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The wood frog ( Rana sylvatica ) possesses remarkable adaptation mechanisms that ensures it survival in extreme environmental conditions, including enduring whole body freezing. The present study investigates the epigenetic mechanisms, specifically histone lysine acetylation and deacetylation, which are critical for regulating gene expression and conserving energy, underlying the wood frog’s ability to endure whole-body freezing. We investigated the expression patterns of lysine acetyltransferases (KATs) and lysine deacetylases (HDACs) in wood frog kidney over the freeze-thaw cycle. Our results reveal a significant downregulation of KATs in kidneys of frozen frogs, with specific KATs showing considerable reductions. This suggests that histone acetylation may play a vital role in suppressing gene expression and conserving energy during freezing. Furthermore, histone acetylation marks, including H2AK5ac, H2BK5ac, H3K9ac, H3K23ac, H3K27ac, and H3K56ac, showed repression under frozen and thawed conditions, indicating a role in silencing specific genes. HDACs exhibited dynamic regulation, with HDAC3 and HDAC11 showing significant repression in frozen frog kidneys, while HDAC5, p-HDAC4, and p-HDAC8 were downregulated during the recovery phase, suggesting their involvement in the thawing process. This research provides crucial insights into the epigenetic control of freeze tolerance in wood frog kidneys and offers a foundation for further exploration of epigenetic modifications that mediate the wood frog’s remarkable adaptations for freezing survival.

When temperatures plummet below 0 °C, wood frogs ( Rana sylvatica ) can endure the freezing of up to ~ 65% of their body water in extracellular ice masses, displaying no measurable brain activity, no breathing, no movement, and a flat-lined heart. To aid survival, frogs retreat into a state of suspended animation characterized by global suppression of metabolic functions and reprioritization of energy usage to essential survival processes that is elicited, in part, by the regulatory controls of microRNAs. The present study is the first to investigate miRNA biogenesis and regulation in the brain of a freeze tolerant vertebrate. Indeed, proper brain function and adaptations to environmental stimuli play a crucial role in coordinating stress responses. Immunoblotting of miRNA biogenesis factors illustrated an overall reduction in the majority of these processing proteins suggesting a potential suppression of miRNA maturation over the freeze–thaw cycle. This was coupled with a large-scale RT-qPCR analysis of relative expression levels of 113 microRNA species in the brains of control, 24 h frozen, and 8 h thawed R. sylvatica . Of the 41 microRNAs differentially regulated during freezing and thawing, only two were significantly upregulated. Bioinformatic target enrichment of the downregulated miRNAs, performed at the low temperatures experienced during freezing and thawing, predicted their involvement in the potential activation of various neuroprotective processes such as synaptic signaling, intracellular signal transduction, and anoxia/ischemia injury protection. The predominantly downregulated microRNA fingerprint identified herein suggests a microRNA-mediated cryoprotective mechanism responsible for maintaining neuronal functions and facilitating successful whole brain freezing and thawing.

Hibernating mammals offer an intriguing example of natural torpor and illustrate the regulatory mechanisms that control metabolic rate depression and the cell preservation strategies that support long-term viability in a hypometabolic state. These suggest applied strategies for improving the hypothermic preservation of human organs for transplant, and guidelines that could aid the development of torpor as an intervention strategy in human medicine. Recent advances in hibernation research have illustrated mechanisms that contribute to metabolic depression by orchestrating the global suppression of ATP-expensive transcription and translation including multiple forms of post-translational modification of proteins/enzymes (phosphorylation, acetylation, SUMOylation), mRNA storage mechanisms, and differential expression of microRNA species. DNA-screening technologies have also contributed new advances in understanding the range of cell functions that are impacted during torpor and point out some critical preservation strategies that aid long-term viability in a torpid state. These include antioxidant defenses, chaperones and the implementation of the unfolded protein response, and the enhancement of serpins (serine protease inhibitors) to control the actions of extracellular proteases in clotting and inflammation responses.

