The Impact of Long-Term Hypoxia on the Antioxidant Defense System in the Siberian Frog Rana amurensis.

The Siberian frog Rana amurensis has a uniquely high tolerance to hypoxia among amphibians, as it is able to withstand several months underwater with almost no oxygen (0.2 mg/liter) vs. several days for other studied species. Since it was hypothesized that hypoxia actives the antioxidant defense system in hypoxia-tolerant animals, one would expect similar response in R. amurensis. Here, we studied the effect of hypoxia in the Siberian frog based on the transcriptomic data, activities of antioxidant enzyme, and content of low-molecular-weight antioxidants. Exposure to hypoxia upregulated expression of three relevant transcripts (catalase in the brain and two aldo-keto reductases in the liver). The activities of peroxidase in the blood and catalase in the liver were significantly increased, while the activity of glutathione S-transferase in the liver was reduced. The content of low-molecular-weight antioxidants (thiols and ascorbate) in the heart and liver was unaffected. In general, only a few components of the antioxidant defense system were affected by hypoxia, while most remained unchanged. Comparison to other hypoxia-tolerant species suggests species-specific adaptations to hypoxia-related ROS stress.

Defining components of early thyroid hormone signalling through temperature-mediated activation of molecular memory in cultured Rana [lithobates] catesbeiana tadpole back skin.

Thyroid hormones (THs) are essential signalling molecules for the postembryonic development of all vertebrates. THs are necessary for the metamorphosis from tadpole to froglet and exogenous TH administration precociously induces metamorphosis. In American bullfrog (Rana [Lithobates] catesbeiana) tadpoles, the TH-induced metamorphosis observed at a warm temperature (24 °C) is arrested at a cold temperature (4 °C) even in the presence of exogenous THs. However, when TH-exposed tadpoles are shifted from cold to warm temperatures (4 â†’ 24 °C), they undergo TH-dependent metamorphosis at an accelerated rate even when the initial TH signal is no longer present. Thus, they possess a "molecular memory" of TH exposure that establishes the TH-induced response program at the cold temperature and prompts accelerated metamorphosis after a shift to a warmer temperature. The components of the molecular memory that allow the uncoupling of initiation from the execution of the metamorphic program are not understood. To investigate this, we used cultured tadpole back skin (C-Skin) in a repeated measures experiment under 24 °C only, 4 °C only, and 4 â†’ 24 °C temperature shifted regimes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RNA-sequencing (RNA-seq) analyses. RNA-seq identified 570, 44, and 890 transcripts, respectively, that were significantly changed by TH treatment. These included transcripts encoding transcription factors and proteins involved in mRNA structure and stability. Notably, transcripts associated with molecular memory do not overlap with those identified previously in cultured tail fin (C-fin) except for TH-induced basic leucine zipper-containing protein (thibz) suggesting that thibz may have a central role in molecular memory that works with tissue-specific factors to establish TH-induced gene expression programs.

Investigation of seasonal changes in lipid synthesis and metabolism-related genes in the oviduct of Chinese brown frog (Rana dybowskii).

A peculiar physiological characteristic of the Chinese brown frog (Rana dybowskii) is that its oviduct dilates during pre-brumation rather than during the breeding season. This research aimed to examine the expression of genes connected with lipid synthesis and metabolism in the oviduct of R. dybowskii during both the breeding season and pre-brumation. We observed significant changes in the weight and size of the oviduct between the breeding season and pre-brumation. Furthermore, compared to the breeding season, pre-brumation exhibited significantly lower triglyceride content and a marked increase in free fatty acid content. Immunohistochemical results revealed the spatial distribution of triglyceride synthase (Dgat1), triglyceride hydrolase (Lpl and Hsl), fatty acid synthase (Fasn), and fatty acid oxidases (Cpt1a, Acadl, and Hadh) in oviductal glandular cells and epithelial cells during both the breeding season and pre-brumation. While the mRNA levels of triglycerides and free fatty acid synthesis genes (dgat1 and fasn) did not show a significant difference between the breeding season and pre-brumation, the mRNA levels of genes involved in triglycerides and free fatty acid metabolism (lpl, cpt1a, acadl, acox and hadh) were considerably higher during pre-brumation. Furthermore, the R. dybowskii oviduct's transcriptomic and metabolomic data confirmed differential expression of genes and metabolites enriched in lipid metabolism signaling pathways during both the breeding season and pre-brumation. Overall, these results suggest that alterations in lipid synthesis and metabolism during pre-brumation may potentially influence the expanding size of the oviduct, contributing to the successful overwintering of R. dybowskii.

