Structural basis of ketamine action on human NMDA receptors.

Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors1. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants2,3. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant4. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.

ZBTB20 Regulates SERCA2a Activity and Myocardial Contractility Through Phospholamban.

BACKGROUND: Intracellular Ca2+ cycling determines myocardial contraction and relaxation in response to physiological demands. SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a) is responsible for the sequestration of cytosolic Ca2+ into intracellular stores during cardiac relaxation, and its activity is reversibly inhibited by PLN (phospholamban). However, the regulatory hierarchy of SERCA2a activity remains unclear. METHODS: Cardiomyocyte-specific ZBTB20 knockout mice were generated by crossing ZBTB20flox mice with Myh6-Cre mice. Echocardiography, blood pressure measurements, Langendorff perfusion, histological analysis and immunohistochemistry, quantitative reverse transcription-PCR, Western blot analysis, electrophysiological measurements, and chromatin immunoprecipitation assay were performed to clarify the phenotype and elucidate the molecular mechanisms. RESULTS: Specific ablation of ZBTB20 in cardiomyocyte led to a significant increase in basal myocardial contractile parameters both in vivo and in vitro, accompanied by an impairment in cardiac reserve and exercise capacity. Moreover, the cardiomyocytes lacking ZBTB20 showed an increase in sarcoplasmic reticular Ca2+ content and exhibited a remarkable enhancement in both SERCA2a activity and electrically stimulated contraction. Mechanistically, PLN expression was dramatically reduced in cardiomyocytes at the mRNA and protein levels by ZBTB20 deletion or silencing, and PLN overexpression could largely restore the basal contractility in ZBTB20-deficient cardiomyocytes. CONCLUSIONS: These data point to ZBTB20 as a fine-tuning modulator of PLN expression and SERCA2a activity, thereby offering new perspective on the regulation of basal contractility in the mammalian heart.

AMPK Attenuation of ß-Adrenergic Receptor-Induced Cardiac Injury via Phosphorylation of ß-Arrestin-1-ser330.

BACKGROUND: ß-adrenergic receptor (ß-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of ß-AR remains unclear. METHODS: Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates ß-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the ß-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between ß-adrenergic insult and ß-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from ß-arrestin-1-S330A/S330D mutation and ß-adrenergic insult. RESULTS: Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to ß-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted ß-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing ß-arrestin-1-S330D (active form) inhibited the ß-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. ß-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the ß-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. CONCLUSIONS: AMPK phosphorylation of ß-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting ß-AR/cAMP/PKA activation. Subsequently, ß-arrestin-1 Ser330 phosphorylation blocks ß-AR-induced cardiac inflammasome activation and remodeling.

Exercise training attenuates cardiac dysfunction induced by excessive sympathetic activation through an AMPK-KLF4-FMO2 axis.

Cardiovascular diseases (CVDs) are a leading cause of mortality worldwide and are associated with an overactivated sympathetic system. Although exercise training has shown promise in mitigating sympathetic stress-induced cardiac remodeling, the precise mechanisms remain elusive. Here, we demonstrate that exercise significantly upregulates cardiac flavin-containing monooxygenase 2 (FMO2) expression. Notably, we find that exercise training effectively counteracts sympathetic overactivation-induced cardiac dysfunction and fibrosis by enhancing FMO2 expression via adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation. Functional investigations employing FMO2 knockdown with adeno-associated virus 9 (AAV9) underscore the necessity for FMO2 expression to protect the heart during exercise in vivo. Furthermore, we identify the krüppel-like factor 4 (KLF4) as a transcriptional mediator of FMO2 that is crucial for the mechanism through which AMPK activation protects against sympathetic overactivation-induced cardiac dysfunction and fibrosis. Taken together, our study reveals a cardioprotective mechanism for exercise training through an AMPK-KLF4-FMO2 signaling pathway that underscores how exercise alleviates cardiac dysfunction induced by excessive sympathetic activation.

SARS-CoV-2 spike protein acts as a ß-adrenergic receptor agonist: A potential mechanism for cardiac sequelae of long COVID.

