OBJECTIVE: To provide a method for the microbial limit test of Anshenning oral liquid and carry out validation of the mothed. METHODS: The validation on the microbial limit test method was conducted in accordance with the counting method of bacteria, mycetes and yeasts and the control bacteria test method stated in the Appendix of China Pharmacopoeia(2010 edition). Culture dilution method was used in bacterium count and total combined molds and yeasts count; while routine method was used in the control bacteria test. RESULTS: The recoveries of test organisms in the control group 3 preparations by routine method were more than 70%. The recoveries of the test group were more than 70%. In the 3 control bacteria tests the negative control bacterias, was not found in the negative control group, while the control bacteria was found in the test group. CONCLUSION: The established method substances can eliminate the antimicrobial in Anshenning oral liquid is accurate, and comprehensive, and can be used for the microbial limit test of Anshenning oral liquid.

Objective To construct the vector that expresses the fusion protein of HIV-Tat protein and red fluorescent protein(mCherry) in mammalian cells,and observe by fluorescence microscopy the intracellular transduction and localization of recombinant protein in cells,in order to obtain a useful tool for the study of the uptake mechanism and intracellular localization of HIV-TAT.Methods With the designed primer coding mCherry sequence,the mCherry gene was amplified by PCR with the vector pmCherry-C2 as template,and inserted into vector pET14b-His-TAT to construct the expression vector pET14b-His-TAT-mCherry.The constructed vector was then transformed into E.coli BL21(DE3),which had been identified by PCR and double digested with restriction endonuclease,followed by sequencing.After IPTG induction,the recombinant protein of His-TAT-mCherry was lyzed and analyzed with SDS-PAGE.Purified His-TAT-mCherry recombinant protein was added to Hela cells and the fluorescence was observed to evaluate the transduction efficiency.Results The results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the vector His-TAT-mCherry was correctly constructed.His-TAT-mCherry fusion protein was expressed in mammalian Hela cell line and purified successfully,and the fusion protein showed cellular transduction activity.It was found by fluorescence microscopy that the red fluorescence protein located mainly over the cytoplasm,and also the membrane to some extent.Conclusion The expression vector is successfully constructed for HIV-TAT labeled with mCherry sequence.Effective expression and purification of this fusion protein is achieved.It has been observed that the constructed vector may be expressed in mammalian Hela cell under active condition.Thus,it might be useful in the study of uptake mechanism and intracellular localization of HIV-TAT.

Objective To evaluate the changes in sagittal profiles and complications during treatment with growing rods (GRs) in hyperkyphotic early-onset scoliosis (EOS).Methods From December 2009 to December 2016,a total of 32 EOS patients who received growing rods treatment in our center,including 8 males and 24 females,were reviewed retrospectively.All the patients had minimum 2-year follow-up and over 2 lengthenings.Based on the reference value of thoracic kyphosis (TK) in T2-12 of normal children,the patients were categorized into an N group (20°≤TK≤50°,15 cases,4 males and 11 females) or K group (TK≥50°,17 cases,4 males and 13 females).The distribution of etiology was similar between the two groups.The average age was (6.2±2.0) years and (6.3±2.3) years respectively,curve flexibility was 34.6％± 10.4％ and 35.8％± 11.2％ before surgery.The precontoured rods were tunneled submuscularly,connecting proximal and distal anchors,and tandem or domino connectors.The rods were then locked after applying direct distraction that allowed appropriate elongation.The connectors were all placed under the deep fascia.Results The mean follow-up in the N and K groups was (5.5±1.9) years and (5.5±2.1) years,respectively.The distribution of proximal and distal anchors was similar between the two groups.The N and K groups,respectively,had an average number of lengthenings of 5.1±2.0 and 5.3±2.3,with mean lengthening intervals of (11.3±2.3) months and (10.9±1.9) months,respectively.In the N group,TK was decreased from 36.0°±9.4° to 30.6°±.8.3° after surgery,and to 32.2°±7.8° at the last follow-up,demonstrating it was maintained within the normal range.In the K group,TK was markedly reduced from 67.6°±11.6° to 41.7°±8.7° after the index surgery,with a correction rate of 38.3％± 14.6％,and the difference was statistically significant.And then it slightly increased to 46.5°±8.4° at the last follow-up,with correction loss of 7.1％±4.2％,and the difference was not statistically significant compared with the postoperatiom.The complication rate in the K group was significantly higher than in the N group (76.5％ vs.33.3％,P=0.031).The most common implant-and alignment-related complication in both groups was rod fracture (15.6％) and proximal junctional kyphosis (21.9％),respectively.The incidence of rod fracture in the N group and K group was 6.7％ and 23.5％,respectively.And the incidence of proximal junctional kyphosis was noted as 13.3％ and 29.4％ in the N group and K group,respectively.Proximal junctional angle (PJA) in the K group was greater than that in the N group preoperatively,postoperatively and at the last follow-up.Moreover,the increasing amount of PJA was significantly greater in the K group compared to the N group (1.6°± 1.0° vs.0.7°±0.8°).Four and seven complication events in the N and K groups,respectively,were evaluated with Grade Ⅰ.Four and seven complication events in the N and K groups,respectively,were classified as Grade Ⅱ A.Conclusion GRs can effectively restore the sagittal profile in hyperkyphotie EOS patients,but with a higher complication rate compared to the patients with normal kyphosis.