Hepatic transcriptome and gut microbiome provide insights into freeze tolerance in the high-altitude frog, Nanorana parkeri.

Among amphibians, freeze tolerance is a low-temperature survival strategy that has been well studied in several species. One influence on animal health and survival under adverse conditions is the gut microbiome. Gut microbes can be greatly affected by temperature fluctuations but, to date, this has not been addressed in high-altitude species. Nanorana parkeri (Anura: Dicroglossidae) lives at high altitudes on the Tibetan plateau and shows a good freeze tolerance. In the present study, we addressed two goals: (1) analysis of the effects of whole body freezing on the liver transcriptome, and (2) assess modifications of the gut microbiome as a consequence of freezing. We found that up-regulated genes in liver were significantly enriched in lipid and fatty acid metabolism that could contribute to accumulating the cryoprotectant glycerol and raising levels of unsaturated fatty acids. The results suggest the crucial importance of membrane adaptations and fuel reserves for freezing survival of these frogs. Down-regulated genes were significantly enriched in the immune response and inflammatory response, suggesting that energy-consuming processes are inhibited to maintain metabolic depression during freezing. Moreover, freezing had a significant effect on intestinal microbiota. The abundance of bacteria in the family Lachnospiraceae was significantly increased after freezing exposure, which likely supports freezing survival of N. parkeri. The lower abundance of bacteria in the family Peptostreptococcaceae in frozen frogs may be associated with the hypometabolic state and decreased immune response. In summary, these findings provide insights into the regulatory mechanisms of freeze tolerance in a high-altitude amphibian at the level of gene expression and gut microbiome, and contribute to enhancing our understanding of the adaptations that support frog survival in high-altitude extreme environments.

Expression of cholesterol synthesis and steroidogenic markers in females of the Chinese brown frog (Rana dybowskii) during prespawning and prehibernation.

The oviduct of the Chinese brown frog (Rana dybowskii) expands in prehibernation rather than in prespawning, which is one of the physiological phenomena that occur in the preparation for hibernation. Steroid hormones are known to regulate oviductal development. Cholesterol synthesis and steroidogenesis may play an important role in the expansion of the oviduct before hibernation. In this study, we investigated the expression patterns of the markers that are involved in the de novo steroid synthesis pathway in the oviduct of R. dybowskii during prespawning and prehibernation. According to histological analysis, the oviduct of R. dybowskii contains epithelial cells, glandular cells, and tubule lumens. During prehibernation, oviductal pipe diameter and weight were significantly larger than during prespawning. 3-Hydroxy-3-methylglutaryl CoA reductase (HMGCR), low-density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) were detected in epithelial cells in prehibernation and glandular cells during prespawning. HMGCR, LDLR, StAR, and P450scc protein expression levels were higher in prehibernation than during prespawning, but the SF-1 protein expression level did not significantly differ. HMGCR, LDLR, StAR, P450scc (CYP11A1), and SF-1 (NR5A1) mRNA expression levels were significantly higher in prehibernation compared with prespawning. The transcriptome results showed that the steroid synthesis pathway was highly expressed during prehibernation. Existing results indicate that the oviduct is able to synthesize steroid hormones using cholesterol, and that steroid hormones may affect the oviductal functions of R. dybowskii.

Effects of perchlorate and exogenous T4 on growth, development and tail resorption of Rana chensinensis.

Endocrine disruptors have been demonstrated to exert adverse effects on growth and development of amphibians by disrupting hormone levels. Tail resorption, which is one of the most remarkable events during amphibian metamorphosis, is closely associated with thyroid hormones levels. However, limited research has been conducted on the effects of endocrine disruptors on tail resorption in amphibians. This study explored the effects of NaClO4 and T4 on the growth, development and tail resorption during the metamorphosis of Rana Chensinensis. The results demonstrated that exposure to NaClO4 led to an increase in body size and a delay in metamorphosis of R. Chensinensis tadpoles. Histological analysis revealed that both NaClO4 and exogenous T4 exposure resulted in thyroid gland injury, and NaClO4 treatment delayed the degradation of notochord and muscles, thereby delaying tail resorption. Moreover, transcriptome sequencing results showed that apoptosis-related genes (APAF1, BAX and CASP6) and cell component degradation-related genes (MMP9 and MMP13) were highly expressed in the T4 exposure group, and the expression of oxidative stress-related genes (SOD and CAT) was higher in the NaClO4 exposure group. Taken together, both NaClO4 and exogenous T4 affect tail resorption in R. Chensinensis, thereby affecting their adaptation to terrestrial life. The present study will not only provide a reference for future experimental research on the effects of other endocrine disruptors on the growth, development and tail resorption of amphibians but will also provide insights into environmental protection and ecological risk assessment.

