Objective:To isolate and identify the exosomes from human hepatocellular carcinoma (HepG2) cells infected with Zika virus (ZKIV), and anaylize the profiles of exosomal miRNA in infectedand uninfected cells, predict and analyze their target genes.Methods:ZIKV(ZJ03 strain) was used to HepG2 cells at a multiplicity of infection (MOI) of 0.5. After 2 days, ultracentrifugation method were used to extract and purify the exosomes from ZIKV infected (Exo-ZIKV) and uninfected HepG2 cells (Exo-NC). The morphology and particle size of exosomes were observed by the transmission electron microscope; the size and concentration of exosomes were determined by Nanoparticle tracking analysis (NTA). Western blot was used to detect protein markers of exosomes. Small RNA was isolated from exosome and analyzed by small RNA deep sequencing. The differentially expressed miRNAs were further detected by bioinformatics analysis. KEGG and GO enriched analysis was used to analyze the target genes of differentially expressed miRNA.Results:Under the transmission electron microscopy, Exo-ZIKV and Exo-NC were spherical or cup-shaped bilayer membrane structures. Exo-ZIKV and Exo-NC shared the similar particle size distribution, wheara Exo-ZIKV displayed the increased concentration (24×10 8 paritcle/ml) than that of Exo-NC (3.06×10 8 paritcle/ml). Typical exosomal markers Hsp70, CD63 and CD9 were detected in both gourps. A total of 20 differentially expressed miRNAs (12 up-rugalated and 8 down-regulated miRNA) were detected. The target genes of differentially expressed miRNAs were suggested to be involved in the pyrimidine and purine metabolism pathways. Conclusions:ZIKV infection could promote the secretion of exosomes in HepG2cells. Furthermore, ZIKV infection cause the change of exosomal miRNA expression profile of HepG2 Cells. This study provides a basis for further elucidation of the role of exosomal miRNA in the mechanisms of ZIKV infection caused hepatocyte injury and provide new ideas for the treatment of ZIKV.

Objective:To compare the DNA methylation levels of DAZL promoter regions in spermatozoa between the normozoospermic and oligoasthenozoospermic males. Methods:A case-control study was performed. Routine semen analysis and morphological analysis were performed in 15 normozoospermic individuals (control group) and 35 subfertile patients with oligoasthenozoospermia(oligoasthenozoospermia group). Semen were purified by density gradient centrifugation. DNA was extracted, treated with bisulfite, amplified by PCR and the purified PCR products were cloned into pCR2.1 vector and then proceeded to chemical transformation, restriction enzyme reaction. The positive clones of each sample were selected for sequencing analysis and DNA methylation analysis.Results:The DNA methylation rate of DAZL promoters was increased significantly in oligoasthenozoospermia group (12.15%±11.35%) compared with control group (0.96%±0.46%) ( P=0.000 4). There was also an obvious difference in the DNA methylation status among oligoasthenozoospermia males. DNA methylation levels were significantly increased in 12 (34.3%) patients with oligoasthenozoospermia. Conclusion:Aberrant methylation of DAZL gene promoters is associated with male infertility, and it may be a biomarker of human spermatogenesis defect.

Objective:To compare the DNA methylation levels of DAZL promoter regions in spermatozoa between the normozoospermic and oligoasthenozoospermic males. Methods:A case-control study was performed. Routine semen analysis and morphological analysis were performed in 15 normozoospermic individuals (control group) and 35 subfertile patients with oligoasthenozoospermia(oligoasthenozoospermia group). Semen were purified by density gradient centrifugation. DNA was extracted, treated with bisulfite, amplified by PCR and the purified PCR products were cloned into pCR2.1 vector and then proceeded to chemical transformation, restriction enzyme reaction. The positive clones of each sample were selected for sequencing analysis and DNA methylation analysis.Results:The DNA methylation rate of DAZL promoters was increased significantly in oligoasthenozoospermia group (12.15%±11.35%) compared with control group (0.96%±0.46%) ( P=0.000 4). There was also an obvious difference in the DNA methylation status among oligoasthenozoospermia males. DNA methylation levels were significantly increased in 12 (34.3%) patients with oligoasthenozoospermia. Conclusion:Aberrant methylation of DAZL gene promoters is associated with male infertility, and it may be a biomarker of human spermatogenesis defect.

