Development and characterization of primary cell culture from the spinal cord of Asian seabass, Lates calcarifer.

Asian seabass, Lates calcarifer, is one of the most important fish species in aquaculture. An attempt was made to develop a primary cell culture from the spinal cord of Lates calcarifer by the enzymatic and mechanical dissociation method. The primary cell culture was sub-cultured for 20 times in Leibovitz's L-15 medium with 20% fetal bovine serum (FBS) and 0.5 nM of human neurotrophin-3 at 28°C. The primary cell culture was cryopreserved at different passage levels and recovery of cells after long-term storage was estimated about 75-85%. The authenticity of origin of primary cell culture from L. calcarifer was confirmed by polymerase chain reaction assay using species-specific mitochondrial 12S rRNA primer. The primary cell culture was designated as seabass spinal cord cells (SBSC). The cells morphologically resembled the neurons due to their neural-like prolongations and star-like structure. Immunophenotypic analysis of the SBSC revealed that they are of neuronal origin. The SBSC were found to be highly susceptible to striped jack nervous necrosis virus (SJNNV) and infection in the cells was confirmed by RT-PCR. In conclusion, this is the first innovative euryhaline fish neuronal primary cell culture of L. calcarifer now available for neurophysiological and neurotoxicological studies.

Studies on In Vitro Antimicrobial, Anticancer, and Anti-oxidative Inflammatory Response of Methanolic Tuber Extracts Derived From Terminalia chebula.

Aim This study aims to investigate the antibacterial, antifungal, and phytochemical properties of methanolic tuber extracts from Terminalia chebula. Additionally, the study seeks to assess the in vitro anticancer effects of these extracts on an oral cancer cell line, as well as their antioxidant and anti-inflammatory activities. Materials and methods The research involves examining the antibacterial and antifungal properties of methanolic tuber extracts from Terminalia chebula. The phytochemical composition will be analyzed using standard techniques. The in vitro anticancer effects will be tested on an oral cancer cell line, while antioxidant and anti-inflammatory activities will be evaluated through appropriate assays. Results The study demonstrated that Terminalia chebula methanolic tuber extracts exhibit cytotoxic effects on the oral cancer cell line (KB-1), reducing cell viability as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A concentration of 30 µg/mL induced notable morphological changes observed under an inverted fluorescence microscope. Antioxidant assays showed a maximum absorption of 85.3% with 50 µL of the extract, while anti-inflammatory tests revealed a 76.0% absorption. Antimicrobial activity, assessed via agar-well diffusion, indicated significant antibacterial effects, especially against Streptococcus mutans and Candida albicans at higher concentrations. The findings suggest promising therapeutic potential for Terminalia chebula extracts. Conclusion Terminalia chebula tuber extracts may treat diseases caused by studied organisms. The study suggests that methanolic extracts from Terminalia chebula tubers have potential commercial value due to their anti-inflammatory, antioxidant, and cytotoxic properties. The extracts induced apoptosis in an oral cancer cell line at 30 µg/mL after 24 hours. Further research is needed to understand the active components and underlying molecular mechanisms.

Systematic Evaluation of Aegle marmelos-Derived Compounds: Potential Therapeutic Agents Against Inflammation and Oxidative Stress.

Aim This study aimed to evaluate the potential antioxidant and anti-inflammatory properties of Aegle marmelos active compounds through a multifaceted approach. The investigation encompasses molecular docking studies, computational pharmacokinetic predictions, and in vitro assessments, with a focus on understanding their physiochemical properties, pharmacokinetics, and molecular interactions. Materials and methods This study was conducted in the Research Department of Biochemistry, Saveetha Medical College & Hospital, Saveetha Institute of Medical and Technical Sciences (SIMATS), Tamilnadu, India. The study employed Soxhlet and methanol extraction techniques to obtain Aegle marmelos extracts, which were then subjected to antioxidant and anti-inflammatory assays. Antioxidant activity was assessed using the H2O2 assay, while anti-inflammatory potential was determined via the egg albumin denaturation assay. Molecular docking studies were conducted with human heme oxygenase 1 (HO-1) and human zanthine oxidoreductase (XO) proteins to elucidate potential therapeutic interactions. Furthermore, computational tools like SwissADME, pkCSM, and ADMETlab 2.0 were utilized to predict physiochemical and pharmacokinetic properties, providing insights into the compound absorption, distribution, metabolism, and excretion profiles. This integrated approach aimed to comprehensively evaluate the therapeutic potential of Aegle marmelos-derived compounds against inflammation and oxidative stress-related disorders, paving the way for future drug development endeavors. Results In the antioxidant assay, Aegle marmelos methanolic tuber extracts showed exceptional absorption of 87.4%, surpassing the reference standard. In the anti-inflammatory assay, the extracts displayed an absorption of approximately 79%, indicating significant anti-inflammatory potential. Auraptene, imperatorin, luvangetin, and psoralen exhibited favorable pharmacokinetic properties and adherence to the Lipinski rule of 5, suggesting promising drug development potential. In molecular docking, imperatorin demonstrated the highest binding affinity to HHO-1 and XO. Conclusion The study on Aegle marmelos highlights its potential as a therapeutic agent due to its potent antioxidant and anti-inflammatory properties. Phytochemical constituents, such as auraptene, imperatorin, luvangetin, and psoralen, show promising pharmacokinetic profiles, suggesting their suitability for drug development. Molecular docking analysis reveals imperatorin as the most effective binder to key enzymes, emphasizing its therapeutic potential against inflammation and oxidative stress-related disorders.

