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MicroRNAs are short non-coding RNA transcripts that act as master cellular regulators with roles in orchestrating virtually all biological functions. The recent affordability and widespread use of high-throughput microRNA profiling technologies has grown along with the advancement of bioinformatics tools available for analysis of the mounting data flow. While there are many computational resources available for the management of data from genomesequenced animals, researchers are often faced with the challenge of identifying the biological implications of the daunting amount of data generated from these high-throughput technologies. In this article, we review the current state of highthroughput microRNA expression profiling platforms, data analysis processes, and computational tools in the context of comparative molecular physiology. We also present RBioMIR and RBioFS, our R package implementations for differential expression analysis and random forest-based gene selection. Detailed installation guides are available at kenstoreylab.com.

The massive accumulation of extrachromosomal ribosomal DNA circles (ERCs) in yeast mother cells has been long cited as the primary driver of replicative ageing. ERCs arise through ribosomal DNA (rDNA) recombination, and a wealth of genetic data connects rDNA instability events giving rise to ERCs with shortened life span and other ageing pathologies. However, we understand little about the molecular effects of ERC accumulation. Here, we studied ageing in the presence and absence of ERCs, and unexpectedly found no evidence of gene expression differences that might indicate stress responses or metabolic feedback caused by ERCs. Neither did we observe any global change in the widespread disruption of gene expression that accompanies yeast ageing, altogether suggesting that ERCs are largely inert. Much of the differential gene expression that accompanies ageing in yeast was actually associated with markers of the senescence entry point (SEP), showing that senescence, rather than age, underlies these changes. Cells passed the SEP irrespective of ERCs, but we found the SEP to be associated with copy number amplification of a region of chromosome XII between the rDNA and the telomere (ChrXIIr) forming linear fragments up to approximately 1.8 Mb size, which arise in aged cells due to rDNA instability but through a different mechanism to ERCs. Therefore, although rDNA copy number increases dramatically with age due to ERC accumulation, our findings implicate ChrXIIr, rather than ERCs, as the primary driver of senescence during budding yeast ageing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zylstra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Caloric restriction increases lifespan and improves ageing health, but it is unknown whether these outcomes can be separated or achieved through less severe interventions. Here, we show that an unrestricted galactose diet in early life minimises change during replicative ageing in budding yeast, irrespective of diet later in life. Average mother cell division rate is comparable between glucose and galactose diets, and lifespan is shorter on galactose, but markers of senescence and the progressive dysregulation of gene expression observed on glucose are minimal on galactose, showing that these are not intrinsic aspects of replicative ageing but rather associated processes. Respiration on galactose is critical for minimising hallmarks of ageing, and forced respiration during ageing on glucose by overexpression of the mitochondrial biogenesis factor Hap4 also has the same effect though only in a fraction of cells. This fraction maintains Hap4 activity to advanced age with low senescence and a youthful gene expression profile, whereas other cells in the same population lose Hap4 activity, undergo dramatic dysregulation of gene expression and accumulate fragments of chromosome XII (ChrXIIr), which are tightly associated with senescence. Our findings support the existence of two separable ageing trajectories in yeast. We propose that a complete shift to the healthy ageing mode can be achieved in wild-type cells through dietary change in early life without caloric restriction., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Horkai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The rapid and reversible nature of microRNA (miRNA) transcriptional regulation is ideal for implementing global changes to cellular processes and metabolism, a necessary asset for the freeze-tolerant gray tree frog (Dryophytes versicolor). D. versicolor can freeze up to 42% of its total body water during the winter and then thaw completely upon more favorable conditions of spring. Herein, we examined the freeze-specific miRNA responses in the gray tree frog using RBiomirGS, a bioinformatic tool designed for the analysis of miRNA-seq transcriptomics in non-genome sequenced organisms. We identified 11 miRNAs differentially regulated during freezing (miR-140-3p, miR-181a-5p, miR-206-3p, miR-451a, miR-19a-3p, miR-101-3p, miR-30e-5p, miR-142-3p and -5p, miR-21-5p, and miR-34a-5p). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggests these miRNAs play roles in downregulating signaling pathways, apoptosis, and nuclear processes while enhancing ribosomal biogenesis. Overall, these findings point towards miRNA inducing a state of energy conservation by downregulating energy-expensive pathways, while ribosomal biogenesis may lead to prioritization of critical processes for freeze-tolerance survival., (© 2023 John Wiley & Sons Ltd.)

Freeze tolerance is an adaptive strategy that wood frogs (Rana sylvatica) use to survive the subzero temperatures of winter. It is characterized by a variety of metabolic and physiological changes that facilitate successful freezing and anoxia. As both mRNA regulation and posttranslation protein modification have been implicated in freeze tolerance, we hypothesized that posttranslational RNA regulation is also involved in coordinating freeze-thaw cycles and metabolic rate depression. As such, we investigated the most abundant RNA modification, adenosine methylation (N 6 -methyladenosine; m 6 A) in wood frog brains during 24 h periods of freezing and anoxia. This was followed by an examination of levels of RNA methyltransferases, demethyltransferases, and the readers of RNA methylation. Despite relative levels of methylation on mRNA remaining constant throughout freezing and anoxia, a significant increase in relative abundance of m 6 A methyltransferases METTL3 and METTL14 was observed. In addition, we investigated the effect of m 6 A RNA methylation on mRNA triaging to stress granules and report a significant increase in stress granule markers TIAR and TIA-1 in both freezing and anoxia. Our findings are the first report of RNA posttranslational regulation during metabolic rate depression in the wood frog brain and suggest that the dynamic RNA methylation observed is not directly linked to mRNA regulation during periods of extreme metabolic reorganization, warranting future investigations., (© 2023 The Authors. Journal of Experimental Zoology Part A: Ecological and Intergrative Physiology published by Wiley Periodicals LLC.)

