Simultaneous determination of eleven volatile components in Cinnamomi Oleum by GC-MS / 中国中药杂志

A gas chromatography-triple quadrupole mass spectrometry(GC-MS) method was established for the simultaneous determination of eleven volatile components in Cinnamomi Oleum and the chemical pattern recognition was utilized to evaluate the quality of essential oil obtained from Cinnamomi Fructus medicinal materials in various habitats. The Cinnamomi Fructus medicinal materials were treated by water distillation, analyzed using GC-MS, and detected by selective ion monitoring(SIM), and the internal standards were used for quantification. The content results of Cinnamomi Oleum from various batches were analyzed by hierarchical clustering analysis(HCA), principal component analysis(PCA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) for the statistic analysis. Eleven components showed good linear relationships within their respective concentration ranges(R~2>0.999 7), with average recoveries of 92.41%-102.1% and RSD of 1.2%-3.2%(n=6). The samples were classified into three categories by HCA and PCA, and 2-nonanone was screened as a marker of variability between batches in combination with OPLS-DA. This method is specific, sensitive, simple, and accurate, and the screened components can be utilized as a basis for the quality control of Cinnamomi Oleum.

Association between age at menarche and prehypertension in female college students / 中国学校卫生

Objective@#To explore the correlation of age at menarche and prehypertension in female college students.@*Methods@#Age at menarche of 558 female college students was collected, while blood pressure, height and weight were measured, and body mass index(BMI) was calculated.@*Results@#Average age at menarche was (12.48±0.95) years, prevalence of prehypertension was 17.56% (98/558). The age of menarche was 8-11, 12, 13, 14, 15-19, and the prevalence of hypertension was 30.95%, 10.00% , 17.31%, 10.81%, 31.58% respectively, U shape association was observed in the association between prehypertension with age at menarche, lowest when age at menarche was <12 years. There was no significant difference in prehypertension between age at menarche was 13, 14 years old with 12-year-old group, those age at menarche 8-11 or 15-19 had a significantly higher risk of developing prehypertension than those 12-year-old( OR =4.03, 4.15, P <0.05).@*Conclusion@#Early or late menarche is associated with high blood pressure, appropriate intervention for girls with early menarche and obesity may be beneficial in reducing their future hypertension.

Influencing factors for the quality of bowel preparation with polyethylene glycol electrolyte powder combined with diet control before colonoscopy in children / 中国当代儿科杂志

OBJECTIVES@#To investigate the influencing factors for the quality of bowel preparation before colonoscopy in children and the association of the interval from the last administration of laxative to the start of colonoscopy (shortly referred to as waiting time) with the quality of bowel preparation.@*METHODS@#A retrospective analysis was performed for the children who were admitted to the Department of Gastroenterology, Children's Hospital of Nanjing Medical University, from January to November 2020, and received bowel preparation with polyethylene glycol electrolyte powder combined with diet control before colonoscopy. According to the score of Boston bowel preparation scale, they were divided into two groups: adequate bowel preparation group (n=337) and inadequate bowel preparation group (n=30). Related data were collected from the children in both groups, including general information, possible influencing factors for the quality of bowel preparation, adverse reactions associated with bowel preparation, duration of colonoscopy, and postoperative diagnosis. Univariate and multivariate analyses were used to explore the influencing factors for the quality of bowel preparation.@*RESULTS@#The univariate analysis showed that age, body weight, and waiting time were associated with inadequate bowel preparation (P<0.05). The multivariate analysis showed that older age (OR=2.155, 95%CI: 1.087-4.273, P=0.028) and longer waiting time (OR=1.559, 95% CI: 1.191-2.041, P=0.001) were independent risk factors for inadequate bowel preparation. The receiver operating characteristic (ROC) curve analysis showed that the cut-off value of waiting time was 5.5 hours in determining whether bowel preparation was adequate or not, with a sensitivity of 90.0%, a specificity of 50.7%, and an area under the ROC curve of 0.708. After grouping based on waiting time, it was found that the incidence rate of inadequate bowel preparation in the ≥5.5 hours group was significantly higher than that in the <5.5 hours group [14.0% (27/193) vs 1.7% (3/174), P<0.001].@*CONCLUSIONS@#For children who use polyethylene glycol electrolyte powder combined with diet control for bowel preparation, older age is an independent risk factor for inadequate bowel preparation before colonoscopy, which may be associated with an insufficient dose of polyethylene glycol in older children. Longer waiting time is also an independent risk factor for inadequate bowel preparation, and it is recommended that the waiting time should not exceed 5.5 hours.

