Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture.

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

Immunoinformatics aided approach for predicting potent cytotoxic T cell epitopes of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is an infectious viral pathogen that causing serious respiratory infection in adults and neonates. The only approved therapies for RSV are the monoclonal antibodies palivizumab and its derivative motavizumab. Both treatments are expensive and require a hospital setting for administration. A vaccine represents a safe, effective and cheaper alternative for preventing RSV infection. In silico prediction methods have proven to be valuable in speeding up the process of vaccine design. In this study, reverse vaccinology methods were used to predict the cytotoxic T lymphocytes (CTL) epitopes from the entire proteome of RSV strain A. From amongst 3402 predicted binders to 12 high frequency alleles from the Immune Epitope Database (IEDB), 567 had positive processing scores while 327 epitopes were predicted to be immunogenic. A thorough examination of the 327 epitopes for possible antigenicity, allergenicity and toxicity resulted in 95 epitopes with desirable properties. A BLASTp analysis revealed 94 unique and non-homologous epitopes that were subjected to molecular docking across the 12 high frequency alleles. The final dataset of 70 epitopes contained 13 experimentally proven and 57 unique epitopes from a total of 11 RSV proteins. From our findings on selected T-cell-specific RSV antigen epitopes, notably the four epitopes confirmed to exhibit stable binding by molecular dynamics. The prediction pipeline used in this study represents an effective way to screen the immunogenic epitopes from other pathogens.Communicated by Ramaswamy H. Sarma.

Selenium: a potent regulator of ferroptosis and biomass production.

Emerging evidence supports the notion that selenium (Se) plays a beneficial role in plant development for modern crop production and is considered an essential micronutrient and the predominant source of plants. However, the essential role of selenium in plant metabolism remains unclear. When used in moderate concentrations, selenium promotes plant physiological processes such as enhancing plant growth, increasing antioxidant capacity, reducing reactive oxygen species and lipid peroxidation and offering stress resistance by preventing ferroptosis cell death. Ferroptosis, a recently discovered mechanism of regulated cell death (RCD) with unique features such as iron-dependant accumulation of lipid peroxides, is distinctly different from other known forms of cell death. Glutathione peroxidase (GPX) activity plays a significant role in scavenging the toxic by-products of lipid peroxidation in plants. A low level of GPX activity in plants causes high oxidative stress, which leads to ferroptosis. An integrated view of ferroptosis and selenium in plants and the selenium-mediated nanofertilizers (SeNPs) have been discussed in more recent studies. For instance, selenium supplementation enhanced GPX4 expression and increased TFH cell (Follicular helper T) numbers and the gene transcriptional program, which prevent lipid peroxidase and protect cells from ferroptosis. However, though ferroptosis in plants is similar to that in animals, only few studies have focused on plant-specific ferroptosis; the research on ferroptosis in plants is still in its infancy. Understanding the implication of selenium with relevance to ferroptosis is indispensable for plant bioresource technology. In this review, we hypothesize that blocking ferroptosis cell death improves plant immunity and protects plants from abiotic and biotic stresses. We also examine how SeNPs can be the basis for emerging unconventional and advanced technologies for algae/bamboo biomass production. For instance, algae treated with SeNPs accumulate high lipid profile in algal cells that could thence be used for biodiesel production. We also suggest that further studies in the field of SeNPs are essential for the successful application of this technology for the large-scale production of plant biomass.

Foldscope microscope, an inexpensive alternative tool to conventional microscopy-Applications in research and education: A review.

Microscope is a device used for the visualization of tiny objects which are not visible to the naked eye. Traditional microscopes have been crucial for the advancement of contemporary science and medicine. Recent advancements in the field of microscopy have fueled its exponential growth rate. However, due to their expensive cost and complicated structure, modern microscopes remain inaccessible to the majority of the public. Nonetheless, the foldscope paper microscope has made it possible for anyone to explore and understand the world of microbes and organisms. In this review, we have listed foldscope-based research projects in various domains, as well as their key properties when compared to traditional research microscopes. In addition, we have briefly explored the impact of a foldscope microscope on public health, clinical diagnostics, forensic science, agriculture, basic science, developmental biology, and education. Moreover, the major drawbacks of paper microscopes and the current steps being taken to upgrade foldscope and its features are discussed in this review. Finally, we have concluded with our perspective that the microscope may be updated to imitate the advancement of a conventional microscope. RESEARCH HIGHLIGHTS: The foldscope, a low-cost instrument for studying the microscopic world. Foldscope applications were compared to conventional microscopes in many sectors. The foldscope microscope's existing limitations and potential prospects are highlighted.