The wood frog (Rana Sylvatica) can endure the sub-zero temperatures of winter by freezing up to 65% of total body water as extracellular ice and retreating into a prolonged hypometabolic state. Freeze survival requires the coordination of various adaptations, including a global suppression of metabolic functions and select activation of pro-survival genes. Transcription factors playing roles in metabolism, stress tolerance, and cell proliferation may assist in making survival in a frozen state possible. In this study, the role of Forkhead box 'other' (FOXO) transcription factors in freeze tolerance, and related changes to the insulin pathway, are investigated. Immunoblotting was used to assess total and phosphorylated amounts of FOXO proteins in wood frogs subjected to freezing for 24 h and thawed recovery for 8 h. Levels of active FOXO3 increased in brain, kidney, and liver during freezing and thawing, suggesting a need to maintain or enhance antioxidant defenses under these stresses. Results implicate FOXO involvement in the metabolic regulation of natural freeze tolerance., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Naked mole-rats are among the few mammals with the ability to endure severe hypoxia. These unique rodents use metabolic rate depression along with various molecular mechanisms to successfully overcome the challenges of oxygen-limitation, which they experience in their underground borrows. While studies have reported that naked mole-rats exhibit inherently higher levels of oxidative damage across their lifespan as compared to mice, it has yet to be determined whether naked mole-rats are vulnerable to oxidative damage during periods of low oxygen exposure. To investigate this phenomenon, we examined cellular oxidative damage markers of macromolecules: DNA oxidation determined as 8-oxo-2'deoxyguanosine (8-OHdG8) levels, RNA oxidation as 8-hydroxyguanosine (8-OHG), protein carbonylation, and lipid peroxidation in normoxic (control), acute (4 h at 7% O 2 ), and chronic (24 h at 7% O 2 ) hypoxia-exposed naked mole-rats. Brain appears to be the most resilient to hypoxia-induced oxidative damage, with both brain and heart exhibiting enhanced antioxidant capacity during hypoxia. Levels of DNA and RNA oxidation were minimally changed in all tissues and no changes were observed in protein carbonylation. Most tissues experienced lipid peroxidation, with liver displaying a 9.6-fold increase during hypoxia. Concomitantly, levels of DNA damage repair proteins were dynamically regulated in a tissue-specific manner, with white adipose displaying a significant reduction during hypoxia. Our findings show that naked mole-rats largely avoid hypoxia-induced oxidative damage, possibly due to their high tolerance to redox stress, or to reduced oxidative requirements made possible during their hypometabolic response when oxygen supply is limited., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mature microRNAs (miRNAs) are short RNA sequences about 18-24 nucleotide long, which provide the recognition key within RISC for the posttranscriptional regulation of target RNAs. Considering the canonical pathway, mature miRNAs are produced via a multistep process. Their transcription (pri-miRNAs) and first processing step via the microprocessor complex (pre-miRNAs) occur in the nucleus. Then they are exported into the cytosol, processed again by Dicer (dsRNA) and finally a single strand (mature miRNA) is incorporated into RISC (miRISC). The sequence of the incorporated miRNA provides the function of RNA target recognition via hybridization. Following binding of the target, the mRNA is either degraded or translation is inhibited, which ultimately leads to less protein production. Conversely, it has been shown that binding within the 5' UTR of the mRNA can lead to an increase in protein product. Regulation of homeostasis is very important for a cell; therefore, all steps in the miRNA-based regulation pathway, from transcription to the incorporation of the mature miRNA into RISC, are under tight control. While much research effort has been exerted in this area, the knowledgebase is not sufficient for accurately modelling miRNA regulation computationally. The computational prediction of miRNAs is, however, necessary because it is not feasible to investigate all possible pairs of a miRNA and its target, let alone miRNAs and their targets. We here point out open challenges important for computational modelling or for our general understanding of miRNA-based regulation and show how their investigation is beneficial. It is our hope that this collection of challenges will lead to their resolution in the near future., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The critical role microRNAs play in modulating global functions is emerging, both in the maintenance of homeostatic mechanisms and in the adaptation to diverse environmental stresses. When stressed, cells must divert metabolic requirements toward immediate survival and eventual recovery and the unique features of miRNAs, such as their relatively ATP-inexpensive biogenesis costs, and the quick and reversible nature of their action, renders them excellent "master controllers" for rapid responses. Many animal survival strategies for dealing with extreme environmental pressures involve prolonged retreats into states of suspended animation to extend the time that they can survive on their limited internal fuel reserves until conditions improve. The ability to retreat into such hypometabolic states is only possible by coupling the global suppression of nonessential energy-expensive functions with an activation of prosurvival networks, a process in which miRNAs are now known to play a major role. In this chapter, we discuss the activation, expression, biogenesis, and unique attributes of miRNA regulation required to facilitate profound metabolic rate depression and implement stress-specific metabolic adaptations. We examine the role of miRNA in strategies of biochemical adaptation including mammalian hibernation, freeze tolerance, freeze avoidance, anoxia and hypoxia survival, estivation, and dehydration tolerance. By comparing these seemingly different adaptive programs in traditional and exotic animal models, we highlight both unique and conserved miRNA-meditated mechanisms for survival. Additional topics discussed include transcription factor networks, temperature dependent miRNA-targeting, and novel species-specific and stress-specific miRNAs., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The goldenrod gall moth (Epiblema scudderiana) is a cold hardy insect that survives subzero temperatures during the winter by supercooling bodily fluids to approximately -40 °C, allowing the insect to remain unfrozen despite the freezing temperatures. This is characterized by a drastic increase of cryoprotectant glycerol along with widespread downregulation of non-essential genes and processes to conserve cellular energy. This study examined the role of epigenetic enzymes in regulating this freeze-avoidant process across a range of freezing temperatures experienced in nature. Cold and subzero temperature exposure in E. scudderiana resulted in upregulation of select DNA methyltransferase (DNMT) enzymes with concurrent decreases in DNMT activity and no change in activity of the Ten-Eleven Translocation (TET) demethylation enzyme activities. Levels of histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity decreased during cold exposures. The increase in DNMT expression and concurrent decrease in HAT activity suggests a role for DNA methylation to assist with transcriptional suppression. These findings propose that epigenetic regulation of genes and histones underpin the winter survival strategies of this insect., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hypothermia is a valuable clinical tool in mitigating against the consequences of ischemia in surgery, stroke, cardiac arrest and organ preservation. Protection is afforded principally by a reduction of metabolism, manifesting as reduced rates of oxygen uptake, preservation of ATP levels, and a curtailing of ischemic calcium overload. The effects of non-ischemic hypothermic stress are relatively unknown. We sought to investigate the effects of clinically mild-to-severe hypothermia on mitochondrial morphology, oxygen consumption and protein expression in normoxic hearts and cardiac cells. Normoxic perfusion of rat hearts at 28-32 °C was associated with inhibition of mitochondrial fission, evidenced by a reduced abundance of the active phosphorylated form of the fission receptor Drp1 (pDrp1 S616 ). Abundance of the same residue was reduced in H9c2 cells subjected to hypothermic culture (25-32 °C), in addition to a reduced abundance of the Drp1 receptor MFF. Hypothermia-treated H9c2 cardiomyocytes exhibited elongated mitochondria and depressed rates of mitochondrial-associated oxygen consumption, which persisted upon rewarming. Hypothermia also promoted a reduction in mRNA expression of the capsaicin receptor TRPV1 in H9c2 cells. When normothermic H9c2 cells were transfected with TRPV1 siRNA we observed reduced pDrp1 S616 and MFF abundance, elongated mitochondria, and reduced rates of mitochondrial-associated oxygen consumption, mimicking the effects of hypothermic culture. In conclusion hypothermia promoted elongation of cardiac mitochondria via reduced pDrp1 S616 abundance which was also associated with suppression of cellular oxygen consumption. Silencing of TRPV1 in H9c2 cardiomyocytes reproduced the morphological and respirometric phenotype of hypothermia. This report demonstrates a novel mechanism of cold-induced inhibition of mitochondrial fission., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Naked mole-rats reduce their metabolic requirements to tolerate severe hypoxia. However, the regulatory mechanisms that underpin this metabolic suppression have yet to be elucidated. 5'-AMP-activated protein kinase (AMPK) is the cellular 'master' energy effector and we hypothesized that alterations in the AMPK pathway contribute to metabolic reorganization in hypoxic naked mole-rat skeletal muscle. To test this hypothesis, we exposed naked mole-rats to 4 h of normoxia (21% O2) or severe hypoxia (3% O2), while indirectly measuring whole-animal metabolic rate and fuel preference. We then isolated skeletal muscle and assessed protein expression and post-translational modification of AMPK, and downstream changes in key glucose and fatty acid metabolic proteins mediated by AMPK, including acetyl-CoA carboxylase (ACC1), glycogen synthase (GS) and glucose transporters (GLUTs) 1 and 4. We found that in hypoxic naked mole-rats (1) metabolic rate decreased ∼80% and fuel use switched to carbohydrates, and that (2) levels of activated phosphorylated AMPK and GS, and GLUT4 expression were downregulated in skeletal muscle, while ACC1 was unchanged. To explore the regulatory mechanism underlying this hypometabolic state, we used RT-qPCR to examine 55 AMPK-associated microRNAs (miRNAs), which are short non-coding RNA post-transcriptional silencers. We identified changes in 10 miRNAs (three upregulated and seven downregulated) implicated in AMPK downregulation. Our results suggest that miRNAs and post-translational mechanisms coordinately reduce AMPK activity and downregulate metabolism in naked mole-rat skeletal muscle during severe hypoxia. This novel mechanism may support tissue-specific prioritization of energy for more essential organs in hypoxia., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Naked mole-rats are among the mammalian champions of hypoxia tolerance. They evolved adaptations centered around reducing metabolic rate to overcome the challenges experienced in their underground burrows. In this study, we used next-generation sequencing to investigate one of the factors likely supporting hypoxia tolerance in naked mole-rat brains, posttranscriptional microRNAs (miRNAs). Of the 212 conserved miRNAs identified using small RNA sequencing, 18 displayed significant differential expression during hypoxia. Bioinformatic enrichment revealed that hypoxia-mediated miRNAs were suppressing energy expensive processes including de novo protein translation and cellular proliferation. This suppression occurred alongside the activation of neuroprotective and neuroinflammatory pathways, and the induction of central signal transduction pathways including HIF-1α and NFκB via miR-335, miR-101, and miR-155. MiRNAs also coordinated anaerobic glycolytic fuel sources, where hypoxia-upregulated miR-365 likely suppressed protein levels of ketohexokinase, the enzyme responsible for catalyzing the first committed step of fructose catabolism. This was further supported by a hypoxia-mediated reduction in glucose transporter 5 proteins that import fructose into the cell. Yet, messenger RNA and protein levels of lactate dehydrogenase, which converts pyruvate to lactate in the absence of oxygen, were elevated during hypoxia. Together, this demonstrated the induction of anaerobic glycolysis despite a lack of reliance on fructose as the primary fuel source, suggesting that hypoxic brains are metabolically different than anoxic naked mole-rat brains that were previously found to shift to fructose-based glycolysis. Our findings contribute to the growing body of oxygen-responsive miRNAs "OxymiRs" that facilitate natural miRNA-mediated mechanisms for successful hypoxic exposures., (© 2020 Wiley Periodicals LLC.)

