Gonadal white adipose tissue is important for gametogenesis in mice through maintenance of local metabolic and immune niches.

Gonadal white adipose tissue (gWAT) can regulate gametogenesis via modulation of neuroendocrine signaling. However, the effect of gWAT on the local microenvironment of the gonad was largely unknown. Herein, we ruled out that gWAT had a neuroendocrine effect on gonad function through a unilateral lipectomy strategy, in which cutting off epididymal white adipose tissue could reduce seminiferous tubule thickness and decrease sperm counts only in the adjacent testis and epididymis of the affected gonad. Consistent with the results in males, in females, ovary mass was similarly decreased by lipectomy. We determined that the defects in spermatogenesis were mainly caused by augmented apoptosis and decreased proliferation of germ cells. Transcriptome analysis suggested that lipectomy could disrupt immune privilege and activate immune responses in both the testis and ovary on the side of the lipectomy. In addition, lipidomics analysis in the testis showed that the levels of lipid metabolites such as free carnitine were elevated, whereas the levels of glycerophospholipids such as phosphatidylcholines and phosphatidylethanolamines were decreased, which indicated that the metabolic niche was also altered. Finally, we show that supplementation of phosphatidylcholine and phosphatidylethanolamine could partially rescue the observed phenotype. Collectively, our findings suggest that gWAT is important for gonad function by not only affecting whole-body homeostasis but also via maintaining local metabolic and immune niches.

[ICSI with testicular or epididymal sperm for patients with obstructive azoospermia: A systematic review].

OBJECTIVE: To assess the effects of testicular sperm and epididymal sperm on the outcomes of ICSI for patients with obstructive azoospermia. METHODS: We searched PubMed, MEDLINE, EMBASE, Cochrane, CNKI, VIP, CBM, and Wanfang Database up to December 2015 for published literature relevant to ICSI with testicular or epididymal sperm for obstructive azoospermia patients. According to the inclusion and exclusion criteria, two reviewers independently conducted literature screening, data extraction and quality assessment of the included trials, followed by meta-analysis with the RevMan 5.3 software. RESULTS: A total of 14 studies were identified, involving 1 278 patients and 1 553 ICSI cycles. ICSI with epididymal sperm exhibited a significantly higher fertilization rate than that with testicular sperm (RR = 1.08, 95% CI 1.05－1.11, P<0.01). No statistically significant differences were observed between the epididymal and testicular sperm groups in the rates of cleavage (RR = 1.04, 95% CI 0.99－1.10, P = 0.13), good-quality embryo (RR = 1.01, 95% CI 0.93－1.09,P = 0.85), implantation (RR = 1.14, 95% CI 0.75－1.73, P = 0.55), clinical pregnancy (RR = 1.14, 95% CI 0.98－1.31, P = 0.08), and miscarriage (RR = 0.86, 95% CI 0.53－1.39,P = 0.54). CONCLUSIONS: ICSI with epididymal sperm yields a markedly higher fertilization rate than that with testicular sperm, but has no statistically significant differences from the latter in the rates of cleavage, good-quality embryo, implantation, clinical pregnancy, and miscarriage in the treatment of obstructive azoospermia.

Tripterygium glycosides impairs the proliferation of granulosa cells and decreases the reproductive outcomes in female rats.

This study was carried out to investigate the impact of tripterygium glycosides (TGs) on ovarian function of female rats in vitro and in vivo. In vitro studies showed that TG induced cells decrease at G1 phase and inhibited cell proliferation in rat granulosa cells. In vivo, female rats were intragastrically administered with TG at the dose of 60 mg/kg/day for consecutive 50 days. TG caused a prolonged estrous cycle, and a significant reduction in ovarian index, serum E2 level, and numbers of secondary and antral follicles (p < 0.05) in these rats. A significant reduction of viable embryos was demonstrated in TG-treated female rats after mating (p < 0.01). Further, we observed observed the reduced expression level of TGF-ß1 after TG treatment in vitro and in vivo. Moreover, the expression of Smad2 and AKT was also decreased after TG treatment. These results suggest that TG can impair ovarian function through Smads-mediated TGF-ß1 signal pathway.