Exosomes serve as vesicles to deliver protein, lipids, nucleic acids or other cellular components, to neighboring or distant cells. Recent studies have highlighted the potential therapeutic effects of stem cell-derived exosomes on cancer and cardiovascular diseases. Our previous studie-shave investigated the role of stem cell-derived exosomes in cardiac protection. Mesenchymalstem cells released miR-22-enriched exosomes after ischemic preconditioning and these exosomes showed protective effects oncardiomyocytes.MiR-21-conaining exosomes were secreted by H2O2-treated cardiac progenitor cells and protected cardiomyocytes from H2O2-induced apoptosis. Heat-shock lead to the production ofheat shock factor 1-enriched exosomes from cardiac stem cells, which reducedapoptosis of cardiomyocytes. Given these important effects of exosomes in intercellular communications, exosomes have been proposed as a vector for drug delivery or other therapeutic purposes. However, cells secretea limited number of exosomes, which has hampered the development of exosomes for research and clinical application.Synthetic exosome-mimics by cellextrusion or cell membrane-cloaked nanoparticles, which canbe fabricated on a large-scale, provide novel platforms fordrug delivery. Two Korean groups fabricated exosome-mimetic nanovesicles by extruding monocytes or macrophages through a serial of filters and utilized these exosome-mimetics for the delivery of anti-tumor drug. Recently,cell membrane-cloaked nanoparticles have emergedas a potential tool for drug delivery with the advantages ofimmunocompatibility, stability and targeting capabilityfor the treatment of cancer. In summary, exosomes or exosome-mimics may serve as potential therapeutic tools for the treatment of cardiovascular diseases.

Objective To investigate the genotype of metallo-?-lactamases (MBL) produced by carbapenem resistant Pseudomonas aeruginosa in pediatric patients.Methods 59 strains of resistance to imipenem or meropenem were collected from December 2003 to November 2005 in Beijing children's hospital.Isolates were further evaluated for MBL production by two screening methods.MBL Etest strips were used to screen the phenotype of MBL production.Molecular screening for blaVIM,blaIMP,blaSPM and blaGIM was carried out using primers targeting the conserved regions of the MBL genes.The PCR fragments obtained with integron primers were sequenced on both strands.The nucleotide sequences were compared with sequences available over the Internet.Results Of the 59 carbapenem resistant Pseudomonas aeruginosa included in this study,29 (49.2%)were MBL positive using Etest methods,and 39 (66.1%) of these tested positive for MBL genes by PCR.35 (89.7%) were positive for blaIMP genes and 4 (10.3%) were positive for blaVIM genes.All isolates were negative for SPM and GIM DNA sequencing revealed that the IMP-1 was detected in 35 IMP-producing isolates,and VIM-2 was detected in 4 VIM-producing isolates.Conclusions This study has demonstrated that MBL-producing strains in pediatric are more common than in adult.IMP-1-producing strains are the main in pediatric,and VIM-2-producing strains concurred.The production of MBL is one of the important reasons of carbapenem resistant Pseudomonas aeruginosa in pediatric.It is very important to monitor the production of MBL.

