R agonist-induced increase in the frequency of spontaneous glutamate inputs onto NTS-CA neurons. Low glucose also blunted 5-HT-induced inward currents in nodose ganglia neurons, which are the cell bodies of vagal afferents. The effect of low glucose in both nodose ganglia cells and in NTS slices was mimicked by the glucokinase inhibitor glucosamine. This study suggests that NTS-CA neurons are glucosensing through a presynaptic mechanism that is dependent on vagal glutamate release, 5-HT3R activity, and glucokinase.

Development of a highly reliable assay for ubiquitin-specific protease 2 inhibitors.

The dynamic modification of proteins with ubiquitin plays crucial roles in major celluar functions, and is associated with a number of pathological conditions. Ubiquitin-specific proteases (USPs) cleave ubiquitin from substrate proteins, and rescue them from proteasomal degradation. Among them, USP2 is overexpressed and plays important roles in various cancers including prostate cancer. Thus, it represents an attractive target for drug discovery. In order to develop potent and selective USP2 inhibitors, a highly reliable assay is needed for in-depth structure-activity relationship study. We report the cloning, expression, and purification of USP2 and UBA52, and the development of a highly reliable assay based on readily available SDS-PAGE-Coomassie systeme using UBA52 as the substrate protein. A number of effective USP2 inhibitors were also identified using this assay.

The dynamic three-dimensional culture of islet-like clusters in decellularized liver scaffolds.

Diabetes mellitus is a worldwide metabolic disease which constitutes a major threat to human health. Stem cells with the ability to differentiate into insulin-producing cells (IPCs) could provide unlimited sources of transplanted cells and solve allogeneic rejection problems. The decellularized scaffolds could provide IPCs with tissue microarchitecture and intact vascular systems. The goal of this study was to engineer intact whole rat liver scaffolds and repopulate the stem cell-derived IPCs into the scaffolds to discover whether the decellularized scaffolds could facilitate the growth and development of IPCs. Decellularized liver scaffolds were obtained using 1 % Triton X-100 with 0.1 % ammonium hydroxide. Architecture and composition of the original extracellular matrix were confirmed by morphologic, histological and immunolabeling examinations. Islet-like clusters were differentiated from Wharton's jelly mesenchymal stem cells (WJMSCs) by a three-step induction procedure. The differentiation was evaluated by morphology, RT-PCR, immunofluorescence and glucose stimulation experiments. The islet-like clusters were recellularized into the decellularized scaffolds by the portal-vein infusion method and cultured by the dynamic circulation perfusion device. After cultivation, hematoxylin-eosin staining, immunofluorescence and RT-PCR were conducted. Our results demonstrated that the decellularized rat liver scaffolds have favorable biochemical properties and could support the survival of WJMSC-derived IPCs. In addition, the three-dimensional decellularized scaffolds could enhance the expression of the insulin gene compared with two-dimensional plate culture. In conclusion, these findings suggested that the decellularized scaffolds could provide a suitable platform for cellular activities of IPCs such as survival, differentiation, proliferation and insulin secretion. This study provides fundamental support for regenerating insulin-secreting organs from the decellularized scaffolds combined with stem cell-derived IPCs as a potential clinical application.

Chalcone-benzoxaborole hybrids as novel anticancer agents.

In this study, we report the synthesis of a series of chalcone-benzoxaborole hybrid molecules and the evaluation of their anticancer activity. Their anticancer potency and toxicity were tested on three human cancer cell lines and two normal cell lines. The 4-fluoro compound 15 was found to be the most potent compound with an IC50 value of 1.4µM on SKOV3 cells. The 4-iodo compound 18 and 3-methyloxy-4-amino compound 47 showed good potency on SKOV3 cells while exhibiting low toxicity on normal cells. This work extended the application of benzoxaboroles to the field of anticancer research.

Decellularization and Recellularization of Rat Livers With Hepatocytes and Endothelial Progenitor Cells.

Whole-organ decellularization has been identified as a promising choice for tissue engineering. The aim of the present study was to engineer intact whole rat liver scaffolds and repopulate them with hepatocytes and endothelial progenitor cells (EPCs) in a bioreactor. Decellularized liver scaffolds were obtained by perfusing Triton X-100 with ammonium hydroxide. The architecture and composition of the original extracellular matrix were preserved, as confirmed by morphologic, histological, and immunolabeling methods. To determine biocompatibility, the scaffold was embedded in the subcutaneous adipose layer of the back of a heterologous animal to observe the infiltration of inflammatory cells. Hepatocytes were reseeded using a parenchymal injection method and cultured by continuous perfusion. EPCs were reseeded using a portal vein infusion method. Morphologic and functional examination showed that the hepatocytes and EPCs grew well in the scaffold. The present study describes an effective method of decellularization and recellularization of rat livers, providing the foundation for liver engineering and the development of bioartificial livers.

