Virus classification for viral genomic fragments using PhaGCN2.

Viruses are the most ubiquitous and diverse entities in the biome. Due to the rapid growth of newly identified viruses, there is an urgent need for accurate and comprehensive virus classification, particularly for novel viruses. Here, we present PhaGCN2, which can rapidly classify the taxonomy of viral sequences at the family level and supports the visualization of the associations of all families. We evaluate the performance of PhaGCN2 and compare it with the state-of-the-art virus classification tools, such as vConTACT2, CAT and VPF-Class, using the widely accepted metrics. The results show that PhaGCN2 largely improves the precision and recall of virus classification, increases the number of classifiable virus sequences in the Global Ocean Virome dataset (v2.0) by four times and classifies more than 90% of the Gut Phage Database. PhaGCN2 makes it possible to conduct high-throughput and automatic expansion of the database of the International Committee on Taxonomy of Viruses. The source code is freely available at https://github.com/KennthShang/PhaGCN2.0.

Transcriptomic analysis reveals the early body wall regeneration mechanism of the sea cucumber Holothuria leucospilota after artificially induced transverse fission.

BACKGROUND: Sea cucumbers exhibit a remarkable ability to regenerate damaged or lost tissues and organs, making them an outstanding model system for investigating processes and mechanisms of regeneration. They can also reproduce asexually by transverse fission, whereby the anterior and posterior bodies can regenerate independently. Despite the recent focus on intestinal regeneration, the molecular mechanisms underlying body wall regeneration in sea cucumbers still remain unclear. RESULTS: In this study, transverse fission was induced in the tropical sea cucumber, Holothuria leucospilota, through constrainment using rubber bands. Histological examination revealed the degradation and loosening of collagen fibers on day-3, followed by increased density but disorganization of the connective tissue on day-7 of regeneration. An Illumina transcriptome analysis was performed on the H. leucospilota at 0-, 3- and 7-days after artificially induced fission. The differential expression genes were classified and enriched by GO terms and KEGG database, respectively. An upregulation of genes associated with extracellular matrix remodeling was observed, while a downregulation of pluripotency factors Myc, Klf2 and Oct1 was detected, although Sox2 showed an upregulation in expression. In addition, this study also identified progressively declining expression of transcription factors in the Wnt, Hippo, TGF-ß, and MAPK signaling pathways. Moreover, changes in genes related to development, stress response, apoptosis, and cytoskeleton formation were observed. The localization of the related genes was further confirmed through in situ hybridization. CONCLUSION: The early regeneration of H. leucospilota body wall is associated with the degradation and subsequent reconstruction of the extracellular matrix. Pluripotency factors participate in the regenerative process. Multiple transcription factors involved in regulating cell proliferation were found to be gradually downregulated, indicating reduced cell proliferation. Moreover, genes related to development, stress response, apoptosis, and cell cytoskeleton formation were also involved in this process. Overall, this study provides new insights into the mechanisms of whole-body regeneration and uncover potential cross-species regenerative-related genes.

Prevalence of Zika virus in blood donations: a systematic review and meta-analysis.

