Effects of hibernation on two important contractile tissues in tibetan frogs, Nanorana parkeri: a perspective from transcriptomics and metabolomics approaches.

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

Endosperm and amyloplast development in waxy wheat cultivars.

The endosperm is an essential part of wheat grains, and the accumulation of amyloplasts in endosperm determines the quality of wheat. Because waxy wheat has a special starch quality, there is a need to understand differences in endosperm and starch morphologies among waxy wheat cultivars. This study investigated differences in the endosperm and amyloplasts of two near-isogenic lines (Shimai19-P and Shimai19-N) and the wheat cultivar Shimai19 during various growth stages using light microscopy and scanning electron microscopy. At 8 days after pollination (DAP), with endosperm development, the amyloplast distributions in the different endosperm regions of the three wheat varieties were in the following order: center of ventral endosperm > subaleurone of ventral endosperm > center of dorsal endosperm > modified aleurone > subaleurone of dorsal endosperm. At 16 DAP, small amyloplasts appeared in the endosperm cells in all three wheat cultivars; subsequently, endosperm cell development until maturity was more rapid in Shimai19-N than in the other varieties. This study revealed variations in amyloplast accumulation among endosperm regions and waxy wheat varieties during wheat grain development, which improved the understanding of nutrient accumulation and nutrient transfer of wheat grains.

Surviving winter on the Qinghai-Xizang Plateau: Extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri.

Changes in protein abundance and reversible protein phosphorylation (RPP) play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes. To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation, we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog, Nanorana parkeri, living on the Qinghai-Xizang (Tibet) Plateau (QTP). In total, 5 170 proteins and 5 695 phosphorylation sites in 1 938 proteins were quantified. Based on proteomic analysis, 674 differentially expressed proteins (438 up-regulated, 236 down-regulated) were screened in hibernating N. parkeri versus summer individuals. Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways, whereas lower expressed proteins were mainly involved in metabolic processes. A total of 4 251 modified sites (4 147 up-regulated, 104 down-regulated) belonging to 1 638 phosphoproteins (1 555 up-regulated, 83 down-regulated) were significantly changed in the liver. During hibernation, RPP regulated a diverse array of proteins involved in multiple functions, including metabolic enzymatic activity, ion transport, protein turnover, signal transduction, and alternative splicing. These changes contribute to enhancing protection, suppressing energy-consuming processes, and inducing metabolic depression. Moreover, the activities of phosphofructokinase, glutamate dehydrogenase, and ATPase were all significantly lower in winter compared to summer. In conclusion, our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.

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.

Polysaccharides from apple pomace exhibit anti-fatigue activity through increasing glycogen content.

The polysaccharides were isolated from apple pomace by hot-water extraction, and their anti-fatigue activity was evaluated in C2C12 muscle myoblasts and male Kunming mice. The purified polysaccharides from apple pomace (PAP) have a molecular weight of 1.74 × 105 Da and were composed of mannose, rhamnose, glucose, galactose and arabinose. In C2C12 myoblasts, PAP showed no cytotoxicity in the concentrations of 0-300 µg/ml. PAP treatment increased the glycogen content, while the ATP content was not affected in C2C12 myoblasts. Further investigation found that the activity and gene expression of glycogen synthase, rather than glycogen phosphorylase, were upregulated by PAP treatment. The studies in vivo showed that PAP treatment did not affect the food intake and weight again in mice. Importantly, PAP prolonged the exhaustive swimming time, increased hepatic and skeletal muscle glycogen levels, and effectively inhibited the accumulation of blood lactic and blood urea nitrogen in mice. Taken together, the results suggested that PAP exhibit anti-fatigue activity in vitro and in vivo through increasing glycogen content.

Effects of acute heat exposure on oxidative stress and antioxidant defenses in overwintering frogs, Nanorana parkeri.

