Development of a novel multi-functional integrated bioconjugate effectively targeting K-Ras mutant pancreatic cancer / 药物分析学报

Folate receptor(FR)overexpression occurs in a variety of cancers,including pancreatic cancer.In addi-tion,enhanced macropinocytosis exists in K-Ras mutant pancreatic cancer.Furthermore,the occurrence of intensive desmoplasia causes a hypoxic microenvironment in pancreatic cancer.In this study,a novel FR-directed,macropinocytosis-enhanced,and highly cytotoxic bioconjugate folate(F)-human serum albumin(HSA)-apoprotein of lidamycin(LDP)-active enediyne(AE)derived from lidamycin was designed and prepared.F-HSA-LDP-AE consisted of four moieties:F,HSA,LDP,and AE.F-HSA-LDP presented high binding efficiency with the FR and pancreatic cancer cells.Its uptake in wild-type cells was more extensive than in K-Ras mutant-type cells.By in vivo optical imaging,F-HSA-LDP displayed prominent tumor-specific biodistribution in pancreatic cancer xenograft-bearing mice,showing clear and lasting tumor localization for 360 h.In the MTT assay,F-HSA-LDP-AE demonstrated potent cytotoxicity in three types of pancreatic cancer cell lines.It also induced apoptosis and caused G2/M cell cycle arrest.F-HSA-LDP-AE markedly suppressed the tumor growth of AsPc-1 pancreatic cancer xenografts in athymic mice.At well-tolerated doses of 0.5 and 1 mg/kg,(i.v.,twice),the inhibition rates were 91.2％and 94.8％,respectively(P＜0.01).The results of this study indicate that the F-HSA-LDP multi-functional bioconjugate might be effective for treating K-Ras mutant pancreatic cancer.

Preparation and anti-tumor activity of a novel antibody-drug conjugate 607-LDM / 药学学报

Antibody-drug conjugates (ADCs) are one of the most important classes of anticancer therapeutics. Human epidermal growth factor receptor-2 (HER2), which is highly expressed in many types of aggressive cancers including breast and ovarian cancer, has been approved as an ideal target for ADCs. Lidamycin (LDM), developed by Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, is an enediyne-containing antibiotic with potent anti-tumor activity. LDM is a promising payload for ADCs. In the present research, using a special site-directed conjugating technology, we made a novel ADC (607-LDM) with a drug-to-antibody ratio (DAR) of 2 and composed of the anti-HER2 antibody 607 and LDM. The new ADC exhibited potent antitumor activity against human ovarian cancer SKOV3 and breast cancer BT-474 cells. It also induced apoptosis and G2/M arrest. In nude mice with SKOV3 xenografts and a tumor volume of 150-200 mm3, a single intravenous injection 607-LDM at 1 mg·kg-1 induced tumor growth inhibition of 72.4%, which was significant compared to either LDM (50.6%) or antibody (30.2%) treatment alone, or both in combination (50.1%, P < 0.05). All animal experiments were performed in accord with National Regulations and approved by the Animal Experiments Ethical Committee of College of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. The novel ADC designed in this study, 607-LDM, is a promising candidate for the treatment of HER2-positive cancers.

Promoting tanshinone synthesis of Salvia miltiorrhiza root by a seed endophytic fungus, Phoma herbarum D603 / 中国中药杂志

The interaction of endophytes and host plant is an effective mean to regulate the growth and secondary metabolism of medicinal plants. Here we want to elucidate the effects and mechanism of Phoma herbarum D603 on the root development and tanshinone synthesis in root of Salvia miltiorrhiza by endophyte-plant coculture system. The mycelium of P. herbarum D603 was colonized in the root tissue space, and formed a stable symbiotic relationship with host plant. The in vitro activities analysis showed that the concentration of IAA produced by D603 can reach(6.45±0.23) μg·mL~(-1), and this strain had some abilities of phosphorus solubilization and siderophore production activities. The coculture experiment showed that strain D603 can significantly promote the synthesis and accumulation of tanshinones in the root of S. miltiorrhiza, in which after 8 weeks of treatment with D603, the content of tanshinone Ⅱ_A in the roots reached up to(1.42±0.59) mg·g~(-1). By the qRT-PCR analysis results, we found that D603 could improve the expression levels of some key genes(DXR, DXS, GGPP, HMGR, CPS) of tanshinone biosynthesis pathway in host plant S. miltiorrhiza, but the promoting effect mainly occurred in the early stage of the interaction, and the enzyme activity level decreased in varying degrees of the later stage. In summary, seed-associated endophyte P. herbarum D603 can promote the growth and root development of S. miltiorrhiza by producing hormones, promoting nutrient absorption and siderophore production, and promote the synthesis and accumulation of tanshinones by regulating the expression level of key genes in the synthetic pathway in S. miltiorrhiza.