BACKGROUND: Currently, pathophysiological mechanisms of post-acute sequelae of coronavirus disease-19-cardiovascular syndrome (PASC-CVS) remain unknown. METHODS AND RESULTS: Patients with PASC-CVS exhibited significantly higher circulating levels of severe acute respiratory syndrome-coronavirus-2 spike protein S1 than the non-PASC-CVS patients and healthy controls. Moreover, individuals with high plasma spike protein S1 concentrations exhibited elevated heart rates and normalized low frequency, suggesting cardiac ß-adrenergic receptor (ß-AR) hyperactivity. Microscale thermophoresis (MST) assay revealed that the spike protein bound to ß1- and ß2-AR, but not to D1-dopamine receptor. These interactions were blocked by ß1- and ß2-AR blockers. Molecular docking and MST assay of ß-AR mutants revealed that the spike protein interacted with the extracellular loop 2 of both ß-ARs. In cardiomyocytes, spike protein dose-dependently increased the cyclic adenosine monophosphate production with or without epinephrine, indicating its allosteric effects on ß-ARs. CONCLUSION: Severe acute respiratory syndrome-coronavirus-2 spike proteins act as an allosteric ß-AR agonist, leading to cardiac ß-AR hyperactivity, thus contributing to PASC-CVS.

The Value of lncRNAs as a Biomarker for the Diagnosis of Gestational Diabetes: A Meta-Analysis.

Gestational diabetes mellitus (GDM) is a common metabolic disorder in pregnancy and leads to serious harm to the mother and the fetus. A variety of lncRNAs play a key role in GDM. This meta-analysis was performed to explore the potential value of lncRNAs in GDM diagnosis. Articles correlated with lncRNA and GDM were screened from Embase, Medline, EBSCO, PubMed, Chinese National Knowledge Infrastructure, and WanFang databases. Summary receiver operator characteristic (SROC) was performed to evaluate the pooled area under curve (AUC). Forest plot was conducted to calculate the sensitivity, specificity, diagnostic likelihood ratio (LR), diagnostic score, and diagnostic odds ratio (DOR). Deeks' funnel plot was utilized to evaluate the publication bias. Eleven articles containing 12 tests (1060 GDM patients and 1066 controls) were included in this meta-analysis. AUC (0.89, 95%CI=0.86-0.92), sensitivity (0.84, 95%CI=0.80-0.87), and specificity (0.81, 95%CI=0.77-0.85)of the SROC curve showed a high diagnostic value of lncRNA for GDM. Positive LR (PLR 4.40, 95%CI=3.45-5.60) and negative LR (NLR 0.20, 95%CI=0.15-0.26) results indicated that the diagnosis of lncRNA for GDM had low clinical utility. Diagnostic score (3.09, 95%CI=2.62-3.57) and DOR (22.04, 95%CI=13.68-35.51) results suggested lncRNAs have good discriminative effect on GDM. Heterogeneity was significantly higher, but not induced by the subgroups. LncRNAs had high diagnostic value and good discriminative effect for GDM, but the clinical utility was not high. This meta-analysis study offers a potential target for GDM diagnosis.

Identification and characterization of STAT family in silver pomfret (Pampus argenteus) involved in different exogenous stresses.

Members of the Signal Transducer and Activator of Transcription (STAT) family function pivotally as transcriptional activators integral to the modulation of inflammatory responses. The aquaculture of silver pomfret is frequently compromised by the imposition of exogenous stressors, which include thermal fluctuations, notably low-temperatures, diminished oxygen levels, and the onslaught of bacterial pathogens. Notwithstanding the critical impact of these stressors, the scientific literature presents a notable gap in our understanding of the STAT pathway's role in the silver pomfret's adaptive response mechanisms. To address this lacuna, we identified stat genes in the silver pomfret-denominated as Pastat1, Pastat2, Pastat3, Pastat4, and Pastat5-through a thorough and systematic bioinformatics analysis. Further scrutiny of the gene configurations and constituent motifs has elucidated that STAT proteins possess analogous structural frameworks and exhibit significant evolutionary preservation. Subsequently, the expression patterns of five stat genes were verified by RT-qPCR in twelve different tissues and four growth periods in healthy fish, showing that the expression of Pastat genes was temporally and spatially specific, with most of the stat genes expressed at higher levels in the spleen, following muscle, gill, and liver. Transcriptomic analysis of exposure to exogenous stressors, specifically formaldehyde and low-temperature conditions, elucidated that Pastat1 and Pastat2 genes exhibited a heightened sensitivity to these environmental challenges. RT-qPCR assays demonstrated a marked alteration in the expression profiles of jak1 and Pastat gene suites in PaS upon prolonged bacterial infection subsequent to these exogenous insults. Moreover, the gene expression of the downstream effectors involved in innate immunity and apoptosis displayed marked deviations. This study additionally elucidated the Pastat gene family's role in modulating the innate immune response and apoptotic regulation within the silver pomfret during exogenous stressors and subsequent pathogenic incursions.

