Profiling microRNAs of earthworm, Perionyx excavatus and deciphering the expression of distinct novel miRNAs regulating epimorphosis regeneration.

Earthworm, P. excavatus, is an ideal model organism for studying regeneration. Due to its prodigious regeneration capability, the amputated head part of the earthworm can regenerate completely within 22 days. MicroRNAs (miRNAs) regulate specific genes and are involved in essential biological processes, including regeneration. In this study, we conducted a comprehensive analysis of miRNA profiling of the earthworm, P. excavatus, during the process of anterior regeneration. Our investigation involved in the identification of 55 miRNAs from 30 distinct miRNA families that exhibit significant relevance to wound healing and regeneration. Notably, we have identified 50 novel miRNAs and predicted their pre-miRNA secondary structures using MIREAP. Both Known and Novel miRNAs are validated using qPCR. In addition, we employed the miRanda algorithm to predict the interactions between these miRNAs and their target mRNA transcripts. Based on the miRanda target prediction results, we identified the target genes such as Wnt, Myc, MAPK, SoxB, IHH, Hox, and Notch. These findings indicate that the potential targets of these miRNAs might play crucial roles in various functions related to wound healing, tissue restoration, and regeneration. Furthermore, the acquisition of these findings provides a unique perspective on understanding the molecular mechanisms driving epimorphosis regeneration in connection with miRNAs for the development of miRNA-based therapeutics.

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.

Understanding the process of angiogenesis in regenerating earthworm.

Developing blood vessels from the existing vasculature is vital for the growth of the organism, as well as for systematic wound healing and the repair process. In this study, we investigated the role of angiogenesis during the regeneration process in the earthworm, Eudrilus eugeniae, animal model. Briefly, the morphological examination of blood vessels in juvenile and mature worms is documented, along with the development of new blood vessels in regenerating blastema. However, in vivo and in vitro experiments with juvenile worms revealed that geraniol retards blastemal regeneration growth with undeveloped blood vessels, as compared to the control. The results of qRT-PCR, western blotting, and immunohistochemistry confirmed a reduced expression of VEGFR2 and WNT5A in the day 3 regenerating blastema of geraniol-treated worms, as compared to the control. We conclude that geraniol acts as a potent natural inhibitor of angiogenesis, thereby retarding the regeneration process in earthworms. In addition, for studying angiogenesis and screening effective angiogenesis inhibitors as drug candidates, the earthworm is an ambient animal model system.

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.

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.