OBJECTIVE To study the distributive properties and susceptibility of yeasts to six antifungal agents. METHODS To analyze the distributive properties of 264 clinical Candida spp isolates and study the susceptibility to amphotericin B,nystatin,fluconazole,ketoconazole,miconazole and clotrimazole.The susceptibility of yeasts was tested according to the National Committee for Clinical Laboratory Standards guideline(NCCLS M27-A2). RESULTS Strains of Candida albicans were the most frequent organism isolated accounted for 62.5% of all the isolates.C.tropicalis,C.glabrata,and C.parapsilosis accounted for 20.8%,12.5%,and 1.9%,the others accounted for only 2.3%.The main infected organs were lungs,urinary tract,and digestive tract;the susceptibility tests showed strains of Candida spp to nystatin,amphotericin B,and fluconazole were more active than to the other antifungal agents.The resistance to triazole antifugal agents could be shown. CONCLUSIONS We should strengthen the diagnosis of Candida spp and strengthen the surveillance on susceptibility of clinical isolates Candida spp so as to help the doctors choose the antifungal agents reasonably.

OBJECTIVE:To establish RP-HPLC fingerprints of Desmodium styracifolium and its preparations in order to provide basis for the quality evaluation of them.METHODS:The separation was performed on ODS-C18(250 mm?4.6 mm,5 ?m) column with mobile phase consisted of methanol-20% phosphoric acid(gradient elution).The detection wavelength was set at 360 nm and flow rate was 1.0 mL?min-1.Column temperature was set at 25 ℃.The RP-HPLC fingerprint of D.styracifolium and its preparations were recorded.RESULTS:There were 13 common peak in RP-HPLC fingerprint of D.styracifolium and its preparations.CONCLUSION:The method is accurate,stable and reproducible for basis of quality evaluation and RP-HPLC finger print of D.styracifolium and its preparations.

Objective To explore the effects of electrical stimulation in different acupoints on the expression of growth associated protein-43 (GAP-43), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) in spinal cord injured rats after the human umbilical cord blood stem cells transplantation.Methods 192 SD rats were injured at T10-11 level with NYU, and divided randomly into groups: Group A received electric stimulation in the scalp of motor area (A1 group received electric stimulation and transplantation of umbilical cord blood stem cell, the rats of A2 group only received electric stimulation); group B received local electric stimulation at damaged site (B1 with electric stimulation and transplantation, B2 only with electric stimulation); group C received electric stimulation in the scalp of motor area and at damaged site (C1 with electric stimulation and transplantation, C2 only with electric stimulation); D1 received transplantation without electric stimulation, D2 neither with transplantation nor electric stimulation. The expression of GAP-43, bFGF and IGF were detected 1, 2, 3, 4, 8 and 12 weeks after operation. Results Electric stimulation increased the expression of GAP-43, bFGF, IGF, stimulating scalp and body were more than stimulated scalp or body alone, combined with the stem cells transplantation were more than no transplantation. Conclusion Both electric stimulation and stem cells transplantation can improve the microenvironment for neural recovery synergistically. Stimulating scalp and body is more effective than alone.

AIM: To further investagate the mechanism of thrombus formation and develop a new remedy of anti-thrombus formation. METHODS: The amplified DNA fragment of vWF-A1 domain was inserted into expression vector with 6?his taq (pQE-31), the recombinant expression vect or was transformed into E coli (strain M15) and induced by IPTG. The recombinant fragment, comprising residues 449-728 of mature vWF subunit, designate rvWF-A1. It was purified by Ni-NTA agarose column and renatured by Tris buffer containin g GSH and GSSG. FACS and platelet aggregometer were employed to analyse the rvWF -A1 function of binding to platelet glycoprotein Ib and inhibiting ristocetin-in duced platelet aggregation. RESULTS: The rvWF-A1 was expressed successfully in E coli, comin g up to 30% of total bacterial protein. Its purify was over 95% through Ni-NTA a garose. It was identified to have ability to bind to GPIb, its biologic activity to inhibit ristocetin-induced platelet aggregation was observed, and the inhibi tive rate was 84 7%. CONCLUSION: The above results indicated that high-level expressi on of rvWF-A1 was successfully achieved in E coli and rvWF-A1 may be an effectiv e antithromotic agent in preventing thrombus formation.

