Dehydration mediated microRNA response in the African clawed frog Xenopus laevis

Exposure to various environmental stresses induces metabolic rate depression in many animal species, an adaptation that conserves energy until the environment is again conducive to normal life. The African clawed frog, Xenopus laevis, is periodically subjected to arid summers in South Africa, and utilizes entry into the hypometabolic state of estivation as a mechanism of long term survival. During estivation, frogs must typically deal with substantial dehydration as their ponds dry out and X. laevis can endure > 30% loss of its body water. We hypothesize that microRNAs play a vital role in establishing a reversible hypometabolic state and responding to dehydration stress that is associated with amphibian estivation. The present study analyzes the effects of whole body dehydration on microRNA expression in three tissues of X. laevis. Compared to controls, levels of miR-1, miR-125b, and miR-16-1 decreased to 37 ± 6, 64 ± 8, and 80 ± 4% of control levels during dehydration in liver. By contrast, miR-210, miR-34a and miR-21 were significantly elevated by 3.05 ± 0.45, 2.11 ± 0.08, and 1.36 ± 0.05-fold, respectively, in the liver. In kidney tissue, miR-29b, miR-21, and miR-203 were elevated by 1.40 ± 0.09, 1.31 ± 0.05, and 2.17 ± 0.31-fold, respectively, in response to dehydration whereas miR-203 and miR-34a were elevated in ventral skin by 1.35 ± 0.05 and 1.74 ± 0.12-fold, respectively. Bioinformatic analysis of the differentially expressed microRNAs suggests that these are mainly involved in two processes: (1) expression of solute carrier proteins, and (2) regulation of mitogen-activated protein kinase signaling. This study is the first report that shows a tissue specific mode of microRNA expression during amphibian dehydration, providing evidence for microRNAs as crucial regulators of metabolic depression.     

Subtropical mouse-tailed bats use geothermally heated caves for winter hibernation

We report that two species of mouse-tailed bats (Rhinopoma microphyllum and R. cystops) hibernate for five months during winter in geothermally heated caves with stable high temperature (20°C). While hibernating, these bats do not feed or drink, even on warm nights when other bat species are active. We used thermo-sensitive transmitters to measure the bats’ skin temperature in the natural hibernacula and open flow respirometry to measure torpid metabolic rate at different ambient temperatures (T_a, 16–35°C) and evaporative water loss (EWL) in the laboratory. Bats average skin temperature at the natural hibernacula was 21.7 ± 0.8°C, and no arousals were recorded. Both species reached the lowest metabolic rates around natural hibernacula temperatures (20°C, average of 0.14 ± 0.01 and 0.16 ± 0.04 ml O₂ g⁻¹ h⁻¹ for R. microphyllum and R. cystops, respectively) and aroused from torpor when T_a fell below 16°C. During torpor the bats performed long apnoeas (14 ± 1.6 and 16 ± 1.5 min, respectively) and had a very low EWL. We hypothesize that the particular diet of these bats is an adaptation to hibernation at high temperatures and that caves featuring high temperature and humidity during winter enable these species to survive this season on the northern edge of their world distribution.     

Comparison of Brain Transcriptome of the Greater Horseshoe Bats (Rhinolophus ferrumequinum) in Active and Torpid Episodes

Hibernation is an energy-saving strategy which is widely adopted by heterothermic mammals to survive in the harsh environment. The greater horseshoe bat (Rhinolophus ferrumequinum) can hibernate for a long period in the hibernation season. However, the global gene expression changes between hibernation and non-hibernation season in the greater horseshoe bat remain largely unknown. We herein reported a comprehensive survey of differential gene expression in the brain between winter hibernating and summer active greater horseshoe bats using next-generation sequencing technology. A total of 90,314,174 reads were generated and we identified 1,573 differentially expressed genes between active and torpid states. Interestingly, we found that differentially expressed genes are over-represented in some GO categories (such as metabolic suppression, cellular stress responses and oxidative stress), which suggests neuroprotective strategies might play an important role in hibernation control mechanisms. Our results determined to what extent the brain tissue of the greater horseshoe bats differ in gene expression between summer active and winter hibernating states and provided comprehensive insights into the adaptive mechanisms of bat hibernation.     

miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandin E₂ (PGE₂) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE₂ levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE₂, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.     