Characteristics and health risk assessment of heavy metals in metro station’s airborne PM2.5 in a South China city / 公共卫生与预防医学

Objective To analyze characteristics of heavy metals in metro station’s airborne PM2.5 and to evaluate its health risk in a South China city. Methods A metro stations were selected for the study. Sampling sites of metro station included ground control, station hall and platform. The PM2.5 sampling was conducted one time per day for three consecutive days. The concentrations of ten heavy metals (As、Cr、Cd、Ni、Hg、Pb、Mn、Sb、Se、Cu) were determined. Inhalation exposure to these heavy metals2.52.5 range from 0.06 ng/m3 to 49.22 ng/m3. The concentrations of Mn、Cr and Ni in metro station’s airborne PM2.5 were respectively 3.75 times, 2.23times and 2.12 times higher than those in ground control. Increased lifetime cancer risk of carcinogenic heavy metal Cr exposure outrange the acceptable level (10-6) when its exposure time exceed 5 hours per day for lifetime. Cancer risk of carcinogenic heavy metal As exposure outrange the acceptable level (10-6) when its exposure time for adult male population exceed 8 hours per day for lifetime. Non-carcinogenic hazards risks of heavy metal Mn、Cu、Pb、Se、Hg and Sb in metro station’s airborne PMPM2.5 were little. Conclusions Airborne particulate matter in metro station has become one of the important sources of heavy metal exposure. Further attention should be paid to the possible carcinogenic risk of heavy metals in metro station’s airborne PM2.5 for long-term exposure.

Elimination of a disulfide bond in the light chain of coagulation factor VIII improves secretion of a BDD-FVIII variant with an engineered inter-chain disulfide / 药学学报

The coagulation VIII factor (FVIII) contains eight pairs of disulfide bonds, which are involved in maintaining its structure and function. It has been demonstrated that the disulfide bond between Cys1899/Cys1903 of the A3 domain in the light chain impedes secretion. In our previous work, an engineered inter-chain disulfide in the B domain-deleted FVIII (BDD-FVIII) promoted heterodimer assembly and secretion of separately expressed heavy and light chains. In this study, we constructed two BDD-FVIII variants, one of which contains an engineered inter-chain disulfide bond (F8C) between Met662 > Cys and Asp1828 > Cys mutations and another contains an endogenous A3 domain with a disrupted disulfide bond from F8C (F8CG) by replacement of Cys1899 and Cys1903 with Gly in F8C. We explored their function and secretion. By transducing F8C and F8CG into HEK293 and COS-7 cells, the formation of disulfide bonds and the secretion and coagulation activity of the two variants in the culture media and their binding affinity for von Willebrand factor (vWF) could be observed. The results show that variants F8C and F8CG are mainly the disulfide bonded heavy and light chain dimer, while the wild type BDD-FVIII (F8) is dominated by the easily dissociated heavy and light chain dimer. The secretion and activity of F8C was significantly higher than that of F8, while the secretion and activity of F8CG was significantly higher than that of F8C. The vWF binding of the two variants is similar to F8. This indicates that the BDD-FVIII variant F8CG may be attractive molecule for protein replacement and as a transgene in gene-therapy strategies. These findings are encouraging for future studies targeting disulfide bond elimination for further enhancement of FVIII secretion.

