Objective To explore the effect of residual UDG on all dU-DNA PCR products. Methods The hybridization percentage of dU-DNA PCR products, which were stored at 37℃ for 20 hours as well as at -20℃,4℃,room temperature and 37℃,respectively, were detected by employing microplate hybridization technique. The effect of inactivating the products at 94 ℃ for 10 minutes on the activity of Taq polymerase was also analysed. Results Compared with the control, A value decreased by 13.281% and 20.557%, respectively after adding 0.2u and 10u UDG (P

Objective To investigate the role of cHSP60 in the pathogenesis of murine cervicitis.Methods Fifty female C3H/HeN mice were randomly and equally classified into 5 groups, including the control group receiving no treatment and 4 groups receiving intravaginal inoculation of cHSP60 (cHSP60 group),live elementary bodies of Chlamydia trachomatis mouse pneumonitis (MoPn group), inactive elementary bodies of MoPn (inactive MoPn group) and growth medium (medium group), respectively. Five days after the inoculation,cervical tissue was resected from these mice and subjected to pathological examination. Results There were varying degrees of inflammatory reaction characterized by neutrophil infiltration, necrosis and shedding of mucosal cells in the cervices of mice in cGSP60 and MoPn groups. No statistical difference was observed in the incidence of cervicitis (90% vs. 80%, P ＞ 0.05), neutrophile count [76.00 (25.0 - 80.0) vs. 25.00 (8.75 -32.5), P＞ 0.05] or inflammation score [12.5 (11.5 - 14.25) vs. 9.00 (8.00 - 11.5), P ＞ 0.05] between the cHSP60 and MoPn group. The inflammatory reaction was weak with decreased incidence of cervicitis (40%),inflammation score [0.00 (0.00- 12.50)] and neutrophile count [0.00 (0.00- 15.50)] in inactive MoPn group compared with the cHSP60 and MoPn groups (all P ＜ 0.05). A small number of neutrophils migrated into the superficial layer of cervical mucosa in only 2 mice in the medium group. Conclusion cHSP60 may be a primary pathogenic factor in chlamydial genital tract infection.

20%ALA cream were applied topically to condylomata acuminata.The cream was kept in place for 4 h.He-Ne laser light at 630nm was used,and the dose of light was 100 J/cm2 for all of the patients.After three treatment,the complete removal rate(CRR)of urethral and other genital mucossa were 93.4%and 88.9%,significantly higher than genital skin 39.1%(P＜0.05).The adverse reactions of ALA-PDT are mainly local minor erosion,short-term pain,but no scar.It showed that ALA-PDT is an effective,minimally invasive treatment for condylomata acuminata,especially for the lesions on urethral and genital mucosa.

Objective To analyze the clinical and histopathological features,immunophenotypes,treatment and prognosis of subcutaneous panniculitis-like T cell lymphoma (SPTL).Methods Clinical and experimental data were collected from 9 cases of SPTL,and retrospectively analyzed.Related pathological and immunohistochemical markers were examined by Envision method.Eight patients were followed up.Results Of the 9 patients,8 had multiple subcutaneous nodules and plaques,which mainly involved the lower limbs in 8 patients and the trunk in 6 patients.Seven patients had fever.Three patients were subjected to the whole-body positron emission tomography-computed tomography (PET-CT),and 7 to bone marrow aspiration.No visceral tumors and hemophagocytic syndrome were found.Histopathological examination of skin lesions showed atypical mononuclear cells with large nuclei and deep staining,which mainly infiltrated the subcutaneous adipose tissue and were arranged in a circular pattern.Among 9 patients,infiltration of tumor cells was observed around skin appendages and blood vessels in the dermis in 5 patients.Immunohistochemical examination showed positive staining for βF1,CD3 and CD8 in tumor cells in 9 cases,positive staining for granzyme B and T-cell-restricted intracellular antigen-1 (TIA-1) in 8 cases,and negative staining for CD4,CD20,CD30 and CD56 in all the patients.Five patients received chemotherapy,including a child and a postpartum woman.One child received methylprednisoloue pulse therapy.During the follow-up,8 patients achieved a complete clinical remission after treatment.Conclusion SPTL is derived from α/β T cells,and histopathological and immunohistochemical examinations can be helpful for its diagnosis and differential diagnosis.

