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.

Cisplatin (CDDP), homoharringtonine (HHT), mitoxantrone (MIT) and hydroxycamptothecin (HCPT) are highly effective anti-tumor drugs. To evaluate their effects in the therapy of leukemia and establish a valuable method to estimate anti-tumor drugs, Annexin V/PI double parameter flow cytometry was used to detect the effects of these drug inducing apoptosis and death in Jurkat cell line. The results showed that MIT and HCTP-induced apoptosis effects on Jurkat cell line were obvious at 4 hours in early phase after adding drug (P < 0.05) and at 8 hours in late phase after adding drug (P < 0.05). HHT had obvious effect on inducing apoptosis of Jurkat cells, but no significant difference from low to high doses. The effect of CDDP on inducing apoptosis of Jurkat cell line was obviously weaker than that of HHT, MIT and HCPT, its weak effect on apoptosis of Jurkat cell line was found only at high concentration of drug for long time. Death effects on Jurkat cell line can not be observed in every experimental group. It is concluded that low dose of MIT can effectively induced apoptosis of Jurkat cell line. Annexin V/PI double parameter flow cytometry can be used as a reliable method for clinical screening anti-tumor drugs.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Camptothecin ; pharmacology ; Cisplatin ; pharmacology ; Drug Screening Assays, Antitumor ; methods ; Harringtonines ; pharmacology ; Humans ; Jurkat Cells ; Mitoxantrone ; pharmacology

BACKGROUNDFolic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos, investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 (HAND2) and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations.

METHODSMorpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression.

RESULTSDHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos (P < 0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos.

CONCLUSIONSDHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; physiology ; Female ; Heart ; embryology ; Heart Defects, Congenital ; etiology ; Tetrahydrofolate Dehydrogenase ; genetics ; physiology ; Zebrafish ; Zebrafish Proteins ; genetics ; physiology

OBJECTIVESTo explore a sensitive and specific method for detection of bcl-2/IgH gene rearrangement in diffuse large B cell lymphoma (DLBCL), and verify the credibility of the established method.

METHODSbcl-2/IgH hemi-nested PCR primers were designed using the professional primer design software. Fifty-two samples of pathologically diagnosed DLBCL and 10 fresh tonsil tissues were amplified using hemi-nested touch down-PCR to detect bcl-2/IgH gene rearrangement. The PCR products were cloned and sequenced.

RESULTSbcl-2/IgH gene rearrangement was detected in 6 of 52 DLBCL samples and 2 of 10 fresh tonsil tissues using one-way method. By using the hemi-nested PCR for the second round amplification, 5 of DLBCL were positive, but all of the fresh tonsil tissues were negative. The positive PCR products were sequenced and analyzed on the Internet, 3 of 8 cases obtained by one-way method were false positive, 5 positive cases amplified using hemi-nested PCR were all bcl-2/IgH gene rearrangement. PCR products of 3 false positive cases were homologous to BAC331191 and LLNLR-245D11 in human chromosome 19 and RP11-498P10 in chromosome 1.

CONCLUSIONThere are false positive results using common primers for detecting bcl-2/IgH gene rearrangement. The mechanism may be that highly homologous sequences to human genome exist in commonly used primers. The specificity of the diagnosis could be improved by hemi-nested PCR using the combination of primers we designed and the traditional ones.

Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Genes, bcl-2 ; genetics ; Humans ; Lymphoma, Large B-Cell, Diffuse ; genetics ; Polymerase Chain Reaction ; methods