The Molecular Landscape of Lung Metastasis in Primary Head and Neck Squamous Cell Carcinomas.

Background Lung metastasis in head and neck cancer (HNC) patients is a critical concern, often indicating an advanced disease stage and a poor prognosis. This study explores the molecular complexities of such metastases, identifying specific genes and pathways that may serve as valuable targets for diagnosis and treatment. The findings underscore the potential for significantly improved patient outcomes through targeted therapeutic strategies. Methodology In this research, we systematically collected raw gene expression data from head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LSCC). By comparing tumorous and normal gene expression profiles from paired patient samples, we identified differentially expressed genes (DEGs). Network analysis helped visualize protein interactions and pinpoint crucial hub genes. Through validation and comparison across several datasets, we identified common DEGs. Additionally, we employed Kaplan-Meier analysis and log-rank testing to examine the relationship between gene expression patterns and patient survival. Result The study identified 145 overlapping DEGs in both HNSCC and LSCC, which are crucial for cancer progression and linked to lung metastasis, offering vital targets for personalized therapy by identifying key genes affecting disease development and patient survival. Pathway analyses linked these to lung metastasis, while protein-protein interaction network construction and hub gene identification highlighted genes crucial for development and patient survival, offering targets for personalized therapy. Conclusion Identifying key genes and pathways in lung metastasis from HNC, this study highlights potential targets for enhanced diagnosis and therapy. It underscores the crucial role of molecular insights in driving forward personalized treatment approaches and improving patient outcomes.

Bacterial Pathogenesis in Various Fish Diseases: Recent Advances and Specific Challenges in Vaccine Development.

Aquaculture is a fast-growing food sector but is plagued by a plethora of bacterial pathogens that infect fish. The rearing of fish at high population densities in aquaculture facilities makes them highly susceptible to disease outbreaks, which can cause significant economic loss. Thus, immunity development in fish through vaccination against various pathogens of economically important aquaculture species has been extensively studied and has been largely accepted as a reliable method for preventing infections. Vaccination studies in aquaculture systems are strategically associated with the economically and environmentally sustainable management of aquaculture production worldwide. Historically, most licensed fish vaccines have been developed as inactivated pathogens combined with adjuvants and provided via immersion or injection. In comparison, live vaccines can simulate a whole pathogenic illness and elicit a strong immune response, making them better suited for oral or immersion-based therapy methods to control diseases. Advanced approaches in vaccine development involve targeting specific pathogenic components, including the use of recombinant genes and proteins. Vaccines produced using these techniques, some of which are currently commercially available, appear to elicit and promote higher levels of immunity than conventional fish vaccines. These technological advancements are promising for developing sustainable production processes for commercially important aquatic species. In this review, we explore the multitude of studies on fish bacterial pathogens undertaken in the last decade as well as the recent advances in vaccine development for aquaculture.

Traditionally used medicinal plants mediate the biosynthesis of silver nanoparticles: methodological, larvicidal, and ecotoxicological approach.

It has been shown that vegetal species constitute an alternative natural source for the biosynthesis of new nanomaterials. Thus, aiming to expand knowledge about the potential use of plants in the fabrication of metallic nanomaterials, we aimed to synthesize silver nanoparticles (AgNPs) from phyto-formulation (PF) of ten commonly used medicinal plants. Our results demonstrate the formation of spherical, stable, polycrystalline AgNPs with a diameter of 8.42 nm to 18.40 nm, whose biosynthesis confirmation was performed via UV-Vis spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction analysis (XRD), field emission scanning electron microscopy (FE-SEM)-energy dispersive X-ray spectroscopy (EDS) mapping, high-resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), and zeta potential studies. Furthermore, we demonstrated that the biosynthesized AgNPs showed larvicidal activity against Aedes aegypti and Anopheles stephensi larvae, with the histopathology findings from the fourth instar larval stage validating such larvicidal toxicity. The histological examinations showed severe degradation of the larvae's hindgut, epithelial cells, midgut, and cortical area. However, the PF extract and the biosynthesized AgNPs showed high ecotoxicity in Danio rerio larvae exposed to different concentrations. The treatments induced changes in hatchability percentage, animal growth, and heartbeat. Therefore, despite supporting the potential of PF (from ten plant species) as a raw material source for AgNPs biosynthesis, our study also sheds light on its ecotoxicological potential, suggesting that more comprehensive assessments of the ecotoxicity of biosynthesized would be performed before its application in different sectors.

Efficacy of recombinant subunit OMP and hly vaccines against Aeromonas hydrophila in Rohu (Labeo rohita).