Systematic Review of Therapeutic Effect of Electroacupuncture for Idiopathic Facial Palsy at Acute Stage / 广州中医药大学学报

Objective To make a systematic review of therapeutic effect of electroacupuncture(EA) for idiopathic facial palsy at acute stage. Methods With reference to the included criteria and excluded criteria, clinical controlled trials of EA for idiopathic facial palsy at acute stage were screened from the databases of CBM, CNKI, Wanfang, VIP by computer retrieval and from the primary domestic academic journals of acupuncture by manual retrieval. Systematic review was performed following the Cochrane Reviewers' Handbook, and RevMan 5.3 was used for Meta-analysis. Results Sixteen clinical trials were included. The Meta-analysis results showed that the cure rate evaluated with 3 kinds of therapeutic effect criteria in the observation group was superior to that of the control group, the difference being significant (P<0.05 or P<0.01); the effects on improving days for cure, cure case number within one month, blink reflex, and case number with complications in the observation group were also superior to that of the control group, the difference being significant (P < 0.01). Conclusion EA exerts certain therapeutic effect for idiopathic facial palsy at acute stage. But for the quantity and quality of the included trials are not satisfactory, the conclusion still needs more high-quality, large-size and long-term follow-up trials to verify.

Expression and activity of cathepsin L2 and its effect on the degradation of melanosomes in skin lesions of seborrheic keratosis / 中华皮肤科杂志

Objective To determine the expression of cathepsin L2 (CTSL2)and evaluate its activity in skin lesions of seborrheic keratosis (SK),to observe the ultrastructural changes of melanosomes in the skin lesions of SK,and to estimate the effect of CTSL2 on the degradation of melanosomes.Methods Twenty patients with SK were enrolled from the Department of Dermatology,Renmin Hospital of Wuhan University.The lesional tissue and the perilesional normal skin were biopsied from each patient.Among 15 of the 20 patients,hematoxylin and eosin (HE)staining and Fontana-Masson silver staining were performed to observe the distribution of melanin granules,transmission electron microscopy (TEM)was conducted to observe the ultrastructural changes of melanosomes,and immunohistochemical staining was performed to estimate the cellular proliferative activity.RT-PCR and fluorogenic substrate cleavage assay were performed in the other 5 patients to determine the mRNA expression of CTSL2 and evaluate its activity,respectively.Sucrose density gradient ultracentrifugation was performed to isolate and purify melanosomes from the retinal pigment epithelium (RPE) harvested from a discarded eyeball of a 35-year old male patient with informed consent.The purified melanosomes were incubated with epidermal lysates of SK lesions,and TEM was used to observe the changes in the membrane structure of melanosomes.Statistical analysis was carried out by paired t test,and a P value ＜ 0.05 was considered statistically significant.Results A large number of melanin granules were deposited in SK lesions,while the linear deposition of melanin granules was only seen in the basal layer of the normal skin.TEM showed that the percentage of damaged melanosomes was much higher in the normal skin (49.00％ ± 4.00％) than in the SK lesions (24.33％ ± 3.06％)(t =8.49,P ＜ 0.05).RT-PCR revealed that the mRNA expression and activity of CTSL2 were both significantly lower in the SK lesions than in the normal skin (mRNA:0.35 ± 0.09 vs.0.43 ± 0.08,t =3.17,P ＜ 0.05;activity:17.46 ± 0.45 vs.28.78 ± 0.58,t =34.29,P ＜ 0.05).Moreover,TEM also showed that the percentage of damaged melanosome was lower in the SK lesion lysate-treated group (32.33％ ± 4.93％) than in the normal skin lysate-treated group (43.00％ ± 2.65％,t =3.30,P ＜ 0.05).Conclusion Decreased expression of CTSL2 in the SK lesions can affect the degradation of melanosomes by keratinocytes.However,whether CTSL2 directly takes part in the pathogenesis of SK or not is still needed to be further confirmed.