Chronic PM2.5 exposure disrupts intestinal barrier integrity via microbial dysbiosis-triggered TLR2/5-MyD88-NLRP3 inflammasome activation.

BACKGROUND: PM2.5, a known public health risk, is increasingly linked to intestinal disorders, however, the mechanisms of its impact are not fully understood. PURPOSE: This study aimed to explore the impact of chronic PM2.5 exposure on intestinal barrier integrity and to uncover the underlying molecular mechanisms. METHODS: C57BL/6 J mice were exposed to either concentrated ambient PM2.5 (CPM) or filtered air (FA) for six months to simulate urban pollution conditions. We evaluated intestinal barrier damage, microbial shifts, and metabolic changes through histopathology, metagenomics, and metabolomics. Analysis of the TLR signaling pathway was also conducted. RESULTS: The mean concentration of PM2.5 in the CPM exposure chamber was consistently measured at 70.9 ± 26.8 µg/m³ throughout the study period. Our findings show that chronic CPM exposure significantly compromises intestinal barrier integrity, as indicated by reduced expression of the key tight junction proteins Occludin and Tjp1/Zo-1. Metagenomic sequencing revealed significant shifts in the microbial landscape, identifying 35 differentially abundant species. Notably, there was an increase in pro-inflammatory nongastric Helicobacter species and a decrease in beneficial bacteria, such as Lactobacillus intestinalis, Lactobacillus sp. ASF360, and Eubacterium rectale. Metabolomic analysis further identified 26 significantly altered metabolites commonly associated with intestinal diseases. A strong correlation between altered bacterial species and metabolites was also observed. For example, 4 Helicobacter species all showed positive correlations with 13 metabolites, including Lactate, Bile acids, Pyruvate and Glutamate. Additionally, increased expression levels of TLR2, TLR5, Myd88, and NLRP3 proteins were noted, and their expression patterns showed a strong correlation, suggesting a possible involvement of the TLR2/5-MyD88-NLRP3 signaling pathway. CONCLUSIONS: Chronic CPM exposure induces intestinal barrier dysfunction, microbial dysbiosis, metabolic imbalance, and activation of the TLR2/5-MyD88-NLRP3 inflammasome. These findings highlight the urgent need for intervention strategies to mitigate the detrimental effects of air pollution on intestinal health and identify potential therapeutic targets.

Immune Response of Silver Pomfret (Pampus argenteus) CC Chemokine Ligand Gene Family to Photobacterium damselae Subsp. Damselae and Nocardia seriolae Infections.

