Heparin and low molecular weight heparin have been widely used in clinical therapy as anticoagulants in cardiovascular disease and in hemodialysis. Crude heparin is usually prepared from porcine intestinal mucosa. Purified heparin is a mixture of polysaccharides consisting mainly of repeating GlcNS(6S)-IdoA2S disaccharides and other disaccharides with different GlcNAc/GlcNS±3S±6S-GlcA/IdoA±2S residues. Heparin injections are drugs prepared from heparin active pharmaceutical ingredient ( API ) that is prepared from crude heparin. Low molecular weight heparins are dominant heparin-based drugs used clinically, which are prepared by degrading heparin into smaller sizes. As a result, low molecular weight heparins are sharing the same major disaccharides but have different reducing and non-reducing ends. In current study, we focused on the disaccharide compositional analysis of clinically used heparin and heparin-based drugs. HeparinaseⅠ,II, and Ⅲ were used to degrade all heparin and heparin-based drugs including heparin sodium injection, Enoxaparin sodium injection, Nadroparin calcium injection, Dalteparin sodium injection, Fondaparinux sodium into disaccharides. All the degraded products were analyzed by strong anion high perforance liquid chromatography ( SAX-HPLC) coupled with an UV-detector. Commercially available unsaturated disaccharide standards were then used for structral identification. Furthermore, unusual disaccharides present in Nadroparin, Dalteparin, and Fondaparinux were confirmed by reversed-phase ion pair HPLC coupled with mass spectrometry. The developed method produced detailed structural information, which should be useful for quality control of heparin and heparin-based drugs.

OBJECTIVE:To provide reference for improving pharmaceutical consultations and the quality of pharmaceutical care. METHODS:The records of pharmaceutical consultations were collected from medication consultation center of Huilong-guan district in our hospital during Jan. 2014-Dec. 2016,and then summarized in terms of consultants composition,consultation form,consulting contents and drugs consulted,while Pareto Diagram was used to analyze the main and minor factors of consult-ing contents. RESULTS:The medication consultation center provided pharmaceutical consultations 20353 cases during 2014-2016. Main consultants were patients (20039 cases,98.5%). A total of 6307 persons were involved,mostly female (3646 persons,57.8%). Face to face was the most common consulting method (19440 cases,95.5%). There were 13 types of consulting contents,among which usage and dosage and guidance for special formulation use were the main factors,including 10392 cases (51.1%) and 3844 cases (18.9%). Among 20353 cases of pharmaceutical consultations,18874 cases of in-volved drugs,and involved 11 categories,mostly respiratory drugs (11756 cases,62.3%). CONCLUSIONS:Usage and dos-age and guidance for special formulation use are the main contents of pharmaceutical consultations in the hospital district. The services of pharmaceutical consultations for patients,physicians and nurses provided by pharmacists can solve the questions on medications,and can promote rational drug use in clinic.

Objective To investigate the role of AcrAB-TolC efflux pump in fluoroquinolones resistance by Shigella. spp and to explore the significance of AcrAB-TolC efflux pump on mutation of acrR, soxS and marOR as well as on drug re?sistence. Methods Drug resistant bacteria were selected by Kirby-Bauer disk diffusion test. After addition of efflux pump inhibitor carbonylcyanide-m-chlorophenylhydrazone (CCCP), change of minimal inhibitory concentration (MIC)s of nilidixic acid, Levofloxacin, ofloxacin, ciprofloxacin and Norfloxacin were examined. The DNA binding region of acrA, acrB, soxS, acrR and marOR gene in these mutants were amplified by PCR and sequenced. Results Among the 159 clinical isolates of Shigella,11 strains are resistant to fluoroquinolone. After the addition of CCCP, MICs of 2 fluoroquinolone resistant strains decreased; the MICs of 7 fluoroquinolone resistant strains did not change; MICs of 2 fluoroquinolone resistant strains in?creased. The corresponding nucleotides C, A, T, T on the 36th to 39th of marOR gene were missing, showing by sequencing, in fluoroquinolone resistent strains which might be regulated by the efflux pump gene AcrAB-TolC. Conclusion Efflux pump inhibitor could restrain the activity of efflux partially. The mutations of marOR might play an important role in fluoroquino?lone resistent by shigella.

