Insights from a vertebrate model organism on the molecular mechanisms of whole-body dehydration tolerance.

Studies on the molecular mechanisms of dehydration tolerance have been largely limited to plants and invertebrates. Currently, research in whole body dehydration of complex animals is limited to cognitive and behavioral effects in humans, leaving the molecular mechanisms of vertebrate dehydration relatively unexplored. The present review summarizes studies to date on the African clawed frog (Xenopus laevis) and examines whole-body dehydration on physiological, cellular and molecular levels. This aquatic frog is exposed to seasonal droughts in its native habitat and can endure a loss of over 30% of its total body water. When coping with dehydration, osmoregulatory processes prioritize water retention in skeletal tissues and vital organs over plasma volume. Although systemic blood circulation is maintained in the vital organs and even elevated in the brain during dehydration, it is done so at the expense of reduced circulation to the skeletal muscles. Increased hemoglobin affinity for oxygen helps to counteract impaired blood circulation and metabolic enzymes show altered kinetic and regulatory parameters that support the use of anaerobic glycolysis. Recent studies with X. laevis also show that pro-survival pathways such as antioxidant defenses and heat shock proteins are activated in an organ-specific manner during dehydration. These pathways are tightly coordinated at the post-transcriptional level by non-coding RNAs, and at the post-translational level by reversible protein phosphorylation. Paired with ongoing research on the X. laevis genome, the African clawed frog is poised to be an ideal animal model with which to investigate the molecular adaptations for dehydration tolerance much more deeply.

MicroRNAs facilitate skeletal muscle maintenance and metabolic suppression in hibernating brown bears.

Hibernating brown bears, Ursus arctos, undergo extended periods of inactivity and yet these large hibernators are resilient to muscle disuse atrophy. Physiological characteristics associated with atrophy resistance in bear muscle have been examined (e.g., muscle mechanics, neural activity) but roles for molecular signaling/regulatory mechanisms in the resistance to muscle wasting in bears still require investigation. Using quantitative reverse transcription PCR (RT-qPCR), the present study characterized the responses of 36 microRNAs linked with development, metabolism, and regeneration of skeletal muscle, in the vastus lateralis of brown bears comparing winter hibernating and summer active animals. Relative levels of mRNA of selected genes (mef2a, pax7, id2, prkaa1, and mstn) implicated upstream and downstream of the microRNAs were examined. Results indicated that hibernation elicited a myogenic microRNA, or "myomiR", response via MEF2A-mediated signaling. Upregulation of MEF2A-controlled miR-1 and miR-206 and respective downregulation of pax7 and id2 mRNA are suggestive of responses that promote skeletal muscle maintenance. Increased levels of metabolic microRNAs, such as miR-27, miR-29, and miR-33, may facilitate metabolic suppression during hibernation via mechanisms that decrease glucose uptake and fatty acid oxidation. This study identified myomiR-mediated mechanisms for the promotion of muscle regeneration, suppression of ubiquitin ligases, and resistance to muscle atrophy during hibernation mediated by observed increases in miR-206, miR-221, miR-31, miR-23a, and miR-29b. This was further supported by the downregulation of myomiRs associated with a muscle injury and inflammation (miR-199a and miR-223) during hibernation. The present study provides evidence of myomiR-mediated signaling pathways that are activated during hibernation to maintain skeletal muscle functionality in brown bears.

Solving Donor Organ Shortage with Insights from Freeze Tolerance in Nature: Activating endogenous antioxidant systems with non-coding RNA to precondition donor organs.

The North American wood frog, Rana sylvatica, endures seasonal whole-body freezing during the winter and thawing during the spring without sustaining any apparent damage from ice or oxidative stress. Strategies from these frogs may solve the shortage of human donor organs, which is a multidisciplinary problem that can be alleviated by eliminating geographical boundaries. Rana sylvatica deploys an array of molecular and physiological responses, such as glucose production and microRNA regulation, to help it survive the cold. These strategies have been adapted in the lab to impart cryotolerance in liver cells, and the non-freezing supercooled storage of transplantable rat livers - milestones that have advanced the field toward cryopreserving human donor organs in the clinic. In this review, a case is presented for the use of non-coding RNAs to decrease oxidative damage of donor organs by activating endogenous antioxidant systems prior to procurement.