Chemokines play a crucial role in immune responses by facilitating the migration of cells expressing corresponding chemokine receptors along concentration gradients. Photobacterium damselae subsp. Damselae (PDD) and Nocardia seriolae (NS) are known to induce substantial mortality in silver pomfret populations, yet there exists a dearth of research regarding the immune response of CCLs in PDD- or NS-infected silver pomfret. In our investigation, we identified 10 PaCCLs, which include one fish-specific CCL (PaCCL44). Phylogenetic analysis revealed considerable diversity in CCL types and copy numbers among various teleost fishes. Notably, silver pomfret lacks specific CCL genes, with most PaCCLs exhibiting heightened expression levels in immune-related organs such as the spleen and kidney, and some being expressed in mucosal immune-related organs like the skin and gills. Transcriptome analysis conducted on silver pomfret infected with NS and PDD elucidated that the expression changes of PaCCLs primarily manifested in the spleen during the initial stages of NS infection, shifting to the kidney in later stages. Conversely, the expression changes of PaCCLs following PDD infection predominantly occurred in the kidney. In vitro studies using silver pomfret spleen cell lines demonstrated an early peak in PaCCLs expression during infection, followed by gradual decline with NS treatment and rapid diminishment with PDD treatment. These findings suggest that PaCCLs primarily support the innate immunity of silver pomfret, potentially exhibiting chemotactic effects in the early infection stages, such as the synergistic action of PaCCL4 and PaCCL25, and later serving as direct antibacterial agents. NS invasion is characterised by a chronic infection affecting multiple organs, whereas PDD primarily inflicts severe damage to the kidney. PaCCL19a and PaCCL19b are specific to PDD, and their expression levels may decrease in the later stages of infection due to PDD immune escape. These data offer initial insights into understanding the mechanism underlying the innate immune response of the CCL gene family in silver pomfret and provide theoretical underpinnings for fish culture practices.

Effects of supplemental medusa (Rhopilema esculentum) on intestinal microbiota and metabolites in silver pomfret (Pampus argenteus).

Pampus argenteus demonstrates a preference for Rhopilema esculentum as prey, yet the ramifications of consuming supplemental medusa on fish microbiota and metabolism remain elusive. To elucidate these effects, 300 juvenile fish were divided into two groups: control group (C, given commercial food only) and supplemental medusa (SM) group (given supplemental medusa + commercial feed). After 15 days, fish in the SM group exhibited a significant increase in fatness, the amylase activity in the intestine significantly increased, and the intestinal microvilli were arranged more neatly. The comprehensive approach involving 16S rRNA amplicon sequencing and metabolomics was employed, leading to the identification of five genera within the SM group, namely Lactococcus, Cohaesibacter, Maritalea, Sulfitobacter, and Carnobacterium. Functional prediction analysis of the microbiota indicated that the consumption of supplemental medusa facilitated processes such as glycolysis/gluconeogenesis and amino acid absorption. Metabolomics analysis revealed significant enrichment of 85 differential metabolites, most of them belonging to fatty acids and conjugates. These differential metabolites primarily participated in processes such as amino acid metabolism, fatty acid synthesis, and disease. Notably, the consumption of medusa resulted in a significant reduction in nine lysophospholipids associated with cardiovascular disease and inflammation. Pearson's correlation coefficient analysis revealed associations between specific microorganisms and metabolites, indicating that Cobetia, Weissella, and Macrococcus exhibited an increased abundance in the SM group, positively correlating with apocynin, 12-Hete, and delta 9-THC-d3. The indicator bacteria Psychrobacter reduced in the SM group, exhibiting a negative correlation with cystathionine (a compound involved in glutathione synthesis). Overall, the supplementation of medusa may confer a beneficial effect on the immunity of the fish. This study contributes to the theoretical framework for fish feed development.

Insulin-like growth factor 1 promotes the gonadal development of Pampus argenteus by regulating energy metabolism†.

Insulin-like growth factor 1 (Igf1) is known to promote ovarian maturation by interacting with other hormones. However, the limited research on the role of Igf1 in the energy metabolism supply of gonads has hindered further exploration. To explore the role of Igf1 in gonadal development of silver pomfret, we analyzed the expression levels and the localization of igf1 mRNA and protein during testicular and ovarian development of silver pomfret. The results of the study showed upregulation of Igf1 in the critical period of vitellogenesis and sperm meiosis, which was found to be mainly expressed in the somatic cells of the gonads. Upon adding E2 and Igf1 to cultured gonadal tissues, the expression of energy-related genes was significantly increased, along with the E2-enhanced effect of Igf1 in the testis. Importantly, stimulation of both ovaries and testes with E2 and Igf1 led to a remarkable increase in the expression of vitellogenesis and meiosis-related genes. Therefore, we conclude that Igf1 promotes vitellogenesis and sperm meiosis by regulating gonadal energy production. Moreover, the expression of Igf1 in gonads is significantly regulated by E2. These findings provide new insights for the research of Igf1 in fish breeding, thus allowing the regulation of energy metabolism between growth and reproduction for successful reproductive outcomes.