[AntiEGFRnano inhibites proliferation and migration of estrogen-dependent Ishikawa cells of human endometrial cancer cell line].

Nanobody is a kind of antibody from camel, which misses light chain. Nanobody has the same antigen binding specificity and affinity as mAb. Moreover, because of its small molecular weight, high stability and easy preparation, nanobody has great value of biomedical applications. In this study, we successfully prepared highly pure antiEGFR nanobody in E.coli using genetic engineering techniques. Cell proliferation assay (CCK-8 assay) and migration experiments (cell scratch test and Transwell assay) indicated that the recombinant antiEGFRnano can significantly inhibit the proliferation and migration of endometrial cancer cells. These results provide a new way of thinking and methods for EGFR-targeted therapy of endometrial cancer.

Pravastatin alleviates lipopolysaccharide-induced placental TLR4 over-activation and promotes uterine arteriole remodeling without impairing rat fetal development.

Preeclampsia is associated with over-activation of the innate immune system in the placenta, in which toll-like receptor 4 (TLR4) plays an essential part. With their potent anti-inflammatory effects, statins have been suggested as potential prevention or treatment of preeclampsia, although evidence remains inadequate. Herewith, we investigated whether pravastatin could ameliorate preeclampsia-like phenotypes in a previously established lipopolysaccharide (LPS)-induced rat preeclampsia model, through targeting the TLR4/NF-κB pathway. The results showed that pravastatin reduced the blood pressure [maximum decline on gestational day (GD) 12, (101.33±2.49) mmHg vs. (118.3±1.37) mmHg, P<0.05] and urine protein level [maximum decline on GD9, (3,726.23±1,572.86) µg vs. (1,991.03±609.37) µg, P<0.05], which were elevated following LPS administration. Pravastatin also significantly reduced the rate of fetal growth restriction in LPS-treated rats (34.10% vs. 8.99%, P<0.05). Further pathological analyses suggested a restoration of normal spiral artery remodeling in preeclampsia rats by pravastatin treatment. These effects of pravastatin were associated with decreased TLR4/NF-κB protein levels in the placenta and IL-6/MCP-1 levels in serum. Additionally, no obvious abnormalities in fetal liver, brain, and kidney were found after administration of pravastatin. These results provide supportive evidence for use of pravastatin in preventing preeclampsia.

FSH acts on the proliferation of type A spermatogonia via Nur77 that increases GDNF expression in the Sertoli cells.

The molecular mechanism responsible for the regulation of GDNF in Sertoli cells remains largely unknown. In the present study, FSH induced the expression of Nur77 and GDNF in mouse testis tissue and human fetal Sertoli cells. Moreover, FSH increased the number of A spermatogonia co-cultured with Sertoli cells. In the additional assays, Nur77 was observed to directly regulate GDNF transcription. Furthermore, overexpression of Nur77 and siRNA-mediated knockdown of Nur77 affected levels of GDNF mRNA and protein in primary human fetal Sertoli cells. These results indicate that FSH-induced Nur77 regulates the expression of GDNF in Sertoli cells to stimulate the proliferation of A spermatogonia in vitro.

Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners.

It has been reported that it is difficult to express cationic antibacterial peptides in engineered bacteria because such peptides are highly toxic to the host bacteria cells and sensitive to intracellular proteases. Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi and tumor cells, which may possibly be used as an antimicrobial agent. Here we tried to express ABP-CM4 in Escherichia coli cells using either the GST fusion system or the intein-mediated fusion expression system. In order to investigate the possible use of these two fusion partners in cationic small peptide expression and purification, a mutant ABP-CMt, which is a highly positively charged peptide with +9 charges at neutral pH, was designed. In the present study, we have shown that both ABP-CM4 and ABP-CMt peptides can be expressed and purified by the intein-mediated expression system but not by the GST fusion expression system. Thus the intein-mediated peptide expression and purification system potentially could be employed for the production of recombinant protease-sensitive and cytotoxic peptides.

[Expression of HCV NS5A gene in HepG2 cell and the effect of the NS5A expression on PI3K in vitro].