FoxO4 activity is regulated by phosphorylation and the cellular environment during dehydration in the African clawed frog, Xenopus laevis.

BACKGROUND: The African clawed frog, Xenopus laevis, is capable of enduring seasonal bouts of severe dehydration stress resulting from transcriptional regulation that facilitates a pro-survival response. Previous studies have shown that dehydration increases antioxidant gene expression in this amphibian. As FoxO4 is known to regulate several antioxidant genes, we sought to understand how differential phosphorylation and environmental factors (urea, temperature) may contribute to its transcriptional regulation during dehydration exposure. METHODS: Immunoblotting was used to quantify relative amount of total FoxO4, of phosphorylated FoxO4, and of the factors in the Ras-Ral pathway that regulate FoxO4 activity in X. laevis skeletal muscle during dehydration. DNA-protein interaction (DPI)-ELISA was used to measure transcription factor-binding to their consensus sequences in the promoters of target genes. Environmental DPI-ELISA was used to assess the effect of the cellular environment on transcription factor binding. RESULTS: FoxO4 protein levels do not change during dehydration, but FoxO4-binding to DNA increases with higher dehydration. The Ras-Ral pathway does not appear to be involved in regulating FoxO4 during dehydration, but Akt-mediated FoxO4 phosphorylation at Ser-193 decreases during high dehydration exposure, which is indicative of increased FoxO4 activity. Further assessment indicated that FoxO4-DNA binding affinity is drastically affected by environmental changes in urea and temperature. CONCLUSION: FoxO4 plays an important role during dehydration stress in X. laevis, and its activity could be regulated through Akt-mediated phosphorylation, and changes in temperature or urea. GENERAL SIGNIFICANCE: Dehydration triggers regulatory mechanisms of transcription by inducing differential phosphorylation and changes to urea in X. laevis.

The role of global histone post-translational modifications during mammalian hibernation.

Mammalian hibernators must cope with hypothermia, ischemia-reperfusion, and finite fuel reserves during days or weeks of continuous torpor. One means of lowering ATP demands during hibernation involves substantial transcriptional controls. The present research analyzed epigenetic regulatory factors as a means of achieving transcriptional control over cycles of torpor-arousal. This study analyzes differential regulation of select histone modifications (e.g. phosphorylation, acetylation, methylation), and identifies post-translational modifications on purified histones using mass spectrometry from thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Post-translational modifications on histone proteins were responsive to torpor-arousal, suggesting a potential mechanism to dynamically alter chromatin structure. Furthermore, proteomic sequencing data of ground squirrel histones identified lysine 19 and 24 acetylation on histone H3, while acetylation sites identified on H2B were lysine 6, 47, 110, and 117. The present study provides a new glimpse into the epigenetic mechanisms which may play a role in transcriptional regulation during mammalian hibernation.

Analysis of microRNA expression during the torpor-arousal cycle of a mammalian hibernator, the 13-lined ground squirrel.

Hibernation is a highly regulated stress response that is utilized by some mammals to survive harsh winter conditions and involves a complex metabolic reprogramming at the cellular level to maintain tissue protections at low temperature. In this study, we profiled the expression of 117 conserved microRNAs in the heart, muscle, and liver of the 13-lined ground squirrel (Ictidomys tridecemlineatus) across four stages of the torpor-arousal cycle (euthermia, early torpor, late torpor, and interbout arousal) by real-time PCR. We found significant differential regulation of numerous microRNAs that were both tissue specific and torpor stage specific. Among the most significant regulated microRNAs was miR-208b, a positive regulator of muscle development that was found to be upregulated by fivefold in the heart during late torpor (13-fold during arousal), while decreased by 3.7-fold in the skeletal muscle, implicating a potential regulatory role in the development of cardiac hypertrophy and skeletal muscle atrophy in the ground squirrels during torpor. In addition, the insulin resistance marker miR-181a was upregulated by 5.7-fold in the liver during early torpor, which supports previous suggestions of hyperinsulinemia in hibernators during the early stages of the hibernation cycle. Although microRNA expression profiles were largely unique between the three tissues, GO annotation analysis revealed that the putative targets of upregulated microRNAs tend to enrich toward suppression of progrowth-related processes in all three tissues. These findings implicate microRNAs in the regulation of both tissue-specific processes and general suppression of cell growth during hibernation.

