Understanding the molecular mechanism of regeneration through apoptosis-induced compensatory proliferation studies - updates and future aspects.

AICP is a crucial process that maintaining tissue homeostasis and regeneration. In the past, cell death was perceived merely as a means to discard cells without functional consequences. However, during regeneration, effector caspases orchestrate apoptosis, releasing signals that activate stem cells, thereby compensating for tissue loss across various animal models. Despite significant progress, the activation of Wnt3a by caspase-3 remains a focal point of research gaps in AICP mechanisms, spanning from lower to higher regenerative animals. This inquiry into the molecular intricacies of caspase-3-induced Wnt3a activation contributes to a deeper understanding of the links between regeneration and cancer mechanisms. Our report provides current updates on AICP pathways, delineating research gaps and highlighting the potential for future investigations aimed at enhancing our comprehension of this intricate process.

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.

Current scenario of solid waste management techniques and challenges in Covid-19 - A review.

Annually, world generates 2.01 billion tonnes of solid wastes and it is expected to generate 2.2 billion tonnes of solid waste by 2025. Globally double the amount of waste generation was anticipated by 2050, hence an urgent action is required for this intricate problem in adopting better management techniques and recycling strategies. Unfortunately, poor management of wastes causes vulnerable effects to the society in terms of health. Waste management is the key infrastructure to be developed in society, but so far it is not recognized as much in many developing countries. Significant innovations and improvements are made in the last few decades globally, but still 2 to 3 billion people around the world lack access to waste collection services. The aim of this present study is to give an overview of different types of waste techniques that are effectively followed by different countries and the action plans need to follow. This review focuses on the global current scenario of waste generation, and its management methods with relevant literatures providing the upgrades in the phases of waste management services like collection and transport, various techniques adopted for waste management, policies and legislation, countries investment in waste management process and the impact of solid waste management during Covid-19. Collectively we conclude that Asian countries need to allot more fund for handling solid waste. Also with the available waste management technique, it is not possible to achieve zero waste. Therefore, more new techniques are needed to be adapted.

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.

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.

Exploring the effect of UV-C radiation on earthworm and understanding its genomic integrity in the context of H2AX expression.

Maintaining genomic stability is inevitable for organism survival and it is challenged by mutagenic agents, which include ultraviolet (UV) radiation. Whenever DNA damage occurs, it is sensed by DNA-repairing proteins and thereby performing the DNA-repair mechanism. Specifically, in response to DNA damage, H2AX is a key protein involved in initiating the DNA-repair processes. In this present study, we investigate the effect of UV-C on earthworm, Perionyx excavatus and analyzed the DNA-damage response. Briefly, we expose the worms to different doses of UV-C and find that worms are highly sensitive to UV-C. As a primary response, earthworms produce coelomic fluid followed by autotomy. However, tissue inflammation followed by death is observed when we expose worm to increased doses of UV-C. In particular, UV-C promotes damages in skin layers and on the contrary, it mediates the chloragogen and epithelial outgrowth in intestinal tissues. Furthermore, UV-C promotes DNA damages followed by upregulation of H2AX on dose-dependent manner. Our finding confirms DNA damage caused by UV-C is directly proportional to the expression of H2AX. In short, we conclude that H2AX is present in the invertebrate earthworm, which plays an evolutionarily conserved role in DNA damage event as like that in higher animals.

Developmental stages of zebrafish (Danio rerio) embryos and toxicological studies using foldscope microscope.

Zebrafish (Danio rerio), is a well-established vertebrate animal model widely used in developmental biology and toxicological research. In the present study, foldscope is used as an innovative tool to study the developmental stages and toxicological analysis of the zebrafish embryos. Briefly, the developmental stages, such as zygote, cleavage, blastula, gastrula, segmentation, and pharyngula formation are observed and documented using simple foldscope. Toxicological parameters upon exposure to different concentration of ethanol extract of Curcuma longa and its lead compound, ar-turmerone along with rhodamine B (bio-coupler) on zebrafish embryos are analyzed upto 72 hr using foldscopes in live condition. The lethal endpoints, such as coagulation, lack of somite formation, non-detachment of tail, and lack of heartbeat are clearly monitored and documented using foldscope. Bio-evaluation of test compounds with the aid of foldscope confirms that the toxicity is directly proportional to the concentration. Our results conclude that, ethanol extract of C. longa, ar-turmerone and rhodamine B exposed embryos remains healthy up to 96, 48, and 24 µg concentrations, respectively. Embryos exposed to higher concentrations become coagulated, however normal physiological active movement of tail lashing and heartbeat are evident in lower concentration exposed embryos. Except coagulation, no other abnormalities are observed and interestingly, the hatching ability is not delayed, when compared with the control embryos. It is confirmed that the test compounds are not highly toxic to zebrafish embryos. Hence it can be used for further analysis, especially for studying the neural-regeneration and its neuronal development in zebrafish embryos.