Objective:To investigate the epidemic trend and risk change of acquired immunodeficiency syndrome (AIDS) complicated with malignant tumors after combination antiretroviral therapy (cART).Methods:The types of malignant tumors in patients with AIDS at different stages of cART were analyzed among anti-human immunodeficiency virus (HIV)-positive population in Hubei Province screened in National AIDS/HIV prevention and control information system from 1st January, 2004 to 31st December, 2018. The standardized incidence ratios(SIR) of malignant tumors in AIDS patients was analyzed based on the incidence of malignant tumors in the general population in Hubei Province or China in 2013. The changes in risks for development of malignant tumors in AIDS patients at different cART stages from 2004 to 2013 and 2014 to 2018 were compared.Chi-square test was used for statistical analysis.Results:Three hundred and twenty-three out of 22 994 AIDS patients were diagnosed with malignant tumors. Non-Hodgkin lymphoma(NHL) and cervical cancer were most common types in acquired immunodeficiency syndrome-defining cancers (ADC), while liver cancers and lung cancers were the most common types in non-acquired immunodeficiency syndrome-defining cancers (NADC). The overall risk of malignancy in AIDS patients was similar to that in the general population (SIR=1.06, χ2=0.62, P=0.426). However, the risks of Kaposi sarcoma, NHL, Hodgkin lymphoma, cervical cancer, and head and face cancers (excepting nasopharyngeal cancer) in AIDS patients were significantly higher than those in the general population (SIR=834.09, 9.65, 13.33, 5.22 and 2.94, respectively, χ2=11 747.27, 625.54, 56.65, 184.21 and 13.66, respectively, all P<0.01). The risks of lung cancer, colorectal anal cancer, stomach cancer and breast cancer in AIDS patients were significantly lower than those in the general population (SIR=0.33, 0.36, 0.43 and 0.45, respectively, χ2=33.43, 12.84, 9.01 and 7.21, respectively, all P<0.05). The SIR of cervical cancer, liver cancer and colorectal anal cancer from 2014 to 2018 were 4.06, 0.43 and 0.10, respectively, which were significantly lower than those from 2004 to 2013 (7.42, 1.96 and 0.84, respectively). The differences were all statistically significant ( χ2=5.39, 19.52 and 10.86, respectively, all P<0.05). Conclusions:At present, there are no significant differences of the incidences of malignant tumors between AIDS patients and general population, but the tumor types are different. The most common malignant tumors in this region are NHL and cervical cancer, which should be noted that HIV screening among patients with such tumors is conducive to comprehensive treatment to improve the efficacy.

The aim of the present study is to investigate the effects of Panax notoginseng saponins (PNS) on pneumocyte apoptosis and apoptosis-related protein, as well as c-Jun N-terminal kinase (JNK) in lung ischemia/reperfusion (I/R) injury. Thirty Wistar rats were randomly divided into control group, I/R group and PNS group. The unilateral lung I/R model was replicated by obstruction of left lung hilus for 30 min and reperfusion for 120 min in vivo. The rats in PNS group were given intraperitoneal injection of PNS at 60 min before ischemia and 10 min before reperfusion. Some lung tissues sampled at the end of the experiment were assayed for wet/dry weight ratio (W/T). The expressions of phosphorylated JNK (p-JNK) and JNK protein were detected by Western blot. The expressions of Bcl-2, Bax and Caspase-3 protein were detected by immunocytochemistry techniques. The pneumocyte apoptotic index (AI) was detected by terminal deoxynuleotidy1 transferase mediated dUTP nick end labeling (TUNEL). The morphological and ultrastructure changes were observed under light microscope and electron microscope, and the injured alveolus rate (IAR) was counted as well. The results showed that compared to control group, I/R group showed increased expressions of p-JNK, Bcl-2, Bax and Caspase-3 protein (all P < 0.01), decreased ratio of Bcl-2/Bax (P < 0.05), and increased values of AI, W/T and IAR (all P < 0.01). Moreover, light microscope and electron microscope showed serious morphological and ultrastructure injury in I/R group. Compared to I/R group, PNS group showed markedly decreased expressions of p-JNK, Bax and Caspase-3 protein (all P < 0.01), increased expression of Bcl-2 protein and ratio of Bcl-2/Bax (both P < 0.01), and lower values of AI, W/T and IAR (all P < 0.01). Meanwhile, light morphological and ultrastructure injury was found to be alleviated in PNS group. These results suggest that PNS can protect lung tissue from I/R injury, and the mechanism may correlate with suppressing JNK signal pathway, up-regulating the ratio of Bcl-2/Bax which results in inhibition of Caspase-3 dependent apoptosis.
Alveolar Epithelial Cells ; drug effects ; Animals ; Apoptosis ; drug effects ; Female ; Ischemia ; physiopathology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; blood supply ; metabolism ; pathology ; Male ; Panax notoginseng ; chemistry ; Rats ; Rats, Wistar ; Reperfusion Injury ; prevention & control ; Saponins ; isolation & purification ; pharmacology ; Signal Transduction ; drug effects