Complete mitochondrial genome sequences of three bats species and whole genome mitochondrial analyses reveal patterns of codon bias and lend support to a basal split in Chiroptera

Order Chiroptera is a unique group of mammals whose members have attained self-powered flight as their main mode of locomotion. Much speculation persists regarding bat evolution; however, lack of sufficient molecular data hampers evolutionary and conservation studies. Of ~ 1200 species, complete mitochondrial genome sequences are available for only eleven. Additional sequences should be generated if we are to resolve many questions concerning these fascinating mammals. Herein, we describe the complete mitochondrial genomes of three bats: Corynorhinus rafinesquii, Lasiurus borealis and Artibeus lituratus. We also compare the currently available mitochondrial genomes and analyze codon usage in Chiroptera. C. rafinesquii, L. borealis and A. lituratus mitochondrial genomes are 16438 bp, 17048 bp and 16709 bp, respectively. Genome organization and gene arrangements are similar to other bats. Phylogenetic analyses using complete mitochondrial genome sequences support previously established phylogenetic relationships and suggest utility in future studies focusing on the evolutionary aspects of these species. Comprehensive analyses of available bat mitochondrial genomes reveal distinct nucleotide patterns and synonymous codon preferences corresponding to different chiropteran families. These patterns suggest that mutational and selection forces are acting to different extents within Chiroptera and shape their mitochondrial genomes.     

Experimental ecology and hibernation of Onchidium struma (Gastropoda: Pulmonata: Systellommatophora)

This study investigated both laboratory-reared and pond-cultured subjects to explore the habit and hibernation patterns of the sea slug Onchidium struma (Mollusca, Gastropoda, Systellommatophora, Onchidioidea, Onchidiidae) from Shanghai and Zhejiang in China. Movement and feeding habits and the process of hibernation were observed in culturing tanks from June 2004 to March 2008. Our results showed that the conditions of movement are as follows: a minimum air temperature of 12 °C; a maximum light intensity of 25 lux and a minimum relative air humidity of 72%. Major movement is usually at dusk and during the night, and the average temperatures for fasting and beginning hibernation are 13.8 °C and 11.4 °C respectively. The analysis showed that the temperature is an essential factor affecting movement and feeding of O. struma and that RH and light intensity also play an important role, but are not necessarily required at the same time. In this study, the survival rate of O. struma through hibernation in high-biodiversity culturing tanks is 77.57% ± 2.86%.     

Hibernation does not affect memory retention in bats

Long-term memory can be critically important for animals in a variety of contexts, and yet the extreme reduction in body temperature in hibernating animals alters neurochemistry and may therefore impair brain function. Behavioural studies on memory impairment associated with hibernation have been almost exclusively conducted on ground squirrels (Rodentia) and provide conflicting results, including clear evidence for memory loss. Here, we for the first time tested memory retention after hibernation for a vertebrate outside rodents—bats (Chiroptera). In the light of the high mobility, ecology and long life of bats, we hypothesized that maintenance of consolidated memory through hibernation is under strong natural selection. We trained bats to find food in one out of three maze arms. After training, the pre-hibernation performance of all individuals was at 100 per cent correct decisions. After this pre-test, one group of bats was kept, with two interruptions, at 7°C for two months, while the other group was kept under conditions that prevented them from going into hibernation. The hibernated bats performed at the same high level as before hibernation and as the non-hibernated controls. Our data suggest that bats benefit from an as yet unknown neuroprotective mechanism to prevent memory loss in the cold brain.     

Suppression of MAPKAPK2 during mammalian hibernation

Metabolic signaling coordinates the transition by hibernating mammals from euthermia into profound torpor. Organ-specific responses by activated p38 mitogen activated protein kinase (MAPK) are known to contribute to this transition. Therefore, we hypothesized that the MAPK-activated protein kinase-2 (MAPKAPK2), a downstream target of p38 MAPK, would also be active in establishing the torpid state. Kinetic parameters of MAPKAPK2 from skeletal muscle of Richardson’s ground squirrels, Spermophilus richardsonii, were analyzed using a fluorescence assay. MAPKAPK2 activity was 27.4 ± 1.27 pmol/min/mg in muscle from euthermic squirrels and decreased by ∼63% during cold torpor, while total protein levels were unchanged (as assessed by immunoblotting). In vitro treatment of MAPKAPK2 via stimulation of endogenous phosphatases and addition of commercial alkaline phosphatase decreased enzyme activity to only ∼3–5% of its original value in muscle extracts from both euthermic and hibernating squirrels suggesting that posttranslational modification suppresses MAPKAPK2 during the transition from euthermic to torpid states. Enzyme S_0.5 and n_H values for ATP and peptide substrates changed significantly between euthermia and torpor, and also between assays at 22 versus 10 °C but, kinetic parameters were actually closely conserved when values for the euthermic enzyme at 22 °C were directly compared with the hibernator enzyme at 10 °C. Arrhenius plots showed significantly different activation energies of 40.8 ± 0.7 and 54.3 ± 2.7 kJ/mol for the muscle enzyme from euthermic versus torpid animals, respectively but MAPKAPK2 from the two physiological states showed no difference in sensitivity to urea denaturation. Overall, the results show that total activity of MAPKAPK2 is in fact reduced, despite previous findings of p38 MAPK activation, and kinetic parameters are altered when ground squirrels enter torpor but protein stability is not apparently changed. The data suggest that MAPKAPK2 suppression may have a significant role in the differential regulation of muscle target proteins when ground squirrels enter torpor.     