Expression of myeloperoxidase, soluble intercellular adhesion molecule-1 and pentraxin-3 in the gingival crevicular fluid during maxillary canine distal movement / 中国组织工程研究

BACKGROUND:To dynamicaly monitor the varying levels of inflammatory factors in the gingival crevicular fluid is helpful to assess the early effect of orthodontic tooth movement. Myeloperoxidase, soluble intercelular adhesion molecule-1, pentraxin 3 are proven to be closely related to inflammation, but it is unclear about the levels of these three kinds of inflammatory factors as wel as association of these three kinds of inflammatory factors with orthodontic tooth. OBJECTIVE:To detect the expression levels of myeloperoxidase, soluble intercelular adhesion molecule-1 and pentraxin-3 in the gingival crevicular fluid during maxilary canine distal movement and to assess their correlation with periodontal disease, canine movement distance and orthodontic force. METHODS:Twenty-one orthodontic patients were enroled and assigned into 150 g (n=12) or 100 g (n=9) groups according to orthodontic force. The gingival crevicular fluid samples of orthodontic patients were colected before and at 4, 12, 24 hours, 7, 14 days after maxilary canine distal movement. Levels of myeloperoxidase, soluble intercelular adhesion molecule-1 and pentraxin-3 in the gingival crevicular fluid were measured and analyzed using ELISA assay. RESULTS AND CONCLUSION: During the distal movement of maxilary canine, under orthodontic force, the level of myeloperoxidase was peaked at 4 hours and then decreased, while the expression level of soluble intercelular adhesion molecule-1 was peaked at 12 hours, and then decreased. Both myeloperoxidase and soluble intercelular adhesion molecule-1 levels returned to normal at 7 days under orthodontic force. The expression level of pentraxin-3 was increased significantly under orthodontic force, peaked at 24 hours, and then decreased gradualy to the normal level at 7 days. In addition, the expression levels of myeloperoxidase, soluble intercelular adhesion molecule-1 and pentraxin-3 in the gingival crevicular fluid were significantly higher under 150 g force than under 100 g force. These findings indicate that detecting varying levels of myeloperoxidase, soluble intercelular adhesion molecule-1 and pentraxin-3 in the gingival crevicular fluid is useful to assess the efficiency of orthodontic treatment and prevent adverse reactions.

Curative effect of continuous positive airway pressure on treatment of patients with obstructive sleep apnea hypopnea syndrome and hypertension: A Meta-analysis / 解放军医学杂志

Objective To systematically evaluate the curative effect of continuous positive airway pressure (CPAP) on treatment of patients with obstructive sleep apnea hypopnea syndrome (OSAHS) and hypertension. Methods The data were retrieved of randomized controlled trials (RCTs) about the curative effect of CPAP on treatment of patients with OSAHS and hypertension from PubMed, Cochrane Library, CNKI, VIP, CBM and WanFang database from inception to Oct. 2015. Literature screening, data extraction and risk bias assessment were performed by two independent reviewers, and meta-analysis was then carried out by using RevMan 5.3 software. Results A total of 16 RCTs involving 2101 patients were included. Meta-analysis revealed that, compared with the antihypertensive drug therapy alone, CPAP plus antihypertensive drug therapy significantly reduced the daytime systolic pressure [MD=–12.60, 95%CI(–17.68 to –7.52), P<0.00001], nighttime systolic pressure [MD=–21.90, 95%CI(–25.94 to –17.86), P<0.00001] and nighttime diastolic pressure [MD=–11.90, 95%CI(–15.44 to –8.36), P<0.00001], while created no significant difference in daytime diastolic pressure, 24-h mean systolic pressure and 24-h mean diastolic pressure in a followingup less than 12 weeks. Whereas in the following-up no less than 12 weeks, compared with the antihypertensive drug therapy alone, CPAP plus antihypertensive drug therapy significantly reduced the 24-h mean systolic pressure [MD=–7.88, 95%CI(–12.09 to –3.66), P=0.00002], 24-h mean diastolic pressure [MD=–5.14, 95%CI(–6.00 to –4.28), P<0.00001], daytime systolic pressure [MD=–5.89, 95%CI(–8.79 to –2.98), P<0.0001], daytime diastolic pressure [MD=–4.34, 95%CI(–6.32 to –2.36), P<0.0001]; nighttime systolic pressure [MD=–7.06, 95%CI(–11.12 to –2.99), P=0.0007] and nighttime diastolic pressure [MD=–4.49, 95%CI (–7.39 to –1.58), P=0.006]. Conclusions The current evidences suggest that on the basis of antihypertensive drug therapy, CPAP may effectively reduce the systolic pressure and diastolic pressure of OSAHS patients with hypertension at long-term follow-up, but the effect in a short-term follow-up is not obvious. For the quality and quantity of included studies limited, more high quality and larger sample studies are needed to verify the above conclusion

