Genome-Wide Identification, Molecular Characterization, and Expression Analysis of the HSP70 and HSP90 Gene Families in Thamnaconus septentrionalis.

Heat shock proteins (HSPs) are a class of highly conserved proteins that play an important role in biological responses to various environmental stresses. The mariculture of Thamnaconus septentrionalis, a burgeoning aquaculture species in China, frequently encounters stressors such as extreme temperatures, salinity variations, and elevated ammonia levels. However, systematic identification and analysis of the HSP70 and HSP90 gene families in T. septentrionalis remain unexplored. This study conducted the first genome-wide identification of 12 HSP70 and 4 HSP90 genes in T. septentrionalis, followed by a comprehensive analysis including phylogenetics, gene structure, conserved domains, chromosomal localization, and expression profiling. Expression analysis from RNA-seq data across various tissues and developmental stages revealed predominant expression in muscle, spleen, and liver, with the highest expression found during the tailbud stage, followed by the gastrula, neurula, and juvenile stages. Under abiotic stress, most HSP70 and HSP90 genes were upregulated in response to high temperature, high salinity, and low salinity, notably hspa5 during thermal stress, hspa14 in high salinity, and hsp90ab1 under low salinity conditions. Ammonia stress led to a predominance of downregulated HSP genes in the liver, particularly hspa2, while upregulation was observed in the gills, especially for hsp90b1. Quantitative real-time PCR analysis corroborated the expression levels under environmental stresses, validating their involvement in stress responses. This investigation provides insights into the molecular mechanisms of HSP70 and HSP90 in T. septentrionalis under stress, offering valuable information for future functional studies of HSPs in teleost evolution, optimizing aquaculture techniques, and developing stress-resistant strains.

Development and characterization of a continuous cell line (EL) from the liver of European eel Anguilla anguilla.

In the present study, a new hepatic tissue-origin cell line from European eel Anguilla anguilla has been developed and characterized. This cell line designated EL has been maintained in Leibovitz L-15 supplemented with 10% fetal bovine serum over 72 months, and subcultured more than 90 times. The EL cell line consisted predominantly of fibroblast-like cells, which could survive over 100 days in vitro, and could grow at 15-32°C. The optimum temperature for growth was 27°C. The chromosome analysis revealed a modal diploid karyotype of 2n = 38. The origin of this cell line was confirmed by the 18S recombinant (r)RNA sequencing. The susceptibility test indicated significant cytopathic effects in the EL cells with regard to the Rana grylio virus and the Herpesvirus anguillae. The viral replication was confirmed by transmission electron microscopy and polymerase chain reaction analysis. Following poly (I:C) exposure, the expression levels of the immune-related molecules interferon regulatory factor-7 (irf7) and transforming growth factor-ß (TGF-ß) were downregulated in EL cells, whereas the expression levels of the rf3 and the cytochrome P450 (CYP450) were upregulated. All four genes were significantly upregulated following inflammation by lipopolysaccharide (LPS). These data suggested the application of EL cell line for viral identification, as well as for immunodiagnosis and pharmacological targeting.

Acute mushroom poisoning of Amanita pseudosychnopyramis: A case report from Fujian, China with exact species identification and descriptive study.

Mushroom poisoning is a deeply concerning food safety problem that affects the public in China every year. Although there are statistics on the number of poisonings and incidents, there is a lack of data on the types of toxic mushrooms, clinical manifestations and toxins. A case of wild mushroom poisoning occurred in Xiamen. Descriptive epidemiological investigation, toxins detection, and morphological and phylogenetic identification were immediately performed. The patients exhibited typical neurotoxic symptoms after consuming wild mushrooms, including chills, vertigo, drowsiness, salivation and coma. The average incubation period was 30 min. Treatments that were adopted included fluid infusion, gastric lavage, catharsis, and liver protection treatment. All patients recovered within 10 days. The species was identified as Amanita pseudosychnopyramis, and its contents of muscarine, muscimol and ibotenic acid were 170.3 ± 5.9 mg/kg, 835.4 ± 43.1 mg/kg and 637.9 ± 54.8 mg/kg in dry weight, respectively, as detected by ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). To our knowledge, this is the first report of Amanita pseudosychnopyramis poisoning worldwide.

Reconstruction of a swine SLA-I protein complex and determination of binding nonameric peptides derived from the foot-and-mouth disease virus.

No experimental system to date is available to identify viral T-cell epitopes in swine. In order to reconstruct the system for identification of short antigenic peptides, the swine SLA-2 gene was linked to the beta(2)m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich sequence (G4S)3, using splicing overlap extension-PCR (SOE-PCR). The maltose binding protein (MBP)-SLA-2-(G4S)3-beta(2)m fusion protein was expressed and purified in a pMAL-p2X/Escherichia coli TB1 system. The purified MBP-SLA-2-(G4S)3-beta(2)m protein was cleaved by factor Xa protease, and further purified by DEAE-Sepharose chromatography. The conformation of the SLA-2-(G4S)3-beta(2)m protein was determined by circular dichroism (CD) spectrum. In addition, the refolded SLA-2-(G4S)3-beta(2)m protein was used to bind three nonameric peptides derived from the foot-and-mouth disease virus (FMDV) O subtype VP1. The SLA-2-(G4S)3-beta(2)m-associated peptides were detected by mass spectrometry. The molecular weights and amino acid sequences of the peptides were confirmed by primary and secondary spectra, respectively. The results indicate that the SLA-2-(G4S)3-beta(2)m was 41.6kDa, and its alpha-helix, beta-sheet, turn, and random coil by CD estimation were 78 aa, 149 aa, 67 aa, and 93 aa, respectively. SLA-2-(G4S)3-beta(2)m protein was able to bind the nonameric peptides derived from the FMDV VP1 region: 26-34 (RRQHTDVSF) and 157-165 (RTLPTSFNY). The experimental system demonstrated that the reconstructed SLA-2-(G4S)3-beta(2)m protein complex can be used to identify nonameric peptides, including T-cell epitopes in swine.