Objective:Osteoprotegerin (OPG) is a key molecule negatively regulating osteoclast differentiation and activation; and the conserved mycobacterial heat shock 70 (HSP70) peptide p111-125 has also been found to inhibit inflammation reactions in chronic arthritis. BaculoDirectTM baculovirus expression system was selected to express recombinant OPG-HSP70 in insect cells.Methods:The human functional fragment (p22-194) of OPG and functional fragment (p111-125) of mycobacterial HSP70 gene were cloned into the transfer vector pENTRTM/SD/D-TOPO. The recombinant plasmid was performed an LR reaction with the BaculoDirectTM Linear DNA to generate recombinant baculovirus DNA. The cultured Sf9 insect cells were directly transferred with the recombinant baculovirus DNA,and the pure recombinant baculovirus was obtained. Then recombinant baculovirus was infected Sf9 insect cells again to express the OPG-HSP70 gene.Results:The target protein was detected at the time of 48h post infection,reached at highest yield at the time of 72h post-infection. A 28kDa protein immunostaining band was detected by Western blotting from lysate of those cells.And the purified protein was obtained by using Ni-NTA system. Functional stuies on the fusion protein showed it significantly reduce osteoclast cell number[(3.10?0.640) cells under each microscope field in treatment group by comparing to (10.70?0.817)cells in the control group] in the osteoclast inhibition test,and reduce the inflammation reaction in a delayed type hypersensitivity (DTH) mice model (P

ObjectiveTo characterize the neural progenitor cell in the human amnion mesenchyme and epithelial layer with specific mark proteins of neural stem cell.MethodsExpressions of specific mark proteins of neural stem cell including nestin, glial fibrillary acidic protein (GFAP), musashi-1, vimentin and PSA-NCAM in human amnion tissue and cultured amniotic cells were determined by immunohistochemistry and immunofluorescence staining.ResultsExpressions of pluripotent neural stem cell specific makers (nestin, musashi-1, vimentin and PSA-NCAM) were detected in the human amnion mesenchyme and epithelial layer. In addition, cultured amniotic cells were expressed several neural stem cell specific markers including nestin, GFAP and PSA-NCAM. Nestin+ and GFAP+ double positive cells were identified in the human amnion tissue and cultured amniotic cells by immunohistochemistry and immunofluorescence staining.ConclusionSpecific mark proteins of neural stem cell are expressed in human amnion tissue and cultured amniotic cells.

Abstract: Objective To screen out a more universally applicable culture medium for the isolation and culturing of pathogenic fungi through comparing the performance of various universal fungal culture media, to optimize the fungal culturomics technique, and to better apply it to the culturomics research of pathogenic fungi. Methods Multiple common fungal culture media Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), modified Dixon (mDixon), modified LeemingNotman agar (MLNA), etc., and a new pan-fungal medium (PF) were used to culture 40 strains of common pathogenic fungi to determine the growth states of strains under different conditions. Based on that, PF, SDA, PDA, mDixon and MLNA, a total of 5 culture media, were used to isolate and culture a simulated sample (suspension of Candida albicans and Aspergillus fumigatus), 10 human samples (4 fecal samples and 6 vaginal secretion samples) and 3 environmental samples. Results The positive growth rates of 40 strains of pathogenic fungi in the 7 media were as follows: PDA 95.0% (38/40), SDA 95.0% (38/40), BHI 95.0% (38/40), YPD 90.0% (36/40), mDixon 95.0% (38/40), MLNA 87.5% (35/40), PF 100.0% (40/40). For the simulated samples, PF could effectively promote the self-limited growth of filamentous fungi, performing better in isolation and culture. For the human samples and environmental samples, PF showed the same versatility as SDA and PDA. Conclusions In the isolation and culturing of pathogenic fungi, PF medium can effectively isolate and culture most fungal species. Meanwhile, PF can make the fast-growing fungi show self-limited growth and clear edges, and not easy to cross-contamination, which indicates it is conducive to the isolation and identification of single colonies. PF medium outperforms other common media in isolating strains from unknown samples in culturomics, which illustrates PF medium can be effectively used for the study of fungal culturomics.

A fused functional gene of human OPG and Mhsp65 was amplified by PCR,and cloned into the prokaryotic expression vector pET-28a.The BL21(DE3) strain of E.coli was transformed using the recombinant plasmid pET-28a-OPG-HSP65 and the expected protein was expressed by induction with IPTG.Result of SDS-PAGE indicated that the expected recombinant protein of 23 kDa was expressed with high yield as inclusion body.The fusion protein could be specifically recognized by both the anti-His antibody and anti-human OPG monoclonal antibody in Western blot analysis.The purified and refolded fusion protein could inhibit osteoclast proliferation and bone absorption in vitro.The results of mouse ear swelling assay and expressions of TNF-?,IFN-? and IL-17 mRNAs detected by real-time quantitative PCR demonstrated that the fusion protein had an anti-inflammation activity.The results suggest that the fusion protein of human OPG and Mhsp65 may act as a potential therapeutics for rheumatoid arthritis.