Decreased acetylation of HDGF improves oviduct production in Rana dybowskii, Rana amurensis, and Rana huanrenensis.

The oviduct of female Rana dybowskii is a functional food and can be used as a component of Traditional Chinese medicine. The differentially expressed genes enriched was screened in cell growth of three Rana species. We quantitatively analyzed 4549 proteins using proteomic techniques, enriching the differentially expressed proteins of Rana for growth and signal transduction. The results showed that log2 expression of hepatoma-derived growth factor (HDGF) was increased. We further verified 5 specific differential genes (EIF4a, EIF4g, HDGF1, HDGF2 and SF1) and found that HDGF expression was increased in Rana dybowskii. Through acetylation modification analysis, we identified 1534 acetylation modification sites in 603 proteins, including HDGF, and found that HDGF acetylation expression was significantly reduced in Rana dybowskii. Our results suggest that HDGF is involved in the development of oviductus ranae, which is regulated by acetylation modification.

Study effect and mechanism of ofloxacin and levofloxacin on development of Rana nigromaculata tadpoles based on the hypothalamus-pituitary-thyroid axis.

Excessive antibiotics transferred into the aquatic environment may affect the development of amphibians. Previous studies on the aquatic ecological risk of ofloxacin generally ignored its enantiomers. The purpose of this study was to compare the effects and mechanisms of ofloxacin (OFL) and levofloxacin (LEV) on the early development of Rana nigromaculata. After 28-day exposure at environmental levels, we found that LEV exerted more severe inhibitory effects on the development of tadpoles than OFL. According to the enrichment results of differentially expressed genes in the LEV and OFL treatments, LEV and OFL had different effects on the thyroid development of tadpoles. dio2 and trh were affected by the regulation of dexofloxacin instead of LEV. At the protein level, LEV was the main component that affected thyroid development-related protein, while dexofloxacin in OFL had little effect on thyroid development. Furthermore, molecular docking results further confirmed that LEV was a major component affecting thyroid development-related proteins, including DIO and TSH. In summary, OFL and LEV regulated the thyroid axis by differential binding to DIO and TSH proteins, thereby exerting differential effects on the thyroid development of tadpoles. Our research is of great significance for comprehensive assessment of chiral antibiotics aquatic ecological risk.

Hepatic citrate synthase suppression in the freeze-tolerant wood frog (Rana sylvatica).

The wood frog, Rana sylvatica endures whole body freezing for weeks/months while overwintering at subzero temperatures. Survival of long-term freezing requires not only cryoprotectants but also strong metabolic rate depression (MRD) and reorganization of essential processes in order to maintain a balance between ATP-producing and ATP-consuming processes. Citrate synthase (CS) (E.C. 2.3.3.1) is an important irreversible enzyme of the tricarboxylic acid (TCA) cycle and forms a crucial checkpoint for many metabolic processes. Present study investigated the regulation of CS from wood frog liver during freezing. CS was purified to homogeneity by a two-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and, notably, demonstrated a significant decrease in the Vmax of the purified form of CS from frozen frogs as compared to controls when assayed at both 22 °C and 5 °C. This was further supported by a decrease in the maximum activity of CS from liver of frozen frogs. Immunoblotting also showed changes in posttranslational modifications with a significant decrease in threonine phosphorylation (by 49 %) for CS from frozen frogs. Taken together, these results suggest that CS is suppressed and TCA flux is inhibited during freezing, likely to support MRD survival of harsh winters.

A frog-derived antimicrobial peptide as a potential anti-biofilm agent in combating Staphylococcus aureus skin infection.