PRMT5 Prevents Dilated Cardiomyopathy via Suppression of Protein O-GlcNAcylation.

[Figure: see text].

Histopathology, immunoenzyme activity and transcriptome analysis of immune response in silver pomfret infected by cryptokaryon (Cryptorchidism irritant).

Cryptorchidism irritant (CI) infection is a major problem in the culturing process of silver pomfret (Pampus argenteus), which can result in rapid and massive death. However, there is limited information available on the immune response of silver pomfret infected by CI. To address this gap, we sampled naturally infected fish and observed milky white translucent oval CI trophozoites on the gills, body surface, and fin rays. Histological analysis showed that CI infection led to vacuolation of epithelial cells and a decrease in blood cells in the gills. We also performed transcriptome profiling of the gill, kidney complex, and spleen, generating 399,616,194 clean reads that assembled into 101,228 unigenes, which were annotated based on public databases. We detected 14,369 differentially expressed genes, and selected several key immune-related genes for further validation using RT-qPCR. The Graft-versus-host pathway and Allograft rejection pathway were enriched in the gills, leading to inflammation and ulceration. CI infection activated the immune system, increasing levels of interleukin-1 beta and MHC class II antigen, and also activated innate and acquired immune genes in silver pomfret. Furthermore, we measured the activities of five immune-related enzymes (SOD, AKP, CAT, CSH and ACP), which all increased to varying degrees after CI infection. Our findings enhance our understanding of the immune response of fish to parasitic infection and may contribute to the development of strategies to prevent high mortality in CI-stimulated fish in aquaculture.

Identification and characterization of toll-like receptor genes in silver pomfret (Pampus argenteus) and their involvement in the host immune response to Photobacterium damselae subsp. Damselae and Nocardia seriolae infection.

Toll-like receptors (TLRs) are vital pattern recognition receptors that play a critical role in the innate immune response against pathogenic attack. Among the bacteria commonly found in the culture process of silver pomfret, Photobacterium damselae subsp. Damselae (PDD, gram-negative) and Nocardia seriolae (NS, gram-positive), can cause large-scale mortality in this fish species. However, there is currently no research on the role of TLRs in mediating the immune response of silver pomfret to these two bacterial infections. Therefore, in this study, we identified nine PaTLRs family members, including several fish-specific TLRs (TLR14 and TLR21). Phylogenetic analysis revealed that these PaTLRs genes could be classified into five subfamilies, namely TLR1, TLR3, TLR5, TLR7, and TLR11, indicating their evolutionary conservation. To further explore the interactions of TLR genes with immune-related mediators, protein and protein interaction network (PPI) results were generated to explain the association of TLR genes with TNF receptor-associated factor 6 (TRAF6) and other relevant genes in the MyD88-dependent pathway and NF-κb signaling pathway. Subsequently, RT-qPCR was conducted to verify the expression patterns of the nine TLR genes in the gills, skin, kidney, liver, and spleen of healthy fish, with most of the TLRs showing high expression levels in the spleen. Following infection with PDD and NS, these PaTLRs exhibited different expression patterns in the spleen, with PaTLR2, PaTLR3, PaTLR5, PaTLR7, PaTLR9, and PaTLR14 being significantly up-regulated. Furthermore, when spleen cells were treated with bacterial compositions, the majority of PaTLRs expression was up-regulated in response to Lipopolysaccharide (LPS) and lipophosphorylcholic acid (LTA) treatment, except for PaTLR21. Finally, changes in the expression levels of TLR-interacting genes were also observed under the stimulation of bacteria and bacterial compositions. The results of this study provide a preliminary reference for further understanding the mechanism of the innate immune response of the TLR gene family in silver pomfret and offer theoretical support for addressing the disease problems encountered during large-scale fish breeding.

Dual-omics reveals temporal differences in acute sympathetic stress-induced cardiac inflammation following α1 and ß-adrenergic receptors activation.