Turn down genes for WAT? Activation of anti-apoptosis pathways protects white adipose tissue in metabolically depressed thirteen-lined ground squirrels.

During hibernation, the metabolic rate of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) can drop to <5 % of normal resting rate at 37 °C, core body temperature can decrease to as low as 1-5 °C, and heart rate can fall from 350-400 to 5-10 bpm. Energy saved by hibernating allows squirrels to survive the winter when food is scarce, and living off lipid reserves in white adipose tissue (WAT) is crucial. While hibernating, some energy must be used to cope with conditions that would normally be damaging for mammals (e.g., low core body temperatures, ischemia) and could induce cell death via apoptosis. Cell survival is largely dependent on the relative amounts and activities of pro- and anti-apoptotic Bcl-2 family proteins. The present study analyzed how anti-apoptotic proteins respond to protect WAT cells during hibernation. Relative levels of several anti-apoptotic proteins were quantified in WAT via immunoblotting over six time points of the torpor-arousal cycle. These included anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL, and Mcl-l, as well as caspase inhibitors x-IAP and c-IAP. Changes in the relative protein levels and/or phosphorylation levels were also observed for various regulators of apoptosis (p-JAKs, p-STATs, SOCS, and PIAS). Mcl-1 and x-IAP protein levels increased whereas Bcl-xL, Bcl-2, and c-IAP protein/phosphorylation levels decreased signifying important roles for certain Bcl-2 family members in cell survival over the torpor-arousal cycle. Importantly, the relative phosphorylation of selected STAT proteins increased, suggesting a mechanism for Bcl-2 family activation. These results suggest that an increase in WAT cytoprotective mechanisms supports survival efforts during hibernation.

Modulation of diabetic kidney disease markers by an antagonist of p75NTR in streptozotocin-treated mice.

Two therapeutic agents targeting p75NTR pathways have been recently developed to alleviate retinopathy and bladder dysfunction in diabetes mellitus (DM), namely the small molecule p75NTR antagonist THX-B and a monoclonal antibody (mAb) that neutralizes the receptor ligand proNGF. We herein explore these two components in the context of diabetic kidney disease (DKD). Streptozotocin-injected mice were treated for 4 weeks with THX-B or anti-proNGF mAb. Kidneys were taken for quantification of microRNAs and mRNAs by RT-qPCR and for detection of proteins by immunohistochemistry, immunoblotting and ELISA. Blood was sampled to measure plasma levels of urea, creatinine, and albumin. DM led to increases in plasma concentrations of urea and creatinine and decreases in plasma albumin. Receptor p75NTR was expressed in kidneys and its expression was decreased by DM. All these changes were reversed by THX-B treatment while the effect of mAb was less pronounced. MicroRNAs tightly linked to DKD (miR-21-5p, miR-214-3p and miR-342-3p) were highly expressed in diabetic kidneys compared to healthy ones. Also, miR-146a, a marker of kidney inflammation, and mRNA levels of Fn-1 and Nphs, two markers of fibrosis and inflammation, were elevated in DM. Treatments with THX-B or mAb partially or completely reduced the expression of the aforementioned microRNAs and mRNAs. P75NTR antagonism and proNGF mAb might constitute new therapeutic tools to treat or slow down the progression of kidney disease in DM, along with other diabetic related complications. The translational potential of these strategies is currently being investigated.

The regulation of Akt and FoxO transcription factors during dehydration in the African clawed frog (Xenopus laevis).

