[Differentially expressed long non-coding RNAs in spermatozoa with different fertilizing abilities].

OBJECTIVE: To search for conception capacity-related long non-coding RNAs (lncRNA) and explore their possible roles in fertilization. METHODS: We obtained 10 semen samples, 5 of high and the other 5 of low fertilizing ability, extracted large RNAs, established a cDNA library, and performed RNA sequencing with the HiSeq 2000 sequencing system. Using the bioinformatics method, we assembled and predicted lncRNAs, screened differentially expressed genes between the two groups by NOIseq, analyzed the lncRNAs with the box plot and volcano plot, and determined their expression patterns by hierarchical cluster analysis. We examined the functional classification of differentially expressed lncRNAs by pathway and gene ontology (GO) enrichment and predicted those of some lncRNAs by lncRNA-mRNA interaction analysis and intersection analysis with up- and down-stream cis-acting elements. RESULTS: A total of 147 1615 lncRNAs were identified in all the semen samples, including 463 596 novel ones and 8 019 known ones, with 4 052 differentially expressed lncRNAs, 985 upregulated and the other 3 067 downregulated. Box plot and volcano plot filtering analyses showed statistically significant differences in the expressions of the lncRNAs between the two groups, and so did hierarchical cluster analysis. GO functional annotations manifested the involvement of the differentially expressed lncRNAs in the metabolic process, biological regulation, membrane and organelle formation, and protein-nucleotide binding. Pathway analysis showed that the differentially expressed lncRNAs were related to transport and catabolism, cell motility, signaling molecular interactions, signaling transduction, and signaling pathways in the development and immune systems. The functions of the 5 lncRNAs predicted were shown to be associated with sperm motility, acrosomal reaction and signal transduction during fertilization. CONCLUSIONS: Differentially expressed lncRNAs may play an important role in fertilization and become biomarkers for the assessment of sperm quality.

Laboratory and Molecular Characterization of Dengue Viruses in a 2014 Outbreak in Guangfo Region, Southern China.

Non-specific symptoms and low viremia levels make early diagnosis of dengue virus (DENV) infection challenging. This study aimed to i) identify laboratory markers that can be used to predict a DENV-positive diagnosis and ii) perform a molecular characterization of DENVs from the 2014 Guangdong epidemic. This retrospective study analyzed 1,044 patients from the Guangdong epidemic who were clinically suspected cases of dengue. Viral RNA was detected by real-time RT-PCR, and viral-specific NS1 antigen was detected using enzyme-linked immuno sorbent assay. A molecular phylogenetic analysis was performed for the with the DENV C-prM gene junction. Patients with dengue infection had leukopenia (2.8 × 109/L), thrombocytopenia (109.0 × 109/L), elevated aspartate aminotransferase (56.0 IU/L) and alanine aminotransferase (43.5 IU/L), and prolonged activated partial thromboplastin time (APTT, 33.5 s) (all P ＜ 0.001) compared to patients without dengue. The positive predictive value of leukopenia and thrombocytopenia for DENV infection were 96.9% and 93.0%, respectively. Leukopenia, thrombocytopenia, elevated aminotransferases, and prolonged APTT were useful predictive markers for an early diagnosis of DENV infection. Phylogenetic analysis indicated that the DENVs from the 2014 epidemic were closely related to a 2010 New Delhi strain and a 2013 Guangzhou strain. The 2014 epidemic consisted of co-circulating DENV-1 genotypes I and V from multiple origins. Efficient dengue surveillance can facilitate rapid response to future outbreaks.

[Effect of TSP-2 antibody against a single epitope of mouse Toll-like receptor 2 extracellular domain on zymosan A-induced peritonitis in mice].