Objective To explore the application values of effect on the expression of growth associated protein-43 (GAP-43) and myelin basic protein(MBP) by electrical stimulation in different sites of spinal cord injured rats after the human umbilical cord blood stem cells transplantation. Methods In this study the subjects adopted are clean grade rats, which were performed with Allen's method by NYU blow device, resulting in SCI models. 192 rats successfully modeled were divided into groups according to the random table. Group A received electric stimulation in the scalp surface projection area of motor area: Group A1 received electric stimulation and transplantation of umbilical cord blood stem cell, while Group A2 only received electric stimulation; Group B received local electric stimulation at damaged site: Group B1 received electric stimulation and transplantation of umbilical cord blood stem cell, while Group B2 only received electric stimulation; Group C received electric stimulation in the scalp surface projection area of motor area and local electric stimulation at damaged site: Group C1 received electric stimulation and transplantation of umbilical cord blood stem cell, while Group C2 only received electric stimulation; Group D are just the SCI models: Group D1 received transplantation of umbilical cord blood stem cell without electric stimulation, while Group D2 neither received cell transplantation nor electric stimulation. The sacrifice time of rats was the 1st week, 2nd week, 3rd week, 4th week, 8th week, and 12th week after operation respectively. The distribution of the remained or regenerative neurons at damaged areas by corticospinal tract anterograde tracer with Biotinylated dextran amine (BDA), and we also detected the content changes of GAP-43, MBP and the MAB1281 specific expression of the human nucleus of human umbilical cord blood stem cells. Results 1). NYU is a blow device for Allen's rat model of SCI, which could quantify the crash potential energy, and gain a stable and repeated model with high successful rate. 2). At each time site in this study, the factors that benefit for the neurofunction restoration after electric stimulation in the microenvironment into which the stem cells have been transplanted are GAP-43 and MBP, and they are all positive; the effects of the combination of head acupuncture with body acupuncture are much better than single head acupuncture or body acupuncture. 3). At each time site in this study, the factors that benefit for the neurofunction restoration after injury are GAP-43 and MBP, and the effects of them are all positive, which do not depend on whether the stem cell transplantation has been done. Conclusion 1). After the stem cell transplantation have been done, the changes of the microenvironment develops in the direction of benefiting for restoring neurofunction, which means that stem cell transplantation can promote the restoration of neurofunction and the stem cell transplantation is successful in this study. 2). Electric stimulation is significantly positive for the factors (GAP-43, MBP) which benefit for restoring neurofunction in the microenvironment after stem cell transplantation, the effects of the combination of head acupuncture with body acupuncture is better than single head acupuncture or body acupuncture. The significantly positive effects of the electric stimulation on microenvironment after injuries are independent, which do not depend on whether the stem cell transplantation has been done. Electric stimulation and stem cell transplantation are significantly synergistic at the microenvironment.

Objective To construct the prokaryotic expression vector of human autophagy-related LC3B gene,obtain the GST-LC3B recombinant plasmid , purify the GST-LC3B fusion protein and identify its activity in vitro.Methods Human LC3B coding region was amplified from the human mammary gland cDNA by PCR and inserted into the prokaryotic expres -sion vector pGEX-KG.The recombinant plasmid pGEX-KG-LC3B was transformed into E.coli Rossate.The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis .The function of the purified protein GST-LC3B was detected by GST pull-down assay.Results About 400 bp of the LC3B coding region was successfully amplified from the mammary gland library by PCR and inserted into pGEX -KG.The result of double diges-tion and sequencing showed that the GST-LC3B recombinant plasmid was successfully obtained .The GST-LC3B fusion pro-tein of about 40 000 (Mr) was successfully purified and identified by SDS-PAGE and Western blotting analysis.GST pull-down assay showed that GST-LC3B could interact with Atg4B, which identified its known function .Conclusion The pro-karyotic expression vector of GST-LC3B is constructed successfully , which will facilitate further research on the function of LC3B in autophagy.