The African clawed frog (Xenopus laevis) withstands prolonged periods of extreme whole-body dehydration that lead to impaired blood flow, global hypoxia, and ischemic stress. During dehydration, these frogs shift from oxidative metabolism to a reliance on anaerobic glycolysis. In this study, we purified the central glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to electrophoretic homogeneity and investigated structural, kinetic, subcellular localization, and post-translational modification properties between control and 30% dehydrated X. laevis liver. GAPDH from dehydrated liver displayed a 25.4% reduction in maximal velocity and a 55.7% increase in its affinity for GAP, as compared to enzyme from hydrated frogs. Under dehydration mimicking conditions (150 mM urea and 1% PEG), GAP affinity was reduced with a K m value 53.8% higher than controls. Frog dehydration also induced a significant increase in serine phosphorylation, methylation, acetylation, beta-N-acetylglucosamination, and cysteine nitrosylation, post-translational modifications (PTMs). These modifications were bioinformatically predicted and experimentally validated to govern protein stability, enzymatic activity, and nuclear translocation, which increased during dehydration. These dehydration-responsive protein modifications, however, did not appear to affect enzymatic thermostability as GAPDH melting temperatures remained unchanged when tested with differential scanning fluorimetry. PTMs could promote extreme urea resistance in dehydrated GAPDH since the enzyme from dehydrated animals had a urea I 50 of 7.3 M, while the I 50 from the hydrated enzyme was 5.3 M. The physiological consequences of these dehydration-induced molecular modifications of GAPDH likely suppress GADPH glycolytic functions during the reduced circulation and global hypoxia experienced in dehydrated X. laevis., (© 2020 Wiley Periodicals LLC.)