Pathogenic surveillance of scarlet fever in Xicheng District of Beijing in 2014 / 国际检验医学杂志

Objective To perform pathogenic surveillance of scarlet fever in Xicheng District of Beijing in 2014 ,and provide sci‐entific data for developing reasonable prevention policy for the disease .Methods The throat swab specimens from 212 patients who were diagnosed with streptococcal infection ,tonsillitis and angina in surveillance hospital were collected ,and from which the patho‐gens were isolated and identified .Results In the 212 samples ,the positive rate of A group hemolytic streptococcus were 21 .2%(45/212) .The patients were mainly 4-15 years old ,especially 6- <8 years old .The positive rate was highest in 6- <7 years old children .The peak of disease incidence was observed in May and June .Conclusion Preschools and schools were the key sites for scarlet fever prevention and control ,therefore ,the surveillance and prevention should be further strengthened to prevent the out‐break .

An overview of antibody-based cancer therapy / 药学学报

The use of monoclonal antibodies (mAbs) for cancer therapy has achieved considerable success in recent years. Approximate 17 monoclonal antibodies have been approved as cancer therapeutics since 1997. Antibody-drug conjugates (ADC) are powerful new treatment options for cancer, and naked antibodies have recently achieved remarkable success. The safety and effectiveness of therapeutic mAbs in oncology vary depending on the nature of the target antigen and the mechanisms of tumor cell killing. This review provides a summary of the current state of antibody-based cancer therapy, including the mechanisms of tumor cell killing by antibodies, tumor antigens as antibody targets, clinical effectiveness of antibodies in cancer patients and nanoparticles-based ADCs.

Sonic hedgehog signaling pathway in tumorigenesis of glioma / 第二军医大学学报

Sonic hedgehog(Shh) is a member of the hedgehog family in vertebrate. The Shh signaling pathway is mainly composed of a secreted glycoprotein, named Shh, two transmembrane proteins (Ptch and Smo) and the downstream transcription factor family Glis. It plays a vital role in the embryo development, especially in the neuronal system. Recent study have demonstrated that the Shh pathway is closely associated with the tumorigenesis of various tumors. Glioma, the most common malignant brain tumor of humans, is characterized by the rapid proliferation, infiltrative growth and high rate of relapse, and it is one of the brain tumors with poorest prognosis. Abnormal activation of multiple signaling pathways has been known to enhance the proliferation ability of glioma cells. Moreover, glioma is composed of various tumor cells and the glioma stem cells were endowed with the ability of self-renewal and unlimited proliferation, which plays a key role in the tumorigenesis, progress and relapse. Evidence has been found that Shh signaling pathway is closely assoicated with tumorigenesis of glioma. Herein we review the current knowledge on the components of Shh signaling pathway and its role in the tumorigenesis of glioma and glioma stem cells.

Study on antibacterial active components from Viola yedoensis / 中国中药杂志

<p><b>OBJECTIVE</b>Study on the antibacterial activity of Viola yedoensis and the antibacterial active compounds.</p><p><b>METHOD</b>The chemical compositions were isolated by means of solvent extraction, column chromatography on silica gel, sephadex LH-20 and crystallization. The antibacterial activities were tested by Neo-Sensitab disk-diffusion method, nephelometric analysis and plating method.</p><p><b>RESULT</b>One new compound (4) along with three known compounds were isolated from this plant for the first time and were identified as aesculetin (1), 6,7-dimethoxycoumarin (2), scopoletin (3) and 5-methoxy-7-hydroxymethylcoumarin (4), respectively. All the compounds showed antibacterial and antibactericidal activities at varying degree on Streptococcus Aureas, S. agalactiae, S. uberis, S. dysgalactiae, E. coli and Salmonella, of which 1 was most active with 0.031- 0.313 g x L(-1) of minimal inhibitory concentrations (MIC) and 0.313 - 0.625 g x L(-1) of minimal bactericidal concentrations (MBC).</p><p><b>CONCLUSION</b>Viola yedoensis has a broad spectrum of antibacterial activity on animal pathogenic bacteria, and coumarins may be the main antibacterial activity ingredients.</p>