Climate warming is intensifying on the Tibetan Plateau and poses a serious threat to amphibians that live there. Although hibernation physiology and ecology of Nanorana parkeri, a frog species native to the Tibetan plateau, has been well studied, little information is available about the physiological and biochemical responses to acute rising temperature. Here, we conducted an acute warming experiment comparing hibernating N. parkeri (acclimated at 4 °C) and frogs acutely warmed to 10 °C for 12 h, comparing indicators of oxidative stress and antioxidant defense between the two groups. Acute warming led to a significant increase in the content of oxidized glutathione and the ratio of oxidized:reduced glutathione in liver and muscle, indicating that rapid warming causing oxidative stress could be a negative factor for frogs inhabiting the Tibetan plateau. Malondialdehyde content increased by 57% only in muscle but decreased significantly in three tissues. Protein carbonyl groups rose by 15% in brain, 28% in liver, and 21% in muscle after heat exposure but vitamin C content in heart decreased by 44%. Acute heat exposure also induced tissue-specific upregulation of antioxidant enzyme activities. Catalase activity increased by 27% in heat-exposed frogs, as compared to controls, and glutathione peroxidase activity rose by 12% in brain, 30% in liver, and 12% in muscle. Glutathione-S-transferase activity was also enhanced in heart and muscle of heat-exposed frogs. Acute warming also resulted in a rise in total antioxidant capacity in all tissues examined except kidney, relative to controls. In summary, our findings show that acute heat exposure to hibernating N. parkeri causes a tissue-specific increase in oxidative damage and antioxidant defenses, with skeletal muscle being the most affected tissue. These results reveal the physiological responses to acute temperature change in overwintering N. parkeri.

Physiological and Biochemical Adaptations to High Altitude in Tibetan Frogs, Nanorana parkeri.

The Xizang plateau frog, N. parkeri (Anura: Dicroglossidae), is endemic to the Tibetan Plateau, ranging from 2,850 to 5,100 m above sea level. The present study explores physiological and biochemical adaptations to high altitude in this species with a particular emphasis on parameters of hematology, oxidative stress, and antioxidant defense in adult and juvenile N. parkeri collected from high (4,600 m a.s.l) and low (3,400 m a.s.l) altitudes. Hematological results showed that hemoglobin concentration ([Hb]), hematocrit (Hct), and red blood cell (RBC) counts were significantly higher in high-altitude N. parkeri. High-altitude juveniles had lower RBC sizes than low-altitude juveniles. Higher levels of GSH and GSSG were indicated only in juveniles from high altitude, not in adults. High-altitude individuals also showed lower oxidative damage, assessed as malondialdehyde (MDA) and carbonyl groups (CG) in the liver. High-altitude adults also showed higher activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione-S-transferase (GST) as well as total antioxidant capacity (T-AOC) in the liver as compared to low-altitude adults. Moreover, higher GPX activity and T-AOC were observed in the heart and brain of high-altitude adults. Liver CAT, GPX, and T-AOC showed significant increases in high-altitude juveniles. Vitamin C content was also higher in the heart of high-altitude frogs compared to low-altitude individuals. In summary, the high-altitude population of N. parkeri showed more robust hematological parameters, less oxidative damage, and stronger antioxidant defenses than the low-altitude population, all contributing to increased protection for survival in high-altitude environments.

Physiological Ecology of Winter Hibernation by the High-Altitude Frog Nanorana parkeri.

AbstractThe Xizang plateau frog, Nanorana parkeri (Anura: Dicroglossidae), enters a dormant state in the winter in response to seasonal cold and lack of food. To investigate the physiological and ecological characteristics of overwintering in this species, we measured habitat conditions (hibernacula temperatures, body temperature, and water quality variables), morphology, metabolite concentrations, total antioxidant capacity (T-AOC), and bacteria-killing ability (BKA) of plasma during summer and winter. We found that N. parkeri hibernates underwater at the bottom of ponds (10-20-cm depth). Dissolved oxygen content in the water decreases significantly (by 12%) in the winter compared with summer, suggesting that overwintering N. parkeri may experience hypoxia. Body mass, body mass index, hepatosomatic index, and hepatic glycogen concentration all increased significantly in winter-collected frogs as compared to summer-collected individuals, indicating that overwintering N. parkeri accumulates high fuel/energy reserves to support prolonged periods of hibernation. A significant reduction in glucose, urea, and lactate concentrations in most organs may be closely related to metabolic depression in overwintering N. parkeri. Liver lactate concentration rose significantly in winter-collected frogs, suggesting that anaerobic metabolism dominates when this species overwinters. The T-AOC of plasma showed a significant reduction in winter, suggesting a reduced need for antioxidant defenses. Oppositely, the BKA of plasma increased significantly in winter versus summer, indicating that innate immunity was enhanced during overwintering. In summary, these behavioral (migrating to caves), physiological, and biochemical adjustments may be key for the successful overwintering of this high-altitude frog.