[Serum amyloid A promotes the inflammatory response via p38-MAPK/SR-BI pathway in THP-1 macrophages].

To investigate the effect and mechanism of serum amyloid A (SAA) on the expression of scavenger receptor class B type I (SR-BI) and inflammatory response in THP-1 macrophages, the human THP-1 cells were treated with SAA and p38-MAPK agonist (anisomycin) or p38-MAPK inhibitor (SB203580). Then, the expressions of SR-BI, phosphorylated p38-MAPK and inflammatory factors (MCP-1, TNF-α, IL-1ß) were examined by real-time quantitative PCR, Western blotting and ELISA, respectively. The results showed that, compared with control group, SAA increased the levels of inflammatory factors (MCP-1, TNF-α, IL-1ß), down-regulated the expressions of SR-BI, and up-regulated the expression of phosphorylated p38-MAPK protein in a concentration- and time-dependent manner in THP-1 cells (P < 0.05). After treatment with SAA and p38-MAPK agonist (anisomycin) in THP-1 cells, the expression of SR-BI was down-regulated, and the levels of inflammatory factors and phosphorylated p38-MAPK protein expression were increased, compared with the group only treated by SAA (P < 0.05). In contrast, the SR-BI expression was up-regulated, whereas inflammatory factors and phosphorylated p38-MAPK protein expressions were decreased after the cells were treated with SAA and p38-MAPK inhibitor (SB203580) (P < 0.05). The results suggest that SAA-promoted inflammatory response in THP-1 macrophages may be through the phosphorylation of p38-MAPK and inhibition of SR-BI expression.

The environmental neurotoxicant PCB 95 promotes synaptogenesis via ryanodine receptor-dependent miR132 upregulation.

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) are widespread environmental contaminants linked to neuropsychological dysfunction in children. NDL PCBs increase spontaneous Ca(2+) oscillations in neurons by stabilizing ryanodine receptor (RyR) calcium release channels in the open configuration, which results in CREB-dependent dendritic outgrowth. In this study, we address the question of whether activation of CREB by NDL PCBs also triggers dendritic spine formation. Nanomolar concentrations of PCB 95, a NDL congener with potent RyR activity, significantly increased spine density and the frequency of miniature EPSCs in primary dissociated rat hippocampal cultures coincident with upregulation of miR132. Inhibition of RyR, CREB, or miR132 as well as expression of a mutant p250GAP cDNA construct that is not suppressed by miR132 blocked PCB 95 effects on spines and miniature EPSCs. PCB 95 also induced spine formation via RyR- and miR132-dependent mechanisms in hippocampal slice cultures. These data demonstrate a novel mechanism of PCB developmental neurotoxicity whereby RyR sensitization modulates spine formation and synaptogenesis via CREB-mediated miR132 upregulation, which in turn suppresses the translation of p250GAP, a negative regulator of synaptogenesis. In light of recent evidence implicating miR132 dysregulation in Rett syndrome and schizophrenia, these findings identify NDL PCBs as potential environmental risk factors for neurodevelopmental disorders.

Leptin-induced spine formation requires TrpC channels and the CaM kinase cascade in the hippocampus.

Leptin is a critical neurotrophic factor for the development of neuronal pathways and synaptogenesis in the hypothalamus. Leptin receptors are also found in other brain regions, including the hippocampus, and a postnatal surge in leptin correlates with a time of rapid growth of dendritic spines and synapses in the hippocampus. Leptin is critical for normal hippocampal dendritic spine formation as db/db mice, which lack normal leptin receptor signaling, have a reduced number of dendritic spines in vivo. Leptin also positively influences hippocampal behaviors, such as cognition, anxiety, and depression, which are critically dependent on dendritic spine number. What is not known are the signaling mechanisms by which leptin initiates spine formation. Here we show leptin induces the formation of dendritic protrusions (thin headless, stubby and mushroom shaped spines), through trafficking and activation of TrpC channels in cultured hippocampal neurons. Leptin-activation of the TrpC current is dose dependent and blocked by targeted knockdown of the leptin receptor. The nonselective TrpC channel inhibitors SKF96365 and 2-APB or targeted knockdown of TrpC1 or 3, but not TrpC5, channels also eliminate the leptin-induced current. Leptin stimulates the phosphorylation of CaMKIγ and ß-Pix within 5 min and their activation is required for leptin-induced trafficking of TrpC1 subunits to the membrane. Furthermore, we show that CaMKIγ, CaMKK, ß-Pix, Rac1, and TrpC1/3 channels are all required for both the leptin-sensitive current and leptin-induced spine formation. These results elucidate a critical pathway underlying leptin's induction of dendritic morphological changes that initiate spine and excitatory synapse formation.