BACKGROUND: Transfusion-Transmitted Zika virus (TT-ZIKV) has become an emerging threat to world blood banks due to the fast spread of ZIKV epidemics and high rate of asymptomatic infections. For the risk assessment of ZIKV infection in blood products, relevant studies in blood donations or blood donors tested for ZIKV were collected and analyzed systematically. The overall prevalence of ZIKV infection were estimated through meta-analysis and potential risk factors were detected. The results will provide important clues for the protocol design of blood screening tests. METHODS: Relevant articles about the rate of ZIKV detected in blood samples were identified from PubMed, Scopus and Web Of Science using key terms search strategy until October 7, 2017. Eligible articles were screened following inclusion and exclusion criteria. Meta-analysis and subgroup analyses were performed by software R3.4.1. Overall postdonation and posttransfusion follow-ups were analyzed. RESULTS: Ten literatures (528,947 blood samples) were included for meta-analysis. The overall pooled prevalence of ZIKV (RNA and antibody) in blood donations was 1.02% (95%CI 0.36-1.99). The pooled prevalence of ZIKV RNA in blood donations was 0.85% (95%CI 0.21-1.88) less than the pooled prevalence of anti-ZIKV antibodies 1.61% (95%CI 0.03-5.21), however the difference was not statistically significant (p = 0.52). The prevalence varied significantly in different geographical regions (p < 0.001). Blood donations were more than two times likely to be infected by ZIKV in Zika epidemic period (1.37, 95%CI 0.91-1.91) than in non-epidemic period (0.61, 95%CI 0-2.55). The prevalence of anti-ZIKV antibodies (1.61, 95%CI 0.03-5.21) was almost twice as much as ZIKV nucleic acid detected in blood donations (0.85, 95%CI 0.21-1.88). However, statistically significant differences were not observed. A total of 122 ZIKV positive blood donors were followed, of which 48 (39%) reported symptoms postdonation, but none of the 13 followed recipients reported any clinical symptoms related to Zika infection posttransfusion. CONCLUSION: The pooled prevalence of Zika infection in blood donations was 1.02%. The prevalence varied greatly and reached to high-risk level in most of the situations. The results suggest that nucleic acid tests (NAT) for blood screening and pathogen reduction/inactivation technology (PRT) should be implemented in Zika-endemic areas and appropriate strategies should be designed according to different conditions. More studies are needed in the future to provide more evidence.

Rapid Detection of Avian Influenza A Virus (H7N9) by Lateral Flow Dipstick Recombinase Polymerase Amplification.

Avian influenza A (H7N9) virus has caused several epidemics and infection in both human and poultry. With mutation, the H7N9 virus gained its fifth endemic in China. Early diagnosis is crucial for the control of viral spread in poultry and prognosis of infected patients. In this study, we developed and evaluated a lateral flow dipstick recombinase polymerase amplification (LFD-RPA) assay for rapid detection of both hemagglutinin and neuraminidase gene of H7N9. Our H7-LFD-RPA and N9-LFD-RPA assay were able to detect 32 fg H7N9 nucleic acid which is more convenient and rapid than previous methods. Through detecting 50 influenza positive samples, cross-reaction was not found with other subtypes of influenza virus. The 100% analytical specificity and sufficient analytical sensitivity results agreed the real time RT-PCR assay. The results data demonstrated that our method performed well and could be applied to the detection of H7N9 virus. This LFD-RPA assay provides a candidate method for rapid point-of-care diagnosis of H7N9.

Fecal Bacteriome and Mycobiome in Bats with Diverse Diets in South China.

Bats can be divided into frugivory, nectarivory, insectivory, and sanguivory based on their diets, and are therefore ideal wild animal models to study the relationship between diets and intestinal microflora. Early studies of bat gut bacteria showed that the diversity and structure of intestinal bacterial communities in bats are closely related to dietary changes. Worthy of note, intestinal microbes are composed of bacteria, fungi, protozoa, and archaea. Although the number of gut fungi is much lower than that of gut bacteria, they also play an important role in maintaining the host homeostasis. However, there are still few reports on the relationship between the gut mycobiota and the dietary habits of the host. In addition, bats have also been shown to naturally transmit pathogenic viruses and bacteria through their feces and saliva, but fungal infections from bat are less studied. Here, we used high-throughput sequencing of bacterial 16S and eukaryotic 18S rRNA genes in the V4 and V9 regions to characterize fecal bacterial and fungal microbiota in phytophagous and insectivorous bats in South China. The results show that the gut microbiota in bats were dominated by bacterial phyla Proteobacteria, Firmicutes, Tenericutes and Bacteroidetes, and fungal phyla Ascomycota and Basidiomycota. There was a significant difference in the diversity of bacterial and fungal microbiota between the groups, in addition to specific bacteria and fungi populations on each of them. Of note, the number of fungi in the feces of herbivorous bats is relatively higher. Most of these fungi are foodborne and are also pathogens of humans and other animals. Thus, bats are natural carriers of fungal pathogens. The current study expands the understanding of the bat gut bacterial and fungal mycobiota and provides further insight into the transmission of fungal pathogens.