Antioxidant and non-specific immune defenses in partially freeze-tolerant Xizang plateau frogs, Nanorana parkeri.

The Xizang plateau frog Nanorana parkeri can tolerate brief and partial freezing of their body. To determine the significant role of antioxidant defense and non-specific immune defense in freezing survival of this species, we assayed parameters of oxidative damage, antioxidant defense and non-specific immune enzymes during freezing exposure (-2 °C for 12 h) in five organs (heart, brain, liver, kidney, and skeletal muscle). The results showed that freezing led to a significant rise in the content of malondialdehyde (MDA) and carbonyl groups (CG) in brain, liver and kidney tissues. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) increased significantly in brain and liver tissues with an augmentation of total antioxidant capacity (T-AOC). Apparent increments in muscle SOD activity and liver GST activity were also observed during freezing exposure. Vitamin C content significantly decreased in liver and kidney but a significant increase occurred in brain. Activities of non-specific immune enzymes, acid phosphatase (ACP) and alkaline phosphatase (AKP), were also assessed. ACP activity was significantly reduced in all five tissues tested whereas AKP activity decreased significantly in four tissues but rose in brain. In summary, freezing is accompanied by oxidative stress in the high-altitude frog, N. parkeri, as documented by increases in the content of MDA and CG in tissues. Freezing exposure also induced tissue-specific changes in the antioxidant defenses showing that activation of antioxidant systems is a part of the survival strategy of this in a high-altitude frog during freezing. Such up-regulation of antioxidant enzymes suggests a particularly important role for them in the liver and brain, serving as an anticipatory mechanism to deal with the ROS challenge during freeze/thaw episodes. Our findings contribute to extending the current understanding of the mechanisms of freeze tolerance in high-altitude frogs.

The effect of long-term cold acclimation on redox state and antioxidant defense in the high-altitude frog, Nanorana pleskei.

Cold hardiness is a key determinant of the distribution and abundance of ectothermic animals, and thermal acclimation can strongly influence stress tolerance phenotypes. However, the effect of cold acclimation on oxidative stress and antioxidant defenses is still not well understood. Here, we investigated the effects of long-term cold exposure (30 days at 4 °C in darkness versus 30 days at 20 °C in natural light) on the redox state and antioxidant defenses of the high-altitude frog, Nanorana pleskei, indigenous to the Tibetan plateau. We found that cold acclimation, under conditions mimicking winter, led to a significant increase in the ratio of oxidized glutathione (GSSG) to its reduced form (GSH) in liver and skeletal muscle tissues, suggesting that cold exposure induced oxidative stress in this species. Furthermore, malondialdehyde (MDA) contents were significantly augmented in heart, liver and muscle, indicating cold-related oxidative damage in these tissues. In the brain, GST activity, total antioxidant capacity (T-AOC), and vitamin C content showed a significant reduction after cold acclimation. In liver, an apparent decrease was also observed in the activities of SOD and GST, as well as T-AOC, whereas CAT and GPX activities showed a prominent increase in cold-acclimated groups. In kidney, there was a significant decrease in most antioxidant enzyme activities except for SOD and GST activity. In skeletal muscle, the activity of SOD, CAT, GR as well as T-AOC significantly decreased but GPX activity showed a significant increase in cold-acclimated frogs. These findings indicate that, in general, cold acclimation induces a suppression of the antioxidant defense system. Overall, our present study systematically describes the responses of antioxidant defenses to long-term cold acclimation and these findings contribute to extending the current understanding of the mechanisms of cold tolerance in high-altitude frogs.

The complete mitochondrial genome of Cochylimorpha cultana (Lederer, 1855) (Lepidoptera: Tortricidae).

The complete mitochondrial genome of Cochylimorpha cultana (Lederer) (Lepidoptera: Tortricidae) was 15,348 bps in size, and comprised 37 genes, which were 13 PCGs, 22 tRNA genes and two rRNA genes. Most PCGs used the conventional ATN start codon, except for cox1 initiating with CGA. Four genes (cox1, cox2, nad4 and nad5) used single T residue as stop condon. 21 out of 22 tRNAs are folded into the cloverleaf secondary structure, except for trnS1. The phylogenetic analysis based on maximum-likelihood (ML) method revealed that the evolutionary status of C. cultana in Tortricinae at the molecular level, which agrees well with the classical taxonomy.

Metabolic responses of plasma to extreme environments in overwintering Tibetan frogs Nanorana parkeri: a metabolome integrated analysis.