AIM: To reveal the influence of HCV-NS5A on PI3K, we probe into the relationship between NS5A and PI3K in vitro. METHODS: Full length NS5A gene of HCV was amplified by PCR, using the plasmid containing HCV full-length open reading frame (ORF) as template, and cloned into the eukaryotic expressing plasmid pcDNA3.0(-) by DNA recombination technique. The recombinant vector was identified by digestion with restriction enzymes and polymerase chain reaction and by directly sequencing. Then both the recombinant vector pcDNA3.0(-)-NS5A and the control vector pcDNA3.0(-) were transfected HepG2 cell using Lipofectamin2000. RESULTS: Expressing NS5A was proven by RT-PCR and Western blot analysis in transfected HepG2 cell. Further more we examined the PI3K protein expressed in the HepG2 cell expressing recombinant NS5A. CONCLUSION: NS5A can activate PI3K in vitro and its signal cascade pathway.

[Detection the coinfection of Hantavirus and Orientia tsutsugamushi in primary cultured mite cells].

OBJECTIVE: To investigate whether HV and Ot can coexist in their host (Leptotrombidium scutellare). METHODS: Collecting the separate Leptotrombidium scutellare and the ones from mice in epidemic area. The cells of mites at larva, nymph, and adult stages were cultured and made into smear. In situ RT-PCR and PCR were used to detect and locate HV RNA and Ot DNA in the primary cultured cells. RESULTS: Positive signals of HV RNA and Ot DNA distributed mostly in epithelial cells of digestive system and ovary cells of larva and nymph. The positive rate increased by the generation of passages. CONCLUSION: Coinfection of HV and Ot did exist in wild Leptotrombidium scutellare.

[Expression and preliminary study of HCV F protein gene].

AIM: To express HCV F protein gene fragment in E.coli and detect if there is its antibody in HCV patients. METHODS: RNA of the virus identified as genetype 1b was choosed as template, the F protein gene was amplified by RT-PCR. This gene fragment was inserted into plasmid vector pGEM simple T. F gene was digested by EcoR I and BamH I from pGEM and cloned into plasmid vector pGEX-4T-2. The ligation mixture was transformed into E.coli. TG1 and F fragment expression was induced by IPTG. The expressed protein was purified from lysates with Glutathione Sepharose 4B. The purified protein was used to identify whether there was anti-F antibody in the patients which HCV RNA was positive by ELISA. RESULTS: After IPTG induction, a positive band about 43 kD was detected. 82 of 120 HCV RNA positive's sera had anti-F antibody by ELISA. CONCLUSION: It is possible to efficiently express the HCV F protein in E.coli. The positive rate of anti-F antibody in HCV RNA positive patients was 68%.

[Study on relationship between HBV post-transcriptional regulatory element and response to IFN-alpha by using a reporter gene luciferase].

AIM: To identify the function of HBV post-transcriptional regulatory element(HPRE) in response to IFN-alpha by using the reporter gene luciferase (LUC). METHODS: The gene encoding LUC was obtained by PCR from the vector pGEM-luc and then cloned into the eukaryotic expression vector pcDNA3.0. The HPRE, HPREalphabeta(1) and HPREbeta(1)beta(2) fragments were amplified by PCR from HBV genome and then cloned into the recombinant vector pcDNA3.0-luc. These constructed plasmids were transfected into the human hepatoma cell line HepG2. The expression of LUC before and after the addition of IFN-alpha was detected by LUC assay system. RESULTS: The DNA sequencing analysis showed that the recombinant plasmids pcDNA3.0-luc, pcDNA3.0-luc-HPRE, pcDNA3.0-luc-HP-REalphabeta(1) and pcDNA3.0-luc-HPREbeta(1)beta(2) were successfully constructed. The results of LUC detection assay showed that HPRE, HPREalphabeta(1) and HPREbeta(1)beta(2) could enhance the expression of LUC before the addition of IFN-alpha. When IFN-alpha was added, only the fragment of HPRE, HPREbeta(1)beta(2) could significantly reduce the expression of LUC, but the expression of LUC was not influenced by HPREalphabeta(1). CONCLUSION: The functional element beta(2) of HPRE plays a more important part in response to IFN-alpha than the element alpha and beta(1), which may be valuable for further research on mechanisms of IFN-alpha therapy in HBV infection and function of HPRE binding protein.

