Identification and classification of the genomes of novel microviruses in poultry slaughterhouse.

Microviridae is a family of phages with circular ssDNA genomes and they are widely found in various environments and organisms. In this study, virome techniques were employed to explore potential members of Microviridae in a poultry slaughterhouse, leading to the identification of 98 novel and complete microvirus genomes. Using a similarity clustering network classification approach, these viruses were found to belong to at least 6 new subfamilies within Microviridae and 3 higher-level taxonomic units. Genome size, GC content and genome structure of these new taxa showed evident regularities, validating the rationality of our classification method. Our method can divide microviruses into about 45 additional detailed clusters, which may serve as a new standard for classifying Microviridae members. Furthermore, by addressing the scarcity of host information for microviruses, the current study significantly broadened their host range and discovered over 20 possible new hosts, including important pathogenic bacteria such as Helicobacter pylori and Vibrio cholerae, as well as different taxa demonstrated different host specificities. The findings of this study effectively expand the diversity of the Microviridae family, providing new insights for their classification and identification. Additionally, it offers a novel perspective for monitoring and controlling pathogenic microorganisms in poultry slaughterhouse environments.

Structural and functional studies of leptins from hibernating and non-hibernating bats.

Leptin, a 16-kDa hormone produced by mature adipocytes, has been shown to regulate the hibernation of mammals. In this study, the leptin gene sequences of both hibernating (Rhinolophus ferrumequinum) and non-hibernating (Rousettus leschenaultii) bats were determined, and the leptin proteins from these two different species of bats were expressed in Escherichia coli for the first time. Results showed that the amino acid sequence of the leptin protein from hibernating bats had a lower degree of identity than that from non-hibernating bats to those of several non-hibernating mammals. The leptin protein of hibernating bats had a stronger growth inhibitory effect on 3T3-L1 cells than that of non-hibernating bats. Structural modeling revealed that the structures of the receptor binding site III, which is critical for signal transduction, of the two bat leptins were very different. Similar to the human leptin, the leptin protein of non-hibernating bats was predicted to have a random loop, whereas that of hibernating bats had a helical structure in this region. This observation provided a clue as to the differential effects of the two different leptins on 3T3-L1 cells.

Differential expression and functional constraint of PRL-2 in hibernating bat.

Circannual hibernation is a biological adaptation to periods of cold and food shortage and the role of the brain in its control is poorly understood. An SSH library of hibernating bat brains (Rhinolophus ferrumequinum) was constructed in order to explore the molecular mechanism of hibernation. An up-regulated gene, PRL-2, was obtained from hibernating bat brains. PRL-2 is a member of PTP family and has an important function in controlling cell growth. Alignment of sequences showed that PRL-2 is highly conserved among species, including two species of hibernating bats (R. ferrumequinum and Myotis ricketti). Moreover, Maximum Likelihood Analysis suggested that it may experience strong selection pressure leading to functional constraint in evolution, which indicated the significance of PRL-2 in normal bio-function. RQ-PCR was performed and statistical analysis suggested that PRL-2 exhibited distinct differential expression patterns in different organs during hibernation. In heart, fat and brain tissue of hibernating bats, the transcriptional level of PRL-2 increased almost 170%, 35% and 12% respectively. However, in muscle it decreased nearly 70%. The change of mRNA level of PRL-2 in heart tissue of hibernating bats was significantly higher than that in heart tissue of active controls (P=0.043). However, the regulation mechanism of differential expression of PRL-2 and the signal pathway involved are still unknown.

Up-regulation of a non-kinase activity isoform of Ca(2+)/calmodulin-dependent protein kinase kinase beta1 (CaMKKbeta1) in hibernating bat brain.

Hibernation is an adaptive strategy that is utilized by some animals to survive the harsh environments of low temperature and food scarce. Hibernators, however, can survive in frequent and dramatic fluctuation of body temperature and blood flow causing by periodic arousals during hibernation without brain insult, and this indicates that it must have some unique adaptive aspects of hibernation physiology. To find out the up-regulated genes of bat brain during hibernation and explore the brain function adaptive mechanism of bat, the suppression subtractive hybridization (SSH) library was constructed from the brain tissue of greater horseshoe bats. Dot blot screening was carried out and the up-regulated genes in hibernating state were obtained. Then RT-PCR and RQ-PCR were performed to test the expression patterns of selected cDNAs. Here we first show that the functional and non-functional isoforms of bat CaMKKbeta1 display distinct expression patterns between hibernating and active states. The up-regulation of non-functional form of CaMKKbeta1 may represent a new neuroprotective strategy adopted by bats or even other hibernators to avoid the CNS damage during hibernation. Our results showed that bat CaMKKbeta1 gene has four transcript isoforms and these transcript variants differ primarily in exons b and d, which are 129 bp and 43 bp respectively. Statistical analyses indicated that these isoforms display distinct expression patterns at different states, in which only isoform 3, the non-functional form, increased 300% at hibernating state. These results suggest that distinct expression patterns of transcript isoforms of a gene, which have different activity, may represent a new potential adaptive mechanism in hibernation, except for the simple up-regulation of selected genes/proteins and the reversible protein phosphorylation.

[Expression and antigenicity analysis of p46000 antigen from newborn larvae of Trichinella spiralis].

OBJECTIVE: To express and analyze p46000 antigen from newborn larvae of Trichinella spiralis. METHODS: p46000 antigen gene was subcloned to the pET28a expression system by PCR. The recombinant transformant was induced by IPTG and the antigenicity was analyzed with ELISA and Western blotting. RESULTS: The molecular weight of the expressed protein was about Mr 48000. ELISA and Western blotting showed that this recombinant protein could be recognized by T. spiralis infected swine serum and rabbit anti-recombinant protein serum, and the rabbit anti-recombinant protein serum could recognize a Mr 46000 protein from newborn larvae of T. spiralis. CONCLUSION: p46000 recombinant antigen from newborn larvae of T. spiralis was expressed which shows a specific antigenicity.

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

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 (Tb) 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 (Myotis ricketti) 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 (P≤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.

Adaptive evolution of Leptin in heterothermic bats.

Heterothermy (hibernation and daily torpor) is a key strategy that animals use to survive in harsh conditions and is widely employed by bats, which are found in diverse habitats and climates. Bats comprise more than 20% of all mammals and although heterothermy occurs in divergent lineages of bats, suggesting it might be an ancestral condition, its evolutionary history is complicated by complex phylogeographic patterns. Here, we use Leptin, which regulates lipid metabolism and is crucial for thermogenesis of hibernators, as molecular marker and combine physiological, molecular and biochemical analyses to explore the possible evolutionary history of heterothermy in bat. The two tropical fruit bats examined here were homeothermic; in contrast, the two tropical insectivorous bats were clearly heterothermic. Molecular evolutionary analyses of the Leptin gene revealed positive selection in the ancestors of all bats, which was maintained or further enhanced the lineages comprising mostly heterothermic species. In contrast, we found evidence of relaxed selection in homeothermic species. Biochemical assays of bat Leptin on the activity on adipocyte degradation revealed that Leptin in heterothermic bats was more lipolytic than in homeothermic bats. This shows that evolutionary sequence changes in this protein are indeed functional and support the interpretation of our physiological results and the molecular evolutionary analyses. Our combined data strongly support the hypothesis that heterothermy is the ancestral state of bats and that this involved adaptive changes in Leptin. Subsequent loss of heterothermy in some tropical lineages of bats likely was associated with range and dietary shifts.