Apart from sharing common ancestry with chordates, sea cucumbers exhibit a unique morphology and exceptional regenerative capacity. Here we present the complete genome sequence of an economically important sea cucumber, A. japonicus, generated using Illumina and PacBio platforms, to achieve an assembly of approximately 805 Mb (contig N50 of 190 Kb and scaffold N50 of 486 Kb), with 30,350 protein-coding genes and high continuity. We used this resource to explore key genetic mechanisms behind the unique biological characters of sea cucumbers. Phylogenetic and comparative genomic analyses revealed the presence of marker genes associated with notochord and gill slits, suggesting that these chordate features were present in ancestral echinoderms. The unique shape and weak mineralization of the sea cucumber adult body were also preliminarily explained by the contraction of biomineralization genes. Genome, transcriptome, and proteome analyses of organ regrowth after induced evisceration provided insight into the molecular underpinnings of visceral regeneration, including a specific tandem-duplicated prostatic secretory protein of 94 amino acids (PSP94)-like gene family and a significantly expanded fibrinogen-related protein (FREP) gene family. This high-quality genome resource will provide a useful framework for future research into biological processes and evolution in deuterostomes, including remarkable regenerative abilities that could have medical applications. Moreover, the multiomics data will be of prime value for commercial sea cucumber breeding programs.

Statistical analysis and data visualization are two crucial aspects in molecular biology and biology. For analyses that compare one dependent variable between standard (e.g., control) and one or multiple independent variables, a comprehensive yet highly streamlined solution is valuable. The computer programming language R is a popular platform for researchers to develop tools that are tailored specifically for their research needs. Here we present an R package RBioplot that takes raw input data for automated statistical analysis and plotting, highly compatible with various molecular biology and biochemistry lab techniques, such as, but not limited to, western blotting, PCR, and enzyme activity assays. The package is built based on workflows operating on a simple raw data layout, with minimum user input or data manipulation required. The package is distributed through GitHub, which can be easily installed through one single-line R command. A detailed installation guide is available at http://kenstoreylab.com/?page_id=2448. Users can also download demo datasets from the same website. By integrating selected functions from existing statistical and data visualization packages with extensive customization, RBioplot features both statistical analysis and data visualization functionalities. Key properties of RBioplot include: -Fully automated and comprehensive statistical analysis, including normality test, equal variance test, Student's t-test and ANOVA (with post-hoc tests);-Fully automated histogram, heatmap and joint-point curve plotting modules;-Detailed output files for statistical analysis, data manipulation and high quality graphs;-Axis range finding and user customizable tick settings;-High user-customizability.

MicroRNAs are small non-coding RNA (18–24 nt long) that fine-tune gene expression at the post-transcriptional level. With the advent of “multi-omics” analysis and sequencing approaches, they have now been implicated in every facet of basic molecular networks, including metabolism, homeostasis, and cell survival to aid cellular machinery in adapting to changing environmental cues. Many animals must endure harsh environmental conditions in nature, including cold/freezing temperatures, oxygen limitation (anoxia/hypoxia), and food or water scarcity, often requiring them to revamp their metabolic organization, frequently on a seasonal or life stage basis. MicroRNAs are important regulatory molecules in such processes, just as they are now well-known to be involved in many human responses to stress or disease. The present review outlines the role of miRNAs in natural animal models of environmental stress and adaptation including torpor/hibernation, anoxia/hypoxia tolerance, and freeze tolerance. We also discuss putative medical applications of advances in miRNA biology including organ preservation for transplant, inflammation, ageing, metabolic disorders (e.g., obesity), mitochondrial dysfunction (mitoMirs) as well as specialized miRNA subgroups respective to low temperature (CryomiRs) and low oxygen (OxymiRs). The review also covers differential regulation of conserved and novel miRNAs involved at cell, tissue, and stress specific levels across multiple species and their roles in survival. Ultimately, the species-specific comparison and conserved miRNA responses seen in evolutionarily disparate animal species can help us to understand the complex miRNA network involved in regulating and reorganizing metabolism to achieve diverse outcomes, not just in nature, but in human health and disease.

Northern Crayfish, Faxonius virilis , displays various strategies that allow them to survive extended periods of oxygen deprivation. However, certain epigenetic adaptations that these crayfish use have not been studied in detail, and the role of specific mechanisms used such as histone modifications remain unknown. Epigenetic studies offer a new perspective on how crayfish can regulate gene expression to redirect energy to essential functions needed for survival. This study investigates the regulation of histone modifications of proteins including acetylation and deacetylation in F. virilis in response to 20-h anoxia exposure. These histone modifications were studied via analysis of writer, reader, and eraser proteins such as lysine acetyltransferases (KATs), bromodomain proteins (BRDs), histone deacetylases (HDAC), and sirtuin proteins (SIRTs). Significant upregulation was seen in one histone protein and one lysine acetyltransferase: H3K14Ac and KAT2A. These proteins are known to be regulated by BRD2; a protein that specifically reads and targets H3K14Ac. In response to anoxia, a larger number of histone deacetylases and sirtuin proteins were upregulated in comparison to lysine acetyltransferases suggesting a focus on suppression of gene expression. The histone deacetylases and sirtuin proteins with significant upregulation were HDAC2, HDAC3, SIRT2, SIRT3, and SIRT6. These proteins have also all been implicated in DNA damage regulation which further suggests that crayfish focus limited energy on ensuring cell survival. This study provides an understanding of how histone acetylation and deacetylation are regulated in crayfish as a component of metabolic rate suppression under anoxia.