No fever and leucocytosis in response to a lipopolysaccharide challenge in an insectivorous bat

Bat immune systems may allow them to respond to zoonotic agents more efficiently than other mammals. As the first line of defence, the taxonomically conserved acute phase immune reaction of leucocytosis and fever is crucial for coping with infections, but it is unknown if this response is a key constituent to bat immunological success. We investigated the acute phase reaction to a standard lipopolysaccharide (LPS) challenge in Pallas''s mastiff bats (Molossus molossus). Challenged bats lost mass, but in contrast to other mammals showed no leucocytosis or fever. There also was no influence on body temperature reduction during torpor. When compared to recent genome-wide assays for constituent immune genes, this lack of a conserved fever response to LPS contributes to a clearer understanding of the innate immune system in bat species and of the coevolution of bats with a wide diversity of pathogens.     

The degree of dietary fatty acid unsaturation affects torpor patterns and lipid composition of a hibernator

Diets rich in unsaturated and polyunsaturated fatty acids have a positive effect on mammalian torpor, whereas diets rich in saturated fatty acids have a negative effect. To determine whether the number of double bonds in dietary fatty acids are responsible for these alterations in torpor patterns, we investigated the effect of adding to the normal diet 5% pure fatty acids of identical chain length (C18) but a different number of double bonds (0, 1, or 2) on the pattern of hibernation of the yellow-pine chipmunk, Eutamias amoenus. The response of torpor bouts to a lowering of air temperature and the mean duration of torpor bouts at an air temperature of 0.5°C (stearic acid C18:0, 4.5±0.8 days, oleic acid C18:1, 8.6±0.5 days; linoleic acid C18:2, 8.5±0.7 days) differed among animals that were maintained on the three experimental diets. The mean minimum body temperatures (C18:0, +2.3±0.3°C; C18:1, +0.3±0.2°C; C18:2,-0.2±0.2°C), which torpid individuals defended by an increase in metabolic rate, and the metabolic rate of torpid animals also differed among diet groups. Moreover, diet-induced differences were observed in the composition of total lipid fatty acids from depot fat and the phospholipid fatty acids of cardiac mitochondria. For depot fat 7 of 13 and for heart mitochondria 7 of 14 of the identified fatty acids differed significantly among the three diet groups. Significant differences among diet groups were also observed for the sum of saturated, unsaturated and polyunsaturated fatty acids. These diet-induced alterations of body fatty acids were correlated with some of the diet-induced differences in variables of torpor. The results suggest that the degree of unsaturation of dietary fatty acids influences the composition of tissues and membranes which in turn may influence torpor patterns and thus survival of hibernation.Abbreviations bm body mass - T _a air temperature - T _b body temperature - FA fatty acid - MR metabolic rate - MUFA monounsaturated fatty acids - PUFA polyunsaturated fatty acids - VO₂ rate of oxygen consumption - SFA saturated fatty acids - UFA unsaturated fatty acids - UI unsaturation index - SNK Student-Newman-Keuls test     

Amyloid beta impairs mitochondrial anterograde transport and degenerates synapses in Alzheimer's disease neurons