AbstractThe limitations that hypoxia imparts on mitochondrial oxygen supply are circumvented by the activation of anaerobic metabolism and prosurvival mechanisms in hypoxia-tolerant animals. To deal with the hypoxia that jumbo squid ( Dosidicus gigas ) experience in the ocean's depth, they depress their metabolic rate by up to 52% relative to normoxic conditions. This is coupled with molecular reorganization to facilitate their daily descents into the ocean's oxygen minimum zone, where they face not only low oxygen levels but also higher pressures and colder frigid waters. Our current study explores the tissue-specific hypoxia responses of three central processes: (1) antioxidant enzymes responsible for defending against oxidative stress, (2) early apoptotic machinery that signals the activation of cell death, and (3) mitogen-activated protein kinases (MAPKs) that act as central regulators of numerous cellular processes. Luminex xMAP technology was used to assess protein levels and phosphorylation states under normoxic and hypoxic conditions in brains, branchial hearts, and mantle muscles. Hypoxic brains were found to activate apoptosis via upregulation of phospho-p38, phospho-p53, activated caspase 8, and activated caspase 9, whereas branchial hearts were the only tissue to show an increase in antioxidant enzyme levels. Hypoxic muscles seemed the least affected by hypoxia. Our results suggest that hypoxic squid do not undergo large dynamic changes in the phosphorylation states of key apoptotic and central MAPK factors, except for brains, suggesting that these mechanisms are involved in squid hypometabolic responses.

The freshwater red-eared slider turtle, Trachemys scripta elegans, experiences weeks to months of anoxia at the bottom of ice-locked bodies of water in the winter. While this introduces anoxia-reoxygenation cycles similar to the ischemia-reperfusion events that mammals experience, T. s. elegans does not suffer any apparent tissue damage. To survive prolonged anoxia and prevent cellular damage associated with reactive oxygen species, these turtles have developed numerous adaptions, including highly effective antioxidant defenses. Herein, we examined the subcellular localization and protein expression of nuclear factor erythroid-2-related factor 2 (Nrf2), a central transcription factor responsible for modulating cellular antioxidant responses, that was found to be upregulated and localized to the nucleus in anoxic turtles. Additionally, we examined protein levels of glutathione S-transferases (GSTs) and manganese superoxide dismutase (MnSOD) antioxidant enzymes in anoxic liver, kidney, heart, and skeletal muscle tissues. MnSOD levels were significantly higher in heart and muscle during anoxia, and the four GST isozymes (GSTK1, GSTT1, GSTP1, and GSTM3) were elevated in a tissue-specific manner during anoxia and/or aerobic recovery. Together, these results indicate that Nrf2 is likely involved in activating downstream antioxidant genes in response to anoxic stress. These results provide a possible Nrf2-mediated transcriptional mechanism that supports existing findings of enhanced antioxidant defenses that allow T. s. elegans to cope with anoxia-reoxygenation cycles, and subsequent oxidative stress., (© 2021 Wiley Periodicals LLC.)