Background In response to seasonal cold and food shortage, the Xizang plateau frogs, Nanorana parkeri (Anura: Dicroglossidae), enter a reversible hypometabolic state where heart rate and oxygen consumption in skeletal muscle are strongly suppressed. However, the effect of winter hibernation on gene expression and metabolic profiling in these two tissues remains unknown. In the present study, we conducted transcriptomic and metabolomic analyses of heart and skeletal muscle from summer- and winter-collected N. parkeri to explore mechanisms involved in seasonal hibernation. Results We identified 2407 differentially expressed genes (DEGs) in heart and 2938 DEGs in skeletal muscle. Enrichment analysis showed that shared DEGs in both tissues were enriched mainly in translation and metabolic processes. Of these, the expression of genes functionally categorized as “response to stress”, “defense mechanisms”, or “muscle contraction” were particularly associated with hibernation. Metabolomic analysis identified 24 and 22 differentially expressed metabolites (DEMs) in myocardium and skeletal muscle, respectively. In particular, pathway analysis showed that DEMs in myocardium were involved in the pentose phosphate pathway, glycerolipid metabolism, pyruvate metabolism, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis. By contrast, DEMs in skeletal muscle were mainly involved in amino acid metabolism. Conclusions In summary, natural adaptations of myocardium and skeletal muscle in hibernating N. parkeri involved transcriptional alterations in translation, stress response, protective mechanisms, and muscle contraction processes as well as metabolic remodeling. This study provides new insights into the transcriptional and metabolic adjustments that aid winter survival of high-altitude frogs N. parkeri .

Ectothermic animals that live in seasonally cold regions must adapt to seasonal variation and specific environmental conditions. During the winter, some amphibians hibernate on land and encounter limited environmental water, deficient oxygen, and extremely low temperatures that can cause the whole body freezing. These stresses trigger physiological and biochemical adaptations in amphibians that allow them to survive. Rana sylvatica , commonly known as the wood frog, shows excellent freeze tolerance. They can slow their metabolic activity to a near halt and endure freezing of 65–70% of their total body water as extracellular ice during hibernation, returning to normal when the temperatures rise again. To investigate the molecular adaptations of freeze-tolerant wood frogs, a comprehensive proteomic analysis was performed on frog liver tissue after anoxia, dehydration, or freezing exposures using a label-free LC–MS/MS proteomic approach. Quantitative proteomic analysis revealed that 87, 118, and 86 proteins were significantly upregulated in dehydrated, anoxic, and frozen groups, suggesting potential protective functions. The presence of three upregulated enzymes, glutathione S-transferase (GST), aldolase (ALDOA), and sorbitol dehydrogenase (SORD), was also validated. For all enzymes, the specific enzymatic activity was significantly higher in the livers of frozen and anoxic groups than in the controls. This study reveals that GST, ALDOA, and SORD might participate in the freeze tolerance mechanism by contributing to regulating cellular detoxification and energy metabolism.

The regulatory role of miRNA in gene expression is an emerging hot new topic in the control of hypometabolism. Sea cucumber aestivation is a complicated physiological process that includes obvious hypometabolism as evidenced by a decrease in the rates of oxygen consumption and ammonia nitrogen excretion, as well as a serious degeneration of the intestine into a very tiny filament. To determine whether miRNAs play regulatory roles in this process, the present study analyzed profiles of miRNA expression in the intestine of the sea cucumber (Apostichopus japonicus), using Solexa deep sequencing technology. We identified 308 sea cucumber miRNAs, including 18 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA) after at least 15 days of continuous torpor, were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to the active state). We identified 42 differentially expressed miRNAs [RPM (reads per million) >10, |FC| (|fold change|) ≥ 1, FDR (false discovery rate) <0.01] during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-200-3p, miR-2004, miR-2010, miR-22, miR-252a, miR-252a-3p and miR-92 were significantly over-expressed during deep aestivation compared with non-aestivation animals. Preliminary analyses of their putative target genes and GO analysis suggest that these miRNAs could play important roles in global transcriptional depression and cell differentiation during aestivation. High-throughput sequencing data and microarray data have been submitted to GEO database.