Loss of synapses and synaptic damage are the best correlates of cognitive decline identified in patients with Alzheimer's disease (AD), and mitochondrial oxidative damage and synaptic pathology have been identified as early events in the progression of AD. The progressive accumulation of amyloid beta (Aβ) in synapses and synaptic mitochondria are hypothesized to cause synaptic degeneration and cognitive decline in patients with AD. However, the precise mechanistic link between Aβ and mitochondria is not well understood. The purpose of this study was to better understand the effects of Aβ on mitochondrial axonal transport and synaptic alterations in AD. Using mouse hippocampal neurons and Aβ_25-35 peptide, we studied axonal transport of mitochondria, including mitochondrial motility, mitochondrial length and size, mitochondrial index per neurite, and synaptic alterations of the hippocampal neurons. In the PBS-treated neurons, 36.4 ± 4.7% of the observed mitochondria were motile, with 21.0 ± 1.3% moving anterograde and 15.4 ± 3.4% moving retrograde and the average speed of movement was 12.1 ± 1.8 μm/min. In contrast, in the Aβ-treated neurons, the number of motile mitochondria were significantly less, at 20.4 ± 2.6% (P < 0.032), as were those moving anterograde (10.1 ± 2.6%, P < 0.016) relative to PBS-treated neurons, suggesting that the Aβ_25-35 peptide impairs axonal transport of mitochondria in AD neurons. In the Aβ-treated neurons, the average speed of motile mitochondria was also less, at 10.9 ± 1.9 μm/min, and mitochondrial length was significantly decreased. Further, synaptic immunoreactivity was also significantly less in the Aβ-treated neurons relative to the PBS-treated neurons, indicating that Aβ affects synaptic viability. These findings suggest that, in neurons affected by AD, Aβ is toxic, impairs mitochondrial movements, reduces mitochondrial length, and causes synaptic degeneration.     

Down-regulation of ether-a-go-go-related gene potassium channel protein through sustained stimulation of AT1 receptor by angiotensin II

We investigated the effects of AT₁ receptor stimulation by angiotensin II (Ang II) on human ether-a-go-go-related gene (hERG) potassium channel protein in a heterogeneous expression system with the human embryonic kidney (HEK) 293 cells which stably expressed hERG channel protein and were transiently transfected with the human AT₁ receptors (HEK293/hERG). Western-blot analysis showed that Ang II significantly decreased the expression of mature hERG channel protein (155-kDa band) in a time- and dose-dependent manner without affecting the level of immature hERG channel protein (135-kDa band). The relative intensity of 155-kDa band was 64.7 ± 6.8% of control (P < 0.01) after treatment of Ang II at 100 nM for 24 h. To investigate the effect of Ang II on the degradation of mature hERG channel protein, we blocked forward trafficking from ER to Golgi with a Golgi transit inhibitor brefeldin A (10 μM). Ang II significantly enhanced the time-dependent reduction of mature hERG channel protein. In addition, the proteasomal inhibitor lactacystin (5 μM) inhibited Ang II-mediated the reduction of mature hERG channel protein, but the lysosomal inhibitor bafilomycin A1 (1 μM) had no effect on the protein. The protein kinase C (PKC) inhibitor bisindolylmaleimide 1 (1 μM) antagonized the reduction of mature hERG channel protein induced by Ang II. The results indicate that sustained stimulation of AT₁ receptors by Ang II reduces the mature hERG channel protein via accelerating channel proteasomal degradation involving the PKC pathway.     

Novel and conserved micrornas in Dalian purple urchin (Strongylocentrotus nudus) identified by next generation sequencing

MicroRNAs are regulators in regulation of broad range of phenotypes. The purple urchin, Strongylocentrotus nudus, is one of the most important marine economic animals that widely distributed in the cold seas along the coasts of eastern pacific area. To date, only 45 microRNAs have been identified in a related species, Strongylocentrotus purpurtus, and there is no report on S. nudus microRNAs. Herein, solexa sequencing technology was used to high throughput sequencing analysis of microRNAs in small RNA library isolated from five tissues of S. nudus. Totally, 8,966,865 reads were yielded, 131,015 of which were related to 415 unique microRNAs including 345 deuterostoma conserved and 70 urchin specific microRNAs, as well as 5 microRNA* sequences. The miRNA features including length distribution, end variations and genomic locations were characterized. Annotation of targets revealed a broad range of biological processes and signal transduction pathways that regulated by urchin miRNAs, of which signal transduction mechanisms was the subgroup containing the maximum targets. In addition, the expression of 100 miRNAs in female gonad was confirmed using microRNA microarray analysis. This study provides a first large scale cloning and characterization of S.nudus miRNAs and their potential targets, providing the foundation for further characterization for their role in the regulation of diversity of physiological processes.