Objective: To isolate, identify and analyze the sex-determining gene fruitless(Anstfru)of malaria-transmitting mosquito Anopheles stephensi. Methods: The full length cDNA of the gene Anstfru was obtained by using bioinformatic and molecular biological method . RT-PCRmethod was used to validate the sex variable splicing pattern and expression time characteristics. The structural features and molecular evolutional features of FRU protein of Anopheles stephensi were analyzed via comparison with FRU protein of known species. Results: The full length of Anstfru gene was isolated and identified, and sex-specific mRNA of the gene could form in female and male mosquitos through variable splicing. The Anstfru began to be expressed from early 1^st-2^nd stage larvae embryo, the quantity of expression increased subsequently and displayed the highest expression level in adult stage. The FRU protein had the sequence-conservative BTB and zinc finger functional domains. Conclusions Anstfru gene showed conservative functional domains and sex-determining gene fru expression features in mosquito and further in-depth studies on which will facilitate the application of techniques separating female mosquitos from male mosquitoes, and sterile insect technique (SIT)/technology in prevention and treatment of mosquito-borne infectious diseases.

Objective To identify and analyze dengue virus-derived endogenous viral elements (EVEs) in Aedes albopictus (Foshan strain).Methods The full sequences of four serotypes of dengue virus (DENV 1-4) were used as queries,and Blast N program was used to scan the genome of Ae.albopictus.The candidates of EVEs were confirmed by PCR method using specific primers with genomic DNA as template from 8 individuals of female and male adult,respectivly.Results Total 5 EVEs candidates displayed the high genetic similarity with DENV genome sequences.Among them,EVE-1 showed 100％ genetic similarity with DENV-2 P8-1407MS strain,whereas EVE-2-5 showed more than 99％ genetic similarity with DENV-1 02-20 strain.Genomic DNA PCR results indicated that EVE-1 exhibit sequence conservation across individuals among population,whereas the EVE-3-5 displayed the genomic sequence variation in individuals.Conclusions Dengue virus-derived EVEs were indentified in Ae.albopictus,that may provide new insight into the interaction between DENV and their vectors.

Several virus families have been shown to encode microRNAs (miRNAs), which have roles in the infection and replication of viruses in host cells. These virus-encoded miRNAs are identified in double-stranded DNA virus (dsDNA virus) and in several RNA virus families, but not in single-stranded DNA virus (ssDNA virus). We used a bioinformatics approach based on VMir, miRNAFold and MaturePred software to predict virus-encoded miRNA-like small RNAs from the genome of a ssDNA virus: Aedes aegypti densovirus (AaeDV). Northern blotting and stem-loop reverse transcription-polymerase chain reaction (RT-PCR) were used to detect predicted small RNAs. A miRNA-like small RNA termed "AaeDVMD" was identified by stem-loop RT-PCR from predicted candidates. This is the first report demonstrating that a ssDNA virus can encode miRNA-like small RNAs. These data will aid further exploration of the interaction between the AaeDV and its mosquito host.
Aedes ; virology ; Animals ; Base Sequence ; Computational Biology ; Densovirinae ; chemistry ; genetics ; metabolism ; MicroRNAs ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; RNA, Viral ; chemistry ; genetics ; metabolism

Objective To screen and analyze Dengue virus-derived vsRNAs and piRNAs in Aedes albopictus.Methods Female adults Aedes albopictus were collected 2-4 days post-emergence and were injected with Dengue type 2 virus NGC strain,while control group were injected with equal volume of physiological saline.Total RNA was isolated,and small RNAs ranging up to 30 nt in length were excised and analyzed using Illumina HiSeq 2000.After removing adaptor sequences and contaminated reads,Clean reads were aligned to Dengue virus genome and its complementary sequence by SOAP2.The length,strand ratio nucleotide bias and genome distribution of Dengue virus-derived vsRNAs and vpiRNAs were further analyzed.Results Total,3835 vsRNAs and 395 vpiRNAs unique tags were obtained,among them 94.99％ vpiRNAs were derived from virus genome strand,and distributed across the viral genome,but with an enrichment at several "hot-spots".Except weak bias for adenine at position 10 (10A)in the sense molecules as the feature of secondary piRNA,other signature piRNA characteristics were not observed,including a preference for a uridine at their 5'-end,which were main characteristics of primary piRNAs,and a significant 10 nt overlap (Ping-Pong)between sense and antisense piRNA.Conclusion Dengue virusderived piRNAs were indentified in Dengue virus infected Ae.albopictus.

OBJECTIVETo isolate, identify and analyze the sex-determining gene Transformer 2 (Aaetra2) of the major vector mosquito Aedes aegypti.

METHODStBLASTn program, RT-PCR and RACE methods were used to obtain the full-length cDNA of Aaetra2. Multiple alignments of nucleotide and amino acid sequences were conducted, and the different domains in tra2 protein were indentified. RT-PCR of the total RNA extracted from different tissue from the mosquitoes in different developmental stages was performed using specific primers.