Designing of cytotoxic T lymphocyte-based multi-epitope vaccine against SARS-CoV2: a reverse vaccinology approach.

SARS-CoV2 is a single-stranded RNA virus, gaining much attention after it out broke in China in December 2019. The virus rapidly spread to several countries around the world and caused severe respiratory illness to humans. Since the outbreak, researchers around the world have devoted maximum resources and effort to develop a potent vaccine that would offer protection to uninfected individuals against SARS-CoV2. Reverse vaccinology is a relatively new approach that thrives faster in vaccine research. In this study, we constructed Cytotoxic T Lymphocytes (CTL)-based multi-epitope vaccine using hybrid epitope prediction methods. A total of 121 immunogenic CTL epitopes were screened by various sequence-based prediction methods and docked with their respective HLA alleles using the AutoDock Vina v1.1.2. In all, 17 epitopes were selected based on their binding affinity, followed by the construction of multi-epitope vaccine by placing the appropriate linkers between the epitopes and tuberculosis heparin-binding hemagglutinin (HBHA) adjuvant. The final vaccine construct was modeled by the I-TASSER server and the best model was further validated by ERRAT, ProSA, and PROCHECK servers. Furthermore, the molecular interaction of the constructed vaccine with TLR4 was assessed by ClusPro 2.0 and PROtein binDIng enerGY prediction (PRODIGY) server. The immune simulation analysis confirms that the constructed vaccine was capable of inducing long-lasting memory T helper (Th) and CTL responses. Finally, the nucleotide sequence was codon-optimized by the JCAT tool and cloned into the pET21a (+) vector. The current results reveal that the candidate vaccine is capable of provoking robust CTL response against the SARS-CoV2.Communicated by Ramaswamy H. Sarma.

The emerging role of selenium metabolic pathways in cancer: New therapeutic targets for cancer.

Selenium (Se) is incorporated into the body via the selenocysteine (Sec) biosynthesis pathway, which is critical in the synthesis of selenoproteins, such as glutathione peroxidases and thioredoxin reductases. Selenoproteins, which play a key role in several biological processes, including ferroptosis, drug resistance, endoplasmic reticulum stress, and epigenetic processes, are guided by Se uptake. In this review, we critically analyze the molecular mechanisms of Se metabolism and its potential as a therapeutic target for cancer. Sec insertion sequence binding protein 2 (SECISBP2), which is a positive regulator for the expression of selenoproteins, would be a novel prognostic predictor and an alternate target for cancer. We highlight strategies that attempt to develop a novel Se metabolism-based approach to uncover a new metabolic drug target for cancer therapy. Moreover, we expect extensive clinical use of SECISBP2 as a specific biomarker in cancer therapy in the near future. Of note, scientists face additional challenges in conducting successful research, including investigations on anticancer peptides to target SECISBP2 intracellular protein.

Point of care diagnosis of plant virus: Current trends and prospects.

Plant viral diseases accounts for major global economic losses in modern-day agriculture. Plant viral disease management is the primary challenge for both farmers and researchers. Detection and identification of plant viruses are of paramount importance for successful management of a viral disease. Recent advancements in molecular biology have contributed to significant progress in the development of new, sensitive, and effective diagnostic methods. However, most techniques are neither time/cost-effective nor user-friendly and require sophisticated labs. Hence, the past few decades of agricultural research have mainly focused on developing farmer-friendly, point-of-care diagnostic tools that provide high-sensitive rapid diagnosis. The current trend in plant virus diagnostic tools is cheaper, easy-to-use portable devices with no compromise on sensitivity and reproducibility.

Strategies and Patterns of Codon Bias in Molluscum Contagiosum Virus.