Selective evolution of Toll-like receptors 3, 7, 8, and 9 in bats.

Previous studies have shown that bats are reservoirs of a large number of viruses, many of which cause illness and mortality in humans and other animals. However, these bat-associated pathogens cause little, if any, clinicopathology in bats. This long-term adaptation should be reflected somewhat in the immune system. Toll-like receptors (TLRs) are the first line of immune defense against pathogens in vertebrates. Therefore, this study focuses on the selection of TLRs involved in virus recognition. The coding sequences of TLR3, TLR7, TLR8, and TLR9 were sequenced in ten bats. The selection pressure acting on each gene was also detected using branch- and site-specific methods. The results showed that the ancestor of bats and certain other bat sublineages evolved under positive selection for TLR7, TLR8, and TLR9. The highest proportion of positive selection occurred in TLR9, followed by TLR8 and TLR7. All of the positively selected sites were located in the leucine-rich repeat (LRR) domain, which implied their important roles in pathogen recognition. However, TLR3 evolved under negative selection. Our results are not in line with previous studies which identified more positively selected sites in TLR8 in mammalian species. In this study, the most positively selected sites were found in TLR9. This study encompassed more species that were considered natural reservoirs of viruses. The positive selection for TLR7, TLR8, and TLR9 might contribute to the adaptation of pathogen-host interaction in bats, especially in bat TLR9.

OB-RL silencing inhibits the thermoregulatory ability of Great Roundleaf Bats (Hipposideros armiger).

Previous studies have shown that the hormone Leptin has an important role in mammalian heterothermy by regulating metabolism and food intake via lipolysis, as well as adaptive evolution of Leptin in heterothermic bats driven by selected pressure. However, the mechanism of Leptin in heterothermic regulation in mammals is unknown. By combining previous results, we speculated that the Leptin signaling pathway mediated by OB-RL (Leptin receptor long form) in the hypothalamus is important. OB-RL is one of the products of db gene and mainly distributed in the hypothalamus. In this study, we used OB-RL as a molecular marker, combining with the RNA interference technology and physiological/molecular analyses with Hipposideros armiger (a hibernating bat species) as an animal model, to explore the mechanism of Leptin in heterothermic regulation. Our data showed that all of four anti-OB-RL shRNA lentivirus significantly inhibited OB-RL expression (>90%), and the interference efficiency of PSC1742 lentivirus reached the highest value. In situ hybridization proved that PSC1742 lentivirus significantly decreased the OB-RL expression in the hypothalamus, especially in the ventromedial hypothalamic nucleus (VHM, 86.6%). Physiological analysis demonstrated that the thermoregulatory ability of bats (e.g., reducing core body temperature and heart rate) was significantly depressed after OB-RL silencing in the hypothalamus, and animals could not enter torpor state. Our study for the first time proved that the knock-down of OB-RL expression in hypothalamus inhibits heterothermic regulation of bats, and also provided the clues for further analyzing the mechanism of Leptin in the heterothermic regulation of mammals.

Advances in zeolitic-imidazolate-framework-based catalysts for photo-/electrocatalytic water splitting, CO2 reduction and N2 reduction applications.

Harnessing electrical or solar energy for the renewable production of value-added fuels and chemicals through catalytic processes (such as photocatalysis and electrocatalysis) is promising to achieve the goal of carbon neutrality. Owing to the large number of highly accessible active sites, highly porous structure, and charge separation/transfer ability, as well as excellent stability against chemical and electrochemical corrosion, zeolite imidazolate framework (ZIF)-based catalysts have attracted significant attention. Strategic construction of heterojunctions, and alteration of the metal node and the organic ligand of the ZIFs effectively regulate the binding energy of intermediates and the reaction energy barriers that allow tunable catalytic activity and selectivity of a product during reaction. Focusing on the currently existing critical issues of insufficient kinetics for electron transport and selective generation of ideal products, this review starts from the characteristics and physiochemical advantages of ZIFs in catalytic applications, then introduces promising regulatory approaches for advancing the kinetic process in emerging CO2 reduction, water splitting and N2 reduction applications, before proposing perspective modification directions.