Effect of ARID2 knockout on proliferation gene expression of hepatocarcinoma cells Hep3B / 解放军医学杂志

Objective To construct ARID2 knockout human liver cancer cell line Hep3B by CRISPR/Cas9 system, explore the effect of ARID2 knockout on the proliferation of Hep3B, and the differences in gene expression between wild type and ARID2 knockout Hep3B cell lines. Methods The plasmid lentiCRISPRv2 was constructed with ARID2 knockout plasmid, and then transfected Hep3B cells; The positive cells were selected by puromycin, sorted by flow cytometry and cultured to obtain the monoclonal cell lines; ARID2 knockout Hep3B cell lines were identified by Western blotting and Sanger sequencing; The effect of ARID2 knockout on the proliferation of Hep3B cell line was detected by CCK-8 method; RNA-seq was used to analyze the differentially expressed genes between wild type Hep3B cells and ARID2 knockout Hep3B cells, and the results of RNA-seq were validated by Real-time quantitative PCR (RT-qPCR). The possible biology functions of ARID2 were explored through Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG Pathway), GO Biological Processes and Gene set enrichment analysis (GSEA). Results Two ARID2 knockout Hep3B cell lines were successfully constructed. Compared with the wild type cell line, the proliferation of ARID2 knockout cell lines was significantly accelerated (P<0.05). A total of 85 differentially expressed genes were identified by RNA-seq analysis between ARID2 knockout cell lines and wild type cell line, of which 17 genes were up-regulated and 68 down-regulated. The mRNA expression of 10 differentially expressed genes were validated by RT-qPCR, the verification results were consistent with that by RNA-seq. Results of KEGG Pathway, GO Biological Processes and GSEA indicated that ARID2 genes might be involved in such biological processes as protein processing and transport, chemokine signaling pathway, Wnt signaling pathway, complement and coagulation cascade reaction, epithelial-mesenchymal transition (EMT), glycolysis, TGF-β signaling pathway, and TNF-α/NF-KB signaling pathway, et al. Conclusion ARID2 knockout can promote proliferation of Hep3B cell line; and ARID2 may play an important role by variety of biological processes in tumor proliferation, invasion, metastasis and tumor microenvironment.

Application of phloroglucinol injection in embryo transfer in patients with recurrent implantation failure / 中国医师杂志

Objective To investigate the effect of phloroglucinol on pregnancy outcome in patients with recurrent implantation failure (RIF).Methods A total of 146 patients with RIF from March 2014 to March 2016 from the reproductive medical center of the Guangxi Zhuang Autonomous Region people's Hospital was randomly divided into two groups,73 cases were included in study group [16 cases of in vitro fertilization and embryo transfer (IVF-ET) and 57 cases of frozen/thawed embryo transfer (FET)].Patients in study group were given intramuscular injection of phloroglucino140mg,two times a day before the transplantation day to three days after transplantation,73 cases without phloroglucinol injection were included as control group.The biochemical pregnancy rate,clinical pregnancy rate,embryo implantation rate,abortion rate,ectopic pregnancy rate,multiple pregnancy rate and live birth rate were compared between two groups.Results The biochemical pregnancy rate in study group of FET was significantly higher than the control group (57.9％ vs 36.8％,P ＜0.05);the biochemical pregnancy rate in study group of IVF-ET was higher than the control group (50％ vs 37.5％,P ＞ 0.05),but there was no significant difference between the study group and control group;compared to the control group,the study group was increased clinical pregnancy rate,implantation rate,live birth rate,and decreased abortion rate (P ＞ 0.05),but the difference was not statistically significant.Conclusions The application of phloroglucinol in women with RIF may improve the biochemical pregnancy rate,especially in FET cycles.

Normal sperm morphology and the outcomes of routine in vitro fertilization / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the application value of morphology assessment of sperm from fresh semen in routine in vitro fertilization (IVF).</p><p><b>METHODS</b>We analyzed the morphology of the sperm from fresh or optimized semen samples and, based on the sperm morphology of the raw semen, allocated 908 IVF cycles due to the pure tubal factor to different groups: morphologically normal sperm (MNS) ≤ 4%, > 4% - ≤ 15%, and > 15% in Trial 1 and MNS ≤ 1%, > 1% - ≤ 2%, > 2% - ≤ 3%, and > 3%-- ≤ 4% in Trial 2. We compared the rates of fertilization, cleavage, high-quality embryo, -blastocyst formation, and pregnancy among different groups.</p><p><b>RESULTS</b>The total fertilization rate was significantly lower in the MNS ≤ 4% than in the MNS > 4% - ≤ 15% and >15% groups (74.40% vs 78.61% and 80.03%, P < 0.01). Compared with the MNS ≤ 1%, > 1% - ≤ 2%, and > 2% - ≤ 3% groups, the MNS > 3% - ≤ 4% group showed remarkably increased rates of 2PN normal fertilization (77.23%, 78.97% and 78.99% vs 85.47%, P < 0.01), cleavage (95.71%, 96.01% and 97.27% vs 98.73%, P < 0.05), and blastocyst formation (53.85%, 49.01% and 49.55% vs 63.41%, P < 0.01). No statistically significant differences were observed in the rates of clinical pregnancy, implantation, early abortion, live birth, or malformation at birth among different groups (P > 0.05).</p><p><b>CONCLUSION</b>MNS ≤ 4% affected the total rate of fertilization while MNS ≤ 3% reduced the rate of normal fertilization in IVF. However, even MNS ≤ 1% did not result in fertilization disorder or failure. Therefore, teratozoospermia alone was not an indicator of ICSI and sperm mor- phology assessment had no obvious value for predicting the rates of embryo quality, clinical pregnancy, and live birth in IVF.</p>