Sympathetic stress is prevalent in cardiovascular diseases. Sympathetic overactivation under strong acute stresses triggers acute cardiovascular events including myocardial infarction (MI), sudden cardiac death, and stress cardiomyopathy. α1-ARs and ß-ARs, two dominant subtypes of adrenergic receptors in the heart, play a significant role in the physiological and pathologic regulation of these processes. However, little is known about the functional similarities and differences between α1- and ß-ARs activated temporal responses in stress-induced cardiac pathology. In this work, we systematically compared the cardiac temporal genome-wide profiles of acute α1-AR and ß-AR activation in the mice model by integrating transcriptome and proteome. We found that α1- and ß-AR activations induced sustained and transient inflammatory gene expression, respectively. Particularly, the overactivation of α1-AR but not ß-AR led to neutrophil infiltration at one day, which was closely associated with the up-regulation of chemokines, activation of NF-κB pathway, and sustained inflammatory response. Furthermore, there are more metabolic disorders under α1-AR overactivation compared with ß-AR overactivation. These findings provide a new therapeutic strategy that, besides using ß-blocker as soon as possible, blocking α1-AR within one day should also be considered in the treatment of acute stress-associated cardiovascular diseases.

Novel roles of an intragenic G-quadruplex in controlling microRNA expression and cardiac function.

Simultaneous dysregulation of multiple microRNAs (miRs) affects various pathological pathways related to cardiac failure. In addition to being potential cardiac disease-specific markers, miR-23b/27b/24-1 were reported to be responsible for conferring cardiac pathophysiological processes. In this study, we identified a conserved guanine-rich RNA motif within the miR-23b/27b/24-1 cluster that can form an RNA G-quadruplex (rG4) in vitro and in cells. Disruption of this intragenic rG4 significantly increased the production of all three miRs. Conversely, a G4-binding ligand tetrandrine (TET) stabilized the rG4 and suppressed miRs production in human and rodent cardiomyocytes. Our further study showed that the rG4 prevented Drosha-DGCR8 binding and processing of the pri-miR, suppressing the biogenesis of all three miRs. Moreover, CRISPR/Cas9-mediated G4 deletion in the rat genome aberrantly elevated all three miRs in the heart in vivo, leading to cardiac contractile dysfunction. Importantly, loss of the G4 resulted in reduced targets for the aforementioned miRs critical for normal heart function and defects in the L-type Ca2+ channel-ryanodine receptor (LCC-RyR) coupling in cardiomyocytes. Our results reveal a novel mechanism for G4-dependent regulation of miR biogenesis, which is essential for maintaining normal heart function.

Establishment and characterization of a liver cell line from silver pomfret (Pampus argenteus) for studying fish health.

Fish cell lines have become a useful tool to study in resource conservation, genetic breeding, diseases control, and environmental pollutants detection. The silver pomfret (Pampus argenteus) is a high-valued marine fish species in aquaculture, which is seriously threatened by various fish diseases. In this study, a new cell line derived from P. argenteus liver (PaL) was established and characterized. PaL cells mainly consisted of fibroblast-like morphology and multiplied well in Leibovitz's L-15 medium supplemented with 15% foetal bovine serum and 3 ng/mL basic fibroblast growth factor at 22°C. Amplification of the Cyt b gene confirmed that the origin of PaL cells as P. argenteus. Chromosome analysis revealed that PaL cells had a diploid Karyotyp. The PaL cells were efficiently transfected with pEGFP-N3 plasmids, indicating its potential application in foreign gene manipulation studies. The PaL cells were found to be susceptible to red sea bream iridovirus (RSIV) and the expression of immune-related gene (TLR5) and apoptosis-related genes (Bax, Cyt c3, CASP9) were upregulated. Furthermore, lipopolysaccharide and palmitic acid (PA) treatments decreased cell viability and up-regulated the expression of inflammation related genes (IL-8, IL-1ß). Meanwhile, PA incubation induced cell apoptosis by Bcl-2-regulated caspase activation. In conclusion, the newly established PaL cell line will be an appropriate in vitro tool for viral propagation and immune response.

Modeling time-varying recruitment rates in multicenter clinical trials.