Staphylococcus aureus (S. aureus), one of the most prevalent bacteria found in atopic dermatitis lesions, can induce ongoing infections and inflammation by downregulating the expression of host defence peptides in the skin. In addition, the emergence of the 'superbug' Methicillin-resistant S. aureus (MRSA) has made the treatment of these infections more challenging. Antimicrobial peptides (AMPs), due to their potent antimicrobial activity, limited evidence of resistance development, and potential immunomodulatory effects, have gained increasing attention as potential therapeutic agents for atopic dermatitis. In this study, we report a novel AMP, brevinin-1E-OG9, isolated from the skin secretions of Odorrana grahami, which shows potent antibacterial activity, especially against S. aureus. Based on the characteristics of the 'Rana Box', we designed a set of brevinin-1E-OG9 analogues to explore its structure-activity relationship. Brevinin-1E-OG9c-De-NH2 exhibited the most potent antimicrobial efficacy in both in vitro and ex vivo studies and attenuated inflammatory responses induced by lipoteichoic acid and heat-killed microbes. As a result, brevinin-1E-OG9c-De-NH2 might represent a promising candidate for the treatment of S. aureus skin infections.

Peptidomic analysis of the host-defense peptides in skin secretions of the Amazon River frog Lithobates palmipes (Ranidae).

Skin secretions of certain frog species represent a source of host-defense peptides (HDPs) with therapeutic potential and their primary structures provide insight into taxonomic and phylogenetic relationships. Peptidomic analysis was used to characterize the HDPs in norepinephrine-stimulated skin secretions from the Amazon River frog Lithobates palmipes (Ranidae) collected in Trinidad. A total of ten peptides were purified and identified on the basis of amino acid similarity as belonging to the ranatuerin-2 family (ranatuerin-2PMa, -2PMb, -2PMc, and-2PMd), the brevinin-1 family (brevinin-1PMa, -1PMb, -1PMc and des(8-14)brevinin-1PMa) and the temporin family (temporin-PMa in C-terminally amidated and non-amidated forms). Deletion of the sequence VAAKVLP from brevinin-1PMa (FLPLIAGVAAKVLPKIFCAISKKC) in des[(8-14)brevinin-1PMa resulted in a 10-fold decrease in potency against Staphylococcus aureus (MIC = 31 µM compared with 3 µM) and a > 50-fold decrease in hemolytic activity but potency against Echerichia coli was maintained (MIC = 62.5 µM compared with 50 µM). Temporin-PMa (FLPFLGKLLSGIF.NH2) inhibited growth of S. aureus (MIC = 16 µM) but the non-amidated form of the peptide lacked antimicrobial activity. Cladistic analysis based upon the primary structures of ranaturerin-2 peptides supports the division of New World frogs of the family Ranidae into the genera Lithobates and Rana. A sister-group relationship between L. palmipes and Warszewitsch's frog Lithobates warszewitschii is indicated within a clade that includes the Tarahumara frog Lithobates tarahumarae. The study has provided further evidence that peptidomic analysis of HDPs in frog skin secretions is a valuable approach to elucidation of the evolutionary history of species within a particular genus.

Characterization of a novel LTA/LPS-binding antimicrobial and anti-inflammatory temporin peptide from the skin of Fejervary limnocharis (Anura: Ranidae).

Septic shock caused by Gram-positive bacteria continues to be a major cause of morbidity and mortality in intensive care units globally. Most Temporins are excellent growth inhibitors of gram-positive bacteria and candidates for developing antimicrobial treatments due to their biological action and small molecular weight. In this study, a novel Temporin peptide from the skin of Fejervarya limnocharis frog, named as Temporin-FL, was characterized. Temporin-FL was found to adopt typical α-helical conformation in SDS solution and to exhibit selective antibacterial activity against Gram-positive bacteria through a membrane destruction mechanism. Accordingly, Temporin-FL showed protective effects against Staphylococcus aureus-induced sepsis in mice. Finally, Temporin-FL was demonstrated to exert anti-inflammatory effects by neutralizing the action of LPS/LTA and by inhibiting MAPK pathway activation. Therefore, Temporin-FL represents a novel candidate for moleculartherapy of Gram-positive bacterial sepsis.

Role of NADP+-dependent isocitrate dehydrogenase from muscle tissue of Rana sylvatica in ROS defense during freeze-tolerance.