Objective To explore carrying rates of class 1, class 2 integrons as well as ISCR1 in Shigella isolates and their connection with drug resistance. Methods Antibiotic sensitivities were detected by K-B disk diffusion in 159 clinical isolates. Total bacteria DNA was prepared through boiling the isolates and the DNA was then used as template for PCR am?plification. PCR, ZSCR1 and sequencing analyse of integrons were applied to all of them. Results were compared by Blast and GenBank. Results Antibiotic sensitivity results showed that in the S. flexneri strains the incidence of resistance to tet?racycline and streptomycin were 88.68%and 81.13%in the S. flexneri strains while the incidence of resistant to chloram?phenicol and trimethoprim-sulfamethoxazol were both 56.60%, and the incidence of multidrug drug resistance was 77.36%. In the sonnei strains, the incidence of resistance to ampicillin, trimethoprim-sulfamethoxazo were 97.17% and 95.28%, 83.96%and 76.42%respectively, and the incidence of multidrug resistance was 98.11%. Among all isolates, 118 were class 1 integron positive , 70 were class 2 integron positive and 89 were double positives. For those 118 isolates that are positive of class 1 integron, 23 were typical while 95 weres atypical. The gene cassettes of typical class 1 integrons contains aadA2, aa?dA1, dfrⅠ, blaoxa-10 and blaoxa-1. IntI1, aadA, blaoxa-1 and IS1 were included in the gene cassetes of the atypical class 1 integrons. Class 2 integrons positive isolates carried gene cassttes which include dfrA1, satl and aadA1. No ISCR1 was found in any isolate. Integron carriage strains were closely associated with higher rate of multiple antibiobic resistance com?pared with the organisms without integrons (90.65%,50%, P<0.05). Conlusion Class 1 and class 2 integrons were widely existence in Shigella isolates and were related to the multidrug resistance.

Objective·To construct recombinant mycobacteriophage TM4-RpfE to lay a foundation for experimental research about how to eradicate Mycobacterium tuberculosis in combination with anti-tuberculosis drugs,and how to shorten treatment for tuberculosis ultimately.Methods·Electrotransformation was used to introduce pJV53 plasmid into Mycobacterium smegmatis to prepare recombinant engineering bacteria.After amplification of hsp60-RpfE fusion gene by overlap PCR,a long gene fragment (homologous +hsp60-RpfE+homologous,HHRH) was amplified by multi-step overlap PCR.The DNA of mycobaeteriophage TM4 and HHRH fragment were cotransfected into the recombinant engineering bacteria by electrotransformation,then the recombinant phage from the single primary plaques were confirmed by PCR and sequencing.SDS-PAGE was used to analyze the protein expression in recombinant phage.Results·The hsp60-RpfE fusion gene at the length of 901 bp and HHRH fragment at the length of 1 873 bp were identified by overlap PCR.The PCR product produced 955 bp and 301 bp DNA bands in the first generation plaques colony.SDS-PAGE analysis showed a specific protein band at 21 000 in the recombinant phages.Conclusion·The recombinant mycobacterium phage TM4-RpfE was successfully constructed and the expression of target gene RpfE was initially verified.

A method was developed to assay α-Hydroxyltriazolam and α-Hydroxyalprazolam,　which are the major metabolites of triazolam and alprazolam respectively,in human urine.　After addition of 2-hydroxyflurazepam　(interal standard) and hydrolysis with β-glucuronidase, the hydroxy-metabolites were extracted with hexane-dichloromethane (1∶1) at pH　10.8, then were derivated with (BSTFA). The analysis was performed on a HP-5 capillary column with electron-capture detector.The detection limits of analysis in urine were about 1μg/L.The method was successfully applied to urine specimens collected from healthy human volunteers who ingested 0.5 mg of triazolam or 0.8 mg alprazolam. The method was enough sensitive to assay urine specimen excreted at 24 h by volunteers after taking the medicine.

Hormones and epigenetic characteristics in a patient with clinically diagnosed adrenal hypoplasia congenita (AHC) were analyzed. Results indicated that plasma ACTH increased, while cortisol, testosterone, LH and FSH decreased. LH, FSH and testosterone did not sufficiently respond to GnRH or hCG stimulation. Gene analysis indicated that C368F mutation was located in exon 1 of DAX-1 gene.