The intertidal marine periwinkle, Littorina littorea, have developed various strategies to deal with cyclic exposures to anoxic and/or freezing stresses when out of water at low tide. With promising translational research potential, evolutionarily conserved microRNAs (miRNAs) have recently become a focus of animal stress response studies. Using RNA-seq, the current study explores the conserved hepatopancreas miRNAs in facilitating snail stress survival. Overall, stress-specific miRNA responses were overserved. Anoxia led to substantial differential miRNA expression patterns, whereas freezing stress showed a relatively high degree of individual variance in miRNA expression. Pathway analysis identified miRNA-related stress survival adaptations, such as cell proliferation. Additionally, machine learning-based gene selection identified seven hepatopancreas miRNAs critical to distinguish between snails under either stress conditions. Our study demonstrated that conserved miRNAs reflect survival adaptations by marine periwinkles under anoxic or frozen conditions, and thus further establishes these snails as an optimal stress model suited for translational research., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hibernators have evolved effective mechanisms to overcome the challenges of torpor-arousal cycling. This study focuses on the antioxidant and inflammatory defenses under the control of the redox-sensitive and inflammatory-centered NFκB transcription factor in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), a well-established model of mammalian hibernation. While hibernators significantly depress oxygen consumption and overall metabolic rate during torpor, arousal brings with it a rapid increase in respiration that is associated with an influx of reactive oxygen species. As such, hibernators employ a variety of antioxidant defenses to combat oxidative damage. Herein, we used Luminex multiplex technology to examine the expression of key proteins in the NFκB transcriptional network, including NFκB, super-repressor IκBα, upstream activators TNFR1 and FADD, and downstream target c-Myc. Transcription factor DNA-binding ELISAs were also used to measure the relative degree of NFκB binding to DNA during hibernation. Analyses were performed across eight different tissues, cerebral cortex, brainstem, white and brown adipose tissue, heart, liver, kidney, and spleen, during euthermic control and late torpor to highlight tissue-specific NFκB mediated cytoprotective responses against oxidative stress experienced during torpor-arousal. Our findings demonstrated brain-specific NFκB activation during torpor, with elevated levels of upstream activators, inactive-phosphorylated IκBα, active-phosphorylated NFκB, and enhanced NFκB-DNA binding. Protein levels of downstream protein, c-Myc, also increased in the brain and adipose tissues during late torpor. The results show that NFκB regulation might serve a critical neuroprotective and cytoprotective role in hibernating brains and selective peripheral tissue., Competing Interests: Declaration of Competing Interest Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

From squid at the bottom of the ocean to humans at the top of mountains, animals have adapted to diverse oxygen-limited environments. Surviving these challenging conditions requires global metabolic reorganization that is orchestrated, in part, by microRNAs that can rapidly and reversibly target all biological functions. Herein, we review the involvement of microRNAs in natural models of anoxia and hypoxia tolerance, with a focus on the involvement of oxygen-responsive microRNAs (OxymiRs) in coordinating the metabolic rate depression that allows animals to tolerate reduced oxygen levels. We begin by discussing animals that experience acute or chronic periods of oxygen deprivation at the ocean's oxygen minimum zone and go on to consider more elevated environments, up to mountain plateaus over 3500 m above sea level. We highlight the commonalities and differences between OxymiR responses of over 20 diverse animal species, including invertebrates and vertebrates. This is followed by a discussion of the OxymiR adaptations, and maladaptations, present in hypoxic high-altitude environments where animals, including humans, do not enter hypometabolic states in response to hypoxia. Comparing the OxymiR responses of evolutionarily disparate animals from diverse environments allows us to identify species-specific and convergent microRNA responses, such as miR-210 regulation. However, it also sheds light on the lack of a single unified response to oxygen limitation. Characterizing OxymiRs will help us to understand their protective roles and raises the question of whether they can be exploited to alleviate the pathogenesis of ischemic insults and boost recovery. This Review takes a comparative approach to addressing such possibilities., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

When food scarcity is coupled with decreased temperatures, gray mouse lemurs (Microcebus murinus) depress their metabolic rates and retreat into bouts of either daily torpor or multi-day hibernation, without dramatically dropping body temperatures like other 'traditional hibernators'. Rapid and reversible mechanisms are required to coordinate the simultaneous suppression of energetically expensive processes and activation of pro-survival pathways critical for successful torpor-arousal cycling. MicroRNAs, a class of endogenous non-coding small RNAs, are effective post-transcriptional regulators that modulate all aspects of cellular function. The present study hypothesizes that miRNAs are intimately involved in facilitating the molecular reorganization events necessary for lemur skeletal muscle torpor. Small RNA-Sequencing was used to compare miRNA profiles from skeletal muscles of torpid and control primates. We characterized 234 conserved miRNAs, of which 20 were differentially expressed during torpor, relative to control. Examples included downregulation of key muscle-specific (myomiR) members, miR-1 and miR-133, suggesting a switch to muscle-specific energy-saving strategies. In silico target mapping and logistic regression-based gene set analysis indicated the inhibition of energy costly pathways such as oxidative phosphorylation and muscle proliferation. The suppression of these metabolic pathways was balanced with a lack of miRNA inhibition of various signaling pathways, such as MAPK, mTOR, focal adhesion, and ErbB. This study identifies unique miRNA signatures and 'biomarkers of torpor' that provide us with primate-specific insights on torpor at high body temperatures that can be exploited for human biomedical concerns., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Naked mole rats are a long-lived animal model that age much like humans, but that can also withstand oxidative damage, cancer, neurodegenerative diseases, and severe hypoxic conditions, which is of particular interest to this study. The conditions of their underground burrows result in competition for oxygen consumption, yet despite this oxygen deprivation they emerge unscathed. To understand the mechanisms in place to facilitate neuronal preservation during hypoxia, we investigated the protein levels of well-known cell-stress factors. We found that under hypoxic conditions, nearly half of the proteins measured increased expression in brain, while only a few decreased. Under hypoxic conditions there appeared to be a HIF1α-centered response, where HIF1α and its interactors carbonic anhydrase 9, CITED2, p21/CIP1, and NFκB1, among others, were upregulated. Concurrently, a hypoxia-induced decrease of cytochrome c was consistent with decreased mitochondrial function and protection from apoptosis. The picture that emerges is one of neuroprotection, cell-cycle arrest, and the promotion of antiapoptotic functions, all of which are consistent with conserving energy and maintaining neural integrity under low oxygen levels. These results suggest how this species may be poised to face hypoxia and contribute to its remarkable ability to deal with myriad of other damaging factors and sets the stage for future work on the neuroprotective facilitators we identified., (© 2019 Wiley Periodicals, Inc.)