Objective: To investigate the genetic screening methods for cryptic acute promyelocytic leukemia (APL) to further explore its clinical prognosis. Methods: From June 2016 to November 2018, we collected 373 newly diagnosed APL cases. The patients were retrospected by the results of PML-RARα detections both by RT-PCR and i-FISH, those who harbored positive PML-RARα detection by RT-PCR and negative by i-FISH were chosen. Metaphase FISH and Sanger sequencing were further performed to verify these results. Results: A total of 7 cryptic APL cases were discovered. These cases had tiny fragment of RARα inserted into PML in chromosome 15, formed ins (15;17) . The 7 cryptic APL cases had no PML-RARα gene subtype specificity, involving 5 cases in L subtype, 1 case in S subtype and 1 case in V subtype respectively. After the treatment of retinoic acid and arsenic or anthracyclines, 6 cases achieved complete remission, 1 case died of intracranial hemorrhage on the 6th day of therapy. Conclusion The size and covering position of PML-RARα probe should be taken into account when PML-RARα was performed by FISH on APL patients. Furthermore, combination with Metaphase FISH could improve the recognition of cryptic APL. There were no differences between the cryptic and common APL patients in terms of clinical features and treatment choices. Cryptic APL patients also had a good response to the therapy of retinoic acid and arsenic or anthracyclines.

Objective To construct the eukaryotic expression vector of cyclin-dependent kinase ( CDK) 7 labeled with Myc tag, obtain the expressed product , and identify its interaction with FLAG-P53 at the protein level .Methods Human CDK7 coding gene region amplified from the mammary cDNA library by PCR was inserted into the pXJ -40 vector.The recombinant plasmid Myc-CDK7 transfected into human 293T cell lines was investigated and examined by SDS-PAGE and Western blotting.In addition,assay was applied to determine the interaction between Myc-CDK7 and FLAG-p53.Results The coding region of CDK7 was successfully amplified by PCR and cloned into pXJ-40 vector, which was identified by double enzyme digestion and gene sequencing .Myc-CDK7 was successfully expressed in human 293T cell lines according to SDS-PAGE and Western blotting assay indicated that Myc-CDK7 could interact with P53 protein, which verified its known function .Conclusion The eukaryotic expression vector Myc-CDK7 is successfully obtained , which will contribute to further research on CDK 7-mediated cell cycle regulation .