Many animals lower their metabolic rate in response to low temperatures and scarcity of food in the winter in phenomena called hibernation or overwintering. Living at high altitude on the Tibetan Plateau where winters are very cold, the frog Nanorana parkeri, survives in one of the most hostile environments on Earth but, to date, relatively little is known about the biochemical and physiological adjustments for overwintering by this species. The present study profiled changes in plasma metabolites of N. parkeri between winter and summer using UHPLC-QE-MS non-target metabolomics in order to explore metabolic adaptations that support winter survival. The analysis showed that, in total, 11 metabolites accumulated and 95 were reduced in overwintering frogs compared with summer-active animals. Metabolites that increased included some that may have antioxidant functions (canthaxanthin, galactinol), act as a metabolic inhibitor (mono-ethylhexylphthalate), or accumulate as a product of anaerobic metabolism (lactate). Most other metabolites in plasma showed reduced levels in winter and were generally involved in energy metabolism including 11 amino acids (proline, isoleucine, leucine, valine, phenylalanine, tyrosine, arginine, tryptophan, methionine, threonine and histidine) and 4 carbohydrates (glucose, citrate, succinate, and malate). Pathway analysis indicated that aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and nitrogen metabolism were potentially the most prominently altered pathways in overwintering frogs. Changes to these pathways are likely due to fasting and global metabolic depression in overwintering frogs. Concentrations of glucose and urea, commonly used as cryoprotectants by amphibians that winter on land, were significantly reduced during underwater hibernation in N. parkeri. In conclusion, winter survival of the high-altitude frog, N. parkeri was accompanied by substantial changes in metabolomic profiles and this study provides valuable information towards understanding the special adaptive mechanisms of N. parkeri to winter stresses.

Enzyme-triggered tyramine-enzyme repeats on prussian blue-gold hybrid nanostructures for highly sensitive electrochemical immunoassay of tissue polypeptide antigen.

A novel sandwich-type electrochemical immunoassay with sensitivity enhancement was developed for quantitative detection of tissue polypeptide antigen (TPA) by coupling with target-induced tyramine signal amplification on prussian blue-gold hybrid nanostructures. The immunosensor was prepared through immobilizing anti-TPA capture antibody on a cleaned screen-printed carbon electrode (SPCE). Prussian blue-gold hybrid nanostructures (PBGNS) labeled with horseradish peroxidase (HRP) and detection antibody were utilized as the signal-transduction tags. Upon target TPA introduction, the sandwiched immunocomplex was formed between capture antibody and detection antibody on the electrode. The carried HRP could trigger the formation of tyramine-HRP repeats on the PBGNS in the presence of H2O2. Using the doped prussian blue as the electron mediator, the conjugated HRP could catalyze the reduction of H2O2. Under the optimal conditions, the catalytic currents increased with the increasing target TPA in the dynamic range from 1.0 pg mL(-1) to 100 ng mL(-1) with a detection limit of 0.3 pg mL(-1). The reproducibility and specificity of the electrochemical immunoassay were acceptable. In addition, the contents of target TPA in nine human serum specimens were evaluated by using the developed electrochemical immunosensor, and the obtained results correlated well with those from commercially enzyme-linked immunosorbent assay (ELISA) method with a correlation coefficient of 0.9975.

[Mechanism of 5'-to-3' degradation of eukaryotic and prokaryotic mRNA].

RNA degradation plays an important role in modulating gene expression and it affects multiple biological processes. There are three common degradation mechanisms of eukaryotic and prokaryotic mRNA: endonucleolytic, 5'-to-3' and 3'-to-5' exonucleolytic degradation. Differences do exist between the two kingdoms. For example, although the 5'-to-3' exoribonucleolytic degradation is the primary degradation mechanism of eukaryotic mRNA, it plays a minimal role in bacteria, and only in Gram-positive bacteria. Recently, novel RNA degradation mechanisms have been revealed, such as a new eukaryotic mRNA decapping mode mediated by 3'-uridylation and a new 3'-to-5' degradation pathway independent of exosome. These accumulating discoveries not only deepen the insight of mRNA degradation mechanisms, but also may contribute to the development of novel therapeutic drugs targeting parasites, viruses or cancer. In this review, we summarize the current knowledge of 5'-to-3' exonucleolytic degradation pathway of eukaryotic and prokaryotic mRNA, and its future therapeutic perspectives.