Genome-wide analysis of ATP-binding cassette (ABC) transporter in Penaeus vannamei and identification of two ABC genes involved in immune defense against Vibrio parahaemolyticus by affecting NF-κB signaling pathway.

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5, PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates.

Molecular Characterization and Expression Changes of the bcl2l13 Gene in Response to Hypoxia in Megalobrama amblycephala.

Hypoxia is a unique environmental stress, which not only reflects the insufficient oxygen supply of cells and tissues, but also occurs in various physiological and pathological environments. Mitophagy as a selective autophagy can recover and utilize damaged organelles and misfolded proteins to ensure normal cell functions and promote cell survival. Bcl2l13 (B-cell lymphoma-2 like 13) is reported to induce mitophagy as a functional mammalian homolog of Atg32. However, the function of the bcl2l13 gene is still unclear in fish. Here the sequence and structure of the bcl2l13 gene in Megalobrama amblycephala were identified and showed that bcl2l13 contained an open reading frame (ORF) of 1458 bp for encoding 485 aa. Amino acid sequence analysis indicated that Bcl2l13, as a typical anti-apoptotic protein of the Bcl2 family, contained four BH domains, one BHNo domain, and one TM domain. Further study showed that Bcl2l13 was mainly located in the mitochondria, while its localization was changed within the whole cell after the TM domain was deleted. Real-time PCR analysis revealed that bcl2l13 showed higher expression levels in early embryos. After hypoxia treatment, the mRNA levels of the bcl2l13 and autophagy-related genes were significantly up-regulated in most detected tissues, and the bcl2l13 transcription was regulated by Hif-1α mediated pathway. Additionally, the transcription activity of the bcl2l13 promoter was further analyzed using luciferase reporter assays and showed the highest activity in the promoter region from -475 to +111. These results indicated that bcl2l13 may play important roles in embryogenesis and hypoxia mediated autophagy in fish.

IL-6 Mutation Attenuates Liver Injury Caused by Aeromonas hydrophila Infection by Reducing Oxidative Stress in Zebrafish.

Interleukin-6 (IL-6), a pleiotropic cytokine, plays a crucial role in acute stress induced by bacterial infection and is strongly associated with reactive oxygen species (ROS) production. However, the role of IL-6 in the liver of fish after Aeromonas hydrophila infection remains unclear. Therefore, this study constructed a zebrafish (Danio rerio) il-6 knockout line by CRISPR/Cas9 to investigate the function of IL-6 in the liver post bacterial infection. After infection with A. hydrophila, pathological observation showed that il-6-/- zebrafish exhibited milder liver damage than wild-type (WT) zebrafish. Moreover, liver transcriptome sequencing revealed that 2432 genes were significantly up-regulated and 1706 genes were significantly down-regulated in il-6-/- fish compared with WT fish after A. hydrophila infection. Further, gene ontology (GO) analysis showed that differentially expressed genes (DEGs) were significantly enriched in redox-related terms, including oxidoreductase activity, copper ion transport, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in pathways such as the PPAR signaling pathway, suggesting that il-6 mutation has a significant effect on redox processes in the liver after A. hydrophila infection. Additionally, il-6-/- zebrafish exhibited lower malondialdehyde (MDA) levels and higher superoxide dismutase (SOD) activities in the liver compared with WT zebrafish following A. hydrophila infection, indicating that IL-6 deficiency mitigates oxidative stress induced by A. hydrophila infection in the liver. These findings provide a basis for further studies on the role of IL-6 in regulating oxidative stress in response to bacterial infections.

Integrated Analysis of DNA Methylome and Transcriptome Reveals Epigenetic Regulation of Cold Tolerance in Litopenaeus vannamei.

DNA methylation is an important epigenetic modification that has been shown to be associated with responses to non-biological stressors. However, there is currently no research on DNA methylation in response to environmental signals in shrimp. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles and differentially expressed genes between two strains of Litopenaeus vannamei with significantly different cold tolerance through whole genome bisulfite sequencing (WGBS) and transcriptome sequencing. Between Lv-C and Lv-T (constant temperature of 28 °C and low temperatures of 18 °C and 10 °C) under cytosine-guanine (CG) environments, 39,100 differentially methylated regions (DMRs) were identified, corresponding to 9302 DMR-related genes (DMRGs). The DMRs were mainly located in the gene body (exons and introns). Gene Ontology (GO) analysis showed that these DMRGs were significantly enriched in cell parts, catalytic activity, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed significant enrichment of these DMRGs in pathways such as proteasome (ko03050), oxidative phosphorylation (ko00190), mTOR signaling pathway (ko04150), fatty acid metabolism (ko01212), and fatty acid degradation (ko00071). The comprehensive results suggested that L. vannamei mainly regulates gene expression in response to low temperatures through hypermethylation or demethylation of some genes involved in thermogenesis, glycolysis, the autophagy pathway, the peroxisome, and drug metabolism pathways. These results provide important clues for studying DNA methylation patterns and identifying cold tolerance genes in shrimp.

Identification of long non-coding RNA MSTRG.5748.1 and MSTRG.7894.1 from Megalobrama amblycephala and their potential roles in innate immunity.

Megalobrama amblycephala is one of the most economically important freshwater fish in China, and the bacterial septicemia caused by Aeromonas hydrophila is a serious threat to the breeding industry of M. amblycephala. Unfortunately, the characterization of long noncoding RNA (lncRNA) in response to A. hydrophila infection has not been performed in M. amblycephala. To better understand the biological significance of lncRNA in the immune system, we identified two lncRNA, named MSTRG.5748.1 and MSTRG.7894.1, as playing critical roles in the antibacterial response of M. amblycephala. After separating the nucleus and cytoplasm of the hepatocytes from M. amblycephala, cellular localization of MSTRG.5748.1 and MSTRG.7894.1 was performed to predict their functions. The results showed that MSTRG.5748.1 was mainly expressed in the nucleus, suggesting that its functions are mostly to regulate the expression of downstream genes through epistasis and transcription. MSTRG.7894.1 existed in both the nucleus and cytoplasm, which indicated that it has many regulatory modes. qPCR analysis showed that MSTRG.5748.1 and MSTRG.7894.1 were expressed in the immune-related organs of M. amblycephala, and significantly changed in the liver after A. hydrophila infection. RNA-seq analysis revealed that differentially expressed genes (DEGs) were mainly enriched in antigen processing and presentation via MHC class I, RIG-I-like receptor (RLR) signaling pathway, and IFN-related pathway, and a large number of pathway-related genes were significantly regulated after lncRNA overexpression in muscle cell of M. amblycephala. Overexpression of MSTRG.5748.1 and MSTRG.7894.1 significantly inhibited the expression of STING and IFN, significantly upregulated muscle cell viability, and promoted cell proliferation by targeting STING and IFN.

Deletion of the foxO4 Gene Increases Hypoxia Tolerance in Zebrafish.

Oxygen homeostasis is an important organizing principle for understanding development, physiology, disease, and evolution. Under various physiological and pathological states, organisms experience oxygen deficiency or hypoxia. FoxO4 has been recognized as an important transcriptional regulator involved in a variety of cellular functions, including proliferation, apoptosis, differentiation, and stress resistance, but its role in hypoxia adaptation mechanisms in animals is not so clear. To explore the role of foxO4 in the hypoxia response, we detected the expression of foxO4 and the regulatory relationship between Hif1α and foxO4 under hypoxic conditions. It was found that the expression of foxO4 was up-regulated in ZF4 cells and zebrafish tissues after hypoxia treatment, and Hif1α could directly target the HRE of the foxO4 promoter to regulate foxO4 transcription, indicating that foxO4 was involved in the hypoxia response by the Hif1α-mediated pathway. Furthermore, we obtained foxO4 knockout zebrafish and found that the disruption of foxO4 increased the tolerance to hypoxia. Further research found that the oxygen consumption and locomotor activity of foxO4-/- zebrafish were lower than those of WT zebrafish, as was true for NADH content, NADH/NAD+ rate, and expression of mitochondrial respiratory chain complex-related genes. This suggests that disruption of foxO4 reduced the oxygen demand threshold of the organism, which explained why the foxO4-/- zebrafish were more tolerant to hypoxia than WT zebrafish. These results will provide a theoretical basis for further study of the role of foxO4 in the hypoxia response.

Janus kinase 1 in Megalobrama amblycephala: Identification, phylogenetic analysis and expression profiling after Aeromonas hydrophila infection.

Janus kinase 1 (JAK1), a member of the JAK family, plays an essential and non-redundant role in the mammalian immune system. However, the potential role of JAK1 in fish immune response remains largely unclear. In the present study, the JAK1 gene of Megalobrama amblycephala (MamJAK1) was identified and characterized. The open reading frame (ORF) of MamJAK1 was 3462 bp, encoding 1153 amino acids. MamJAK1 consists of four common domains of the JAK family, including B41, SH2, STyrKc (a pseudo kinase domain), and TyrKc (a kinase domain). Phylogenetic analysis showed that JAK1s are divided into two evolutionary clades, one containing fish JAK1s, and the other containing JAK1s from other vertebrates. The results of quantitative real-time PCR (qPCR) showed that in healthy M. amblycephala, MamJAK1 mRNA was highest expressed in blood, followed by spleen, intestine and mid-kidney, and lowly expressed in other tissues including gill, liver, head kidney, muscle, brain and heart. After Aeromonas hydrophila infection, the expression of MamJAK1 mRNA was significantly induced in four selected tissues including spleen, mid-kidney, liver and intestine, reaching a peak at 24 hpi (hour post infection) in spleen and mid-kidney, at 12 hpi in liver and at 4 hpi in intestine, and then the expression level was restricted to control levels at 72 or 120 hpi. In addition, the results of Western blot showed that the phosphorylation level of MamJAK1 protein in spleen and mid-kidney increased significantly after A. hydrophila infection, although MamJAK1 protein did not change obviously. Further, the JAK1 phosphorylation in Ctenopharyngodon idellus kidney (CIK) cells was found to be significantly induced by LPS stimulation and IL-6R over-expression. The results above suggest that MamJAK1 may play an essential role in the immune response against bacterial infection through the IL-6R mediated JAK1/STAT signaling pathway, which further deepen our understanding of JAK1 and provides a potential target for the treatment and prevention of bacterial diseases in teleost.

6His-tatritin promotes antimicrobial defense via regulating immune ability and intestinal microbial community in grass carp (Ctenopharyngodon idella).

Antimicrobial peptides are small, cationic, and amphiphilic peptides found in most organisms, and many of these peptides have broad antimicrobial activity against Gram-negative, -positive bacteria and fungi. In the present study, a derivative of antimicrobial peptide Tatritin, 6His-Tatritin, was designed and expressed by Pichia pastoris using a constitutive vector pGAPZαA with the promoter of pGAP. The 6His-Tatritin had a broad-spectrum antibacterial activity based on the Oxford cup method and the micro broth dilution test. In addition, to explore the role of 6His-Tatritin in vivo, grass carps (Ctenopharyngodon idellus) were infected with Aeromonas hydrophila after they were fed with 6His-Tatritin as feed additives for 28 days. The results revealed that 6His-Tatritin could significantly up-regulate the expression levels of Hepcidin, Leap-2b, Nrf-2, CuZn-SOD and LZM (P < 0.05). In addition, 6His-Tatritin could significantly reduce the mortality (P < 0.05) and the intestinal injury of grass carps infected with bacteria. The 16S sequencing analysis showed that the structure of microbial community in intestine of fish was more diversified compared with control after treatment with 6His-Tatritin. In summary, the peptide of 6His-Tatritin could promote antimicrobial defense via regulating immune ability and intestinal microbial community in grass carp. This study provides an effective method and approach for the application of antimicrobial peptide Tatritin in aquaculture, and also provides insights into the function of antimicrobial peptides in immunity against pathogens in fish.

Brief Report: Sex Differences in the Association Between Cerebrovascular Function and Cognitive Health in People Living With HIV in Urban China.

BACKGROUND: Cardiometabolic and cerebrovascular disease are strong independent contributors to cognitive impairment in people living with HIV. Data suggest that cardiovascular risk may play a greater role in cognitive health in women than in men with HIV. METHODS: We performed a cross-sectional study of 104 participants with virologically suppressed HIV from 2 clinics in urban China. Participants underwent neuropsychological testing from which we calculated T scores globally and in 5 cognitive domains. We assessed cerebral vasoreactivity of the middle cerebral arteries in response to breath holding. We constructed linear regression models to determine associations between cerebrovascular and cognitive function overall and stratified by sex. RESULTS: Women were younger than men (48 versus 51 years, P = 0.053), had fewer years of education (9 years versus 12 years, P = 0.004), and fewer cardiometabolic risk factors (0 versus 1 factor, P = 0.008). In a model with all participants, cerebrovascular function was significantly associated with global cognition (2.74 higher T score per 1-point higher cerebral vasoreactivity [SE 1.30], P = 0.037). Cerebrovascular function remained significantly associated with global cognition among women (4.15 higher T score [SE 1.78], P = 0.028) but not men (1.70 higher T score [SE 1.74], P = 0.33). The relationships between cerebrovascular function and specific cognitive domains followed a similar pattern, with significant associations present among women but not men. CONCLUSIONS: Women with well-controlled HIV may be more vulnerable to the effect of cerebrovascular injury on cognitive health than men. Studies evaluating strategies to protect against cognitive impairment in people living with HIV should include adequate representation of women and stratification of analyses by sex.

Development and validation of tools for predicting the risk of death and ICU admission of non-HIV-infected patients with Pneumocystis jirovecii pneumonia.

Introduction: The mortality rate of non-HIV-infected Pneumocystis jirovecii pneumonia (PCP) is high. This research aimed to develop and validate two clinical tools for predicting the risk of death and intensive care unit (ICU) admission in non-HIV-infected patients with PCP to reduce mortality. Methods: A retrospective study was conducted at Peking Union Medical College Hospital between 2012 and 2021. All proven and probable non-HIV-infected patients with PCP were included. The least absolute shrinkage and selection operator method and multivariable logistic regression analysis were used to select the high-risk prognostic parameters. In the validation, the receiver operating characteristic curve and concordance index were used to quantify the discrimination performance. Calibration curves were constructed to assess the predictive consistency compared with the actual observations. A likelihood ratio test was used to compare the tool and CURB-65 score. Results: In total, 508 patients were enrolled in the study. The tool for predicting death included eight factors: age, chronic lung disease, respiratory rate, blood urea nitrogen (BUN), lactate dehydrogenase (LDH), cytomegalovirus infection, shock, and invasive mechanical ventilation. The tool for predicting ICU admission composed of the following factors: respiratory rate, dyspnea, lung moist rales, LDH, BUN, C-reactive protein/albumin ratio, and pleural effusion. In external validation, the two clinical models performed well, showing good AUCs (0.915 and 0.880) and fit calibration plots. Compared with the CURB-65 score, our tool was more informative and had a higher predictive ability (AUC: 0.880 vs. 0.557) for predicting the risk of ICU admission. Conclusion: In conclusion, we developed and validated tools to predict death and ICU admission risks of non-HIV patients with PCP. Based on the information from the tools, clinicians can tailor appropriate therapy plans and use appropriate monitoring levels for high-risk patients, eventually reducing the mortality of those with PCP.

Changes in serum angiogenic factors among patients with acute pain and subacute pain.

Screening serum biomarkers for acute and subacute pain is important for precise pain management. This study aimed to examine serum levels of angiogenic factors in patients with acute and subacute pain as potential biomarkers. Serum samples were collected from 12 healthy controls, 20 patients with postherpetic neuralgia (PHN), 4 with low back pain (LBP), and 1 with trigeminal neuralgia (TN). Pain intensity in these patients was evaluated using the visual analog scale (VAS). The serum concentrations of 11 angiogenic biomarkers were examined by Milliplex Map Human Angiogenesis Magnetic Bead Panel 2. The pain assessment from VAS showed that all patients showed moderate and severe pain. Among 11 angiogenic factors, osteopontin (OPN), thrombospondin-2 (TSP-2), soluble platelet endothelial cell adhesion molecule-1 (sPECAM-1), soluble urokinase-type plasminogen activator receptor (suPAR), and soluble epidermal growth factor receptors (sErbB2) were up-regulated and soluble interleukin-6 receptor α (sIL-6Rα) were down-regulated in patients with pain compared to the healthy participants (all P-values were < 0.005). Moreover, a linear regression model showed that the serum OPN concentration was correlated with pain intensity in patients with PHN (P = 0.03). There was no significant difference between the serum concentration of soluble epidermal growth factor receptors, sErbB3, soluble AXL, tenascin, and soluble neuropilin-1 in patients with acute and subacute pain and that of healthy controls. The results of this study provided new valuable insights into our understanding of angiogenic factors that may contribute to as mechanistic biomarkers of pain, and reveal the pathophysiological mechanism of pain. Clinical Trial Registration: www.chictr.org.cn, identifier ChiCTR2200061775.

Characterization of myogenic regulatory factors, myod and myf5 from Megalobrama amblycephala and the effect of lipopolysaccharide on satellite cells in skeletal muscle.

Myod and Myf5 are muscle-specific basic helix-loop-helix (bHLH) transcription factors that play essential roles in regulating skeletal muscle development and growth. In order to investigate potential function of myod and myf5 of Megalobrama amblycephala, an economically important freshwater fish species, in the present study, we characterized the sequences and expression profiles of M. amblycephala myod and myf5. The open reading frame (ORF) sequences of myod and myf5 encoded 275 and 240 amino acids, respectively, possessing analogous structure with the highly conserved domains, bHLH and C-terminal helix III domains. Spatio-temporal expression patterns revealed that myod and myf5 were predominant in skeletal muscle with the highest expression in white muscle, and the highest at 10 days post-hatching (dph) and the segmentation period, respectively. Furthermore, we evaluated the effects of lipopolysaccharide (LPS) on the expression of muscle-related genes in white and red muscle, and proliferation and differentiation of satellite cells. The myod, myf5 and pax-7 expression generally increased and then decreased with increase of LPS concentration and treatment time in red muscle, while these genes showed inconsistent expression patterns in white muscle. In addition, LPS administration caused the frequency increase of satellite cells in red and white muscle especially at 3 and 7 days after LPS-injection.

Identification of four STAT3 isoforms and functional investigation of IL-6/JAK2/STAT3 pathway in blunt snout bream (Megalobrama amblycephala).

Signal transducer and activator of transcription 3 (STAT3) is a major regulator of immune response and chronic inflammatory, which can be activated by interleukin-6 (IL-6). In mammals, STAT3 has multiple isoforms, and its function has been well studied. In teleost, a single stat3 has been cloned and identified in several species, but studies on its function are limited. In the present study, four stat3 isoforms including mastat3α1, mastat3α2, mastat3ß1 and mastat3ß2 were identified from blunt snout bream (Megalobrama amblycephala). The results of quantitative PCR (qPCR) showed that four mastat3 transcripts were ubiquitously expressed in all 10 tissues examined. After Aeromonas hydrophila challenge, the expression patterns of mastat3a1, mastat3a2 and mastat3ß2 were similar, but significantly different from that of mastat3ß1. In addition, western blot showed that rmaIL-6+rmasIL-6R (IL-6 trans-signaling) significantly up-regulated phosphorylation levels of the four maSTAT3 isoforms and mRNA levels of the il-10, il-11, tnf-a, socs3a and socs3b genes, while rmaIL-6 (IL-6 classical signaling) only significantly up-regulated phosphorylation levels of the two maSTAT3α isoforms and mRNA levels of the il-10, socs3a and socs3b genes. Meanwhile, overexpression or inhibition of JAK2 could significantly change the STAT3 phosphorylation. Finally, JAK2 and STAT3 inhibitors could significantly inhibit the up-regulation of il-10, il-11, tnf-a, socs3a and socs3b induced by rmaIL-6+rmasIL-6R or rmaIL-6. To sum up, this study reveals the functional distinctions and overlaps among the four maSTAT3 isoforms in blunt snout bream and reveals the differential regulation of IL-6 classical signaling and trans-signaling on downstream immune genes via the JAK2/STAT3 pathway, enriching our knowledge of fish's defense mechanisms against pathogens.

Factors influencing the prescription pattern of essential medicines from the perspectives of general practitioners and patients: a qualitative study in China.

OBJECTIVES: This qualitative study aimed to explore the factors influencing the prescription patterns of essential medicines (EMs) from the perspectives of general practitioners (GPs) and patients in Beijing, China. DESIGN: The qualitative study was conducted using individual in-depth interviews. SETTING: This study was conducted from January to August 2020, in community health service centres (CHSCs) across six urban districts of Beijing, China. PARTICIPANTS: A total of 17 GPs from 17 CHSCs in 6 urban districts and 22 patients with non-communicable diseases from three CHSCs in the three urban districts of Beijing were recruited using the purposive sampling method and a three-stage sampling strategy, respectively. RESULTS: Five major themes were identified among factors influencing the prescription pattern of EMs: (1) efficacy and safety of medicines, (2) prescription recommendations from physicians in tertiary or secondary hospitals, (3) patients' medication preference, (4) financial status of patients and (5) minimum requirement for the prescription of EMs. CONCLUSION: The findings of this study contribute to our understanding of the factors influencing the prescription patterns and utilisation of EMs from the perspectives of GPs and patients, respectively. Policymakers should implement policies and measures to promote the National Essential Medicines System in China.

The mechanism of Megalobrama amblycephala muscle injury repair based on RNA-seq.

Skeletal muscle myogenesis and injury-induced muscle regeneration contribute to muscle formation. Skeletal muscle stem cells, termed satellite cells (SCs), proliferate to repair injured muscle. To identify the molecular mechanism of regeneration after muscle injury as well as the genes related to muscle development in fish, in this study, the immunohistochemistry and the high-throughput RNA sequencing (RNA-seq) analysis were performed after Megalobrama amblycephala muscle was injured by needle stab. The results showed that paired box7-positive (Pax7+) SCs increased, and peaked at 96 to 144 h-post injury (hpi). The 6729 differentially expressed genes (DEGs), including 2125 up-regulated and 4604 down-regulated genes were found. GO terms significantly enriched by DEGs contained intercellular connections, signaling transduction and enzyme activity. KEGG enrichment analysis showed that most of the pathways were related to immunity, metabolism and cells related molecules, including actin skeleton regulation, Epstein Barr virus infection and plaque adhesion. The WGCNA results revealed that actin cytoskeleton and lipid metabolism related genes probably played crucial roles during repair after muscle injury. Collectively, all these results will provide new insights into the molecular mechanisms underlying muscle injury repair of fish.

Identification of a Novel Antimicrobial Peptide From the Ancient Marine Arthropod Chinese Horseshoe Crab, Tachypleus tridentatus.

Humoral immunity is the first line of defense in the invertebrate immune system, and antimicrobial peptides play an important role in this biological process. A novel antimicrobial peptide, termed Tatritin, was identified and characterized in hemolymph of Chinese horseshoe crab, Tachypleus tridentatus, infected with Gram-negative bacteria via transcriptome analysis. Tatritin was significantly induced by bacterial infection in hemolymph and gill. The preprotein of Tatritin consists of a signal peptide (21 aa) and a mature peptide (47 aa) enriched by cysteine. The putative mature peptide was 5.6 kDa with a theoretical isoelectric point (pI) of 9.99 and showed a α-helix structure in the N-terminal and an anti-parallel ß-sheet structure in the cysteine-stabilized C-terminal region. The chemically synthesized peptide of Tatritin exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Furthermore, Tatritin may recognize and inhibit pathogenic microorganisms by directly binding to LPS, DNA, and chitin. In addition, administration of Tatritin reduced the mortality of zebrafish after bacterial infection. Due to its broad-spectrum antimicrobial activity in vivo and in vitro and the sensitivity to drug-resistant bacterial strains, Tatritin peptide can be used as a new type of drug for infection treatment or as an immune enhancer in animals.

miR-731 modulates the zebrafish heart morphogenesis via targeting Calcineurin/Nfatc3a pathway.

BACKGROUND: Zebrafish miR-731 is orthologous of human miR-425, which has been demonstrated to have cardio-protective roles by a variety of mechanisms. The miR-731 morphants show pericardium enlargement, and many DEGs (differentially expressed genes) are enriched in 'Cardiac muscle contraction' and 'Calcium signaling pathway', implying that miR-731 plays a potential role in heart function and development. However，the in vivo physiological role of miR-731 in the heart needs to be fully defined. METHODS: Zebrafish miR-731 morphants were generated by morpholino knockdown, and miR-731 knockout zebrafish was generated by CRISRP/Cas9. We observed cardiac morphogenesis based on whole-mount in situ hybridization. Furthermore, RNA-seq and qRT-PCR were used to elucidate the molecular mechanism and analyze the gene expression. Double luciferase verification and Western blot were used to verify the target gene. RESULTS: The depletion of miR-731 in zebrafish embryos caused the deficiency of cardiac development and function, which was associated with reduced heart rate, ventricular enlargement and heart looping disorder. In addition, mechanistic study demonstrated that Calcineurin/Nfatc3a signaling involved in miR-731 depletion induced abnormal cardiac function and developmental defects. CONCLUSION: MiR-731 regulates cardiac function and morphogenesis through Calcineurin/Nfatc3a signaling. GENERAL SIGNIFICANCE: Our studies highlight the potential importance of miR-731 in cardiac development.

The effects of blunt snout bream (Megalobrama amblycephala) IL-6 trans-signaling on immunity and iron metabolism via JAK/STAT3 pathway.

Interleukin-6 (IL-6) is a pleiotropic inflammatory cytokine, which plays a dual role in mammalian inflammation through both classical signaling (IL-6 binds to IL-6 receptor/IL-6R) and trans-signaling (IL-6 binds to soluble IL-6R). However, the function of IL-6, especially the regulatory mechanism of IL-6 trans-signaling in immunity and iron metabolism remains largely unclear in teleost. Here, L8824 cells (Ctenopharyngodon idella hepatic cells) were stimulated with blunt snout bream (Megalobrama amblycephala) IL-6 combination with sIL-6R protein (rmaIL-6+rmasIL-6R/maIL-6 trans-signaling) or STAT3 inhibitor (c188-9), and RNA-sequencing, global transcriptional analyses. The enrichment analysis of GO and KEGG showed that maIL-6 trans-signaling is mainly involved in stress and inflammation response, and the activation of STAT3 is mainly related to cell proliferation, apoptosis and immune regulation. Furthermore, after treated L8824 cells with JAK2 inhibitors, it was found that the induction of IL-6 trans-signaling on the selected immune-related genes could be inhibited. These results implied that in early stage after rmaIL-6+rmasIL-6R treatment, the maIL-6 trans-signaling played an important role in the immune regulation through the JAK2/STAT3 pathway. By extending the rmaIL-6+rmasIL-6R treatment time, it was found that maIL-6 trans-signaling could promote the expression of iron metabolism related genes (ft, tf, tfr1, hamp and fpn1) in L8824 cells, indicating that maIL-6 trans-signaling may be involved in iron metabolism in the non-acute immune phase. Finally, after treated L8824 cells with JAK2 and STAT3 inhibitors, it was found that only tf and fpn1 were regulated by maIL-6 trans-signaling through the JAK2/STAT3 pathway. These findings provided novel insights into IL-6 trans-signaling regulatory mechanism in teleost, enriching our knowledge of fish immunity and iron metabolism.