The wood frog, Rana sylvatica, employs freeze tolerance as a winter survival strategy in seasonally cold environments. At subzero temperatures, up to 65-70% of total body water can freeze in extracellular spaces, halting vital functions (breathing, heartbeat) and causing ischemia that, in turn, can have numerous consequences including the generation of damaging reactive oxygen species (ROS). NADPH serves as a key donor of reductive power for most ROS detoxifying enzymes and can be generated by several metabolic pathways. One source of NADPH reducing power is the NADP-dependent isocitrate dehydrogenase (IDH) reaction. The present study evaluated the properties and regulation of IDH from skeletal muscle of R. sylvatica when frogs were exposed to stress conditions: freezing, dehydration or anoxia. Purified IDH exhibited higher affinity for isocitrate under all stress conditions as compared to controls, suggesting that the enzyme is primed to synthesize NADPH relative to the control state. Immunoblotting showed reduced serine and threonine phosphorylation of muscle IDH from frozen frogs and decreased serine phosphorylation on IDH from dehydrated frogs relative to control and anoxic states, demonstrating a reversible phosphorylation regulatory mechanism for IDH activity during freezing stress. Taken together, these results suggest activation and maintenance of IDH activity despite hypometabolic conditions. This initiation in activity of IDH during freezing may play a role in antioxidant defense by contributing to maintenance of the NADPH pool under stress conditions.

TXNIP shuttling - a key molecular link in regulating inflammation and mitochondrial dysfunction in freeze tolerant wood frogs.

Amphibians such as the wood frogs,Rana sylvatica, are a primary example of a freeze-tolerant vertebrate that undergoes whole body freezing. Multiple adaptations including sequestering 65-70% of total body water as extracellular/extra organ ice and producing massive amounts of glucose as a cryoprotectant support this. Interestingly, the high glucose levels induced in response to freezing can amplify oxidative stress's effects (reactive oxygen species, ROS) and induce inflammation and mitochondrial dysfunction. Since both freezing and dehydration stress (independent of freezing) can render wood frogs hyperglycemic, this study focussed on these two stresses to elucidate the role of a scaffold protein thioredoxin interacting protein (TXNIP), which localizes in multiple compartments inside the cell under hyperglycemic conditions and mediate diverse stress responses. The results from this study suggest a stress-specific response of TXNIP in inducing the cell-damaging pathway of inflammasome activation via its cytoplasmic localization during freezing. Interestingly, mitochondrial localization of TXNIP did not leads to increase in its binding to thioredoxin 2 (TRX-2) and activating the dysfunction of this organelle by releasing a mitochondrial protein cytochrome c (Cyt c) in cytoplasm under both freezing and dehydration stresses. Post-translational modifications of TXNIP hinted on changes in the regulating proteins involved in the inflammasome and mitochondrial dysfunction pathways, whereas sequential differences (cytosine residues) of amphibian TXNIP (compared to mammalian) assessed via 3D-modeling attributed to its weak binding to TRX-2. Overall, this study summarizes differential role of proteins activated under freeze and dehydration induced hyperglycemic response in freeze tolerant wood frogs.

mRNA m6 A methylation in wood frog brain is maintained during freezing and anoxia.

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 (N6 -methyladenosine; m6 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 m6 A methyltransferases METTL3 and METTL14 was observed. In addition, we investigated the effect of m6 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.

Role of FOXO transcription factors in the tolerance of whole-body freezing in the wood frog, Rana sylvatica.

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.

Freeze-induced suppression of pyruvate kinase in liver of the wood frog (Rana sylvatica).

The wood frog (Rana sylvatica) undergoes physiological and metabolic changes to withstand subzero temperatures and whole body freezing during the winter months. Along with metabolic rate depression, high concentrations of glucose are produced as a cryoprotectant by liver and distributed to all other tissues. Pyruvate kinase (PK; EC:2.7.1.40), the final enzyme of glycolysis, plays an important role in the modulation of glucose metabolism and, therefore, overall metabolic regulation. The present study investigated the functional and kinetic properties of purified PK from liver of control (5 °C acclimated) and frozen (-2.5 °C for 24 h) wood frogs. Liver PK was purified to homogeneity by a two-step chromatographic process, followed by analysis of enzyme properties. A significant decrease in the affinity of PK for its substrates, phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) at 22 °C and 5 °C was noted in liver from frozen frogs, as compared with controls. Immunoblotting also revealed freeze-responsive changes in posttranslational modifications with a significant increase in serine and threonine phosphorylation by 1.46-fold and 1.73- fold for PK from frozen frogs as compared with controls. Furthermore, a test of thermal stability showed that PK from liver of frozen wood frogs showed greater stability as compared with PK from control animals. Taken together, these results suggest that PK is negatively regulated, and glycolysis is suppressed, during freezing. This response acts as an important survival strategy for maintaining continuously elevated levels of cryoprotectant in frogs while they remain in a hypometabolic frozen state.

Hypoxia inducible factor-1α responds to freezing, anoxia and dehydration stresses in a freeze-tolerant frog.