The African clawed frog (Xenopus laevis) naturally tolerates severe dehydration using biochemical adaptation, one of which is the elevation of antioxidant defenses during whole-body dehydration. The present study investigated the role and regulation of a pathway known to regulate oxidative stress response, the Akt-FoxO signaling pathway, in clawed frog skeletal muscle, responding to medium (15%) and high (30%) dehydration. Protein levels of total and phosphorylated Akt, FoxO1, and FoxO3 were assessed via immunoblotting, in addition to the levels of the E3 ubiquitin ligase known to be associated with muscle atrophy, MAFbx. Akt activity/phosphorylation in addition to its total protein levels were decreased in the skeletal muscle during dehydration, and this corresponded with decreases in the relative phosphorylation of FoxO1 and FoxO3 as well on several residues. Akt is an inhibitor of FoxO1 and FoxO3 activity via phosphorylation, suggesting that FoxO activities were increased during dehydration stress. Furthermore, MAFbx showed decreased protein expression during high dehydration as well, suggesting that the clawed frog may exhibit some natural resistance to skeletal muscle atrophy during severe dehydration conditions. In addition to identifying that the suppression of Akt could lead to an activation of FoxO transcription factors in X. laevis during dehydration, these investigations suggest that X. laevis dehydration may implicate FoxO1 and FoxO3 in controlling skeletal muscle atrophy in X. laevis exposed to dehydration. This study implicates the Akt signaling pathway, its regulation of FoxO transcription factors, and FoxO-controlled targets, in stress adaptation against dehydration.

The complete mitochondrial genome of Dryophytes versicolor: Phylogenetic relationship among Hylidae and mitochondrial protein-coding gene expression in response to freezing and anoxia.

Dryophytes versicolor is one of the most extreme freeze-tolerant frogs from eastern North America. In this study, the mitochondrial genome of D. versicolor was sequenced to analyze the phylogenetic relationships among Hylidae and investigate mitochondrial gene expression in response to freezing and anoxia. The total length of the D. versicolor mitogenome is the longest known to date among the available family members of Hylidae. Both maximum likelihood (ML) and Bayesian inference (BI) analyses strongly supported D. versicolor as a sister clade to (D. japonica + D. ussuriensis) + (D. suweonensis + D. immaculata (KP212702)), and indicated that Dryophytes is monophyletic. Using the mitochondrial genome, gene expression analysis was performed using RT-qPCR in skeletal muscle samples, and determined that relative levels of D. versicolor COX2 increased by 2.40 ±â€¯0.23 fold in response to anoxia, but did not change with exposure to freezing. In addition, ND3 transcript levels decreased in response to anoxia but remained constant during freezing. By contrast, COX1 transcript levels decreased with exposure to freezing, but did not change under anoxic conditions. These results suggest that modulations of protein-coding mitochondrial genes of D. versicolor may play a role in the molecular response to freezing and anoxia tolerance.

The regulation of heat shock proteins in response to dehydration in Xenopus laevis.

African clawed frogs (Xenopus laevis) endure bouts of severe drought in their natural habitats and survive the loss of approximately 30% of total body water due to dehydration. To investigate molecular mechanisms employed by X. laevis during periods of dehydration, the heat shock protein response, a vital component of the cytoprotective stress response, was characterized. Using western immunoblotting and multiplex technology, the protein levels of HSP27, HSP40, HSP60, HSP70, HSC70, and HSP90 were quantified in the liver, skeletal muscle, kidney, lung, and testes from control frogs and those that underwent medium or high dehydration (~16 or ~30% loss of total body water). Dehydration increased HSP27 (1.45-1.65-fold) in the kidneys and lungs, and HSP40 (1.39-2.50-fold) in the liver, testes, and skeletal muscle. HSP60 decreased in response to dehydration (0.43-0.64 of control) in the kidneys and lungs. HSP70 increased in the liver, lungs, and testes (1.39-1.70-fold) during dehydration, but had a dynamic response in the kidneys (levels increased 1.57-fold with medium dehydration, but decreased to 0.56 of control during high dehydration). HSC70 increased in the liver and kidneys (1.20-1.36-fold), but decreased in skeletal muscle (0.27-0.55 of control) during dehydration. Lastly, HSP90 was reduced in the kidney, lung, and skeletal muscle (0.39-0.69 of control) in response to dehydration, but rose in the testes (1.30-fold). Overall, the results suggest a dynamic tissue-specific heat shock protein response to whole body dehydration in X. laevis.

Preadolescent Phthalate (DEHP) Exposure Is Associated With Elevated Locomotor Activity and Reward-Related Behavior and a Reduced Number of Tyrosine Hydroxylase Positive Neurons in Post-Adolescent Male and Female Rats.