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.

Sea Cucumber Derived Triterpenoid Glycoside Frondoside A: A Potential Anti-Bladder Cancer Drug.

Bladder cancer is a highly recurrent disease and a common cause of cancer-related deaths worldwide. Despite recent developments in diagnosis and therapy, the clinical outcome of bladder cancer remains poor; therefore, novel anti-bladder cancer drugs are urgently needed. Natural bioactive substances extracted from marine organisms such as sea cucumbers, scallops, and sea urchins are believed to have anti-cancer activity with high effectiveness and less toxicity. Frondoside A is a triterpenoid glycoside isolated from sea cucumber, Cucumaria frondosa. It has been demonstrated that Frondoside A exhibits anti-proliferative, anti-invasive, anti-angiogenic, anti-cancer, and potent immunomodulatory effects. In addition, CpG oligodeoxynucleotide (CpG-ODN) has also been shown to have potent anti-cancer effects in various tumors models, such as liver cancer, breast cancer, and bladder cancer. However, very few studies have investigated the effectiveness of Frondoside A against bladder cancer alone or in combination with CpG-ODN. In this study, we first investigated the individual effects of both Frondoside A and CpG-ODN and subsequently studied their combined effects on human bladder cancer cell viability, migration, apoptosis, and cell cycle in vitro, and on tumor growth in nude mice using human bladder cancer cell line UM-UC-3. To interrogate possible synergistic effects, combinations of different concentrations of the two drugs were used. Our data showed that Frondoside A decreased the viability of bladder cancer cells UM-UC-3 in a concentration-dependent manner, and its inhibitory effect on cell viability (2.5 µM) was superior to EPI (10 µM). We also showed that Frondoside A inhibited UM-UC-3 cell migration, affected the distribution of cell cycle and induced cell apoptosis in concentration-dependent manners, which effectively increased the sub-G1 (apoptotic) cell fraction. In addition, we also demonstrated that immunomodulator CpG-ODN could synergistically potentiate the inhibitory effects of Frondoside A on the proliferation and migration of human bladder cancer cell line UM-UC-3. In in vivo experiments, Frondoside A (800 µg/kg/day i.p. for 14 days) alone and in combination with CpG-ODN (1 mg/kg/dose i.p.) significantly decreased the growth of UM-UC-3 tumor xenografts, without any significant toxic side-effects; however, the chemotherapeutic agent EPI caused weight loss in nude mice. Taken together, these findings indicated that Frondoside A in combination with CpG-ODN is a promising therapeutic strategy for bladder cancer.

Anti-tumor target screening of sea cucumber saponin Frondoside A: a bioinformatics and molecular docking analysis.

