Objective To prepare monoclonal antibody(McAb) against human tissue kallikrein (HK) and develop an ELISA kit allows for the in vitro quantitative determination of human tissue kallikrein in urine. Methods To generate a monoclonal antibody specific for TK, the synthetic TK peptide consisting of 12 amine acids(12P), was fused to keyhole limpet hemocyanin(KLH) and used for immunization. Using hybridoma screening, monoclonal secreting cell lines were identified and used to generate ascites in BALB/c mouse. Antibody was purified by affinity column chromatography. 12% SDS-PAGE and Western blot were used to visualize the purified antibody. This kit employs indirect competitive ELISA technique and BiotinAvidin System. 12P was fused to bovine serum albumin(BSA) and has been pre-coated onto a microplate at first. Standards and samples were added to the appropriate microplate wells with a biotin-conjugated McAb croplate well. A TMB substrate solution is added to each well. The enzyme-substrate reaction is terminating by the addition of a sulphuric acid solution and the color change is measured spectrotometrically at a wavelength of 450 nm. The concentration of tissue kallikrein in the samples is then determined by comparing the O.D. of the samples to the standard curve. Results 8 hybridoma cell lines secreting mAbs special to HK,SDS-PAGE and Western blot demonstrated successful preparing and purification of McAb( 100% ). The linearity of this ELISA kit is demonstrated(r =0. 990). The range of detection of the assay is 0.008 μg/ml to 0. 5 μg/ml. The assay remained stable, with no change in the values measured, over five cycles of freezing and thawing. Conclusion 8 McAbs against HK have been prepared successfully and possess high titer and specificity. The development of an ELISA kit for detecting HK can meet the needs of detection of HK in urine samples.

Until the recent emergence/re-emergence of human-pathogenic viruses in ticks, tick-borne viruses have been neglected as causative agents of human disease (particularly in China). To gain insight into the diversity of tick-borne viruses in Xinjiang Uygur Autonomous Region (northwestern China), we conducted illumina deep sequencing-based screening for virus-derived small RNAs in field-collected Ixodes persulcatus ticks. We found 32, 631 unique virus-matched reads. In particular, 77 reads mapped to the tick-borne group within the genus of Flavivirus, and covered 3.8%-2.4% viral genomes. In addition, 32 unique reads were specific to the Siberian subtype of tick-borne encephalitis viruses (TBEV-Sib) which have never been reported in Chinese TBE loci. We confirmed the potential existence of TBEV-Sib by amplification (using reverse transcription-polymerase chain reaction) of genomic fragments from the envelope gene or 3' genomic terminus from the pools of examined ticks. Both sequences demonstrated high homology to TBEV-Sib strains attached geographically to southern Siberia with nucleotide identity of 97.2%-95.5% and aminoacid identity of 99.4%-98.3%, respectively. In conclusion, we report, for the first time, detection of TBEV-Sib in the natural TBE loci of China. These novel data may provide genetic information for further isolation and epidemiologic investigation of TBEV-Sib.
Animals ; Arachnid Vectors ; virology ; China ; Encephalitis Viruses, Tick-Borne ; classification ; genetics ; isolation & purification ; Encephalitis, Tick-Borne ; transmission ; virology ; Genome, Viral ; Humans ; Ixodes ; virology ; Molecular Sequence Data ; Phylogeny

Objective To investigate the diversity of mosquito-borne viruses in Xinjiang , China, and to identify mosquitos-borne viruses of medical importance rapidly .Methods The virus-derived RNAs in mosquitos captured in wild were screened and confirmed by using Illumina deep sequencing approach and reverse transcription PCR , respectively .The alignment analysis was performed by using gene sequences from GenBank.Results One hundred and forty-four Culex Flavivirus ( CxFV, Flavivirus genus, Flaviviridae) specific sequences were identified .The overlapping reads were assembled into 7 uncontinuous viral genomic contigs.The gaps between the contigs were further filled by RT-PCR products, which resulted in reconstruc-tion of viral genomic 5′and 3′terminus (687 nt and 411 nt).Phylogenetic analysis showed that the newly identified CxFV belonged to America/Asian genotype , which specifically clustered into a clade with other CxFV strains from China mainland ,sharing 98.2%-99.5%homologies in nucleotide sequences and 99.5%in amino acids sequences among them .Conclusion Illumina deep sequencing approach was successfully applied to arthropod-borne virus surveillance .The recently emerged Culex Flavivirus was detected for the first time in Xinjiang, China.