Recently, organ transplant therapy has received a major boost from a change in perspective - a move away from damaging, cold static organ storage to the use of warm normothermic perfusion. The concept for warm preservation is one that has been borrowed from Nature, and it is only fitting that we go back to the wild for more 'tricks' to further improve warm organ stabilization. Current warm preservation strategies are designed to mimic natural conditions in the human body as closely as possible, but what if we could mimic these conditions while simultaneously inducing a reversible state of torpor that would further extend the viability window of donor organs? Indeed, the original driver for using cold organ storage was its ability to strongly reduce metabolic rate many-fold when organs were cooled from 37 to 5 °C. Herein, we discuss the adaptations that allow warm hibernators such as bears and lemurs (fellow primates) to naturally depress their metabolic rate and retreat into states of suspended animation, and how these can be applied to improve organ transplant therapy. Can we look to Nature for instructions to induce torpor in human organs? This article discusses the possibilities., (© 2018 Federation of European Biochemical Societies.)

A degree of regulation continues into death according to post-mortem transcriptome studies, which have identified 'zombie genes' that come alive hours and days after organismal death. We hypothesized that hibernation, representing the closest natural mammalian phenomenon to death, would display similar gene expression profiles. Exploring zombie genes using qPCR and available transcriptomic resources from multiple torpid tissues in 13-lined ground squirrels showed little in common with gene profiles observed following death. Hibernators repress transcription, surviving only on the transcripts required during profound slowdowns of metabolic rate and of most physiological functions, therefore not requiring zombie gene expression that could be the cell's last resort during stress. This is the first study to explore zombie gene responses to a near-death situation in a living system., (© 2019 Federation of European Biochemical Societies.)

When faced with extreme environmental conditions, the milk snail ( Otala lactea ) enters a state of dormancy known as estivation. This is characterized by a strong reduction in metabolic rate to <30% of normal resting rate that is facilitated by various behavioural, physiological, and molecular mechanisms. Herein, we investigated the regulation of microRNA in the induction of estivation. Changes in the expression levels of 75 highly conserved microRNAs were analysed in snail foot muscle, of which 26 were significantly upregulated during estivation compared with controls. These estivation-responsive microRNAs were linked to cell functions that are crucial for long-term survival in a hypometabolic state including anti-apoptosis, cell-cycle arrest, and maintenance of muscle functionality. Several of the microRNA responses by snail foot muscle also characterize hypometabolism in other species and support the existence of a conserved suite of miRNA responses that regulate environmental stress responsive metabolic rate depression across phylogeny., Competing Interests: Kenneth B. Storey is an Academic Editor for PeerJ.

Mitochondria are not just the powerhouses of the cell; these 'end of function' organelles are crucial components of cellular physiology and influence many central metabolic and signaling pathways that support complex multicellular life. Not surprisingly, these organelles play vital roles in adaptations for extreme survival strategies including hibernation and freeze tolerance, both of which are united by requirements for a strong reduction and reprioritization of metabolic processes. To facilitate metabolic rate depression, adaptations of all aspects of mitochondrial function are required, including; energetics, physiology, abundance, gene regulation, and enzymatic controls. This review discusses these factors with a focus on the stress-specific nature of mitochondrial genes and transcriptional regulators, and processes including apoptosis and chaperone protein responses. We also analyze the regulation of glutamate dehydrogenase and pyruvate dehydrogenase, central mitochondrial enzymes involved in coordinating the shifts in metabolic fuel use associated with extreme survival strategies. Finally, an emphasis is given to the novel mitochondrial research areas of microRNAs, peptides, epigenetics, and gaseous mediators and their potential roles in facilitating hypometabolism. © 2018 IUBMB Life, 70(12):1260-1266, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Some animals must endure prolonged periods of oxygen deprivation to survive. One such extreme model is the northern crayfish ( Orconectes virilis ), that regularly survives year-round hypoxic and anoxic stresses in its warm stagnant summer waters and in its cold, ice-locked winter waters. To elucidate the molecular underpinnings of anoxia resistance in this natural model, we surveyed the expression profiles of 76 highly conserved microRNAs in crayfish hepatopancreas and tail muscle from normoxic, acute 2 h anoxia, and chronic 20 h anoxia exposures. MicroRNAs are known to regulate a diverse array of cellular functions required for environmental stress adaptations, and here we explored their role in anoxia tolerance. The tissue-specific anoxia responses observed herein, with 22 anoxia-responsive microRNAs in the hepatopancreas and only four in muscle, suggest that microRNAs facilitate a reprioritization of resources to preserve crucial organ functions. Bioinformatic microRNA target enrichment analysis predicted that the anoxia-downregulated microRNAs in hepatopancreas targeted Hippo signalling, suggesting that cell proliferation and apoptotic signalling are highly regulated in this liver-like organ during anoxia. Compellingly, miR-125-5p , miR-33-5p and miR-190-5p , all known to target the master regulator of oxygen deprivation responses HIF1 (hypoxia inducible factor-1), were anoxia downregulated in the hepatopancreas. The anoxia-increased transcript levels of the oxygen-dependent subunit HIF1α highlight a potential critical role for miRNA-HIF targeting in facilitating a successful anoxia response. Studying the cytoprotective mechanisms in place to protect against the challenges associated with surviving in oxygen-poor environments is critical to elucidating the vast and substantial role of microRNAs in the regulation of metabolism and stress in aquatic invertebrates., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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.