Objective To investigate the expression and effect of high mobility group box 1(HMGB1) and its signaling pathway(HMGB1 RAGE/TLR4-NF-κB-cytokines)in rats with dilatd cardiomyopathy(DCM).Methods The rats were divided into two groups:normal control group (control,n=20) which treated with physiological saline,and DCM group(n=22) which treated with adriamycin(1 mg/kg twice a week)for 6 weeks,and then observed for 2 weeks.Echocardiography was performed at the end of the study.Plasma IL-1,IL-6,TNF-α level were measured by the flow cytometry.The CRP,BNP concentrations were measured by enzyme linked immunosorbent assay (ELISA).The expression of HMGB1 mRNA,TLR4 mRNA,RAGE mRNA,NF κB mRNA were measured by real time PCR.Results There were four rats dead in the DCM group;two rats were randomly selected from the DCM group to certified modeled successfully by echocardiography and pathological examination.Left ventricular end-diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) in DCM group were significantly higher than those in the normal control group(P＜0.05);left ventricular ejection fraction (LVEF),left ventricular short axis contractility(FS) in DCM group was significantly lower than that in normal control group(P＜0.05).The expression of H MGB1 mRNA,TLR4 mRNA,RAGE mRNA and NF-κB mRNA in myocardial tissue were significantly increased in DCM group than in the normal control saline group (P＜ 0.05),The expression of HMGB1 mRNA were positively correlated with TLR4 mRNA,RAGE mRNA and NF κB mRNA(r=0.873,P=0.005;r=0.949;P=0.000;r=0.898,P=0.002).The serum levels of IL-1,IL 6,TNF α and CRP were significantly higher in DCM group.The expression of HMGB1 mRNA in myocardial tissue was positively correlated with IL 1,IL-6,TNF-α and CRP(r=0.944,P=0.002;r=0.988,P=0.000;r=0.968,P=0.000;r=0.961,P=0.000).Conclusion HMGB1 and it's inflammation signaling pathway (HMGB1-TLR4/RAGE-NF-κB-cytokines) were highly expressed in dilated cardiomyopathy,and have relationship with left ventricular diameter and cardiac function,they may be involved in the development of DCM.

Objective To evaluate the value of D-dimer in assessing severity and predicting longterm prognosis in patients with community acquired pneumonia (CAP).Methods From June 2009 to December 2010,a total of 189 patients with CAP were enrolled.After admission,D-dimer,procalcitonin (PCT) and C-reactive protein (CRP) were measured,and Pneumonia Severity Index (PSI) was calculated.They were assigned into two groups according to their D-dimer levels:high D-dimer levels group (D-dimer levels≥500 μg/L) and normal D-dimer levels group (D-dimer levels ＜ 500 μg/L).The followup time was one year.A Kaplan-Meier survive curve was constructed to assess the 1-year mortality,and multivariate logistic regression analysis were used to assess the value of D-dimer for predicting long-term prognosis.Results D-dimer levels increased with increasing PSI class [class Ⅰ-Ⅲ:378.37 μg/L (216.74,649.50) μg/L; class Ⅳ:673.41 μg/L (544.77,866.85) μg/L; class Ⅴ:831.58 μg/L (591.78,1066.39) μg/L,x2 =56.58,P ＜ 0.01].The Kaplan-Meier survival curve showed that 1-year mortality rate of high D-dimer levels group was higher than normal D-dimer levels group (log-rank test,x2 =52.51,P ＜ 0.01).The multivariate logistic regression analysis showed an independent relationship between higher D-dimer levels and long-term mortality (OR =2.05,95％ CI:1.48-2.61,P ＜ 0.01).Conclusion D-dimer is an independent predictor of severity and long-term prognosis in patients with CAP.

Objective To evaluate the saline and ethanol for effects of disinfecting penicillin skin test.Methods Searching CNKI,VIP,CBM and Wanfang Data to collect case-control studies suiting enrolled conditions.Meta-analysis was conducted using Revman 5.0.0 software.Results Five case-control studies with 9 481 patients were enrolled.Meta-analysis demonstrated there was significant difference between saline and ethanol in penicillin skin test disinfection.Conclusions Saline has lower positive results than ethanol,and can better represent the result of penicillin skin test.