Phthalate administration to male rats has been shown to negatively impact neural development while development of the female rat brain is less affected. Because a number of exogenous agents have been shown to interfere with dopamine function, we evaluated post-adolescent behavioral (operant conditioning for food reward and locomotor activity), histological (tyrosine hydroxylase; TH), and genetic (mRNA levels) outcomes of preadolescent (postnatal days [PND] 16-22) phthalate exposure. Male and female Long-Evans rats were administered 4 doses (0, 1, 10, or 20 mg/kg) of di-(2-ethylhexyl)phthalate (DEHP) i.p. from PND16 to 22. Rats were trained on an operant task to bar press for chocolate-flavored pellets from PND55-63 then euthanized on PND78. The 10 mg/kg DEHP dose was associated with elevated bar pressing for food reward during acquisition and extinction while the 20 mg/kg dose was associated with elevated locomotor activity in both males and females. Stereological analysis revealed reduced TH+ densities in the SNc in DEHP- (10 and 20 mg/kg) treated male and female rats. In the VTA, TH+ staining was reduced in male rats treated with 10 or 20 mg/kg DEHP while in females, the TH: CV ratio was higher at the 10 mg/kg dose compared with controls. An examination of Th mRNA showed a main effect of sex with females showing increased Th expression at all DEHP doses. The present results show that preadolescent phthalate exposure results in detrimental dopaminergic system development impacting neurobehavioral function in post-adolescent rats.

Metabolic suppression in the pelagic crab, Pleuroncodes planipes, in oxygen minimum zones.

The pelagic red crab, Pleuroncodes planipes, is abundant throughout the Eastern Tropical Pacific in both benthic and pelagic environments to depths of several hundred meters. The oxygen minimum zones in this region reaches oxygen levels as low as 0.1kPa at depths within the crabs vertical range. Crabs maintain aerobic metabolism to a critical PO2 of ~0.27±0.2kPa (10°C), in part by increasing ventilation as oxygen declines. At subcritical oxygen levels, they enhance anaerobic ATP production slightly as indicated by modest increases in lactate levels. However, hypoxia tolerance is primarily mediated via a pronounced suppression of aerobic metabolism (~70%). Metabolic suppression is achieved, primarily, via reduced protein synthesis, which is a major sink for metabolic energy. Posttranslational modifications on histone H3 suggest a condensed chromatin state and, hence, decreased transcription. Under hypoxia, p-H3S10, Ac-H3K9, Ac-H3K14 were 39, 68, and 36% of control values, respectively. We also report a net decrease in protein translation. In particular, eEF2 activity is reduced due to a ~5-fold increase in inhibitory phosphorylation and a significant decrease in protein level. Elevated heat shock proteins suggest that, despite impressive tolerance, the cellular stress response is triggered during hypoxia. We discuss the implications for pelagic ecology and biogeochemical cycles.

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 4. Regulation of pyruvate dehydrogenase complex and metabolic fuel selection.

Mammalian hibernation is characterized by extensive adjustments to metabolism that typically include suppression of carbohydrate catabolism and a switch to triglycerides as the primary fuel during torpor. A crucial locus of control in this process is the pyruvate dehydrogenase complex that gates carbohydrate entry into the tricarboxylic acid cycle. Within the complex, the E1 enzyme pyruvate dehydrogenase (PDH) is the main regulatory site and is subject to inhibitory phosphorylation at three serine residues (S232, S293, S300). To determine if marsupial hibernators show a comparable focus on PDH to regulate fuel metabolism, the current study explored PDH control by site-specific phosphorylation in the South American marsupial, monito del monte (Dromiciops gliroides). Luminex multiplex technology was used to analyze PDH responses in six tissues comparing control and hibernating (4days continuous torpor) animals. Total PDH content did not change significantly during hibernation in any tissue but phospho-PDH content increased in all. Heart PDH showed increased phosphorylation at all three sites by 8.1-, 10.6- and 2.1-fold for S232, S293 and S300, respectively. Liver also showed elevated p-S300 (2.5-fold) and p-S293 (4.7-fold) content. Phosphorylation of S232 and S293 increased significantly in brain and lung but only S232 phosphorylation increased in kidney and skeletal muscle. The results show that PDH suppression via enzyme phosphorylation during torpor is a conserved mechanism for inhibiting carbohydrate catabolism in both marsupial and eutherian mammals, an action that would also promote the switch to fatty acid oxidation instead.