Objective To investigate differences in Rab protein expressions in McCoy cells with acute versus persistent Chlamydia trachomatis (Ct) infection.Methods Cultured McCoy cells were infected with different amounts (400,500,550 μl/well) of Ct strain D suspensions,then cultured with the medium containing 100 U/ml penicillin G (persistent Ct infection groups) or that without penicillin G (acute Ct infection groups).Ct-uninfected McCoy cells receiving no penicillin G treatment served as the blank control group,and those receiving penicillin G treatment as the penicillin group.Mter 48-hour culture,McCoy cells were lysed,proteins were collected,and total RNA was extracted from the cells.Enzyme-linked immunosorbent assay (ELISA) was conducted to measure protein levels of Rab4A,Rab6A,Rab10,Rab11A and Rab14,and fluorescence-based quantitative PCR to quantify mRNA expressions of Rab4A and Rab14 (expressed as 2-ΔΔα).Results Protein levels of Rab4A,Rab6A,Rab10,Rab11A and Rab14 were all significantly lower in the acute than in the persistent Ct infection groups (all Z =3.621,P ＜ 0.001),and lower in the persistent and acute Ct infection groups than in the blank control group (all P ＜ 0.008 3),but insignificantly different between the blank control group and penicillin group (all P ＞ 0.05).In addition,the expressions of Rab4A and Rab14 mRNAs were consistent with those of their proteins in these groups.Conclusion The transcriptional and expression levels of Rab proteins are higher in McCoy cells persistently infected with Ct than in those acutely infected with Ct.

Objective To clone and express Chlamydia trachomatis (Ct) heat shock protein 60 (hsp60) gene. Methods The hsp60 gene fragment was amplified from Ct chromosomal DNA by PCR. After purification and digestion with enzymes Sal I and Not I , the hsp60 gene fragment was inserted into the compatible site of prokaryotic expression vector pET-28a. The constructed recombinant plasmid was identified by PCR, restriction enzyme cleavage and sequencing, then, it was transfected into an expression strain Escherichia coli BL21 (DE3). The expression of fusion protein was induced by isopropy-β-D- thiogalactoside (IPTG) in the host bacteria, and the expressed product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blot. Results PCR and restriction enzymes cleavage analysis confirmed that the hsp60 gene was successfully cloned into the recombinant plasmid. DNA sequencing showed that the sequence of cloned gene was fully consistent with the published sequence in Genebank. As revealed by SDS-PAGE, the size of expressed fusion protein approximated 60 kilodaltons, and Western-blot confirmed the expressed product to be the expected protein. The final concentration of fusion protein was 17.85 mg/L with a purity of more than 90%. Conclusions A recombinant expression plasmid pET-28a-hsp60 is successfully constructed in this study, and soluble hsp60 protein is expressed by the recombinant plasmid-transfected E. coli.