OBJECTIVETo explore the relation of the anogenital distance (AGD) with cryptorchidism in male newborns.

METHODSThis study included 350 male infants delivered in two community hospitals between September 2013 and September 2014. Within 24 hours after birth, a pediatric surgeon measured the AGD of the neonates and determined whether they had cryptorchidism. According to the testicular position, we divided the undescended testes into three types: upper scrotal, inguinal, and non-palpable.

RESULTSTotally 39 cases of cryptorchidism were found in the 350 newborns. The AGD of the cryptorchidism infants was significantly shorter than that of the normal neonates ([2.01 ± 0.22] vs [2.35 ± 0.19] cm, P < 0.01), and statistically significant differences remained even when preterm and low birth-weight infants were excluded ([2.32 ± 0.14] vs [2.06 ± 0.19] cm; (2.37 ± 0.17) cm vs (2.12 ± 0.12) cm, all P < 0.01). The newborns with higher-position cryptorchidism had a shorter AGD, though with no significant difference (F = 0.434, P > 0.05). No significant differences were observed in the AGD between unilateral and bilateral cryptorchidism ([1.96 ± 0.13] vs [2.02 ± 0.17] cm, P > 0.05).

CONCLUSIONShorter AGD is associated with a higher incidence of cryptorchidism in male newborns. AGD could serve as a potential biomarker for disruption of androgen action during the male programming window period.

Androgens ; physiology ; Cryptorchidism ; diagnosis ; Humans ; Infant, Low Birth Weight ; Infant, Newborn ; Infant, Premature ; Male ; Perineum ; abnormalities

Objective To investigate the expression of caspase-10 in differentiated thyroid carcinoma and association with its development and metastasis. Methods Thyroid samples from 37 patients in a period from January 2006 to December 2007, with differentiated thyroid carcinoma were retrospectively analyzed for caspase-10 by immunohistocbemistry(streptavidin-perosidase, S-P), compared to control group of 46 cases with nodtdar goiter. The relationship between the expression of caspase-10 and the clinical pathologic characteristics of thyroid carcinoma were also explored simultaneously. Results caspase-10 were observed as brown or yellow particles located in the cytoplasm or cell membrane of nodular goiter but there were no significant evidence for its positive expression in thyroid carcinoma, caspase-10 expression was markedly down-regulated in differentiated thyroid carcinoma(29.73%,11/37) compared with benign nodules(71.74%,33/46, χ2=14.528, P<0.01). The positive expression in 18 cases with lymph node metastasis(11.11%,2/18) was significantly lower than those in 19 patients without lymph node metastasis(47.37%,9/19; χ2=4.210, P<0.01). There was no significant correlation(P> 0.05) between the expression of caspase-10 and the clinical pathologic characteristics including male, age, TNM stage and pathologic type. Conclusion Down-regulation of caspase-10 may play a critical role in carcinogenesis and development of differentiated thyroid carcinoma.

Objective To evaluate clinical features,therapeutic effects and outcomes of patients with non-human immunodeficiency virus(HIV)-infected cryptococcal meningitis treated with fluconazole or fluconazole and flucytosine.Methods Twenty-four cases of non-HIV-infected cryptococcal meningitis(fluconazole with or without flucytosine as initial therapy)in Huashan Hospital,Fudan University from 1997 to 2007 were retrospectively reviewed.Clinical manifestations,therapeutic effects and outcomes of the patients were collected.Results Fluconazole was administered with median dosage of 400 mg/d,for a median duration of 20.5 days.After fluconazole initial therapy for 2 weeks,16.7% showed partial response,83.3% showed no response,and the overall response rate was 16.7%.After 10 weeks,33.3% showed partial response,29.2% showed complete response,16.7% showed no response,and the overall response rate was 62.5%.Mortality at week 10 was 20.8%.Twenty-two patients who failed to respond to initial therapy were switched to other antifungal drugs(amphotericin B,amphotericin B colloidal dispersion,itraconazole)or other fluconazole containing combined therapy.Eleven out of the 24 patients died during one-year follow-up,8 of whom died of eryptococcal meningitis,and 3 died of other diseases.Conclusions The initial therapy of fluconazole with or without flucytosine is inefficient,and most of the patients need other antifungal drugs because of initial therapy failure.Therefore,fluconazole might not be appropriate for initial therapy in non-HIV-infected cryptococcal meningitis.

OBJECTIVETo explore the differential proteomic expression in human liver cells L-02 after exposure to HQ.