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.

Understanding the Role of the Clitellum in the Regeneration Events of the Earthworm Eudrilus eugeniae.

Regeneration is a complex mechanism to restore lost or damaged body parts. In earthworms, regeneration capability varies among different species, and it is important to explore the mechanism behind the regeneration process. Interestingly, regeneration in earthworms is either dependent or independent of clitellum segments. In the present study, juvenile earthworms (Eudrilus eugeniae) were amputated at 3 different sites, namely the head, clitellum, and tail segments (at segments 10, 15, and 30, respectively), and their regeneration ability was documented using a foldscope. The amputated segments having the intact clitellum were able to heal the wounds and form the regenerative blastema. The smaller portions of the amputated segments (segments 1-10 and 1-15) without intact clitellum were unable to heal the wound, and death occurs within 12-24 h. The larger portions of the amputated segments (segments 15 and 30 to anus) without intact clitellum were able to heal the wound but lacked the regeneration capability. In control worms, alkaline phosphatase (ALP) signals were observed at the anterior tip, clitellum, and gut epithelium tissues, whereas, upon amputation, the enriched signals from the clitellum diminished, but profound signals were observed at the amputation site and regenerative blastema. Interestingly, on days 3 and 4, blastemal tips lacked ALP signals due to initiation of the differentiation process in the regeneration blastema. In summary, using a foldscope microscope, the role of the clitellum in the regeneration mechanism was indicated by ALP activity.

Function of translationally controlled tumor protein (TCTP) in Eudrilus eugeniae regeneration.

TCTP (Translationally Controlled Tumour Protein) is a multifunctional protein that plays a role in the development, immune system, tumour reversion, and maintenance of stem cells. The mRNA of the Tpt1 gene is over-expressed during liver regeneration. But, the function of the protein in regeneration is not known. To study the role of the protein in regeneration, the earthworm Eudrilus eugeniae was chosen. First, the full length cDNA of the Tpt1 gene was sequenced. The size of the cDNA is 504 bp and the protein has 167 amino acids. The highest level of TCTP expression was documented in the worm after three days of regeneration. The protein was found to be expressed specifically in the epithelial layer of the skin. During regeneration, the protein expression was found to be the highest at the tip of blastema. The pharmacological suppression of TCTP using nutlin-3 and TCTP RNAi experiments resulted in the failure of the regeneration process. The suppression of TCTP caused the arrest of proliferation in posterior amputated worms. The severe cell death was documented in the amputated region of nutlin-3 injected worm. The silencing of TCTP has blocked the modification of clitellar segments. The experiments confirm that TCTP has major functions in the upstream signalling of cell proliferation in the early regeneration process in E. eugeniae.

Studies on regeneration of central nervous system and social ability of the earthworm Eudrilus eugeniae.

Earthworms are segmented invertebrates that belong to the phylum Annelida. The segments can be divided into the anterior, clitellar and posterior parts. If the anterior part of the earthworm, which includes the brain, is amputated, the worm would essentially survive even in the absence of the brain. In these brain amputee-derived worms, the nerve cord serves as the primary control center for neurological function. In this current work, we studied changes in the expression levels of anti-acetylated tubulin and serotonin as the indicators of neuro-regenerative processes. The data reveal that the blastemal tissues express the acetylated tubulin and serotonin from day four and that the worm amputated at the 7th segment takes 30 days to complete the regeneration of brain. The ability of self-assemblage is one of the specific functions of the earthworm's brain. The brain amputee restored the ability of self-assemblage on the eighth day.