OBJECTIVE: To observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the inflammation in mice with zymosan A-induced peritonitis. METHODS: In mice with peritonitis induced by intraperitoneal injection of zymosan A, pretreatments with PBS, normal rabbit IgG and TSP-2 antibody at two different doses (2.5 and 5.0 mg/kg) were administered via the tail vein. Six hours after intraperitoneal injection of zymosan A, Evans blue was injected through the tail vein, and the frequency of writhing of the mice within 20 min were recorded. The mice were then sacrificed for peritoneal lavage, and the lavage fluid was collected to assess the exudation of Evans blue in the supernatant. The peritoneal leukocyte count, mast cell degranulation and release of such inflammatory mediators as platelet activating factor (PAF) and tumor necrosis factor-alpha (TNFalpha) in the lavage fluid were observed by cell counting, specific cell staining, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with PBS or rabbit IgG groups, TSP-2 treatment resulted in significantly reduced writhing response of the mice and lowered Evans blue exudation and leukocyte count in the peritoneal lavage, with also decreased degranulation of the mast cells induced by C48/80. CONCLUSION: TSP-2 antibody against a single epitope of mTLR2ECD inhibits the inflammatory response in mice with zymosan A-induced peritonitis.

Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses.

An alphavirus, M-1 strain, was isolated from a pool of culicine mosquitoes collected in Hainan island of China during an arbovirus survey in 1964. In the present study, we determined the complete nucleotide sequence of the M-1 strain using RT-PCR and RACE techniques. The M-1 genome is 11,690 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding four nonstructural proteins and five structural proteins, respectively. Searches using Blast and comparison analyses suggested that M-1 is closely linked to Sagiyama virus (SAGV, AB032553) with 98% identity and Getah viruse (GETV, AY702913) with 97.8% identity in the full-length nucleotide sequence. However, compared with SAGV, there is 1 deletion (3 nucleotides in length) in the Capsid region, a deletion in the 3' untranslated region (10 nucleotides in length) and 2 insertions in the 3' untranslated region involving a total of 5 nucleotides. Interestingly, from the 5' UTR to the end of coding region, M-1 share the highest identity with GETV, even though the identity of 3' UTR drops dramatically to 76.2%. Furthermore, phylogenetic analysis based on the complete genomic sequences and sequences for structural or non-structural proteins of M-1 and 15 alphaviruses showed that M-1 grouped with GETV first and then grouped together with SAGV. Based on the comparison analysis and phylogenetic analysis, we conclude that M-1 strain can be considered as a strain that is a Chinese isolate of Getah-like virus.

[Cloning and expression of murine Toll-like receptor-2 N terminal and preparation of its antibody].

OBJECTIVE: To prepare the recombinant murine Toll-like receptor-2 N-terminal (mTLR-2N) fusion protein and obtain anti-mTLR-2N polyclonal antibody. METHODS: The gene encoding 153 amino acids of mTLR-2N was amplified by PCR and cloned into pET32A vector with sequence verification. The recombinant fusion protein was expressed in E. coli and purified by Probond resin column. Rabbits were immunized with fusion protein to obtain the polyclonal anti-sera, and the antibodies were identified by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. RESULTS: The recombinant fusion protein was efficiently expressed and purified. The polyclonal antibodies could bind to the fusion protein expressed in different vectors as the antigens in ELISA, and also bind with RAW264.7 cells expressing mTLR-2 and CHO cells transfected with full-length mTLR-2 gene. CONCLUSION: The recombinant mTLR-2N fusion protein is obtained and the anti-mTLR-2N polyclonal antibody can recognize natural mTLR-2 on the cell surface.

[Expression of human Toll-like receptor-9 in E.coli and the preparation of antibody].

AIM: To express N terminal fragment of human Toll-like receptor-9 (hTLR-9) in E.coli and prepare its antiserum. METHODS: The thioredoxin fusion expression plasmid of terminal fragment of hTLR-9 gene (707-1 167 bp) was constructed and then transformed into E.coli BL21 (DE3). The fusion protein was expressed in E.coli BL21 (DE3) under induction of 0.5 mmol/L IPTG. Inclusion body dissolved in urea was used as immunogen to prepare mouse anti-hTLR-9 antibody. RESULTS: The fusion protein was expressed in the form of inclusion body with molecular mass of about 31kD. The purity of fusion protein reached to 90%; The result of immunohistochemical staining showed that the antisera against recombinant fusion protein could react specifically to HEK293 cells transfected with hTLR-9. CONCLUSION: The fusion protein of hTLR-9 N-terminal fragment was expressed in E.coli BL21 (DE3). The prepared anti-hTLR-9 antibody can react specifically to HEK293 cells transfected with full length hTLR-9.