Objective To evaluate the influences of susceptibility interpretation of Escherichia coli,Klebsiella pneumonia and Proteus mirabilis in China mainland according to the old and new ceftazidime,cefotaxime and ceftriaxone breakpoints in CLSI M100-S20 and CLSI M100-S19. Methods First, We analyzed the antibacterial susceptibility results of the three bacteria by agar dilution method in the SEANIR surveillance item, which were collected from 15 national hospitals between the year of 2005 and 2007 and excluded the AmpC enzyme positive isolates according to the PGR-DNA sequencing method and/or the antibacterial susceptibility phenotype. ESBL phenotype was confirmed by the CLSI phenotypic confirmatory test. Antibacterial susceptibility of the total 2733 Escherichia coli, Klebsiella pneumonia, Proteus mirabilis isolates was retrospectively analyzed by WHONET 5. 4 software according to the breakpoints of the CLSI M100-S19 (S19) and CLSI M100-S20 (S20). Second, 207 isolates of Peking Union Medical College Hospital with the results of both agar dilution method and disk diffusion method were performed by recurrent analysis. Then we observed the inter-method agreement through the scatter diagram according to the breakpoints of S19 and S20. Results First, as to the ESBL positive Escherichia coli, Klebsiella pneumonia and Proteus mirabili.s, the resistant rate of cefotaxime increased from 65.2% , 55.5%, 14. 6% under S19 (64 μg/ml) to 99. 7%, 96. 2% , 93. 8% under S20 (4 μg/ml). The susceptibility rates decreased from 6. 0%, 11.5%, 33.3% under S19 (8 μg/ml) to 0%, 0. 2%, 0% under S20 ( 1 μg/ml). Ceftriaxone had the same trend as cefotaxime. Though ceftazidime was more active than cefotaxime and ceftriaxone, as to the ESBL positive Escherichia coli and Klebsiella pneumonia, the resistant rates slightly increased from 30. 3%,43. 2% under S19 (32 μg/ml) to42.0%, 56. 0% under S20 (16 μg/ml). The susceptibility rates slightly decreased from 58. 1%, 44. 1% under S19 (8 μg/ml) to 44. 7%, 28.0% under S20 (4 μg/ml). Second,as to the ESBL negative Escherichia coli, Klebsiella pneumonia and Proteus mirabilis, all the susceptibility rates of ceftazidime, cefotaxime and ceftriaxone were between 99. 2%-100. 0%, the resistant rate were between 0%-0. 4%. Third, the S20 MIC breakpoints had a good correspondence with the ESBL phenotype.Fourth, according to the recurrent analysis of MIC testing and disk dilution method, r value was 0. 67,0. 79, 0. 77 for ceftazidime, cefotaxime and ceftriaxone, respectively, and all P value were under 0. 01. The intermethod rates of S19 and S20 were both acceptable. Conclusions If the cefotaxime and ceftriaxone S20 new breakpoints were used, the concordance of antibacterial susceptibility results and ESBL phenotype would increase greatly. The clinician could select proper antibiotics according to the antibacterial susceptibility results and clinical symptoms. It is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins from susceptible to resistant. However, until laboratories implement the new interpretive criteria,ESBL testing should be performed as described in Supplemental Table 2A-S1. The relationship between the new breakpoints of ceftazidime and clinical outcomes need to be further evaluated.

Objective To investigate the effect and the mechanism of epithelial-mesenchymal transition (EMT) in renal tubular cells induced by uric acid.Methods Normal rat kidney tubular cell line (NRK-52E) were exposed to different concentrations of uric acid (100,200,400,600,800 μmol/L UA) for 48 hours to induce EMT.Morphological changes of the NRK-52E cells were examined under an inverted phase contrast microscope.The protein expression of E-cadherin,α-SMA,p-Akt and Akt were detected by Western blotting.The distribution of E-cadherin and α-SMA were detected by immunofluorescence.NRK-52E cells were pretreated by different concentrations of LY294002(0,2.5,5,10,15 μmol/L),the inhibitor of PI3K/p-Akt signaling pathway,and then processed by uric acid (400 μmol/L) for 48 hours.Western blotting was used to detect the protein expression of p-Akt and Akt.NRK-52E cells were then divided into four groups:normal group (N),uric acid group (UA),LY294002 group (LY),uric acid with LY294002 group (UA + LY).The protein expression of E-cadherin and α-SMA were detected by Western blotting,the distribution of E-cadherin,α-SMA and p-Akt were detected by immunofluorescence.Results There was abundant cellular expression of E-cadherin in unstimulated renal tubular cells whereas its expression was significantly decreased in uric acidstimulated cells (P ＜ 0.05).In addition,uric acid induced de novo expression of α-SMA in contrast to almost negative staining in untreated cells (P ＜ 0.05).p-Akt were obviously increased in high uric acid group (P ＜ 0.05) and Akt changed not significantly (P ＞ 0.05).NRK-52E cells transformed into elongated fibroblast-like cells from cuboidal clustered epithelial cells.These indicated that uric acid has induced EMT and activated PI3K/p-Akt signaling pathway in NRK-52E cells.However,the above effects of uric acid were abolished when p-Akt was blocked by the PI3K inhibitor (10,15 μmol/L LY294002),indicated that LY294002 has reversed the trend of EMT.Conclusions High uric acid induces phenotypic transition of renal tubular cells probably via activating PI3K/Akt signaling pathway.