Down but Not Out: The Role of MicroRNAs in Hibernating Bats

<div><p>MicroRNAs (miRNAs) regulate many physiological processes through post-transcriptional control of gene expression and are a major part of the small noncoding RNAs (snRNA). As hibernators can survive at low body temperatures (T_b) for many months without suffering tissue damage, understanding the mechanisms that enable them to do so are of medical interest. Because the brain integrates peripheral physiology and white adipose tissue (WAT) is the primary energy source during hibernation, we hypothesized that both of these organs play a crucial role in hibernation, and thus, their activity would be relatively increased during hibernation. We carried out the first genomic analysis of small RNAs, specifically miRNAs, in the brain and WAT of a hibernating bat (<i>Myotis ricketti</i>) by comparing deeply torpid with euthermic individual bats using high-throughput sequencing (Solexa) and qPCR validation of expression levels. A total of 196 miRNAs (including 77 novel bat-specific miRNAs) were identified, and of these, 49 miRNAs showed significant differences in expression during hibernation, including 33 in the brain and 25 in WAT (<i>P</i>≤0.01 &│logFC│≥1). Stem-loop qPCR confirmed the miRNA expression patterns identified by Solexa sequencing. Moreover, 31 miRNAs showed tissue- or state-specific expression, and six miRNAs with counts >100 were specifically expressed in the brain. Putative target gene prediction combined with KEGG pathway and GO annotation showed that many essential processes of both organs are significantly correlated with differentially expressed miRNAs during bat hibernation. This is especially evident with down-regulated miRNAs, indicating that many physiological pathways are altered during hibernation. Thus, our novel findings of miRNAs and Interspersed Elements in a hibernating bat suggest that brain and WAT are active with respect to the miRNA expression activity during hibernation.</p></div

DataSheet1_Exploring the deactivation mechanism of human β2 adrenergic receptor by accelerated molecular dynamic simulations.PDF

The β2 adrenergic receptor (β2AR), one of important members of the G protein coupled receptors (GPCRs), has been suggested as an important target for cardiac and asthma drugs. Two replicas of Gaussian accelerated molecular dynamics (GaMD) simulations are performed to explore the deactivation mechanism of the active β2AR bound by three different substrates, including the agonist (P0G), antagonist (JTZ) and inverse agonist (JRZ). The simulation results indicate that the Gs protein is needed to stabilize the active state of the β2AR. Without the Gs protein, the receptor could transit from the active state toward the inactive state. During the transition process, helix TM6 moves toward TM3 and TM5 in geometric space and TM5 shrinks upwards. The intermediate state is captured during the transition process of the active β2AR toward the inactive one, moreover the changes in hydrophobic interaction networks between helixes TM3, TM5, and TM6 and the formation of a salt bridge between residues Arg3.50 and Glu6.30 drive the transition process. We expect that this finding can provide energetic basis and molecular mechanism for further understanding the function and target roles of the β2AR.</p

Overview of small RNA gene expression in four bat libraries generated by Solexa deep sequencing.

<p>(A) Length distribution of perfectly matched small RNA reads. The percentage of total or distinct (unique) small RNA reads that perfectly matched the reference genome are shown. (B-F) Breakdown of the proportions (in percentage) of various classes of small RNAs detected by sequencing of total/all combined, brain (HB, AB), and WAT (HA, AA). Various classes of small RNAs are shown by percentages. The miRNA family comprises the majority of small RNAs (47.7% in total). snoRNA, small nucleolar RNA; rRNA, ribosomal RNA; tRNA, transfer RNA; unknown, derived from unannotated/intergenic regions.</p

49 differentially expressed miRNAs identified by Solexa sequencing in the brain or adipose tissues during hibernation.

<p>49 differentially expressed miRNAs identified by Solexa sequencing in the brain or adipose tissues during hibernation.</p

Illustration of genomic repeat-derived reads in four libraries.

<p>LINE, Long INterspersed Elements; SINE, Short INterspersed Elements; LTR, Transposable elements with Long Terminal Repeats; DNA, DNA transposons; SSR, Simple Sequence Repeats; Low, Low Complexity Sequences; unknown, derived from unannotated/intergenic regions.</p

Co-regulation of miRNAs and target mRNAs in the adipocytokine signaling pathway.

<p>(A) Expression pattern and relationship of differentially expressed miRNAs (left) and mRNAs involved in the adipocytokine signaling pathway (right, identified by DGE, unpublished data). The expression profile of a miRNA or gene in the brain (HB/AB) and adipose tissue (HA/AA) was calculated by sequence counts (TPM). The red and green colors represent high and low gene expression after comparing the hibernating state vs. the active state. Differentially expressed miRNAs and mRNAs (<i>P</i>≤0.01) during hibernation are linked with red lines; (B) Network of adipocytokine signaling pathway. Red box, predicted target genes of differentially expressed miRNAs that were also identified by DGE; Blue box, predicted target genes of differentially expressed miRNAs, but that were not identified by DGE; Green box, genes identified by DGE.</p

MiRNA expression patterns in four bat libraries.

<p>The expression of miRNAs identified in this study were calculated by sequence counts (Transcript Per Million, TPM). Heat maps represent the clustering of miRNAs (up) and the Venn diagram shows the number of miRNAs in each library (down). (A) 119 conserved miRNAs match to the Metazoan mature miRNAs in Sanger miRBase; (B) 77 Novel miRNAs identified by manual screening. Details of differentially expressed miRNAs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135064#pone.0135064.s010" target="_blank">S7 Table</a>. HB: hibernating state brain; AB: active state brain; HA: hibernating state adipose tissue; AA: active state adipose tissue.</p

Identification of Proteasome Subunit Beta Type 6 (PSMB6) Associated with Deltamethrin Resistance in Mosquitoes by Proteomic and Bioassay Analyses

<div><p>Deltamethrin (DM) insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (<i>PSMB6</i>) is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of <i>PSMB6</i> in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of <i>PSMB6</i>. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, <i>PSMB6</i> knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that <i>PSMB6</i> is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control.</p></div

The Profile of 27 Proteins Identified by 2-DE.

<p>Profile of differentially expressed proteins in DM-resistant cells. Acc. no.: Swiss-Prot or TrEMBL database accession number; Protein description: name of protein in the Swiss-Prot or TrEMBL database; No of matched peptides: number of peptides matched to the candidate protein (the number of observed peptides); Score: Mowse Score, scores greater than 67 are considered statistically significant (<i>p</i><0.05); Sequence coverage: identified sequence as a percentage of the complete sequence; Mr: molecular weight; pI: theoretical isoelectric point; Folds of resistant/susceptible: describes the fold changes of the protein expression level in resistant cell compared with that in susceptible cell; <i>P</i>-value: Statistical significance of the fold-change in protein expression;</p>a<p>Up-regulated proteins.</p>b<p>Down-regulated proteins.</p>c<p>This protein spot contains more than one protein.</p

Proteasome inhibitor enhanced cell susceptibility to DM.

<p>Resistant and susceptible mosquito cells were exposed to DM at the indicated concentrations after pre-treatment with 1 µM MG-132 (A) or 0.1 µM bortezomib (B) and cell viability was measured. *<i>P</i><0.05, **<i>P</i><0.01 compared with the control group. The results shown are representative of three independent experiments.</p