Multicenter phase II/III clinical trials are large-scale operations that often include hundreds of recruiting centers in several countries. Therefore, the operational aspects of a trial must be thoroughly planned and closely monitored to ensure better oversight and study conduct. Predicting patient recruitment plays a pivotal role in trial monitoring as it informs how many people are expected to be recruited on a given day. As such, study teams may rely on predictions to assess progress and detect any deviations from the original plan that might put the study and potentially, patients at risk. Recruitment predictions are often based on a Poisson-Gamma model that assumes that centers have a constant recruitment rate throughout the trial. The model has useful features and has extensively been used, yet its main assumption is often unrealistic. A nonhomogeneous Poisson process has been recently proposed that can incorporate time-varying recruitment rates. In this work, we predict patient recruitment using both approaches and assess the impact of said assumption in a real-world setting where studies may not necessarily have constant center-specific recruitment rates. The paper showcases experience from modeling recruitment in trials sponsored by AstraZeneca between 2005 and 2018. In these data, there is evidence of time-varying recruitment rates. The predictive performance of models that account for both constant and time-varying recruitment effects is presented. Following a descriptive analysis of data, we assess model performance and investigate the impact of model misspecification.

Exercise Training Attenuates Acute ß-Adrenergic Receptor Activation-Induced Cardiac Inflammation via the Activation of AMP-Activated Protein Kinase.

Exercise has proven cardiac benefits, but the underlying mechanisms of exercise that protect the heart from acute sympathetic stress injuries remain unknown. In this study, adult C57BL/6J mice and their AMP-activated protein kinase α2 knockout (AMPKα2-/-) littermates were either subjected to 6 weeks of exercise training or housed under sedentary conditions and then treated with or without a single subcutaneous injection of the ß-adrenergic receptor (ß-AR) agonist isoprenaline (ISO). We investigated the differences in the protective effects of exercise training on ISO-induced cardiac inflammation in wild-type (WT) and AMPKα2-/- mice using histology, enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. The results indicated that exercise training alleviated ISO-induced cardiac macrophage infiltration, chemokines and the expression of proinflammatory cytokines in wild-type mice. A mechanism study showed that exercise training attenuated the ISO-induced production of reactive oxygen species (ROS) and the activation of NLR Family, pyrin domain-containing 3 (NLRP3) inflammasomes. In cardiomyocytes, the ISO-induced effects on these processes were inhibited by AMP-activated protein kinase (AMPK) activator (metformin) pretreatment and reversed by the AMPK inhibitor (compound C). AMPKα2-/- mice showed more extensive cardiac inflammation following ISO exposure than their wild-type littermates. These results indicated that exercise training could attenuate ISO-induced cardiac inflammation by inhibiting the ROS-NLRP3 inflammasome pathway in an AMPK-dependent manner. Our findings suggested the identification of a novel mechanism for the cardioprotective effects of exercise.

Morphological and Molecular Functional Evidence of the Pharyngeal Sac in the Digestive Tract of Silver Pomfret, Pampus argenteus.

The pharyngeal sac is a comparatively rare organ in the digestive tract among teleost fishes. However, our understanding of this remarkable organ in the silver pomfret (Pampus argenteus) is limited. In the present study, we examined the various morphological and histological characteristics of the pharyngeal sac using histochemical techniques and electron microscopy. The pharyngeal sac showed unique characteristics such as well-developed muscular walls, weakly keratinized epithelium, numerous goblet cells, and needle-like processes on the papillae. The porous cavity of the papillae contained numerous adipocytes and was tightly enveloped by type I collagen fibers. These structures might provide mechanical protection and excellent biomechanical properties for grinding and shredding prey. A comparison of gene expression levels between the pharyngeal sac and esophagus using RNA-seq showed that phenotype-associated genes (epithelial genes and muscle genes) were upregulated, whereas genes related to nutrient digestion and absorption were downregulated in the pharyngeal sac. These results support the role of the pharyngeal sac in shredding and predigesting food. Overall, these findings provide a clearer understanding of the pharyngeal sac morphology and explain the morphological adaptations of the digestive tract for feeding on gelatinous prey. To our knowledge, this is the first report on pharyngeal sac gene expression in P. argenteus.