Cancer remains the leading cause of death worldwide. In spite of significant advances in targeted and immunotherapeutic approaches, clinical outcomes for cancer remain poor. The aim of the present study was to investigate the potential mechanisms and therapeutic targets of Frondoside A for the treatment of liver, pancreatic, and bladder cancers. The data presented in our study demonstrated that Frondoside A reduced the viability and migration of HepG2, Panc02, and UM-UC-3 cancer cell in vitro. Moreover, we utilized the GEO database to screen and identify for differentially expressed genes (DEGs) in liver, pancreatic, and bladder cancers, which resulted in the identification of 714, 357, and 101 DEGs, respectively. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation were performed using the Metascape database for DEGs that were significantly associated with cancer development. The protein-protein interaction (PPI) networks of the identified DEGs in liver, pancreatic, and bladder cancers were analyzed using Cytoscape 3.9.0 software, and subsequently identified potential key genes that were associated with these networks. Subsequently, their prognostic values were assessed by gene expression level analysis and Kaplan-Meier survival analysis (GEPIA). Furthermore, we utilized TIMER 2.0 to investigate the correlation between the expression of the identified key gene and cancer immune infiltration. Finally, molecular docking simulations were performed to assess the affinity of Frondoside A and key genes. Our results showed a significant correlation between these DEGs and cancer progression. Combined, these analyses revealed that Frondoside A involves in the regulation of multiple pathways, such as drug metabolism, cell cycle in liver cancer by inhibiting the expression of CDK1, TOP2A, CDC20, and KIF20A, and regulates protein digestion and absorption, receptor interaction in pancreatic cancer by down-regulation of ASPM, TOP2A, DLGAP5, TPX2, KIF23, MELK, LAMA3, and ANLN. While in bladder cancer, Frondoside A regulates muscle contraction, complement and coagulation cascade by increase FLNC expression. In conclusion, the present study offers valuable insights into the molecular mechanism underlying the anticancer effects of Frondoside A, and suggests that Frondoside A can be used as a functional food supplement or further developed as a natural anti-cancer drug.

Cancer Cell Inhibiting Sea Cucumber (Holothuria leucospilota) Protein as a Novel Anti-Cancer Drug.

Cancer remains the primary cause of death worldwide. To develop less toxic anti-cancer drugs to relieve the suffering and improve the survival of cancer patients is the major focus in the anti-cancer field. To this end, marine creatures are being extensively studied for their anti-cancer effects, since extracts from at least 10% of the marine organisms have been shown to possess anti-tumor activities. As a classic Chinese traditional medicine, sea cucumbers and compounds extracted from the sea cucumbers, such as polysaccharides and saponins, have recently been shown to exhibit anti-cancer, anti-inflammatory, and anti-oxidant effects. Holothuria leucospilota (H. leucospilota) is a tropical edible sea cucumber species that has been successfully cultivated and farmed in large scales, providing a readily available source of raw materials to support the development of novel marine anti-cancer drugs. However, very few studies have so far been performed on the biological activities of H. leucospilota. In this study, we first investigated the anti-cancer effect of H. leucospilota protein on three cancer cell lines (i.e., HepG2, A549, Panc02) and three normal cell lines (NIH-3T3, HaCaT, 16HBE). Our data showed that H. leucospilota protein decreased the cell viabilities of HepG2, A549, HaCaT, 16HBE in a concentration-dependent manner, while Panc02 and NIH-3T3 in a time- and concentration-dependent manner. We also found that the inhibitory effect of H. leucospilota protein (≥10 µg/mL) on cell viability is near or even superior to EPI, a clinical chemotherapeutic agent. In addition, our data also demonstrated that H. leucospilota protein significantly affected the cell cycle and induced apoptosis in the three cancer cell lines investigated; in comparison, it showed no effects on the normal cell lines (i.e., NIH-3T3, HaCaT and 16HBE). Finally, our results also showed that H. leucospilota protein exhibited the excellent performance in inhibiting cell immigrations. In conclusion, H. leucospilota protein targeted the cancer cell cycles and induced cancer cell apoptosis; its superiority to inhibit cancer cell migration compared with EPI, shows the potential as a promising anti-cancer drug.

A discussion of RNA virus taxonomy based on the 2020 International Committee on Taxonomy of Viruses report.

RNA viruses have a higher mutation rate than DNA viruses; however, RNA viruses are insufficiently studied outside disease settings. The International Committee on Taxonomy of Viruses (ICTV) is an organization set up by virologists to standardize virus classification. To better understand ICTV taxonomy and the characteristics and rules of different RNA virus families, we analyzed the 3,529 RNA viruses included in the 2020 ICTV report using five widely used metrics: length, host, GC content, number of predicted ORFs, and sequence similarity. The results show that host type has a significant influence on viral genome length and GC content. The genome lengths of virus members within the same genus are quite similar: 98.28% of the genome length differences within any particular genus are less than 20%. The species within those genera containing segmented viruses also have a similar length and number of segments. The number of predicted ORFs in the RNA viral genomes also shows a strong, statistically significant correlation with genome length. We suggest that due to the high mutation rate of RNA virus genomes, current RNA virus classification should mainly rely on protein similarities rather than nucleic acid similarities.