[Inhibition of fibrosarcoma growth in mice by antibody against B cell epitope on mouse Toll-like receptor-2 extracellular domain].

OBJECTIVE: To observe the effect of the antibody to a B cell epitope on mouse Toll-like receptor-2 (mTLR-2) extracellular domain on the growth of murine fibrosarcoma. METHODS: An immunogen was prepared by conjugating a 20-mer peptide, synthesized on the basis of prediction for B cell dominant epitope of mTLR-2, to a carrier protein. Rabbit polyclonal antibodies against B cell epitope of mTLR-2 were prepared and purified, and characterized by Western blotting, immunohistochemistry and flow cytometry. Murine fibrosarcoma S180 cells were inoculated into the left hindlimbs of Swiss mice, and in the treatment group, the injected cells were mixed with 100 microg anti-mTLR-2 antibody TSP-1, with the same dose of irrelevant rabbit IgG in the IgG control group and with buffer solution only in the blank control group. The mice in each group were anesthetized and sacrificed on days 10, 14, and 18 following the injections, respectively, and the weight of the tumors was measured and the inhibition rate calculated. RESULTS: A distinct band for the antibody TSP-1 was shown at the relative molecular mass of 95 000 by Western blotting. The antibody TSP-1 could also react with native mTLR-2 expressed on J774A.1 cells, as identified by immunohistochemistry and flow cytometry. The growth of the fibrosarcoma S180 was obviously inhibited by injections with the antibody TSP-1 mixture, and the weight of tumor in mice treated with TSP-1 was significantly lower than that in the two control groups (P<0.05), with an inhibition rate of 77% on day 10, 51% on day 14 and 53% on day 18. CONCLUSION: Local application of the antibody against B cell epitope on mTLR-2 extracellular domain can inhibit the growth of murine fibrosarcoma, especially in the early stage of tumor proliferation.

[Cloning of mouse TLR-2 gene and its expression in Pichia pastroris].

AIM: To clone the mouse TLR-2 gene and to express it in Pichia pastroris. METHODS: Full-length gene encoding mouse Toll-like receptor 2 (mTLR-2) was amplified by RT-PCR, cloned into pUCm-T vector, and confirmed by sequencing. The target gene was then inserted into Pichia pastroris expression vector pPICZalphaC, which was transformed into Pichia pastroris. The recombinant Pichia pastroris was confirmed by PCR and RT-PCR. Expressed protein was identified by SDS-PAGE and Western blot. RESULTS: The full-length mTLR-2 gene(GenBank accession No.AY179346) was cloned. The homology of the cloned gene to published mTLR-2 gene reached 99.84%. The recombinant expression plasmid pPICZ- mTLR-2 was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass(M(r)) of recombinant protein was about 97 000. Western blot analysis showed expressed product can react to rabbit anti- mTLR-2 antibody. CONCLUSION: The full-length mTLR-2 gene is cloned and the recombinant protein can be expressed in Pichia pastroris correctly.

[Preparation and characterization of anti-morphine antiserum].

OBJECTIVE: To prepare the holoantigens of morphine and morphine-6-succinyl, and to evaluate the efficacy of both of the haptens in eliciting immune responses and their effects on morphine withdrawal symptoms in male mice. METHODS: Morphine-6-succinyl was prepared using acid anhydride mixture, and conjugated, along with morphine, with Blue carrier in the presence of carbodi imide. Immunization of mice with both the holoantigens and Freund adjuvant was performed, followed by determination of the antibody titer in the mouse serum with competitive ELISA and morphine dependence evaluation. RESULTS: The titers of the antibodies exceeded 1:8 000 in the serum of mice immunized with both holoantigens, and morphine- 6-succinyl induced a higher titer that was maintained for a longer period. The inhibition rates of the antisera to morphine and morphine-6-auccinyl had both reached 50%, and increased in a dose-dependent manner. CONCLUSION: Both morphine carrier conjugates can elicit high-titer antibody response in mice and relieve morphine addiction.