To investigate the antitumor mechanism of artemisninin, a flexible docking analysis was used to score all kinds of functions of 11 Qinghaosu derivatives and transferrin with different resolutions. The distances of Asp-63, Tyr-188, His-249, Arg-124 and Lys-296 with Qinghaosu were less than 0.5 nm, separately. Meanwhile, the higher is the activity of Qinghaosu derivatives the higher is the score. Our model explains that Fe2+ is more feasible to react with Qinghaosu, and not involved in other metabolism in presence of transferrin. Docking results unveil that Iron(II)-transferrin increased the cytotoxicity of Qinghaosu derivatives and provide a rational basis for further design and synthesis of novel Qinghaosu derivatives.
Antineoplastic Agents, Phytogenic ; chemical synthesis ; chemistry ; pharmacology ; Artemisinins ; chemical synthesis ; chemistry ; pharmacology ; Catalytic Domain ; Drug Discovery ; Models, Chemical ; Molecular Structure ; Protein Binding ; Transferrin ; chemistry

Objective To define the maximum-tolerated dnse(MTD)and observe the side effect of escalating cisplatin with 5-fluorouracil in concurrent chemoradiotherapy for esophageal carcinoma in Chinese,with toxicity studied.Methods Previously untreated fifteen Chinese patients suffering from esophageal carcinoma received conventional fractionafiun radiotherapy,with 5 daily fractions of 2.0 Gy per week.The total radiation dose was 60 Gy.Concurrent chemotherapy dose escalation was given by the relatively safe and kidney-sparing modified Fibonacci sequence.The starting dose was cisplatin 37.5 mg/m~2 D1 and 5-fluorouracil 500 mg/m~2 D1-5, respectively.This regimen was repeated 4 times every 28 days.Escalation dose was eisplatin 7.5mg/m~2 and 5- fluorouracil 100mg/m~2.Every cohort contained at least 3 patients.If no dose-limiting toxicity(DLT)was observed, the next dose level was opened for entry.These courses were repeated until DLT appeared.MTD was declared as one dose level below which DLT appeared.Results DLT was defined as grade 3 radiation-induced esophngitis at the level of cisplatin 60 mg/m~2,5-fluorouracil 700 mg/m~2.MTD was defined as eisplafin 52.5 mg,/m~,5- fluorouracil 700 mg/m~2.The major side effect were radiation-induced esophagitis,leucopenia,nausea,vomiting and anorexia.Conclusion Maximun tolerated dose of cisplatin with 5-fluorouracil in concurrent chemoradiotherapy in the Chinese people with esophageal carcinoma were eisplatin 52.5 mg/m~2 D1,5-fluorouracil 700 mg/m~2 D1-5,repeated 4 times every 28 days.

Objective To establish an extensively drug-resistant Acinetobacter baumannii (XDR-AB) infection model using Caenorhabditis elegans (C.elegans),and evaluate the effect of efflux pump inhibitors(EPIs) on reversal of ciprofloxacin resistance in XDR-AB.Methods XDR-AB infection model of C.elegans was established,six EPIs(CCCP,PAβN,NMP,omeprazole,reserpine,and verapamil)combined with ciprofloxacin were used to treat the infected model,the survival rate of C.elegans was recorded to evaluate the in vivo activities of drugs,toxicity test and in vitro drug susceptibility test were also performed.Results Lethal effect of different concentrations of XDR-AB on C.elegans was varied,5 × 106 CFU/mL of XDR-AB was selected to infect C.elegans.C.elegans survival test showed that survival curves of C.elegans infected with XDR-AB for 3 hours and curves of control group (polymixin B was added) were not significantly different (x2 =3.154,P＞0.05);compared with control group,survival curves of C.elegans infected with XDR-AB for 6 hours or 9 hours were significantly different (both P＜0.001),but 6 hours and 9 hours were not significantly different(x2 =0.669,P＞0.05),6 hours was chosen as the duration of infection,36 hours was appropriate for the duration of antimicrobial therapy.Ciprofloxacin with EPIs for infection model revealed that low concentration of PAβN,NMP,omeprazole,and reserpine could improve the survival rate of C.elegans by 30％-40％,15％-20％,20％-30％,and 20％ respectively,high concentration of verapamil could improve the survival rate of infected C.elegans by about 30％.In vitro susceptibility test and toxicity test results showed that ciprofloxacin combined respectively with CCCP,omeprazole,and verapamil could reduce minimum inhibitory concentration(MIC) to the original 1/4,combined respectively with PAβN,NMP,and reserpine could reduce MIC to the original 1/2,CCCP had the best bacterial inhibitory effect in vitro,but the toxicity was large,and was not suitable for the study of pharmacodynamics in vivo.Conelusion The infection model of C.elegans XDR-AB is initially and successfully established,which is used to evaluate the efficiency of six EPIs for reversing ciprofloxacin resistance.