At night, Humboldt squid (Dosidicus gigas) rise to the ocean's surface to feed, but come morning, they descend into the ocean's oxygen minimum zone where they can avoid predators but must deal with severe hypoxia, high pressure, and very cold water. To survive this extreme environment, squid use various adaptations to enter a hypometabolic state characterized by metabolic rate suppression by 35-52%, relative to normoxic conditions. The molecular mechanisms facilitating this metabolic flexibility have yet to be elucidated in hypometabolic squid. Herein, we report the first investigation of the role of microRNAs, a rapid and reversible post-transcriptional master regulator of virtually all biological functions, in cephalopods. We examined expression levels of 39 highly-conserved invertebrate microRNAs in D. gigas brain, mantle muscle, and branchial heart, comparing hypoxic and normoxic conditions. Hypoxia-inducible microRNAs are potentially involved in facilitating neuroprotection, anti-apoptosis, and regenerative mechanisms in brain; inhibiting apoptosis and cell proliferation while conserving energy in heart; and limiting damage by reactive oxygen species and apoptosis in muscle. Rather than orchestrate global metabolic rate depression, the majority of hypoxia-inducible microRNAs identified are involved in promoting cytoprotective mechanisms, suggesting a regulatory role for microRNA in hypoxic marine invertebrates that sets the stage for mechanistic analyses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The wood frog ( Rana sylvatica ) can endure freezing of up to 65% of total body water during winter. When frozen, wood frogs enter a dormant state characterized by a cessation of vital functions (i.e., no heartbeat, blood circulation, breathing, brain activity, or movement). Wood frogs utilize various behavioural and biochemical adaptations to survive extreme freezing and component anoxia and dehydration stresses, including a global suppression of metabolic functions and gene expression. The stress-responsive myocyte enhancer factor-2 (MEF2) transcription factor family regulates the selective expression of genes involved in glucose transport, protein quality control, and phosphagen homeostasis. This study examined the role of MEF2A and MEF2C proteins as well as select downstream targets (glucose transporter-4, calreticulin, and muscle and brain creatine kinase isozymes) in 40% dehydration and 24 h anoxia exposure at the transcriptional, translational, and post-translational levels using qRT-PCR, immunoblotting, and subcellular localization. Mef2a/c transcript levels remained constant during dehydration and anoxia. Total, cytoplasmic, and nuclear MEF2A/C and phospho-MEF2A/C protein levels remained constant during dehydration, whereas a decrease in total MEF2C levels was observed during rehydration. Total and phospho-MEF2A levels remained constant during anoxia, whereas total MEF2C levels decreased during 24 h anoxia and P-MEF2C levels increased during 4 h anoxia. In contrast, cytoplasmic MEF2A levels and nuclear phospho-MEF2A/C levels were upregulated during anoxia. MEF2 downstream targets remained constant during dehydration and anoxia, with the exception of glut4 which was upregulated during anoxia. These results suggest that the upregulated MEF2 response reported in wood frogs during freezing may in part stem from their cellular responses to surviving prolonged anoxia, rather than dehydration, leading to an increase in GLUT4 expression which may have an important role during anoxia survival., Competing Interests: Kenneth B. Storey is an Academic Editor for PeerJ.