Secreted expression of dengue virus type 2 envelope glycoprotein in eukaryotic cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly.</p><p><b>METHODS</b>The entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, SA14-14-2 strain) was introduced using fusion PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3' terminus and one chimeric gene, which was generated by replacing the 3' terminal 20% region of E gene with the corresponding sequence of JEV (SA14-14-2 strain). The PCR segments were inserted into the NheI and NotI sites of pcDNA5/FRT vector or into the NheI and XhoI sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay (IFA) and Western Blot.</p><p><b>RESULTS</b>After transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis.</p><p><b>CONCLUSION</b>Signal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.</p>

Secreted expression of dengue virus type I envelope glycoprotein in 293T cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To expression prM/E gene of dengue virus type I in mammalia cells.</p><p><b>METHODS</b>The full-length prM/E gene of dengue virus type I strain GZ01/95 was amplified by RT-PCR, the signal peptide preceding the prM gene was added or the carboxyl-terminal 20% of DEN-1 E was replaced with the corresponding JE sequence in the meanwhile, and three of the constructions were cloned into the pcDNA5/FRT.Then they were transfected into 293T cells by lipofectamine respectively. The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA) as well as Western blot.</p><p><b>RESULTS</b>In the cytoplasm of 293T cells transfected with all the recombinant plasmids DNA, the expressed products for gene of dengue virus type I were confirmed by IFA. The secreted expression products for gene of dengue virus type I specific protein bands were confirmed by Western blot only existing in the cell supernatants transfected with the modified recombinant plasmids DNA.</p><p><b>CONCLUSION</b>The prM/E protein of dengue virus type 1 were expressed in 293T cells transfected with all the three recombinant plasmids DNA. The prM/E protein was obtained secretion after transfecting the modified recombinant plasmids adding a signal peptide preceding the prM gene or replacing the carboxyl-terminal 20% of E with the corresponding JE sequence.</p>

Association of non-alcoholic fatty liver with plasma homocysteine and the methylenetetrahydrofolate reductase gene polymorphism in patients of type 2 diabetes mellitus in Shanxi, China / 中华全科医师杂志

To investigate the relationship between plasma level of homocysteine(Hcy) and the methylenetetrahydrofolate reductase ( MTHFR ) gene polyroorphism with non-alcoholic fatty liver in patients with type 2 diabetes mellitus (T2DM). Methods In a case-control study, plasma levels of Hcy, folic acid (FA), vitamin B12 (VitB12), glycosylated hemoglobin Alc (HbAlc), fasting blood glucose (FBG), total cholesterol and triglyceride were measured in 159 T2DM patients with and without non-alcoholic fatty liver ( NAFL), as well as 52 normal controls. Mutation of the C677T of MTHFR gene was determined by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) for all of them. Results Patients of T2DM both without NAFL (96 case) and with NAFL had higher prevalence of hyperhomocysteinemia (Hhcy) (49% and 21%, respectively ) than normal controls did (4 cases, 8% ) (P<0.05), while patients of T2DM with NAFL had higher prevalence of Hhcy than those without it did (P <0. 05). Plasma level of Hey positively correlated to genotype frequency of the MTHFR gene, plasma 0levels of HbAlc and FBG in patients of T2DM, with coefficients of correlation of 0.248, 0.423 and 0.242, respectively (P < 0.05). Results of multiple logistic regression analysis showed that course of the disease, body mass index, plasma levels of FBG and Hcy all were independent risk factors for non-alcoholic fatty liver in patients with T2DM. Conclusions Hhey was an independent risk for non-alcoholic fatty liver and plasma level of Hey was influenced by frequency of the TT genotype of the MTHFR gene, plasma levels of FA and VitB12, as well as metabolic disturbance in patients with T2DM.

Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv / 药学学报

Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

Dynamic measurements of serum inhibin B and estradiol: a predictive evaluation of ovarian response to gonadotrophin stimulation in the early stage of IVF treatment / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>We dynamically measured serum inhibin B and estradiol in the early stage of hormonal stimulation to predict the ovarian response in in vitro fertilization (IVF) treatment.</p><p><b>METHODS</b>A total of 57 patients (<40 years of age) who underwent the first cycle of long protocol IVF or introcytoplasmic sperm injection (ICSI) treatment were included. Serum inhibin B, estradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels were measured four times: (1) on Day 3 of the menstrual cycle (basal); (2) on the day before the first administration of gonadotrophin (Gn) (Day 0); (3) on Day 1 of Gn therapy; and (4) on Day 5 of Gn therapy. Comparisons of these measurements with ovarian responses and pregnancy outcomes were made and analyzed statistically.</p><p><b>RESULTS</b>(1) On Day 1 and Day 5 of recombinant FSH (rFSH) stimulation, ovarian response, i.e., numbers of follicles, oocytes, fertilized oocytes, and embryos, had a positive correlation (r(s)=0.46~0.61, P=0.000) with raised inhibin B and estradiol concentrations, but a negative correlation (r(s)=-0.67~-0.38, P=0.000 or P<0.01) with total rFSH dose and total days of rFSH stimulation. (2) No significant variation (P>0.05) between the pregnant and non-pregnant groups on the basis of mean age or on all hormone concentrations at four times of the IVF cycle was observed. However, all the seven patients aged >35 years did not reach pregnancy.</p><p><b>CONCLUSIONS</b>(1) Serum inhibin B and estradiol concentrations obtained shortly after Gn therapy may offer an accurate and early prediction of ovarian response; (2) Low levels of serum inhibin B and estradiol obtained shortly after Gn stimulation indicate the need for a longer period of Gn treatment and a higher daily dosage; (3) No obvious pregnancy difference among patients of age <35 years was found; however, IVF pregnancy outcome is significantly lower in women of age >35 years.</p>

Construcion of a chimeric Japanese encephalits virus/dengue virus-2 / 病毒学报

The prM/E gene of DV2 was cloned into the JEV (SA14-14-2 strain) replicon vector which had been constructed previously, and the resulting recombinant plasmid was named pPartialdeltaprM/E. The constructed chimeric clone was linearized and then was transcripted into RNA in vitro. The produced RNA was transfected into BHK-21 cells. Five to seven days later, CPE could be observed on the transfected BHK-21cells, and then the supernatant containing the chimeric virus was collected. The Supernatant was inoculated to BHK-1 cells and C6/36 cells, respectively. CPE could be observed about 4 days post the infection of C6/36cell with the chimeric virus. The results from RT-PCR, IFA, Western blot showed that the virus contained the chimeric RNA and the envelop protein of DV2. However, the chimeric virus could not be passaged in BHK-21 cell. The successful construction of the infectious clone JE/DEN-2 laid the basis for the further research of the DV vaccine.

Recombinant envelope glycoprotein domain III of dengue virus inhibit virus infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the ability of dengue virus recombinant envelope protein domain expressed in E. coli to inhibit virus infection and induce the neutralizing antibody.</p><p><b>METHODS</b>E III protein of Dengue virus serotypes 1-4 were expressed in E. coli BL21(DE3) then purified. Recombinant proteins were tested to inhibit DV2 from infecting BHK-21 cell. Rabbits were immunized with recombinant proteins to produce anti-E III serum. Antibody titers were determined by neutralizing assay.</p><p><b>RESULTS</b>The recombinant E III proteins of Dengue virus serotypes 1-4 were expressed in E. coli. They effectively protected BHK cells in culture against DV2 infection. All four type anti-E III sera can neutralize DV2 but their efficacies are different.</p><p><b>CONCLUSION</b>proteins of dengue virus expressed in E. coli can directly inhibit DV2 infection. Neutralizing antibodies were induced by E III proteins. Both E III protein of dengue virus and the neutralizing antibodies they induced are more efficient in inhibiting homologous dengue serotypes infection than heterologous serotypes.</p>

Molecular typing of isolates from two dysentery outbreaks in Chengdu, through pulsed field gel electrophoresis technology / 中华流行病学杂志

<p><b>OBJECTIVE</b>Using pulsed field gel electrophoresis (PFGE) and polymerase chain reaction (PCR) typing to analyze strains isolated from two outbreaks caused by Shigella sonnei and to trace the source of infection.</p><p><b>METHODS</b>Virulence genes ipaH and ial were detected by PCR and PFGE was used to subtype the isolates. Patterns were compared, using the software BioNumerics.</p><p><b>RESULTS</b>Within the 54 isolates, all were ipaH positive with 48 as ial positive. Strains from the Chongzhou outbreak were clustered into 4 PFGE patterns, with the predominant pattern accounted for 72% of the analyzed strains. The pattern of strains isolated from the cold pork with sauce was identical to the predominant pattern. The strains from Dayi outbreak were clustered into 8 PFGE patterns and the predominant pattern accounted for 56% of the test strains.</p><p><b>CONCLUSION</b>Strains from the two outbreaks were quite different and the 'cold pork with sauce' seemed to be the major source of infection, causing the outbreak of diarrhea in Chongzhou. The sources of infection of the Dayi outbreak might be complicated whereas PFGE showed a discriminatory and reproducible laboratory tool in the epidemiologic investigation on outbreaks of diarrhea.</p>