Strategies of biochemical adaptation for hibernation in a South American marsupial Dromiciops gliroides: 1. Mitogen-activated protein kinases and the cell stress response.

Hibernation is a period of torpor and heterothermy that is typically associated with a strong reduction in metabolic rate, global suppression of transcription and translation, and upregulation of various genes/proteins that are central to the cellular stress response such as protein kinases, antioxidants, and heat shock proteins. The current study examined cell signaling cascades in hibernating monito del monte, Dromiciops gliroides, a South American marsupial of the Order Microbiotheria. Responses to hibernation by members of the mitogen-activated protein kinase (MAPK) pathways, and their roles in coordinating hibernator metabolism were examined in liver, kidney, heart and brain of control and versus hibernating (4days continuous torpor) D. gliroides. The targets evaluated included key protein kinases in their activated phosphorylated forms (p-ERK/MAPK 1/2, p-MEK1, p-MSK1, p-p38, p-JNK) and related target proteins (p-CREB 2, p-ATF2, p-c-Jun and p-p53). Liver exhibited a strong coordinated response by MAPK members to hibernation with significant increases in protein phosphorylation levels of p-MEK1, p-ERK/MAPK1/2, p-MSK1, p-JNK and target proteins c-Jun, and p-ATF2, all combining to signify a strong activation of MAPK signaling during hibernation. Kidney also showed activation of MAPK cascades with significant increases in p-MEK1, p-ERK/MAPK1/2, p-p38, and p-c-Jun levels in hibernating animals. By contrast, responses by heart and brain indicated reduced MAPK pathway function during torpor with reduced phosphorylation of targets including p-ERK/MAPK 1/2 in both tissues as well as lower p-p38 and p-JNK content in heart. Overall, the data indicate a vital role for MAPK signaling in regulating the cell stress response during marsupial hibernation.

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 2. Control of the Akt pathway and protein translation machinery.

When faced with harsh environmental conditions, the South American marsupial, monito del monte (Dromiciops gliroides), reduces its body temperature and uses either daily torpor or multiday hibernation to survive. This study used ELISA and multiplex assays to characterize the responses to hibernation by three regulatory components of protein translation machinery [p-eIF2α(S51), p-eIF4E(S209), p-4EBP(Thr37/46)] and eight targets involved in upstream signaling control of translation [p-IGF-1R(Tyr1135/1136), PTEN(S380), p-Akt(S473), p-GSK-3α(S21), p-GSK-3ß(S9), p-TSC2(S939), p-mTOR(S2448), and p70S6K(T412)]. Liver, brain and kidney were analyzed comparing control and hibernation (4days continuous torpor) conditions. In the liver, increased phosphorylation of IGF-1R, Akt, GSK-3ß, TSC2, mTOR, eIF2α, and 4EBP (1.60-1.98 fold compared to control) occurred during torpor suggesting that the regulatory phosphorylation cascade and protein synthesis remained active during torpor. However, responses by brain and kidney differed; torpor resulted in increased phosphorylation of GSK-3ß (2.15-4.17 fold) and TSC2 (2.03-3.65 fold), but phosphorylated Akt decreased (to 34-62% of control levels). Torpor also led to an increase in phosphorylated eIF2α (1.4 fold) content in the brain. These patterns of differential protein phosphorylation in brain and kidney were indicative of suppression of protein translation but also could suggest an increase in antioxidant and anti-apoptotic signaling during torpor. Previous studies of liver metabolism in hibernating eutherian mammals have shown that Akt kinase and its downstream signaling components play roles in facilitating hypometabolism by suppressing energy expensive anabolic processes during torpor. However, the results in this study reveal differences between eutherian and marsupial hibernators, suggesting alternative actions of liver Akt during torpor.

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 3. Activation of pro-survival response pathways.