[Screening and identification of a strain with lipolytic activity against Jatropha oil and its catalytic capacity].

OBJECTIVE: To screen lipases applied to biodiesel production, a lipase-producing microorganism was isolated and the enzyme was characterized. METHODS: A strain with lipolytic activity against Jatropha oil was isolated from the soil pretreated by Jatropha curcas L. seed and cultivated on Jatropha oil as sole carbon source. The organic solvent tolerance of the isolated strain and its lipase were measured. The esterification and transesterification catalyzed by the isolated lipase were surveyed. The isolated strain was identified according to the physiological and biochemical characteristics as well as 16S rDNA sequences analysis. RESULTS: The lipolytic activity of the strain LP-2 was 3.03 U/mL. The relative biomass of strain LP-2 in the media containing 5% (v/v) methanol was 87.3%. The residual activity of LP-2 lipase in 10% (v/v) hexane was 80.9%. LP-2 lipase could catalyze esterification between lauric acid or palmitic acid and n-butanol, n-octanol, dodecanol or glycerol; stearic acid and n-octanol, dodecanol or glycerol; oleic acid and methanol, n-butanol, n-octanol or dodecanol. The transesterification of Jatropha oil with methanol could be catalyzed by LP-2 lipase. Strain LP-2 was identified as Staphylococcus epidermidis and named Staphylococcus epidermidis LP-2. CONCLUSION: S. epidermidis LP-2 lipase had the ability to catalyze esterification and transesterification reactions, which suggested that it had potential of producing biodiesel.

New genotype invasion of dengue virus serotype 1 drove massive outbreak in Guangzhou, China.

BACKGROUND: Dengue fever is a mosquito-borne infectious disease that has caused major health problems. Variations in dengue virus (DENV) genes are important features of epidemic outbreaks. However, the associations of DENV genes with epidemic potential have not been extensively examined. Here, we assessed new genotype invasion of DENV-1 isolated from Guangzhou in China to evaluate associations with epidemic outbreaks. METHODOLOGY/PRINCIPAL FINDINGS: We used DENV-1 strains isolated from sera of dengue cases from 2002 to 2016 in Guangzhou for complete genome sequencing. A neighbor-joining phylogenetic tree was constructed to elucidate the genotype characteristics and determine if new genotype invasion was correlated with major outbreaks. In our study, a new genotype invasion event was observed during each significant outbreak period in 2002-2003, 2006-2007, and 2013-2014. Genotype II was the main epidemic genotype in 2003 and before. Invasion of genotype I in 2006 caused an unusual outbreak with 765 cases (relative risk [RR] = 16.24, 95% confidence interval [CI] 12.41-21.25). At the middle and late stages of the 2013 outbreak, genotype III was introduced to Guangzhou as a new genotype invasion responsible for 37,340 cases with RR 541.73 (95% CI 417.78-702.45), after which genotypes I and III began co-circulating. Base mutations occurred after new genotype invasion, and the gene sequence of NS3 protein had the lowest average similarity ratio (99.82%), followed by the gene sequence of E protein (99.86%), as compared to the 2013 strain. CONCLUSIONS/SIGNIFICANCE: Genotype replacement and co-circulation of multiple DENV-1 genotypes were observed. New genotype invasion was highly correlated with local unusual outbreaks. In addition to DENV-1 genotype I in the unprecedented outbreak in 2014, new genotype invasion by DENV-1 genotype III occurred in Guangzhou.

Interleukin-2 enhancer binding factor 2 (ILF2) in pacific white shrimp (Litopenaeus vannamei): Alternatively spliced isoforms with different responses in the immune defenses against vibrio infection.