RESULTSTwo genes, namely Aaetra2-α and Aaetra2-β, were identified in different supercontig locations. The multi-transcripts were expressed by means of alternative promoters or terminators. The different domains in tra2 protein were defined as RS-rich N-terminal region, RNA recognition motif-RRM, linker region, and RS-rich C-terminal region. Both Aaetra2-α and Aaetra2-β showed sustained expression throughout the developmental stages of Ae.aegypti, and in all the tissues without a sex specificity.

CONCLUSIONAaetra2 gene has multiple isoforms and is mapped to multiple locations in the genome. Aaetra2 has conservative functional domains of the sex-determining gene tra2. For Ae.agypti, Aaetra2 shows the potential as a new target for release of insects carrying a dominant lethal (RIDL) technology based on transgenic mosquitoes.

Aedes ; genetics ; growth & development ; Amino Acid Sequence ; Animals ; Drosophila Proteins ; genetics ; Gene Expression Regulation, Developmental ; Genes, Insect ; Insect Proteins ; genetics ; isolation & purification ; Nerve Tissue Proteins ; genetics ; Phylogeny ; RNA-Binding Proteins ; genetics ; Ribonucleoproteins ; genetics ; Sequence Alignment ; Serine-Arginine Splicing Factors ; Sex Differentiation ; genetics

Objective To study the feasibility of using silk fibroin/calcium phosphate cement (SF/CPC) as an injectable bone augmentation filling material for defected vertebrae in a sheep model.Methods Bone defects were created on L3,L4 and L5 in 24 adult sheep through the lateral retroperitoneum approach.CPC,SF/CPC,and polymethylmethacrylate (PMMA) were injected into the defects of 3 vertebrae randomly.Twelve sheep were sacrificed at 1 and 6 months postoperation,respectively.Un-decalcified sections were made from the specimens from any 4 sheep and stained with Van-Gieson method.The microcosmic changes of bone-material interface were observed,and the amounts of new bone formation and cement residue were evaluated by histomorphometric analysis.Biomechanical testing was performed on the specimens from the other 8 sheep,in which the strength and stiffness were determined on the vertebrae with L6 as a control.Results Histologically,CPC and SF/CPC contacted the bone directly but the absorption and bone formation were superficial at one month postoperation.At 6 months postoperation,the absorption and bone formation were limited on the surface of CPC while the absorption and bone ingrowth were accelerated in SF/CPC group.In PMMA group where no significant changes were observed between 1 and 6 months,the material contacted the bone loosely,with membrane structure at partial interface but no new bone formation on the material.Histological quantitative analysis showed that new bone formation was significantly more and cement residue significantly less in SF/CPC group than in CPC group at 6 months (P ＜0.05).Biomechanical testing showed that the compressive strength and stiffness were significantly enhanced at 6 months compared with one month in CPC and SF/CPC groups but significantly decreased in PMMA group (P ＜ 0.05).At the 2 time points,SF/CPC,PMMA and intact groups showed equivalent compressive strength and stiffness(P ＞ 0.05).Conclusions The SF/CPC composite has advantages of satisfactory bioactivity and osteoconduction,and relatively faster cement degradation and bone formation during which biomechanical function of vertebrae can be maintained.Therefore it may become a new kind of vertebral augmentation filling material to replace PMMA.

Objective To isolate, identify and analyze the sex-determining gene Transformer 2 (Aaetra2) of the major vector mosquito Aedes aegypti. Methods tBLASTn program, RT-PCR and RACE methods were used to obtain the full-length cDNA of Aaetra2. Multiple alignments of nucleotide and amino acid sequences were conducted, and the different domains in tra2 protein were indentified. RT-PCR of the total RNA extracted from different tissue from the mosquitoes in different developmental stages was performed using specific primers. Results Two genes, namely Aaetra2-α and Aaetra2-β, were identified in different supercontig locations. The multi-transcripts were expressed by means of alternative promoters or terminators. The different domains in tra2 protein were defined as RS-rich N-terminal region, RNA recognition motif-RRM, linker region, and RS-rich C-terminal region. Both Aaetra2-α and Aaetra2-β showed sustained expression throughout the developmental stages of Ae.aegypti, and in all the tissues without a sex specificity. Conclusion Aaetra2 gene has multiple isoforms and is mapped to multiple locations in the genome. Aaetra2 has conservative functional domains of the sex-determining gene tra2. For Ae.agypti, Aaetra2 shows the potential as a new target for release of insects carrying a dominant lethal (RIDL) technology based on transgenic mosquitoes.