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 reconstitute a transactivation system in yeast (yeast model) for screening the pesticides acting on ecdysone metabolism route and eventually influencing the process of ecdysis. Methods The fragment of 5 times repeated EcRE from Drosophila melanogaster was synthesized and the HSP27 promoter from D. melanogaster genome was amplified with PCR. The two sequences were connected and followed by a reporting gene——green fluorescence protein(GFP) gene. The EcRE-HSP27 promoter-GFP fragment was inserted into the expression plasmid pPIC3.5 and integrated into the yeast chromosome to construct yeast A. EcR and USP coding sequences of Aedes albopictus were synthesized, and these two fragments were inserted into Pichia pastoris expression plasmid pGAPZ as two respective reading frames. The two reading frames were integrated into Pichia pastoris chromosome in another recombinant site(pGAPZ and pPIC3.5k share different recombinant sites while being integrated into Pichia pastoris yeast chromosome). EcR and USP were constituted and expressed in the yeast. This recombinant yeast was called yeast B. The model yeast was thus constructed. A known ecdysone agonist-tebufenozide was used to test the yeast model. The effect of tebufenozide on the model yeast was observed under fluorescent microscope. Semi-quantitative RT-PCR was used to test the transcrip-tion level of GFP in the tebufenozide affected yeast and the control. Results In the model yeast, the intracellular expressed EcR and USP constituted EcR/USP heterodimer interacting with EcRE, the expression of GFP was activated, and green fluorescence was observed in model yeast under fluorescent microscope. Tebufenozide affected model yeast showed less fluorescence in comparison to the control model yeast, indicating that the transcription of GFP was suppressed by tubufenozide. Yeast housekeeping gene Actin-1 was used as inner control, semi-quantitative RT-PCR was operated and the result was scanned. The ratio of the brightness of GFP to Actin-1 was calculated automatically, and that of tubufenozide added yeast and the control yeast was 0.614 and 1.134 respectively. This result showed a low transcription level of GFP in tebufenozide affected model yeast, comparing to that of the control. Conclusion The ecdysone-related transacting system in yeast has been constructed, and the model yeast can be used to screen the ecdysone agonists which can act on the ecdysone metabolic route.

To prepare a large amount of pure alive Schistosoma japonicum eggs, rabbit was infected with 2000 cercariae and its liver was taken aseptically 38-45 days after infection and homogenized. The homogenate was screened through different sieves(60, 120, 200, 300, 360 meshes per inch respectively), and washed with 1.2% NaCl. The eggs and leftover were then digested with 0.25% trypsin for 2 hours, sieved over 360 meshes per inch and washed with RPMI 1640 medium. The collected eggs reached to (95.1?6.4)% of live eggs, with a high efficiency.

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.

Public hospitals reform is a key roadblock for the ongoing health reform.By means of such experiments as Three openings and three mechanisms,Beijing is practicing a separation of hospital regulation and management and separation of clinic and pharmacy,while building the mechanism of financial subscription for pricing,that of medical insurance adjustment,and that of hospital corporate governance.These measures aim at building a new management structure,operation mechanism and medical service model focusing on quality of care,efficiency and satisfaction.Separation of clinic and pharmacy has lowered drug proportion,average outpatient expense and out of-pocket payment of patients,as well as producing higher patient satisfaction,quality of care and hospital income.Other benefits include better management efficiency indirectly caused by separation of clinic and pharmacy,higher acceptance of the corporate governance,and service model innovation to better serve the people.

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

This research was carried out to investigate the effect of basic fibrous growth factor (bFGF) controlled release hydrogel nanoparticles on the proliferation and differentiation of mesenchymal stem cells. The dex-GMA-bFGF-NPs were prepared by an improved emulsion polymerization method; their morphology, size and encapsulated ratio were assessed by routine procedure. Dynamic dialysis method was used to determine the release characteristics of dex-GMA-bFGF-NPs in vitro. The secondary culture MSCs were divided into four groups according the different ingredients being added into the DMEM culture medium: free bFGF group (A), blank dex-GMA nanoparticles group (B), dex-GMA-bFGF nanoparticles group (C), nothing group (D). The proliferation of cultured MSCs was measured by using cell counting method, MTT method and flow cytometry. ALP kit was used to evaluate the ALP activity of the MSCs to show the differentiation of the cells by adding the dex-GMA-bFGF-NPs to the DMEM culture medium (C group) or bFGF only (A group). B group and D group were taken as the controls. The results were analyzed by statistical analysis software (SPSS11.0). All results showed that the shape of dex-GMA-bFGF-NPs was spherical. The encapsulated ratio was 88% and more than 85% of the encapsulated bFGF could be released during 14 days. The in vitro cellular study showed the control release of bFGF from nanoparticles could promote the proliferation of MSCs. After 12 days, the cell number in groups A, B and C was (21.97 +/- 0.25) x 10(4) cells/ml, (12.43 +/- 0.13) x 10(4) cells/ml, (27.45 +/- 0.78) x 10(4) cells/ml and (12.03 +/- 0.43) x 10(4) cells/ml, with the difference being statistically significant among them (P < 0.05). The flow cytometry revealed that the G2/M+S percentage in group C was the highest at 4-8 days after plate culture(P < 0.05). During the first 3 days, the proliferation and differentiation of BMSCs between group A and group B were of no significance (P > 0.05), but were much faster than those of group C and D. After 7 days, dex-GMA-bFGF-NPs could enhance BMSCs proliferation and differentiation continually, but bFGF had no enhancement any more, the difference between group A and group B became more significant (P < 0.05). So we made the conclusions the bFGF loading dex-GMA hydrogel nanoparticles can release bFGF more than 21 days and can promote the proliferation and differentiation of the BMSCs through a long period of controlled release of bFGF. Dex-GMA-bFGF-NPs may be an ideal controlled release carrier for bioactive growth factors.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; Male ; Mesenchymal Stromal Cells ; cytology ; Nanoparticles ; Rats ; Rats, Sprague-Dawley

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.