BACKGROUND: Glial cell-derived neurotrophic factor (GDNF) and transforming growth factor-beta 1 (TGF-β1)co-subordinate to TGF-β family. Both of them play very important roles in the development and differentiation of central and peripheral nervous system, and regulation of cell cycle in mammals.OBJECTIVE: To observe the differentiation of spinal cord-derived neural stem cells(NSCs) induced by GDNF combined with TGF-β1, and make a comparison of differentiation results with GDNF or TGF-β1 culture fluid.DESIGN: Controlled observation.SETTING: Central Laboratory, Shenzhen Hospital Affiliated to Southern Medical University.MATERIALS: Ten SD rats of clean grade, which were at conception for 16 days, were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technplogy. Main reagents and materials:DMEM/F12,B27(GIBCO); basic fibroblast growth factor (bFGF), GDNF; TGF-β1(PeproTech);fetal bovine serum (FBS,Hyclone); nestin multiple antibody (Boster, Wuhan); glial fibrillary acidic protein (GFAP) multiple antibody; neurofilament protein (NF-200) monoclonal antibody (Sigma).METHODS: This experiment was carried out in the Central Laboratory, Shenzhen Hospital Affiliated to Southern Medcial University between October 2005 and September 2006. Under the aseptic condition, rat fetus was isolated for isolation and culture of spinal cord-derived neural stem cells. In this study, five groups were divided: basal medium group, control group, bFGF group, TGF-β1 group, GDNF+ TGF-β1 group. In the basal medium group, DMEM/F12 containing penicillin,streptomycin, amphotericin (AMPH) B and 0.02 volume fraction of B27 annex solution. At 1 week after primary culture, rat spinal cord-derived NSC clones proliferated in vitro stably were harvested. In the control group, 0.1 volume fraction of FBS was added into basal medium. In the later three groups, induced medium was exchanged, i.e. 20 μg/L bFGF, 2 μg/L TGF-β1, and 10 μg/L GDNF+2 μg/L TGF-β1 were added into the basal medium, respectively. ①The differentiation of spinal cord-derived NSCs induced by different factors were observed under the optical microscope. ②The expressions of neurons and astrocytes were detected by immunocytochemical staining labeling. ③ The differentiated cells were counted by sorting technique by means of fluorescence excitation flow cytometer, and the percentage of NSCs differentiating into neurons and astrocytes were detected under the different induction environments.MAIN OUTCOME MEASURES: ① Morphological feature of cell differentiation in each group. ② Immunohistochemical detection of NSCs in each group. ③ The percentage of NSCs differentiating into neurons and astrocytes in each group.RESULTS: ① Cell morphology during differentiation: At the early stage of differentiation, lots of cells creeped to all the directions, and one week later, most of the migrated cells adhered to the wall entirely. Neuron-like cells, astrocyte-like cells and oligodendrocyte-like cells could be identified in the low-density cell region. ②Immunohistochemical detection results: A lot of GFAP- positive astrocytes were found in the control group and TGF-β1 group; Many differentiated neurons and NF-200 staining positive were found in the bFGF group and GDNF+ TGF-β1 group. ③Percentage of stained neuron and astrocyte: at one week of induction, the percentage of stained neurons was higher in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.15,19.56,25.32,P ＜ 0.05-0.01), and the percentage of stained astrocytes was lower in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.45,23.79,P ＜ 0.01 ).CONCLUSION: The combined in vitro induction of GDNF and TGF-β1 contributes to the neuronal differentiation of spinal cord-derived NSCs, indicating that both of them have synergistic effect.

Objective To determine the prevalent herpes simplex virus (HSV) strain in patients with genital herpes (GH),and to evaluate the sensitivity,specificity,positive predictive value (PPV) and negative predictive value (NPV) of anti-herpes simplex virus type 2 (HSV2) IgG and IgM antibodies in the diagnosis of genital herpes (GH) before in vitro fertilization (IVF).Methods Totally,193 HSV2 clinical strains isolated in cell culture from the lesions of patients with GH in the Department of Dermatology,First Affiliated Hospital,Sun Yat-sen University between 2009 and 2011 were typed by using type-specific fluorescein isothiocyanate (FITC)-labelled anti-HSV monoclonal antibodies.Serum samples were obtained from 57 anti-HSV2 IgM/IgG antibody-positive females with suspected GH as well as their husbands (clinical observation group),68 HSV culture-positive patients diagnosed with GH (positive control group),and 120 children aged 8-12 years (negative control group).Enzyme-linked immunosorbent assay (ELISA) was performed to detect anti-HSV1/HSV2 IgG/IgM antibodies in these serum samples.Statistical analysis was carried out using chi-square test.Results There was a significant difference between the positive control group and negative control group in the positivity rate of anti-HSV1 IgG (89.71％ (61/68) vs.40.80％ (49/120),P ＜ 0.01) and anti-HSV2 IgG (91.18％ (62/68) vs.0,P ＜ 0.01),but not in that of anti-HSV1 IgM (20.59％ (14/68) vs.21.70％ (26/120),P ＞ 0.05) or anti-HSV2 lgM (13.24％ (9/68)vs.13.30％ (16/120),P ＞ 0.05).In the clinical observation group,the positivity rate of anti-HSV1 and anti-HSV2 IgM antibodies,anti-HSV1 and anti-HSV2 IgG antibodies was 80.70％ (46/57),91.23％ (52/57),84.21％ (48/57) and 14.04％ (8/57) respectively in the females,19.30％ (11/57),8.77％ (5/57),87.71％ (50/57),12.28％ (7/57)respectively in the males,with significant differences in the positivity rate of anti-HSV1 and-HSV2 IgM antibodies (both P ＜ 0.01),but not in that of anti-HSV 1 or-HSV2 IgG antibodies (both P ＞ 0.05).The sensitivity,specificity,PPV and NPV were 13.24％ (9/68),86.67％ (104/120),36.00％ (9/25) and 63.80％ (104/163) respectively for anti-HSV2 IgM antibody in the diagnosis of GH,91.18％ (62/68),100.00％ (120/120),100.00％ (62/62),and 95.24％ (120/126) respectively for anti-HSV2 IgG antibody.Conclusions HSV2 prevails in the patients with GH in this region,while HSV1 only amounts to 5.18％.The type-specific anti-HSV2 IgG antibody shows a higher specificity,sensitivity,PPV and NPV in the diagnosis of GH than anti-HSV2 IgM antibody,hence,the type-specific anti-HSV2 IgG antibody is superior to anti-HSV2 IgM antibody in diagnosing GH before assisted reproduction.