Trends associated with codon usage in molluscum contagiosum virus (MCV) and factors governing the evolution of codon usage have not been investigated so far. In this study, attempts were made to decipher the codon usage trends and discover the major evolutionary forces that influence the patterns of codon usage in MCV with special reference to sub-types 1 and 2, MCV-1 and MCV-2, respectively. Three hypotheses were tested: (1) codon usage patterns of MCV-1 and MCV-2 are identical; (2) SCUB (synonymous codon usage bias) patterns of MCV-1 and MCV-2 slightly deviate from that of human host to avoid affecting the fitness of host; and (3) translational selection predominantly shapes the SCUB of MCV-1 and MCV-2. Various codon usage indices viz. relative codon usage value, effective number of codons and codon adaptation index were calculated to infer the nature of codon usage. Correspondence analysis and correlation analysis were performed to assess the relative contribution of silent base contents and significance of codon usage indices in defining bias in codon usage. Among the tested hypotheses, only the second and third hypotheses were accepted.

Alternative to FBS in animal cell culture - An overview and future perspective.

Fetal bovine serum (FBS) is a widely used growth supplement in the in vitro culturing of animal and human cells, tissues and organs, notably due to the occurrence of abundant micro- and macronutrients, along with growth factors. Over the years, increasing demand, high price, batch-to-batch variability in quality and composition, increasing ethical concerns lead to the search for an alternative to FBS. Several approaches have been suggested and employed in the past, but none is implemented as widely as FBS, and each supplement has its own disadvantages. In this review, we described the importance of FBS in cell culture, discussed the issues associated with FBS use and presented the efforts made in the recent past to reduce or replace FBS. The potential of four different alternative sources to FBS, namely, bovine ocular fluid, sericin protein, human platelet lysate and earthworm heat inactivated coelomic fluid was evaluated. In the end, we present the conceptual perspective using the Human Platelet Lysate (HPL) and earthworm Heat Inactivated Coelomic Fluid (HI-CF) combination to alternate FBS and its context in scientific and economic impacts.

Identification of SARS-CoV-2 CTL epitopes for development of a multivalent subunit vaccine for COVID-19.

An immunoinformatics-based approach was used to identify potential multivalent subunit CTL vaccine candidates for SARS-CoV-2. Criteria for computational screening included antigen processing, antigenicity, allergenicity, and toxicity. A total of 2604 epitopes were found to be strong binders to MHC class I molecules when analyzed using IEDB tools. Further testing for antigen processing yielded 826 peptides of which 451 were 9-mers that were analyzed for potential antigenicity. Antigenic properties were predicted for 102 of the 451 peptides. Further assessment for potential allergenicity and toxicity narrowed the number of candidate CTL epitopes to 50 peptide sequences, 45 of which were present in all strains of SARS-CoV-2 that were tested. The predicted CTL epitopes were then tested to eliminate those with MHC class II binding potential, a property that could induce hyperinflammatory responses mediated by TH2 cells in immunized hosts. Eighteen of the 50 epitopes did not show class II binding potential. To our knowledge this is the first comprehensive analysis on the proteome of SARS-CoV-2 for prediction of CTL epitopes lacking binding properties that could stimulate unwanted TH2 responses. Future studies will be needed to assess these epitopes as multivalent subunit vaccine candidates which stimulate protective CTL responses against SARS-COV-2.

Heat-inactivated coelomic fluid of the earthworm Perionyx excavatus is a possible alternative source for fetal bovine serum in animal cell culture.

Fetal Bovine Serum (FBS) is used as a major supplement in culturing animal cells under in vitro conditions. Due to ethical concern, high cost, biosafety, and geographical as well as batchwise result variations, it is important to reduce or replace the use of FBS in animal cell culture. The major objective of this work is to evaluate the feasibility of heat-inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a possible alternative for FBS in animal cell culture experiments. The coelomic fluid (CF) was extruded from the earthworm using electric shock method and used for the experiments. Electric shock method is a simple non-invasive technique, which has no harmful effect on earthworms. Mouse primary fibroblast and HeLa cell lines were used in this study. Among HI-CF, autoclaved CF and crude CF, the supplement of medium with HI-CF shows positive results. The processed HI-CF (90°C for 5 min) at 10% supplement in cell culture medium promote maximum cell growth but cells need the initial support of FBS for the attachment to the culture flask. Microscopic observation and immunofluorescence assay with actin and lamin A confirm that the cellular and molecular morphology of the cells is maintained intact. The HI-CF of earthworm, P. excavatus has shown better cellular viability when compared with FBS and making it possible as an alternative supplement to minimize the use of FBS.