OBJECTIVETo investigate the protective effects and mechanism of SP600125-specificity inhibitor of c-Jun N-terminal kinase (JNK)on lung ischemia /reperfusion injury in rats.

METHODSThe unilateral lung ischemia/reperfusion model was replicated in vivo. Rats were randomly divided into three groups (n = 10): control group, ischemia/reperfusion group ( I/R group) and ischemia/reperfusion + SP600125 group (SP600125 group). The lung tissues sampled at the end of each experiment were assayed for wet/dry weight ratio (W/D),the injured alveoli rate (IAR), the expression of phosphorylation JNK (p-JNK) and JNK protein were detected by Western blot, the expression of Bcl-2, Bax, Caspase3 protein were detected by immunocytochemistry techniques, the pneumocyte apoptosis index (AI) was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end abeling(TUNEL), the ultrastructure changes were observed under electron microscope.

RESULTSCompared to I/R group, the expression of p-JNK, Bcl-2, Bax and caspase-3 protein were markedly decreased (all P < 0.01), the expression of Bcl-2 protein and the ratio of Bcl-2/Bax were markedly increased in SP600125 group(all P < 0.01). The value of AI, W/D, IAR showed significantly lower than those in I/R group (all P <0.01). Meanwhile, light morphological and ultrastructure injury were found in SP600125 group.

CONCLUSIONSP600125 can suppress JNK signal pathway, up-regulate the ratio of Bcl-2/Bax to inhibit Caspase-3 dependent apoptosis, so that it protects lung tissue from ischemia/reperfusion injury.

Animals ; Anthracenes ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Lung ; blood supply ; metabolism ; pathology ; MAP Kinase Signaling System ; Phosphorylation ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; pathology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVEIn order to develop the diagnostic genechip for specific detection of Schistosoma japonicum (Chinese mainland strain).

METHODSProbe and primers were designed based on the Schistosoma japonicum 5D gene encoding an immunogenic miracidial antigen. The probe for the conservative and specific gene sequence was spotted onto the specially treated glass slides by pin-based spotting robot Pixsys 5500 and was employed to make genechips. A polymerase chain reaction (PCR) protocol was designed to effectively amplify the 5D gene fragment containing the probe sequence from cercaria, egg, adult worm and infected Oncomelania DNA as well as other flukes DNA, respectively. After 35 cycles by PCR, the products were then labeled with fluorescent Cy3-labeled primer, using dissymmetrical PCR. The labeled PCR products of the target genes were hybridized to the diagnostic genechips for detection of Schistosoma japonicum and a fluorescent scanner (ScanArray 3000) was used to observe and record the hybridization signals.

RESULTSThe result obtained from the study showed that a 262 bp DNA fragment was amplified from cercaria, egg and adult worm with the designed primers and enable the genechip be applied to detect a single cercaria, egg and adult worm. When the genechip was used to detect Clonorchis sinensis, Fasciolopsis busk, and Paragonimus westermani DNA, the results showed negative, indicating that the genechip had good specificity.

CONCLUSIONThe genchip technique for detection of Schistosoma japonicum was established successfully and having the characteristics of high sensitivity and specificity.

Animals ; China ; DNA, Helminth ; genetics ; Genes, Helminth ; genetics ; Genetic Techniques ; Polymerase Chain Reaction ; methods ; Schistosoma japonicum ; genetics ; Sensitivity and Specificity