Frequency-dependent facilitation of synaptic throughput via postsynaptic NMDA receptors in the nucleus of the solitary tract.

Hindbrain NMDA receptors play important roles in reflexive and behavioural responses to vagal activation. NMDA receptors have also been shown to contribute to the synaptic responses of neurons in the nucleus of the solitary tract (NTS), but their exact role remains unclear. In this study we used whole cell patch-clamping techniques in rat horizontal brain slice to investigate the role of NMDA receptors in the fidelity of transmission across solitary tract afferent-NTS neuron synapses. Results show that NMDA receptors contribute up to 70% of the charge transferred across the synapse at high (>5 Hz) firing rates, but have little contribution at lower firing frequencies. Results also show that NMDA receptors critically contribute to the fidelity of transmission across these synapses during high frequency (>5 Hz) afferent discharge rates. This novel role of NMDA receptors may explain in part how primary visceral afferents, including vagal afferents, can maintain fidelity of transmission across a broad range of firing frequencies. Neurons within the nucleus of the solitary tract (NTS) receive vagal afferent innervations that initiate gastrointestinal and cardiovascular reflexes. Glutamate is the fast excitatory neurotransmitter released in the NTS by vagal afferents, which arrive there via the solitary tract (ST). ST stimulation elicits excitatory postsynaptic currents (EPSCs) in NTS neurons mediated by both AMPA- and NMDA-type glutamate receptors (-Rs). Vagal afferents exhibit a high probability of vesicle release and exhibit robust frequency-dependent depression due to presynaptic vesicle depletion. Nonetheless, synaptic throughput is maintained even at high frequencies of afferent activation. Here we test the hypothesis that postsynaptic NMDA-Rs are essential in maintaining throughput across ST-NTS synapses. Using patch clamp electrophysiology in horizontal brainstem slices, we found that NMDA-Rs, including NR2B subtypes, carry up to 70% of the charge transferred across the synapse during high frequency stimulations (>5 Hz). In contrast, their relative contribution to the ST-EPSC is much less during low (<2 Hz) frequency stimulations. Afferent-driven activation of NMDA-Rs produces a sustained depolarization during high, but not low, frequencies of stimulation as a result of relatively slow decay kinetics. Hence, NMDA-Rs are critical for maintaining action potential generation at high firing rates. These results demonstrate a novel role for NMDA-Rs enabling a high probability of release synapse to maintain the fidelity of synaptic transmission during high frequency firing when glutamate release and AMPA-R responses are reduced. They also suggest why NMDA-Rs are critical for responses that may depend on high rates of afferent discharge.

FAM3A activates PI3K p110α/Akt signaling to ameliorate hepatic gluconeogenesis and lipogenesis.

UNLABELLED: FAM3A belongs to a novel cytokine-like gene family, and its physiological role remains largely unknown. In our study, we found a marked reduction of FAM3A expression in the livers of db/db and high-fat diet (HFD)-induced diabetic mice. Hepatic overexpression of FAM3A markedly attenuated hyperglycemia, insulin resistance, and fatty liver with increased Akt (pAkt) signaling and repressed gluconeogenesis and lipogenesis in the livers of those mice. In contrast, small interfering RNA (siRNA)-mediated knockdown of hepatic FAM3A resulted in hyperglycemia with reduced pAkt levels and increased gluconeogenesis and lipogenesis in the livers of C57BL/6 mice. In vitro study revealed that FAM3A was mainly localized in the mitochondria, where it increases adenosine triphosphate (ATP) production and secretion in cultured hepatocytes. FAM3A activated Akt through the p110α catalytic subunit of PI3K in an insulin-independent manner. Blockade of P2 ATP receptors or downstream phospholipase C (PLC) and IP3R and removal of medium calcium all significantly reduced FAM3A-induced increase in cytosolic free Ca(2+) levels and attenuated FAM3A-mediated PI3K/Akt activation. Moreover, FAM3A-induced Akt activation was completely abolished by the inhibition of calmodulin (CaM). CONCLUSION: FAM3A plays crucial roles in the regulation of glucose and lipid metabolism in the liver, where it activates the PI3K-Akt signaling pathway by way of a Ca(2+) /CaM-dependent mechanism. Up-regulating hepatic FAM3A expression may represent an attractive means for the treatment of insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD).