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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., Competing Interests: Declaration of competing interest The authors declared no financial and commercial conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs), as important regulators of post-transcriptional gene expression, play important roles in the occurrence and function of organs. In this study, morpholino (MO) knockdown of miR-462/miR-731 was used to investigate the potential mechanisms of the miR-462-731 cluster during zebrafish liver development. The results showed significant reduction of digestive organs, especially liver and exocrine pancreas after the miR-462/miR-731 knockdown, and those phenotypes could be partially rescued by corresponding miRNA duplex. Acinar cells of the exocrine pancreas were also severely affected with pancreatic insufficiency. In particular, knockdown of miR-462 caused pancreas morphogenesis abnormity with specific bilateral exocrine pancreas. Additionally, it was found that miR-731 played a role in liver and exocrine pancreas development by directly targeting dkk3b, instead of the down-regulation of Wnt/β-catenin signaling. These findings contribute significantly to our understanding of molecular mechanisms of miR-462-731 cluster in development of digestive organs., Competing Interests: Declaration of competing interest, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nitrite accumulation in aquatic environments is a potential risk factor that disrupts multiple physiological functions in aquatic animals. In this study, the physiology, transcriptome and metabolome of the control group (LV-C), nitrite-tolerance group (LV-NT) and nitrite-sensitive group (LV-NS) were investigated to identify the stress responses and mechanisms underlying the nitrite tolerance of Litopenaeus vannamei. After LV-NT and LV-NS were subjected to nitrite stress, the hemocyanin contents were significantly decreased, and hepatopancreas showed severe histological damage compared with LV-C. Likewise, the antioxidant enzymes were also significantly changed after nitrite exposure. The transcriptome data revealed differentially expressed genes associated with immune system, cytoskeleton remodeling and apoptosis in LV-NT and LV-NS. The combination of transcriptomic and metabolomic analysis revealed nitrite exposure disturbed metabolism processes in L. vannamei, including amino acid metabolism, nucleotide metabolism and lipid metabolism. The multiple comparative analysis implicated that higher nitrite tolerance of LV-NT than LV-NS may be attributed to enhanced hypoxia inducible factor-1α expression to regulate energy supply and gaseous exchange. Moreover, LV-NT showed higher antioxidative ability, detoxification gene expression and enhanced fatty acids contents after nitrite exposure in relative to LV-NS. Collectively, all these results will greatly provide new insights into the molecular mechanisms underlying the stress responses and tolerance of nitrite exposure in L. vannamei., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: The aim of this study was to investigate the efficacy of combination therapy of caspofungin and TMP/SMZ (trimethoprim/sulfamethoxazole) in moderate to severe pneumocystis jirovecii pneumonia (PJP) in patients without human immunodeficiency virus infection (HIV) and the relationship between therapeutic effect and plasma (1, 3) Beta-d-Glucan (BDG) levels., Methods: We retrospectively reviewed HIV-negative patients with PJP diagnosed in our department, who were treated with combination therapy of caspofungin and TMP/SMZ or monotherapy of TMP/SMZ during a six and a half year period., Results: A total of 126 moderate to severe PJP patients were enrolled in the study. In the multivariate analysis, low lymphocyte counts, high serum lactate dehydrogenase levels at the diagnosis of PJP and progression to shock were significant risk factors for death. In all patients, there was no significant difference in risk of death at 3 months. In the group of BDG≥800pg/m, patients receiving combination therapy was associated with a significantly decreased risk of death at 3 months, whereas in the group of BDG<800pg/ml, there were no statistically significant difference in survival rate between the two treatment regimens., Conclusion: High initial plasma (1, 3) Beta-d-Glucan concentration may be a predictor of satisfactory caspofungin response to HIV-negative patients with PJP. Based on our findings, we suggest the choice of combination therapy with caspofungin and TMP/SMZ as the initial treatment when BDG≥800pg/ml in moderate to severe HIV-negative patients with PJP., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ammonia, one of the major limiting environment factors in aquaculture, may pose a threat to the shrimp growth, reproduction and survival. In this study, to understand molecular differences of transcriptomic and metabolomic responses and investigate the tolerance mechanisms underlying ammonia stress in Litopenaeus vannamei, ammonia-tolerant family (LV-AT) and ammonia-sensitive family (LV-AS) of these two extreme families were exposed to high-concentration (NH 4 Cl, 46 mg/L) ammonia for 24 h. The comparative transcriptome analysis between ammonia-treated and control (LV-C) groups revealed involvement of immune defense, cytoskeleton remodeling, antioxidative system and metabolic pathway in ammonia-stress response of L. vannamei. Likewise, metabolomics analysis showed that ammonia exposure could disturb amino acid metabolism, nucleotide metabolism and lipid metabolism, with metabolism related-genes changed according to RNA-seq analysis. The comparison of metabolite and transcript profiles between LV-AT and LV-AS indicated that LV-AT used the enhanced glycolysis and tricarboxylic acid (TCA) cycle strategies for energy supply and ammonia excretion to adapt high-concentration ammonia. Furthermore, some of genes involved in the detoxification and ammonia excretion were highly expressed in LV-AT. We speculate that the higher ability of ammonia excretion and detoxification and the accelerated energy metabolism for energy supplies might be the adaptive strategies for LV-AT relative to LV-AS after ammonia stress. Collectively, the combination of transcriptomics and metabolomics results will greatly contribute to incrementally understand the stress responses on ammonia exposure to L. vannamei and supply molecular level support for evaluating the environmental effects of ammonia on aquatic organisms. The results further constitute new sights on the potential molecular mechanisms of ammonia adaptive strategies in shrimps at the transcriptomics and metabolomics levels., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: Hypoxia-inducible factor-1α (HIF-1α) is a transcriptional regulator of cellular responses to hypoxic stress. MicroRNAs (miRNAs) play an essential role in hypoxia-mediated cellular responses. Previous studies have identified some let-7 family members as hypoxia-regulated miRNAs (HRMs). In the present study, we aimed to investigate whether zebrafish let-7b/7f contribute cellular hypoxic response in a Hif-1α-dependent manner., Main Methods: Stable suppression of zebrafish hif-1α was achieved by microinjection of an optimized short-hairpin RNA (shRNA) expression vector. Next-generation sequencing was conducted to characterize miRNA and mRNA expression profiles. MiRNA promoter analysis and target detection was performed by dual-luciferase assay. Quantitative real-time PCR (qRT-PCR) and western blot were used to determine the expression of let-7b/7f, Hif-1α and Foxh1. Proliferation of ZF4 cells was examined using Cell Counting Kit-8 (CCK-8) and cell cycle progression was analyzed by flow cytometry assay., Key Findings: Correlation between 7 miRNAs and 76 putative targets was identified based on integrated analysis of miRNA-mRNA profiles. Let-7b and let-7f were further considered as potential HRMs, with let-7b further validated as Hif-1α up-regulated. In addition, Forkhead-box H1 (Foxh1) was confirmed as a bona fide downstream target of let-7b. Furthermore, overexpression of both let-7b and let-7f repressed cell proliferation through blocking cell cycle progression of the G1-S transition., Significance: Our findings for the first time suggest zebrafish let-7b acts downstream of Hif-1α to assist in hypoxia-mediated cell proliferation and cell cycle regulation at least in part through the downregulation of foxh1. We also identified 4 novel potential HIF-1α-regulated miRNAs in zebrafish., (Copyright © 2017 Elsevier Inc. All rights reserved.)

PHD3 is a hydroxylase that hydroxylates prolyl residues on hypoxia-inducible factors (HIFs) in mammals. In this study, the full-length cDNA and promoter sequences of Megalobrama amblycephala PHD3 gene were isolated by a modified RACE method. PHD3 cDNA was 1622 bp in length, including an ORF of 717 bp encoding 238 amino acid residues. The semi-quantitative PCR results suggested that PHD3 was highly expressed in liver in the normal condition, while after hypoxia treatment this gene was significantly increased in all analyzed tissues. PHD3 was detected only in the initial stages of M. amblycephala embryo development. In addition, the presence of another alternatively processed PHD3 transcript, designated PHD3Δ1 was observed in the process of analyzing the expression of PHD3. Both PHD3 and PHD3Δ1 were up-regulated under hypoxia, and had five the hypoxia response elements (HREs) by in silico scanning on the promoter. Further luciferase assay indicated that all HREs significantly responded to hypoxia. Taken together, these results suggest that PHD3 plays important roles in hypoxia response and early embryo development of M. amblycephala., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The expression of heat shock protein 70 (Hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. In this study, Megalobrama amblycephala HSP70 promoter was cloned, and characteristic heat shock elements (HSEs) were identified in the promoter region. The recombinant M. amblycephala Hsp70 protein (rMaHsp70) was expressed and purified from Escherichia coli BL21 (DE3). To evaluate in vivo immune response of rMaHsp70, we administered intraperitoneal (IP) injection, and demonstrated that rMaHsp70 stimulated M. amblycephala immune activity by inducing the expression of HSP70, HIF-1α, HSC70, CXCR4b, TNF-α and IL-1β mRNAs in liver, headkidney, spleen and gill, as well as SOD, glutathione, lysozyme and interferon alpha proteins in serum and liver. The effect of rMaHsp70 as adjuvant against Aeromonas hydrophila was assessed by injecting a mixed vaccine of rMaHsp70 and A. hydrophila (A. hydrophila/Hsp70) into M. amblycephala, and the relative percent survival (RPS) in the A. hydrophila/Hsp70 group was 75% compared to 50% in the A. hydrophila/PBS group. Furthermore, rMaHsp70 also promoted the proliferation and suppressed apoptosis in M. amblycephala fin cells (MAF) in a dose-dependent manner. Taken together, these results suggest that rMaHsp70 can induce organic immune response and improve environmental tolerance., (Copyright © 2014 Elsevier GmbH. All rights reserved.)