The South American marsupial, monito del monte (Dromiciops gliroides) uses both daily torpor and multi-day hibernation to survive in its southern Chile native environment. The present study leverages multiplex technology to assess the contributions of key stress-inducible cell cycle regulators and heat shock proteins to hibernation in liver, heart, and brain of monito del monte in a comparison of control versus 4day hibernating conditions. The data indicate that MDM2, a stress-responsive ubiquitin ligase, plays a crucial role in marsupial hibernation since all three tissues showed statistically significant increases in MDM2 levels during torpor (1.6-1.8 fold). MDM2 may have a cytoprotective action to deal with ischemia/reperfusion stress and is also involved in a nutrient sensing pathway where it could help regulate the metabolic switch to fatty acid oxidation during torpor. Elevated levels of stress-sensitive cell cycle regulators including ATR (2.32-3.91 fold), and the phosphorylated forms of p-Chk1 (Ser345) (1.92 fold), p-Chk2 (Thr68) (2.20 fold) and p21 (1.64 fold) were observed in heart and liver during hibernation suggesting that the cell cycle is likely suppressed to conserve energy while animals are in torpor. Upregulation of heat shock proteins also occurred as a cytoprotective strategy with increased levels of hsp27 (2.00 fold) and hsp60 (1.72-2.76 fold) during hibernation. The results suggest that cell cycle control and selective chaperone action are significant components of hibernation in D. gliroides and reveal common molecular responses to those seen in eutherian hibernators.

The roles of hippocampal microRNAs in response to acute postnatal exposure to di(2-ethylhexyl) phthalate in female and male rats.

Previous studies have shown that di(2-ethylhexyl) phthalate (DEHP) exposure impairs the normal development of pre- and post-synaptic elements of the male, but not female, rat hippocampus. While males seem to be vulnerable to the neurodevelopmental deficits resulting from DEHP exposure, females appear to show a protective response. The purpose of the present study was to characterize hippocampal microRNAs in female and male rats exposed to DEHP to assess whether any patterns emerged that would be consistent with vulnerability in males and resilience in females. Male and female rats were treated with 0, 1, 10, or 20mg/kg of DEHP by intraperitoneal injections from postnatal day 16 (PND16) - PND22 and brains were removed and flash frozen on PND78. A group of 85 microRNAs which have been previously shown to play a role in the development and maintenance of hippocampal neurons was assessed with RT-qPCR. In response to DEHP exposure, there were 19 microRNAs that increased in females and 52 that decreased in males. The strongest microRNA response in females occurred in conjunction with the 10mg/kg of DEHP dose, whereas suppression of microRNAs in males appeared to be dose-dependent. Select hippocampal microRNAs (such as miR-132-3p and miR-191-5p), previously shown to regulate dendrite morphology, were modulated by DEHP exposure in this study. The results suggest that DEHP exposure has the potential to regulate microRNAs in a sex-specific manner which may interfere with proper hippocampal development in males and preserve hippocampal development in females.

MicroRNA regulation in heart and skeletal muscle over the freeze-thaw cycle in the freeze tolerant wood frog.

The North American wood frog, Rana sylvatica, is one of just a few anuran species that tolerates whole body freezing during the winter and has been intensely studied to identify the biochemical adaptations that support freeze tolerance. Among these adaptations is the altered expression of many genes, making freeze-responsive changes to gene regulatory mechanisms a topic of interest. The present study focuses on the potential involvement of microRNAs as one such regulatory mechanism and aims to better understand freeze/thaw stress-induced microRNA responses in the freeze-tolerant wood frog. Using quantitative PCR, relative levels of 53 microRNAs were measured in heart and skeletal muscle of control, 24 h frozen, and 8 h thawed frogs. MicroRNAs showed tissue specific expression patterns: 21 microRNAs decreased in the heart during thawing, whereas 16 microRNAs increased during freezing stress in skeletal muscle. These findings suggest that select genes may be activated and suppressed in heart and skeletal muscle, respectively, in response to freezing. Bioinformatics analysis using the DIANA miRPath program (v.2.0) predicted that the differentially expressed microRNAs may collectively regulate tissue-specific cellular pathways to promote survival of wood frogs undergoing freezing and thawing.