Epigenetics is the study of heritable changes in gene expression other than changes in the underlying DNA sequence. Such changes include DNA methylation, histone modification, chromatin remodeling, genomic imprinting, X chromosome inactivation and non-coding RNA regulation. Recent progresses on epigenetics open new possibilities in tackling these challenging problems in forensic science, including identification of fetal paternity testing in embryonic period, determination of the necessary allele in paternity testing, discrimination of identical twins, origination analysis of micro tissue, verification of forged DNA. This review focuses on epigenetics concept and its latest application in the field of paternity testing, age estimation, discrimination between the twins, identification of tissue of origin, and estimation of postmortem interval.
Alleles ; DNA Methylation ; Epigenesis, Genetic ; Epigenomics ; Forensic Sciences ; Gene Expression ; Genomic Imprinting ; Humans ; Twins, Monozygotic

OBJECTIVETo investigate the efficacy of pressure reduction by peritoneal catheterization in patients with malignant ascites-induced abdominal compartment syndrome (ACS).

METHODSClinical data of 29 patients with malignant ascites-induced ACS from October 2002 to October 2008 were analyzed retrospectively. Intra-abdominal pressure (IAP) was reduced by peritoneal catheterization. Changes of intra-abdominal pressure and ascites volume were observed during treatment. Clinical signs and urinary volume were monitored.

RESULTSIAP was less than 25 cm H(2)O in 2 cases, 25 to 35 cm H(2)O in 21 cases, more than 35 cm H(2)O in 6 cases. IAP decreased significantly after drainage of 1000 to 1500 ml of ascites, then IAP curve leveled off. With all the ascites drained, IAP maintained at 11 to 12 cm H(2)O and at 6 to 8 cm H(2)O after 24 hours. Blood pressure was stable without significant changes before and after IAP reduction (P>0.05). The breathing rate and heart rate were improved, and 24 h urinary volume increased significantly after IAP reduction (P<0.01).

CONCLUSIONEarly peritoneal catheterization can improve the cardiac, pulmonary, and renal function in malignant ascites-induced ACS.

Adult ; Aged ; Aged, 80 and over ; Ascites ; complications ; etiology ; Catheterization ; Compartment Syndromes ; etiology ; surgery ; Decompression, Surgical ; methods ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo observe the effect of electroacupuncture (EA) on cytokines in the cardiac surgical patient and to evaluate the application of combined acupuncture anesthesia to cardiac surgery.

METHODSThirty patients with atrial septal defect were divided into 3 groups, general anesthesia group (A), acupuncture anesthesia group (B) and combined general anesthesia and EA group (C). Peripheral blood samples were collected before anesthesia, before cardiopulmonary bypass (CPB), 30 min after CPB and 24 h after operation to determine the levels of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-6 and IL-10.

RESULTSThe levels of IFN-gamma and IL-2 decreased in the 3 groups after CPB and further decreased 24 h after operation, and in the group C were higher than those in the group B. The levels of IL-6 and IL-10 significantly increased 24 h after operation in the 3 groups with no significant difference among the 3 groups.

CONCLUSIONThe general anesthesia combined with EA can not completely improve the decrease of IFN-gamma and IL-2 induced by CPB, indicating that the good response of the general anesthesia combined with EA to stress can partially improve the immunosupression induced by CPB. Acupuncture does not have significant effect on inflammatory cytokine reaction induced by cardiac surgery.

Acupuncture Analgesia ; Adult ; Cardiac Surgical Procedures ; Electroacupuncture ; Female ; Humans ; Interferon-gamma ; blood ; Interleukin-10 ; blood ; Interleukin-2 ; blood ; Interleukin-6 ; blood ; Male ; Middle Aged