[Study on the coinfection of Hantavirus and Orientia tsutsugamushi in tissue cell culture].

OBJECTIVE: To investigate the possibility of Hantavirus (HV) and Orientia tsutsugamushi (Ot) coinfection in their hosts. METHODS: HV and Ot were used to infect Vero E6 cells cultured in vitro singly, simultaneously or successively. Genes of HV and Ot were identified in different generation cells with RT-PCR. RESULTS: Five experiment groups of infected Vero E6 cells were tested, the results were as follows: HV and Ot were both positive in infected Vero E6 cells passaged 2 times and the positive rate increased following the passaged times in HV and Ot infection groups, simultaneously or successively. However, in the groups which were infected with HV and Ot separately, the gene of HV or Ot could be detected in infected Vero E6 cells passaged only once and the positive rate increased following the times of the passaged. The positive rate was higher in the singly infected groups than in those infected simultaneously or successively. CONCLUSION: Coinfection of HV and Ot did exist in the hosts while HV and Ot could inhibit each other in the initial infection stage.

Production and characterization of a bacterial single-chain antibody fragment specific to B-cell-activating factor of the TNF family.

An active form of a single-chain antibody fragment (scFv) from the murine monoclonal antibody ABL-1, which is specific for B-cell-activating factor of the TNF family, was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker, using an assembly polymerase chain reaction. The construct VH-linker-VL was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli BL21(DE3) as inclusion bodies. After extraction from the E. coli cells, the inclusion bodies were solubilized and denatured in the presence of 8M urea. The expressed scFv fusion proteins were purified by Ni(2+)-IDA His-bind resin and finally renatured by dialysis. The purity and activity of the purified scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and enzyme-linked immunosorbent assay. The result revealed that the ABL-1 scFv retains the specific binding activity to BAFF with an affinity constant of 0.9x10(-8)molL(-1).

Expression, refolding, and characterization of human soluble BAFF synthesized in Escherichia coli.

The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Here, a recombinant form of the extracellular domain of the BAFF (hsBAFF) was expressed in Escherichia coli BL21(DE3) under the control of a T7 promoter. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 8 M urea. A rapid and simple on-column refolding procedure was developed. It was applied and then the refolded hsBAFF was purified by anion-exchange. The purified final product was >98% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 17.5 kDa, which equalled the theoretically expected mass. The N-terminal sequencing of refolding hsBAFF showed the sequence corresponded to the designed protein. The correct refolding of the recombinant protein was verified in the recovery of its secondary and tertiary structures as assessed by circular dichroism and fluorescence emission spectra. The renatured protein displayed its immunoreactivity with the antibodies to BAFF protein by Western blotting. The final purified material was biologically active in a validated induced human B lymphocyte proliferation bioassay. The expression and in vitro refolding of hsBAFF resulted in production of an active molecule in a yield of 15 mg/L flask cultivation.

[Study on hepatitis C virus genotyping in Yixing area, Jiangsu province].

OBJECTIVE: To investigate the distribution of hepatitis C virus (HCV) genotypes in Yixing, Jiangsu province. METHODS: Genotypes identification on sera samples were obtained from 158 donors who had already been anti-HCV positive through PCR method with type specific primer designed according to the sequence of 5'non-coding region (5'NCR). 5'NCR was also sequenced and compared with published date. Genotypes distribution was investigated in patients with different sex and clinical types of hepatitis C. RESULTS: Of the total 158 patients, 95 were HCV RNA positive in which 80 patients having genotype 1b (80/95; 84.4%), 5 patients having genotype 2(5/95; 5.3%), 5 patients with 1b/2 mixed genotypes (5/ 95; 5.3%) and another 5 patients whose genotype undetermined. The difference on the distribution of HCV genotypes was significant between female and male patients (P < 0.05) but not in different kinds of hepatitis C patients. CONCLUSION: Type 1b was the predominant HCV genotype in Yixing area.