Expression of Attractin in male reproductive tract of human and mice and its correlation with male reproduction / 华中科技大学学报（医学）（英德文版）

The expression of Attractin mRNA and protein in testis and semen of human and male mice was investigated. Human testis and semen samples were all collected from Reproductive Center of Renmin Hospital, Wuhan University in December, 2012. Testis samples were collected from 7 cases of obstructive azoospermias when they were subjected to diagnosed testis biopsy, and 30 normal human semen samples were obtained from those cases of semen analysis. Adult mice testis tissues were obtained from 10 2-month-old male BALB/c mice, and 60 male mice at different ages were classified into 10 groups (day 1, 5, 10, 15, 21, 28, 35, 42, 56, and 120 respectively, n=6 each). The expression of Attractin mRNA and protein in testis was detected by RT-PCR and Western blotting respectively. Human semen samples were centrifuged into sperm plasma (SP) and sperm extract (SE), and mice sperm samples were collected from the epididymis of 10 adult male BALB/c mice. Western blotting was used to determine the Attractin protein expression level. Attractin mRNA and protein were expressed in the testis of both patients with obstructive azoospermias and adult Bcl/B mice. Quantitative RT-PCR revealed that no Attractin mRNA was detectable in day 1 male BALB/c mice group. The Attractin mRNA and protein levels were low on the day 10, and increased with age until day 56. On the day 120, the expression levels of Attractin were decreased. As for human semen samples, Attractin protein was expressed in both SP and SE, but didn't exist in samples from the epididymis of male BALB/c mice. It was suggested that Attractin acted as a novel active substance and was involved in male reproduction in both human and BALB/c mice, but it exerted a different expression profile in different mammal species.

Expression of Attractin in male reproductive tract of human and mice and its correlation with male reproduction / 华中科技大学学报（医学）（英德文版）

The expression of Attractin mRNA and protein in testis and semen of human and male mice was investigated. Human testis and semen samples were all collected from Reproductive Center of Renmin Hospital, Wuhan University in December, 2012. Testis samples were collected from 7 cases of obstructive azoospermias when they were subjected to diagnosed testis biopsy, and 30 normal human semen samples were obtained from those cases of semen analysis. Adult mice testis tissues were obtained from 10 2-month-old male BALB/c mice, and 60 male mice at different ages were classified into 10 groups (day 1, 5, 10, 15, 21, 28, 35, 42, 56, and 120 respectively, n=6 each). The expression of Attractin mRNA and protein in testis was detected by RT-PCR and Western blotting respectively. Human semen samples were centrifuged into sperm plasma (SP) and sperm extract (SE), and mice sperm samples were collected from the epididymis of 10 adult male BALB/c mice. Western blotting was used to determine the Attractin protein expression level. Attractin mRNA and protein were expressed in the testis of both patients with obstructive azoospermias and adult Bcl/B mice. Quantitative RT-PCR revealed that no Attractin mRNA was detectable in day 1 male BALB/c mice group. The Attractin mRNA and protein levels were low on the day 10, and increased with age until day 56. On the day 120, the expression levels of Attractin were decreased. As for human semen samples, Attractin protein was expressed in both SP and SE, but didn't exist in samples from the epididymis of male BALB/c mice. It was suggested that Attractin acted as a novel active substance and was involved in male reproduction in both human and BALB/c mice, but it exerted a different expression profile in different mammal species.