Alternative splicing is an essential molecular mechanism that increase the protein diversity of a species to regulate important biological processes. As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates the functions of interleukin-2 (IL-2) at the levels of transcription, splicing and translation, and plays other critical roles in the immune system. ILF2 is well-documented in vertebrates, while little is currently known in crustacean species such as the Pacific white shrimp (Litopenaeus vannamei). In the present study, five cDNA for spliced isoforms of Lv-ILF2 were identified, in which four of them are the full-length long isoforms (Lv-ILF2-L1, Lv-ILF2-L2, Lv-ILF2-L3 and Lv-ILF2-L4) and one of them is a truncated short isoform (Lv-ILF2-S). The whole sequence of ILF2 gene from L. vannamei was obtained, which is 11,680 bp in length with 9 exons separated by 8 introns. All five isoforms contain a domain associated with zinc fingers (DZF). Two alternative splicing types (alternative 5' splice site and alternative 3' splice site) were identified in the five isoforms. The Lv-ILF2 mRNA showed a broad distribution in all detected tissues, and the Lv-ILF2-L transcript levels were higher than those of Lv-ILF2-S in corresponding tissues. The mRNA levels of Lv-ILF2-S in the hepatopancreas, heart, muscle and stomach, but not in the eyestalk, were significantly increased after challenges with Vibrio harveyi or lipopolysaccharide (LPS), while no significant changes were observed for the transcript levels of Lv-ILF2-L in these tissues under the same immune stimulants. On the contrary, the transcript levels of neither Lv-ILF2-S nor Lv-ILF2-L were affected by challenges of polyinosinic: polycytidylic acid [Poly (I:C)]. In addition, after knockdown of the Lv-ILF2 mRNA level by siRNA, the mortality of shrimp and the hepatopancreatic bacterial numbers were significantly increased under V. harveyi challenge, indicating that Lv-ILF2 might participate in the immune defenses against V. harveyi invasion. Collectively, our study here supplied the first evidence for a novel splicing mechanism of ILF2 transcripts, and provided a functional link between the Lv-ILF2 isoforms and the capacity against pathogenic Vibrio in penaeid shrimp.

Adaptive evolution of 5'HoxD genes in the origin and diversification of the cetacean flipper.

The homeobox (Hox) genes Hoxd12 and Hoxd13 control digit patterning and limb formation in tetrapods. Both show strong expression in the limb bud during embryonic development, are highly conserved across vertebrates, and show mutations that are associated with carpal, metacarpal, and phalangeal deformities. The most dramatic evolutionary reorganization of the mammalian limb has occurred in cetaceans (whales, dolphins, and porpoises), in which the hind limbs have been lost and the forelimbs have evolved into paddle-shaped flippers. We reconstructed the phylogeny of digit patterning in mammals and inferred that digit number has changed twice in the evolution of the cetacean forelimb. First, the divergence of the early cetaceans from their even-toed relatives coincided with the reacquisition of the pentadactyl forelimb, whereas the ancestors of tetradactyl baleen whales (Mysticeti) later lost a digit again. To test whether the evolution of the cetacean forelimb is associated with positive selection or relaxation of Hoxd12 and Hoxd13, we sequenced these genes in a wide range of mammals. In Hoxd12, we found evidence of Darwinian selection associated with both episodes of cetacean forelimb reorganization. In Hoxd13, we found a novel expansion of a polyalanine tract in cetaceans compared with other mammals (17/18 residues vs. 14/15 residues, respectively), lengthening of which has previously been shown to be linked to synpolydactyly in humans and mice. Both genes also show much greater sequence variation among cetaceans than across other mammalian lineages. Our results strongly implicate 5'HoxD genes in the modulation of digit number, web forming, and the high morphological diversity of the cetacean manus.

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.

The Apoptosis Mechanism of Epirubicin Combined with BCG on Human Bladder Cancer Cells.