The wood frog survives frigid North American winters by retreating into a state of suspended animation characterized by the freezing of up to 65% of total body water as extracellular ice and displaying no heartbeat, breathing, brain activity, or movement. Physiological and biochemical adaptations are in place to facilitate global metabolic depression and protect against the consequences of whole body freezing. This study examined the myocyte enhancer factor 2 (MEF2) transcription factor family, proteins responsible for coordinating selective gene expression of a myriad of cellular functions from muscle development and remodelling to various stress responses. Immunoblotting, subcellular localization, and RT-PCR were used to analyze the regulation of MEF2A and MEF2C transcription factors and selected downstream targets under their control at transcriptional, translational, and post-translational levels in skeletal and cardiac muscles from control, frozen and thawed frogs. Both MEF2A/C proteins were freeze-responsive in skeletal muscle, displaying increases of 1.7-2 fold for phosphorylated MEF2A Thr312 and MEF2C Thr300 during freezing with an enrichment of nuclear phosphorylated MEF2 proteins (by 1.7-2.1 fold) observed as early as 4h post-freezing. Despite the reduced response of total and phosphorylated MEF2A/C protein levels observed in cardiac muscle, the MEF2 downstream gene targets (glucose transporter-4, calreticulin, and creatine kinase brain and muscle isozymes) displayed similar increases in transcript levels (1.7-4.8 fold) after 24h freezing in both muscle types. This study describes a novel freeze-responsive function for MEF2 transcription factors and further elaborates our understanding of the molecular mechanisms underlying natural freeze tolerance. This novel freeze-responsive regulation suggests a role for MEF2s and downstream genes in cryoprotectant glucose distribution, calcium homeostasis, and maintenance of energy reserves vital for successful freeze tolerance., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The wood frog, Rana sylvatica, survives sub-zero winter temperatures by undergoing full body freezing for weeks at a time, during which it displays no measurable brain activity, no breathing, and a flat-lined heart. Freezing is a hypometabolic state characterized by a global suppression of gene expression that is elicited in part by transcription factors that coordinate the activation of vital pro-survival pathways. Smad transcription factors respond to TGF-β signalling and are involved in numerous cellular functions from development to stress. Given the identity of genes they regulate, we hypothesized that they may be involved in coordinating gene expression during freezing. Protein expression of Smad1/2/3/4/5 in response to freezing was examined in 24h frozen and 8h thawed wood frog tissues using western immunoblotting, with the determination of subcellular localization in muscle and liver tissues. Transcript levels of smad2, smad4 and downstream genes (serpine1, myostatin, and tsc22d3) were measured by RT-PCR. Tissue-specific responses were observed during freezing where brain, heart, and liver had elevated levels of pSmad3, and skeletal muscle and kidneys had increased levels of pSmad1/5 and pSmad2 during freeze/thaw cycle, while protein and transcript levels remained constant. There were increases in nuclear levels of pSmad2 in muscle and pSmad3 in liver. Transcript levels of serpine1 were induced in heart, muscle, and liver, myostatin in muscle, and tsc22d3 in heart, and liver during freezing. These results suggest a novel freeze-responsive activation of Smad proteins that may play an important role in coordinating pro-survival gene networks necessary for freeze tolerance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

When faced with adverse environmental conditions, the marsupial Dromiciops gliroides uses either daily or seasonal torpor to support survival and is the only known hibernating mammal in South America. As the sole living representative of the ancient Order Microbiotheria, this species can provide crucial information about the evolutionary origins and biochemical mechanisms of hibernation. Hibernation is a complex energy-saving strategy that involves changes in gene expression that are elicited in part by microRNAs. To better elucidate the role of microRNAs in orchestrating hypometabolism, a modified stem-loop technique and quantitative PCR were used to characterize the relative expression levels of 85 microRNAs in liver and skeletal muscle of control and torpid D. gliroides. Thirty-nine microRNAs were differentially regulated during torpor; of these, 35 were downregulated in liver and 11 were differentially expressed in skeletal muscle. Bioinformatic analysis predicted that the downregulated liver microRNAs were associated with activation of MAPK, PI3K-Akt and mTOR pathways, suggesting their importance in facilitating marsupial torpor. In skeletal muscle, hibernation-responsive microRNAs were predicted to regulate focal adhesion, ErbB, and mTOR pathways, indicating a promotion of muscle maintenance mechanisms. These tissue-specific responses suggest that microRNAs regulate key molecular pathways that facilitate hibernation, thermoregulation, and prevention of muscle disuse atrophy.

Bacillus subtilis cyclic lipopeptides are known to have various antimicrobial effects including different types of interactions with the cell membranes of plant pathogenic fungi. The various spectra of activities of the three main lipopeptide families (fengycins, iturins, and surfactins) seem to be linked to their respective mechanisms of action on the fungal biomembrane. Few studies have shown the combined effect of more than one family of lipopeptides on fungal plant pathogens. In an effort to understand the effect of producing multiple lipopeptide families, sensitivity and membrane permeability of spores from four fungal plant pathogens (Alternaria solani, Fusarium sambucinum, Rhizopus stolonifer, and Verticillium dahliae) were assayed in response to lipopeptides, both individually and as combined treatments. Results showed that inhibition of spores was highly variable depending on the tested fungus-lipopeptide treatment. Results also showed that inhibition of the spores was closely associated with SYTOX stain absorption suggesting effects of efficient treatments on membrane permeability. Combined lipopeptide treatments revealed additive, synergistic or sometimes mutual inhibition of beneficial effects., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Brevinin-1SY is the only described antimicrobial peptide (AMP) of Rana sylvatica. As AMPs are important innate immune molecules that inhibit microbes, this study examined brevinin-1SY regulation during development and in adult frogs in response to environmental stress. The brevinin-1SY nucleotide sequence was identified and used for protein modeling. Brevinin-1SY was predicted to be an amphipathic, hydrophobic, alpha helical peptide that inserts into a lipid bilayer. Brevinin-1SY transcripts were detected in tadpoles and were significantly increased during the later stages of development. Effects of environmental stress (24 h anoxia, 40% dehydration or 24 h frozen) on the mRNA levels of brevinin-1SY in the dorsal and ventral skin were examined. The brevinin-1SY mRNA levels were increased in dorsal and ventral skin of dehydrated frogs, and in ventral skin of anoxic frogs, compared with controls (non-stressed). Brevinin-1SY protein levels in peptide extracts of dorsal skin showed a similar, but not significant, trend to that of brevinin-1SY mRNA levels. Antimicrobial activity of skin extracts from control and stressed animals were assessed for Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Botrytis cinerea, Rhizopus stolonifer and Pythium sulcatum using disk diffusion assays. Peptide extracts of dorsal skin from anoxic, frozen and dehydrated animals showed significantly higher inhibition of E. coli and P. sulcatum than from control animals. In ventral skin peptide extracts, significant growth inhibition was observed in frozen animals for E. coli and P. sulcatum, and in anoxic animals for B. cinerea, compared with controls. Environmental regulation of brevinin-1SY may have important implications for defense against pathogens.