Effect of the 5-HT4 receptor and serotonin transporter on visceral hypersensitivity in rats

Visceral hypersensitivity plays an important role in motor and sensory abnormalities associated with irritable bowel syndrome, but the underlying mechanisms are not fully understood. The present study was designed to evaluate the expression of the 5-HT4 receptor and the serotonin transporter (SERT) as well as their roles in chronic visceral hypersensitivity using a rat model. Neonatal male Sprague-Dawley rats received intracolonic injections of 0.5% acetic acid (0.3-0.5 mL at different times) between postnatal days 8 and 21 to establish an animal model of visceral hypersensitivity. On day 43, the threshold intensity for a visually identifiable contraction of the abdominal wall and body arching were recorded during rectal distention. Histological evaluation and the myeloperoxidase activity assay were performed to determine the severity of inflammation. The 5-HT4 receptor and SERT expression of the ascending colon were monitored using immunohistochemistry and Western blot analyses; the plasma 5-HT levels were measured using an ELISA method. As expected, transient colonic irritation at the neonatal stage led to visceral hypersensitivity, but no mucosal inflammation was later detected during adulthood. Using this model, we found reduced SERT expression (0.298 ± 0.038 vs 0.634 ± 0.200, P < 0.05) and increased 5-HT4 receptor expression (0.308 ± 0.017 vs 0.298 ± 0.021, P < 0.05). Treatment with fluoxetine (10 mg·kg-1·day-1, days 36-42), tegaserod (1 mg·kg-1·day-1, day 43), or the combination of both, reduced visceral hypersensitivity and plasma 5-HT levels. Fluoxetine treatment increased 5-HT4 receptor expression (0.322 ± 0.020 vs 0.308 ± 0.017, P < 0.01) but not SERT expression (0.219 ± 0.039 vs 0.298 ± 0.038, P = 0.654). These results indicate that both the 5-HT4 receptor and SERT play a role in the pathogenesis of visceral hypersensitivity, and its mechanism may be involved in the local 5-HT level.

Effect of trimebutine maleate on ion channels in colon smooth muscle cell of cold restraint stress induced rats / 中华消化杂志

Objective To investigate the effects of trimebutine maleate (TM) on the expression of large conductance calcium-activated potassium channel (BKCa) and ryanodine receptors (RyR)channels at mRNA and protein level in colonic smooth muscle cell of cold restraint stress(CRS)induced rats.Methods A total of 24 Wistar rats were divided into CRS group,CRS with TM group and control group equally.The rats of CRS group were gavaged with 0.9％NaCl (6 ml/kg) daily; the rats of CRS with TM group were gavaged with 15 g/L TM (6 ml/kg) daily and activity was restricted in wire cage at 4 ℃ for two hours,continuously for five days.The rats of control group were gavaged with 0.9 ％ NaCl (6 ml/kg) once without CRS.The amount and characteristics of stool of rats in each group were observed.The colonic smooth muscle was isolated to detect the expression of BKCa and RyR at mRNA and protein level by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot.Results The median of rats defecation particles of CRS group was six,control group was one and CRS with TM group was five.Compared with control group,the defecation appearance of CRS group and CRS with TM group was looser and wetter observed by naked eyes.Compared with control group,there was no obvious pathological changes in CRS and CRS with TM group.There was no significant difference in the mRNA expression of BKCa and RyR channels between control group and CRS group.Compared with control group,the BKCa expression at mRNA level of CRS with TM group increased 1.45 fold.Compared with control group,the RyR2 expression at mRNA level of CRS with TM group increased 1.32 fold.Compared with control group,the BKCa expression at protein level of CRS with TM group increased 1.39 fold,and there was no RyR2 expression band at protein level.Conclusion TM might affect colonic smooth muscle contraction through the upregulation of BKCa expression at mRNA and protein level and RyR expression at mRNA level.

Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system / 药学学报

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.

Trans-splicing of Cys mutated coagulation factor VIII / 药学学报

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.