ObjectiveTo observe the ultrastructural changes of Chlamydia trachomatis after treatment with azithromycin.Methods The Chlamydia trachomatis laboratory strain (D/UW-3/Cx) was cultured in McCoy cells with or without the presence of azithromycin of 0.0667,0.1340,0.1900,0.2680 and 0.3330 mg/L for 48 hours.The ultrastructural changes of host cells andChlamydia trachomatis were observed by transmission electron microscopy.ResultsAfter 48-hour culture,vesicles increased in number both inside and outside of the inclusion bodies with the rise in azithromycin concentration; there were abnormally large reticulate bodies,some of which experienced abnormal division and even necrosis or breakdown; the number of elementary bodies was decreased,while their size was enlarged,with a more wrinkled outer membrane.No inclusionbodieswereseenwhentheconcentrationofazithromycinwas0.333mg/L. Conclusions Azithromycin can induce an increment in the outer membrane of Chlamydia trachomatis,formation of vesicles,abnormal enlargement or breakdown of reticulate bodies,and a decrease in elementary bodies.

A 22-year-old patient was admitted to the hospital with a solitary,gradually growing and painless mass in the left shoulder for 2 years.Physical examination revealed no abnormality except for the skin lesion.Skin examination showed an elevated lesion measuring about 3.5 cm× 2.0 cm × 1.5 cm with smooth surface and normal color,which was located in the subcutaeous tissue,indurated and movable.Resection of the tumor was performed under local anesthesia.On visual observation during operation,the tumor was sited in the subcutaneous fat tissue,nodular-like and surrounded locally by fibrous pseudocapsules with a grey incisal surface and mild texture.Microscopicalty,the tumor was extremely similar to breast fibroadenoma with multiple lobuli,and each of the lobuli was composed of tubiform structures,basal cell-like epithelial cell trabs and fibromyxoid stroma abundant in fibroblast-like spindle cells.No hair bulb or primitive dermal papillae were observed in the lobuli,which were separated by compact collagen fibers infiltrated by a few scattered inflammatory cells.Fibromyxoid strotma was surrounded by basal cell-like epithelial cell strabs in most lobuli,and some tubiform structures were filled with a little thin lightly eosinophilic material in a concentric arrangement.Immunohistochemistry showed that intralobular epithelial cells were strongly positive for cytokeratin 5/6,but negative for CAM5.2 or carcinoembryonic antigen (CEA).In addition,the lightly eosinophilic material in lumens was negative for periodic acid-Schiff (PAS) staining.These results suggested that the tubiform structures were immature follicles,but not sweat ducts.The patient was diagnosed with nodular fibrofolliculoma (NFF) based on the clinical manifestations,morphological features,immunohistochemical and PAS staining results.No relapse was observed in more than 3 months of postoperative follow-up.As a benign trichogenic adnexal neoplasm with unique clinicopathological manifestations,NFF may be a new entity of cutaneous adnexal neoplasm.