Alternative splicing variants of stimulator of interferon genes (STING) from Asian seabass (Lates calcarifer) and their immune response against red spotted grouper nervous necrosis virus (RGNNV).

The Stimulator of Interferon Genes (STING, also known as MITA/ERYS/MPYS) is an adaptor molecule that plays a crucial role in the RLR pathway and responds to DNA and RNA viruses. In the present study, we have identified two novel isoforms of STING (the canonical form named as LcSTINGa and its alternative splicing isoform named as LcSTINGb) from teleost Lates calcarifer. LcSTINGa has an ORF of 1230 bp, encoding a 409 amino acid protein, while its alternative splicing variant, LcSTINGb, features an ORF of 987 bp, encoding 328 amino acids. LcSTINGa is predicted to contain four transmembrane helices, whereas LcSTINGb has only two. The Lates STING protein showed about 86.85% identity with Perca flavescens, 86.45% with Seriola and 39.51% with Homo sapiens. The tissue distribution studies revealed that the STING variants were constitutively expressed in all the tissues examined, with the highest expression in blood. In-vivo upregulation of LcSTINGa and LcSTINGb mRNA following immune challenge with poly (I:C), Red-spotted grouper nervous necrosis virus (RGNNV) and zymosan A suggests its significance in the immune response.

Characterization of two tripartite motif-containing genes from Asian Seabass Lates calcarifer and their expression in response to virus infection and microbial molecular motifs.

OBJECTIVE: We identified two tripartite motif (TRIM) genes, LcTRIM21 and LcTRIM39, from the Asian Seabass Lates calcarifer, and examined their responses to experimental betanodavirus infection and stimulation with microbial pathogen-associated molecular patterns. METHODS: Genes encoding LcTRIM21 and LcTRIM39 were identified, cloned, and sequenced from the Asian Seabass. We analyzed the sequence using a variety of bioinformatics tools to determine protein structure, localization, and establish a phylogenetic tree. By using quantitative real-time PCR, we analyzed expression profiles of the LcTRIM21 and LcTRIM39 genes in response to betanodavirus challenge as well as molecular pathogen-associated molecular patterns like poly(I:C) and Zymosan A. The tissue distribution pattern of these genes was also examined in healthy animals. RESULT: Asian Seabass homologues of the TRIM gene, LcTRIM21 and LcTRIM39, were cloned, both encoding proteins with 547 amino acids. LcTRIM21 is predicted to have an isoelectric point of 6.32 and a molecular mass of 62.11 kilodaltons, while LcTRIM39 has an isoelectric point of 5.57 and a molecular mass of 62.11 kilodaltons. LcTRIM21 and LcTRIM39 homologues were predicted to be localized in cytoplasm by in silico protein localization. Structurally, both proteins contain an N-terminal really interesting new gene (RING) zinc-finger domain, B-box domain, coiled-coil domain and C-terminal PRY/SPRY domain. Most tissues and organs examined showed constitutive expression of LcTRIM21 and LcTRIM39. Upon poly(I:C) challenge or red-spotted grouper nervous necrosis virus infection, LcTRIM21 and LcTRIM39 mRNA expression was significantly upregulated, suggesting that they may play a critical antiviral role against fish viruses. LcTRIM21 and LcTRIM39 expression were also upregulated by administration of the glucan Zymosan A. CONCLUSION: The TRIM-containing gene is an E3 ubiquitin ligase that exhibits antiviral activity by targeting viral proteins via proteasome-mediated ubiquitination. TRIM proteins can be explored for the discovery of antivirals and strategies to combat diseases like viral nervous necrosis, that threaten seabass aquaculture.

Applications of Actinobacteria in aquaculture: prospects and challenges.

Disease outbreaks due to improper culture management, poor water quality, and climate change are major concerns in aquaculture. Most of the aquatic pathogens are opportunistic and any imbalance in the host-pathogen-environment triad will result in a disease outbreak. The indiscriminate use of chemotherapeutics such as antibiotics to prevent diseases in aquaculture will lead to antimicrobial resistance in aquaculture. Hence, the demand for natural microbial strains which can be used as beneficial probiotics and bioaugmentors in fish farming systems has increased to ensure one health in aquaculture. Studies have proved the probiotic and bioremediation potential of several Actinobacterial species that can be applied in aquaculture. Actinobacteria, especially Streptomyces, can be applied in aquaculture for disease prevention, treatment, and bioremediation of organic and inorganic waste in the culture systems. The growth, immunity, and resistance towards aquatic pathogens in cultured organisms also get enhanced through their capability to release potent antimicrobial compounds, bioactive molecules, and novel enzymes. Their broad-spectrum antimicrobial and quorum quenching activity can be well exploited against quorum sensing biofilm forming aquatic pathogens. Even though they impart specific adverse effects like the production of off-flavour compounds, this could be controlled through proper management strategies. This review discusses the applications, challenges, and prospects of Actinobacteria in aquaculture. Research gaps are also highlighted, which may shed light on the existing complexities and should pave the way for their better understanding and utilisation in aquaculture.