Expression of heme oxygenase enzyme in the testis tissue and azoospermia / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the location of heme oxygenase (HO) enzyme in the human testis, and explore the correlation of the expression of HO enzyme with azoospermia by analyzing its different expression levels in the testes of nonobstructive azoospermia, obstructive azoospermia and normal men.</p><p><b>METHODS</b>We detected the location of the cells expressing HO enzyme in the human testis tissue using immunohistochemistry, determined the mRNA and protein expression levels of HO-1 and HO-2 in the testes of azoospermia patients and normal healthy men by RT-fluorescence quantitative PCR (RT-FQ-PCR) and Western blot, and explored the correlation of HO expressions with the pathogenesis of azoospermia.</p><p><b>RESULTS</b>HO-1 enzyme was expressed mainly in the Sertoli cells and HO-2 enzyme chiefly in the germ cells of the testis tissue. RT-FQ-PCR showed that the expression of HO-1 in the testis tissue was significantly lower in the nonobstructive azoospermia than in the normal and obstructive azoospermia groups (P < 0.05), with no significant difference between the latter two. Western blot revealed no obvious difference between the expression level of HO-1 protein and that of HO-1 mRNA. There were no differences in the expression level of HO-2 protein among the three groups.</p><p><b>CONCLUSION</b>The expression level of HO enzyme is significantly decreased in the testis tissue of nonobstructive azoospermia patients, and the expression of HO-1 protein is consistent with that of HO-1 mRNA. As HO-1 protects the testis tissue against various stress injuries through its antioxidant, anti-inflammatory and anti-apoptotic effects, its decreased expression level may be correlated with spermatogenic dysfunction, and therefore considered as a possible mechanism of nonobstructive azoospermia.</p>

Enhancing effect of deoxynivalenol-mediated GRP78 down-regulation on heavy chain secretion and bioactivity of two-chain FVIII gene co-transfected cells / 药学学报

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.

Molecular properties of grass carp reovirus in southern China and establishment of a duplex PCR detection method / 病毒学报

A strain of grass carp reovirus was isolated from sick grass carp with symptoms of haemorrhage in Guangdong province in 2009. The strain was tentatively named as GCRV-GD108 because it was isolated from grass carp and possessed 11 segments of dsRNA. Complete genome sequence analysis showed that significant differences existed between GCRV-GD108 and GCRV, as well as other known species of aquareovirus. In this study, molecular characteristics of diseased grass carp collected from different farms in Guangdong, Fujian and Hunan provinces were assayed. Based on the sequences of the 11 segments of GCRV-GD108, PCR primers corresponding to each of the segments were designed and synthesized. Total RNA of the diseased fish was extracted and used as templates of RT-PCR reaction. Specific amplification bands were obtained from all of the samples whereas no band was produced from GCRV standard strain. While using the primers specific to GCRV produced specific band in GCRV standard strain rather than in these collected samples. Sequencing of the amplification products showed that these samples displayed high similarities with each other (95.2%-99.4%), and they also shared high sequence similarities with that of GCRV-GD108 (95.0%-99.8%), suggesting that these samples shared similar molecular characteristics with those of GCRV-GD108, and were quite different from GCRV as well as the known species of genus aquareovirus. The results indicated that there are different molecular types of reovirus existed in the pond-cultured grass carp in China, and GCRV-GD108 is a representative strain in southern China, therefore great attention should be paid in order to control the disease efficiently, especially in vaccine preparation. Two pairs of primers were chosen to establish a duplex PCR assay method by combining each pair of the primers specific to GCRV-GD108 with the GCRV primer pair respectively. The duplex PCR assay method will enable the identification of GCRV-GD108 or GCRV by only a single PCR reaction.

vWF improves secretion and activity of intein spliced BDD-FVIII / 药学学报

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion / 药学学报

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.

Glycosylation and L303e/F309S mutations improve intein-mediated splicing of the split coagulation factor VIII / 药学学报

We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity. The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to (149 +/- 23) ng x mL(-1) and (1.12 +/- 0.14) u x mL(-1) respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells [(99 +/- 14) ng x mL(-1) and (0.77 +/- 0.13) u x mL(-1)] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity. A cellular mechanism-independent BDD-FVIII splicing was also observed. It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.