The main aim of the current study was to clone and express a new outer membrane protein (OMP) and haemolysin (hly) from a pathogenic Aeromonas hydrophila and to investigate their potential as a vaccine candidate against A. hydrophila infection in Rohu (Labeo rohita). The OMP and hly genes were cloned in pET-30b vector and recombinant plasmids pET-30b-OMP and pET-30b-hly were constructed, which were then transferred into Escherichia coli BL21 (DE3). The recombinant E. coli BL21 (DE3) was induced by IPTG, and the OMP and hly proteins were expressed highly. The proteins OMP and hly were estimated in 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their molecular weights were found to be 40 kD and 68 kD. The expressed proteins OMP and hly were purified by Ni-NTA His-Bind Resin column, and the immunogenicity was confirmed by Western blotting. The fishes (L. rohita) were divided into IV groups, and the group I fishes were treated with phosphate saline, the II and III group were immunized with the purified OMP and hly recombinant proteins, and the fishes were treated IV group with combined OMP and hly for 10 days. After 10 days of treatment, the fishes of all the four groups were challenged with virulent A. hydrophila. The results revealed that vaccinated fish showed significantly improved haematological profile, phagocytic activity, myeloperoxidase activity and total immunoglobulin levels on the 5th and 10th days. The non-vaccinated group (Group I) showed 100% mortality, whereas the mixture of recombinant OMP (r-OMP) and hly (r-hly) protein-treated groups (Group IV) exhibited higher survival rate (80%). Relatively, expression of pro- and anti-inflammatory cytokines (IL-1ß, IL-10 and TGF-ß), c-type and g-type lysozymes were significantly up-regulated in heart and kidney of vaccinated groups compared with the non-vaccinated group. Our results revealed that OMP and hly genes were effective vaccine candidates in the aquaculture system and could be used as recombinant subunit vaccine for diseases caused by pathogenic A. hydrophila.

Oral delivery of pVAX-OMP and pVAX-hly DNA vaccine using chitosan-tripolyphosphate (Cs-TPP) nanoparticles in Rohu, (Labeo rohita) for protection against Aeromonas hydrophila infection.

The present study examines the effectiveness of DNA vaccine against Aeromonas hydrophila through oral route using chitosan-tripolyphosphate (Cs-TPP) nanoparticles encapsulation. The virulent gene of outer membrane protein (OMP) and hemolysin (hly) related to pathogenicity of A. hydrophila was used to construct a DNA vaccine using pVAX1, and the construct was named as pVAX-OMP and pVAX-hly DNA vaccines. The pVAX-OMP and pVAX-hly DNA vaccines were encapsulated by Cs-TPP nanoparticles and size measured by field emission scanning electron microscopy (FE-SEM). The encapsulation efficiency of Cs-TPP nanoparticles was found to be 79.6% for pVAX-OMP DNA and 82.3% for pVAX-hly DNA binding with Cs-TPP nanoparticles. The stability and invitro release profile of plasmid DNA was also determined after encapsulation using DNase and chitosanase. DNA vaccines distribution in tissues was investigated in fish fed with the pVAX-OMP, pVAX-hly and pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles and confirmed by PCR and multiplex PCR. The results suggest that Cs-TPP nanoparticles encapsulated DNA vaccine delivered into fish by feeding. After oral vaccination of Labeo rohita were challenged with A. hydrophila by intraperitoneal injection. Relatively, gene expression of c- and g-type lysozyme followed by pro- and anti-inflammatory cytokines (Interlukin-10 and Tumor Growth Factor ß) was up-regulated in heart and kidney for pVAX-OMP+pVAX-hly vaccinated group. Moreover, fish fed with pVAX-OMP+pVAX-hly encapsulated in Cs-TPP nanoparticles had a significantly higher survival rate (76.2%) against A. hydrophila. This study concludes that pVAX-OMP and pVAX-hly DNA vaccines can be delivered orally using Cs-TPP nanoparticles for protection against A. hydrophilainfection.

In vitro propagation of tilapia lake virus in cell lines developed from Oreochromis mossambicus.

Tilapia lake virus (TiLV)-suspected samples of tilapia were collected from grow-out ponds located with clinical signs and mortality ranged from 5% to 50%. The reverse transcription-polymerase chain reaction (RT-PCR) assay revealed the presence of TiLV in the disease outbreak ponds. Cell lines were developed from heart, gill and eye of Mozambique tilapia and characterized. Morphologically, these cell lines are composed of epithelioid cells. The optimum growth of these cells was observed at 28°C and 20% concentration of FBS. After cryopreservation, 70%-90% of cells were found to be viable. The cells of all three cell lines were found to be positive to fibronectin and pancytokeratin. PCR amplification of 16S rRNA and COI of O. mossambicus confirmed the origin of these cell lines from O. mossambicus. Heart and gill cell lines were found to be highly susceptible to TiLV and found to be useful for its isolation from infected fish samples. The experimental infection was carried out in O. niloticus and O. mossambicus using the TiLV propagated in susceptible cell lines. The RT-PCR results revealed the presence of TiLV in brain, gill, liver, kidney, spleen, eye, muscle, intestine and heart of experimentally infected O. niloticus and O. mossambicus.