Antiviral radical SAM enzyme viperin homologue from Asian seabass (Lates calcarifer): Molecular characterisation and expression analysis.

The host response to virus infection is mediated by the interferon system and its workhorse effector proteins like Interferon-stimulated genes (ISGs). Viperin is an interferon-inducible antiviral protein. In the present study, an antiviral radical SAM enzyme, viperin homologue, was cloned and characterised from teleost, Asian seabass (Lates calcarifer). This cloned viperin cDNA encodes 351 amino acid protein with predicted N-terminal amphipathic alpha-helix, conserved radical S-adenosyl l-methionine (SAM) domain with CxxxCxxC motif and a highly conserved C-terminal domain. Lcviperin gene consists of six exons and five introns. The secondary structure contains nine alpha helices and beta sheets. Viperin from Lates is evolutionarily conserved and shares about 89% identity with Seriola dumerili and 70% identity with human orthologue. Poly(I:C) and RGNNV upregulated Lcviperin during in-vivo challenge studies, providing insight into its antiviral properties. Lates antiviral effector genes like viperin could help in elucidating the host-virus protein interactions and allow the development of improved antiviral strategies against pathogens like betanodavirus that devastate aquaculture of the species.

Targeted Isolation of Two New Anti-inflammatory and UV-A Protective Dipyrroloquinones from the Sponge-associated Fungus Aspergillus tamarii MCCF102.

In following up on observed in vitro anti-inflammatory activity of the organic extract of the marine sponge-derived fungus Aspergillus tamarii MCCF102, two new dipyrrolobenzoquinones, terreusinone B and C (1: and 2: ), were discovered along with the known analogue, terreusinone (3: ). The structures of 1: -3: were determined by spectroscopic and spectrometric analyses, along with chemical inter-conversion. In vitro testing on lipopolysaccharide (LPS) stimulated RAW 264.7 murine macrophage cells revealed that 1: -3: exhibit anti-inflammatory activity by inhibiting nitric oxide production in a dose-dependent manner (IC50 < 1 µM) without any cytotoxicity observed at the same concentrations. Due to this and the UV-A absorptive properties imparted by the highly conjugated structures of these molecules, the potential for using 1: -3: or related analogues as natural sunscreen components is suggested. Gene sequencing and informatics biosynthetic gene cluster comparisons were insufficient to confidently elucidate the biosynthetic origins of these compounds, possibly suggesting the occurrence of a gene cluster not detected in the initial sequencing or a non-canonical pathway that should be further investigated.

Folate functionalized chitosan nanoparticles as targeted delivery systems for improved anticancer efficiency of cytarabine in MCF-7 human breast cancer cell lines.

Anticancer drug cytarabine, has been widely used for treating haematological malignancies while it has minimal activity against solid tumours, which demands continuous infusion leading to high dose cytarabine toxicity. In this study, folate conjugated chitosan nanoparticles (FCCNP) were used for targeted delivery of cytarabine in breast adenocarcinoma cell lines by making use of the overexpressed folate receptors on the surface of MCF-7. Folate was conjugated to chitosan using carbodiimide. FCCNPs show spherical morphology with a size of<50 nm. Zeta potential of + 45.2 mV and PDI of 0.98 from DLS measurement confirms a stable monodisperse nanoformulation. Cytotoxicity was studied in folate receptor positive, MCF-7 and folate receptor negative, A-549 cell lines. Increased cellular uptake of the drug incorporated nanoparticles was confirmed in MCF-7 cells with fluorophore, squaraine 650 compared to A-549 cells. The relative fold of expression of genes involved in apoptosis such as bax, cyt c and cas 9 were upregulated. The present in vitro study confirms improved cytotoxicity of cytarabine folate conjugated chitosan nanoparticles in MCF-7 cells.