METHODSSubcultured L-02 cells were treated by HQ for 24 h at a 1 x 10(-4) mol/L concentration and a blank group was set as the control. Immediately after the treatment, total cellular proteins were extracted and separated by 2-DE, and the images were analyzed by PDQuest software. The experiment was totally repeated 3 times with 3 repetitions for each group every time. The well repeated spots were identified by MALDI-TOF MS and then searched in NCBI human protein database with Mascot.

RESULTSAbout 1,000 spots per gel were found. Compared with the control group, 17, 18 and 24 spots were significantly altered in 3 separate experiments. The 4 well repeated spots were identified by MALDI-TOF MS as Rho GDP dissociation inhibitor GDI alpha, 6-phosphogluconolactonase, erbB3 binding protein EBP1 and lamin A/C, isoform 1 precursor. They were involved in cell skeleton, signal transduction and energy metabolization in functional classification.

CONCLUSIONHydroquinone can change the protein expression in liver cells, which provides clues for exploring the toxic mechanism.

Cell Line ; Electrophoresis, Gel, Two-Dimensional ; Hepatocytes ; drug effects ; metabolism ; Humans ; Hydroquinones ; toxicity ; Mass Spectrometry ; Proteomics ; Reproducibility of Results

BACKGROUND: Active compression-decompression cardiopulmonary resuscitation (ACD-CPR) has been popular in the treatment of patients with cardiac arrest (CA). However, the effect of ACD-CPR versus conventional standard CPR (S-CRP) is contriversial. This study was to analyze the efficacy and safety of ACD-CPR versus S-CRP in treating CA patients. METHODS: Randomized or quasi-randomized controlled trials published from January 1990 to March 2011 were searched with the phrase "active compression-decompression cardiopulmonary resuscitation and cardiac arrest" in PubMed, EmBASE, and China Biomedical Document Databases. The Cochrane Library was searched for papers of meta-analysis. Restoration of spontaneous circulation (ROSC) rate, survival rate to hospital admission, survival rate at 24 hours, and survival rate to hospital discharge were considered primary outcomes, and complications after CPR were viewed as secondary outcomes. Included studies were critically appraised and estimates of effects were calculated according to the model of fixed or random effects. Inconsistency across the studies was evaluated using the I2 statistic method. Sensitivity analysis was made to determine statistical heterogeneity. RESULTS: Thirteen studies met the criteria for this meta-analysis. The studies included 396 adult CA patients treated by ACD-CPR and 391 patients by S-CRP. Totally 234 CA patients were found out hospitals, while the other 333 CA patients were in hospitals. Two studies were evaluated with high-quality methodology and the rest 11 studies were of poor quality. ROSC rate, survival rate at 24 hours and survival rate to hospital discharge with favorable neurological function indicated that ACD-CPR is superior to S-CRP, with relative risk (RR) values of 1.39 (95% CI 0.99–1.97), 1.94 (95%CI 1.45–2.59) and 2.80 (95% CI 1.60–5.24). No significant differences were found in survival rate to hospital admission and survival rate to hospital discharge for ACD-CPR versus S-CRP with RR values of 1.06 (95% CI 0.76–1.60) and 1.00 (95% CI 0.73–1.38). CONCLUSION: Quality controlled studies confirmed the superiority of ACD-CPR to S-CRP in terms of ROSC rate and survival rate at 24 hours. Compared with S-CRP, ACD-CPR could not improve survival rate to hospital admission or survival rate to hospital discharge.

Objective To explore the effects of coriafia lactone (CL)-activated astrocytes (Ast) conditioned medium (ACM) on the expressions of glutamate (Glu) and GluR2 in the brain of rat. Methods Asts of hippocampus were cultured according to the McCarthy and DeVellis's method, and then the ACM was collected. Forty-eight male adult Sprague-Dawley (SD) rats were randomly divided into the control group (n=16) and the CL group (n=32). Rats in the control group were administered 10 μL ACM I. C. V., which was not added any stimulating substance. Rats of the CL group were injected I. C. V. 10 μL CL-activated ACM. The rats in both groups were subdivided into post-injection 2,4,8,12h subgroups, 4 in each subgroup in the control group and 8 in each subgroup in the CL group. The behaviors of the rats were observed and the expressions of Glu and GluR2 in the cerebral cortex and hippocampus were detected with immunohistochemistry and immunofluorescence. The content of GluR2 was tested with Western blot. Results The rats injected with CL-activated ACM showed seizure activities, whereas the rats of the control group showed no seizure activities. The expression of Glu in cerebral cortex and hippocampus in the brains injected with CL-activated ACM was increased compared with the control group at 4h (P<0.05), but the expression of GluR2 was attenuated compared with the control group at 4h(P<0.05). The results of GluR2 in the cerebral cortex and hippocampus detected with Western blot were different significantly with control group (P<0.05). Conclusion CL-activated ACM can enhance the expression of Glu and reduce the expression of GluR2 in the brain of rat, resulting in the activation of AMPA pathway and the Ca2+ influx, and then induce seizure activities.