Classical Signaling and Trans-Signaling Pathways Stimulated by Megalobrama amblycephala IL-6 and IL-6R.

Interleukin-6 (IL-6) is a multipotent cytokine. IL-6 plays a dual role in inflammation through both classical signaling (IL-6 binds membrane IL-6 receptor/IL-6R) and trans-signaling (IL-6 binds soluble IL-6R). However, the regulation of IL-6 activity, especially the regulation of signaling pathways and downstream genes mediated by IL-6 trans-signaling, remains largely unclear in teleost. Grass carp (Ctenopharyngodon idellus) hepatic (L8824) cells, kidney (CIK) cells, and primary hepatocytes were used as test models in this study. First, the biological activity of recombinant blunt snout bream (Megalobrama amblycephala) IL-6 (rmaIL-6) and sIL-6R (rmasIL-6R) was verified by quantitative PCR (qPCR) and western blot. The western blot results showed that rmaIL-6 significantly upregulated signal transducer and activator of transcription 3 (STAT3) phosphorylation in L8824 cells and primary hepatocytes, while rmaIL-6 in combination with rmasIL-6R (rmaIL-6+rmasIL-6R) significantly upregulated STAT3 phosphorylation in all types of cells. Furthermore, maIL-6 and maIL-6+rmasIL-6R could only induce extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation in L8824 cells and CIK cells, respectively. Therefore, IL-6 mainly acts by activating the janus kinase (JAK)/STAT3 pathway rather than the mitogen-activated protein kinase (MEK)/ERK pathway. Finally, the activation of the JAK2/STAT3 pathway was shown to be essential for the generation of socs3a and socs3b induced by IL-6 trans-signaling after treatment by JAK2/STAT3 pathway inhibitors (c188-9 and TG101348). These findings provide functional insights into IL-6 classical signaling and trans-signaling regulatory mechanisms in teleost, enriching our knowledge of fish immunology.

Evolution and expression analysis of STAT family members in blunt snout bream (Megalobrama amblycephala).

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is involved in regulating the body's immunity, cell proliferation, differentiation, and apoptosis. Members of the STAT family have been extensively studied in different mammalian species. However, there are few studies on the STAT family genes in farmed economic fish. In this study, eight STAT genes including STAT1a, STAT1b, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6, in blunt snout bream (Megalobrama amblycephala), an economically important fish in China, were identified and characterized. Analyses of gene location, phylogeny and conserved synteny were conducted to infer the evolutionary origin of these STAT family genes. Furthermore, the evolutionary origin model of STATs was constructed based on the 2R hypothesis and teleost genome duplication (TGD) hypothesis, which clarified the evolutionary origin of the eight STATs in blunt snout bream. Besides, expression of the eight STATs was detected in 10 tissues of healthy blunt snout bream, which showed different expression patterns, and all had the highest level in the blood. In addition, expression of the STATs was significantly induced in the spleen, liver, and kidney after infection of Aeromonas hydrophila, suggesting that they play an important role in protecting the host from pathogens. In general, the evolution of cytokine-related genes parallels that of the immune system, which has likely been a main evolutionary driver. Therefore, the evolutionary model of STAT genes, constructed in the non-model organism pioneeringly, may provide some enlightenment for the evolution of the fish STAT family genes and their involvement in the immune function.