The wood frog, Rana sylvatica (aka Lithobates sylvaticus) is the main model for studies of natural freeze tolerance among amphibians living in seasonally cold climates. During freezing, ∼65% of total body water can be converted to extracellular ice and this imposes both dehydration and hypoxia/anoxia stresses on cells. The current study analyzed the responses of the alpha subunit of the hypoxia-inducible transcription factor (HIF-1), a crucial oxygen-sensitive regulator of gene expression, to freezing, anoxia or dehydration stresses, examining six tissues of wood frogs (liver, skeletal muscle, brain, heart, kidney, skin). RT-PCR revealed a rapid elevation hif-1α transcript levels within 2 h of freeze initiation in both liver and brain and elevated levels of both mRNA and protein in liver and muscle after 24 h frozen. However, both transcript and protein levels reverted to control values after thawing except for HIF-1 protein in liver that dropped to ∼60% of control. Independent exposures of wood frogs to anoxia or dehydration stresses (two components of freezing) also triggered upregulation of hif-1α transcripts and/or HIF-1α protein in liver and kidney with variable responses in other tissues. The results show active modulation of HIF-1 in response to freezing, anoxia and dehydration stresses and implicate this transcription factor as a contributor to the regulation of metabolic adaptations needed for long term survival of wood frogs in the ischemic frozen state.

Role of unfolded protein response and ER-associated degradation under freezing, anoxia, and dehydration stresses in the freeze-tolerant wood frogs.

The North American amphibian, wood frogs, Rana sylvatica are the most studied anuran to comprehend vertebrate freeze tolerance. Multiple adaptations support their survival in frigid temperatures during winters, particularly their ability to produce glucose as natural cryoprotectant. Freezing and its component consequences (anoxia and dehydration) induce multiple stresses on cells. Among these is endoplasmic reticulum (ER) stress, a condition spawned by buildup of unfolded or misfolded proteins in the ER. The ER stress causes the unfolded protein response (UPR) and the ER-associated degradation (ERAD) pathway that potentially could lead to apoptosis. Immunoblotting was used to assess the responses of major proteins of the UPR and ERAD under freezing, anoxia, and dehydration stresses in the liver and skeletal muscle of the wood frogs. Targets analyzed included activating transcription factors (ATF3, ATF4, ATF6), the growth arrest and DNA damage proteins (GADD34, GADD153), and EDEM (ERAD enhancing α-mannosidase-like proteins) and XBP1 (X-box binding protein 1) proteins. UPR signaling was triggered under all three stresses (freezing, anoxia, dehydration) in liver and skeletal muscle of wood frogs with most tissue/stress responses consistent with an upregulation of the primary targets of all three UPR pathways (ATF4, ATF6, and XBP-1) to enhance the protein folding/refolding capacity under these stress conditions. Only frozen muscle showed preference for proteasomal degradation of misfolded proteins via upregulation of EDEM (ERAD). The ERAD response of liver was downregulated across three stresses suggesting preference for more refolding of misfolded/unfolded proteins. Overall, we conclude that wood frog organs activate the UPR as a means of stabilizing and repairing cellular proteins to best survive freezing exposures.

The regulation of m6A-related proteins during whole-body freezing of the freeze-tolerant wood frog.

Rana sylvatica (also known as Boreorana sylvatica) is one of the few vertebrates that spend extreme winters showing no physiological signs of life. Up to 70% of the total body water of the wood frog freezes as extracellular ice. Survival in extreme conditions requires regulation at transcriptional and translational levels to activate prosurvival pathways. N6-methyladenosine (m6A) methylation is one of the most common RNA modifications, regulating transcript processing and translation by executing important functions that affect regulatory pathways in stress conditions. In the study, regulation of m6A-related proteins in the liver of R. sylvatica was analyzed during 24 h frozen and 8 h thaw conditions. Decreases in the activity of demethylases of 28.44 ± 0.4% and 24.1 ± 0.9% of control values in frozen and thaw tissues, respectively, were observed. Total protein levels of m6A methyltransferase complex components methyltransferase-like 14 and Wilm's tumor associated protein were increased by 1.28-fold and 1.42-fold, respectively, during freezing. Demethylase fat mass and obesity, however, showed a decreasing trend, with a significant decrease in abundance during recovery from frozen conditions. Levels of mRNA degraders YTHDF2 and YTHDC2 also decreased under stress. Overall, increased levels of m6A methylation complex components, and suppressed levels of readers/erasers, provide evidence for the potential role of RNA methylation in freezing survival and its regulation in a hypometabolic state.