AIMS: The purpose of our study was to explore the combination effect of epirubicin and Bacillus Calmette Guerin (BCG) and its mechanism. BACKGROUND: Bladder cancer is a threat to human health worldwide. Commonly used chemotherapy drugs and biotherapy have significant therapeutic effects on bladder cancer, but the mechanism and combined effects are still unclear. OBJECTIVE: To evaluate the anti-cancer effect of epirubicin combined with BCG on human bladder cancer cells, our studies were carried out. METHODS: The viability of human bladder cancer cells with epirubicin and/or BCG treatments was examined by Cell Counting Kit-8 (CCK-8) assay. Apoptosis and cell cycle phase were determined by flow cytometry analysis. Pre-apoptosis factors of caspase-3, p53, B-cell lymphoma 2 associated X protein (Bax) and anti-apoptosis factor of B-cell lymphoma 2 (Bcl-2) were detected by western blot. RESULTS: The viability of human bladder cancer with epirubicin or BCG treatment was decreased and the viability with epirubicin combined with BCG treatment was decreased more, which were determined by CCK-8 assay. Both epirubicin and BCG increased the apoptosis rate of human bladder cancer and arrested more cells into G0/G1 phase, which were tested by flow cytometry. The expression of caspase-3, p53 and Bax was increased and the expression of Bcl-2 was decreased with epirubicin treatment on human bladder cells, which were analyzed by western blot. The expression of caspase-3 and p53 was increased with BCG treatment, which was examined by western blot. CONCLUSION: Epirubicin induced apoptosis in human bladder cancer cells by up-regulating the expression of proapoptotic factors (caspase-3, p53 and Bax) and down-regulating the expression of anti-apoptotic factor (Bcl-2). BCG promoted apoptosis of human bladder cancer cells by up-regulating the expression of caspase-3 and p53. BCG plays a potential role at the time of the combination of epirubicin and BCG on bladder cancer cells in early stage. Both epirubicin and BCG affected cell cycle distribution via arresting more bladder cancer cells at G0/G1 phase, which ultimately led bladder cancer proliferation in vitro and promoted apoptosis.

CpG Oligodeoxynucleotides Induces Apoptosis of Human Bladder Cancer Cells via Caspase-3-Bax/Bcl-2-p53 Axis.

OBJECTIVE: To evaluate the anti-cancer effect of unmethylated cytosine-phosphorothioate-guanine (CpG)-containing oligodeoxynucleotides (ODNs) on human bladder cancer UM-UC-3 cells, our study was carried out. METHODS: The viability of cells (UM-UC-3, T24 and SV-HUC-1) with CpG ODN treatments was examined by cell counting kit-8 (CCK-8) assay. Apoptosis and cell cycle phase were determined by flow cytometry analysis. Pre-apoptosis factors of caspase-3, p53, B-cell lymphoma 2 associated X protein (Bax) and anti-apoptosis factor of B-cell lymphoma 2 (Bcl-2) were detected by western blot. RESULTS: Experimental results showed that the viability of human bladder cancer cells (UM-UC-3 and T24) with CpG ODN treatment was decreased and the viability of human normal urothelial cells (SV-HUC-1) with CpG ODN treatment was increased with time-dependance manner. Moreover, CpG ODN increased the apoptosis rate of UM-UC-3 cells and arrested more cells in G0G1 phase. Furthermore, the expression of caspase-3, p53 and Bax were increased and the expression of Bcl-2 was decreased with CpG ODN treatment on UM-UC-3 cells. CONCLUSION: CpG ODN promoted the proliferation of normal urinary transitional epithelial cells (SV-HUC-1) and inhibited the cell viability of human bladder cancer cells (UM-UC-3 and T24) in vitro. CpG ODN induced the apoptosis of human bladder cancer (UM-UC-3) cells in a cascade progress via enhancing the expression of caspase-3, p53 and Bax, and inhibiting the expression of Bcl-2 with significant time-dependancy. CpG ODN inhibited cell cycle distribution of human bladder cancer (UM-UC-3) cells with more cells were arrested in G0G1 phase. This study suggested that the CpG ODN is the potential candidate on human bladder cancer.