Retrieval of ancient DNA (aDNA) sequences from organism remains provide direct view of their evolutionary history. However, researches on aDNA have suffered from lots of technical problems. Specifically, discredited sequences were generated from damaged aDNA templates, and expensive and time-consuming methods were employed. Here, a method which could recover the endogenous aDNA as well as to reduce the cost and research period is described. This is achieved by improving the ancient DNA extraction method of isopropanol precipitation, and reevaluating the method of PCR after N-glycosylase (UNG) treatment, which could remove the damaged DNA from the aDNA extract. The efficiency of these methods were tested by comparing with traditional methods using ancient specimens of pig teeth aged between 4 300 years before present (BP) and 3 900 BP. The results showed that: firstly, the extraction efficiency of the improved method of isopropanol precipitation and current method with silica-based spin column were all 60%. Furthermore, the research period at least could be reduced by half with the application of the improved methods and the cost to 1/10 of the current method. Secondly, sequences obtained through the method of PCR after UNG treatment were 100% authentic. In contrast, 66%～ 88% sequences were authentic based on the results obtained with the method of multiple PCRs without UNG treatment. And the research cost and period needed by the method with UNG treatment were only half of the later one. These results demonstrate that the improved extraction method of isopropanol precipitation combined with the method of PCR after UNG treatment could increase the success rate of authentic DNA amplified and at least reduce the research cost and period by half. Therefore, this method can be applied in the large-scale detection of ancient specimens.

AIM:To observe whether modified epitopes from pancreatic tumor antigen mucin 4 (MUC4) have HLA-A2-restricted antitumor ability.METHODS:RT-PCR and Western blot were used to identify the expression of MUC4 in the pancreatic tumor cell lines CAPAN-2 and ASPC-1.HLA-A2 epitopes from MUC4 protein were predicted by the software of NetCTL 1.2, BIMAS, SYFPEITHI and IEDB.The modified peptides from MUC4 containing HLA-A2 were obtained by replacing anchor residues of the binding anchor motifs.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecule was evaluated by T2 binding assay.ELISPOT assay was used to investigate the ability of the peptide to induce specific restricted cytotoxic T-lymphocytes (CTLs) and release of IFN-γ.The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro.RESULTS:The expression of MUC4 was observed in the CAPAN-2 cells and ASPC-1 cells.The candidate peptides P1944-1Y, P1944-2L, P1944-1Y2L, P2004 and P2004-1Y9V showed moderate affinity toward HLA-A2 molecule.T2 binding assay showed that P1944-1Y2L and P2004-1Y9V had significantly higher affinity for HLA-A2 than the native peptides.ELISPOT assay showed P1944, P1944-1Y2L, P2004 and P2004-1Y9V were able to induce specific CTLs and more amounts of IFN-γ were released.ELISPOT assay showed that significantly more amounts of IFN-γ released by P1944-1Y2L and P2004-1Y9V were observed than the native peptides.The CTLs induced by P1944, P1944-1Y2L, P2004 and P2004-1Y9V lyzed the CAPAN-2 cells.P1944-1Y2L and P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell line CAPAN-2 than the native peptide-specific CTLs.CONCLUSION:Compared with the native peptides, modified epitopes P1944-1Y2L and P2004-1Y9V have higher binding affinity with HLA-A2 and retain immunogenecity.In addition, the anti-tumor immunity of modified epitopes P1944-1Y2L and P2004-1Y9V is stronger than that of the native peptides.The peptides P1944-1Y2L and P2004-1Y9V are excellent HLA-A2-restricted CTL epitopes from tumor antigen MUC4, which could serve as new candidates towards antitumor peptide vaccines.

Objective To explore the change in heme oxygenase-1 (HO-1) level in cerebrospinal fluid(CSF)and serum in patients with mild cognitive impairment(MCI),and the correlation between HO-1 and the rating scale,to provide a new marker for the diagnosis of MCI.Methods The HO-1 levels in CSF and serum in 45 MCI patients (MCI group) and 85 normal cases (control group) were analyzed with sensitive enzyme linked immunosorbent assays (ELISA).MMSE and MoCA scores were evaluated.Results The level of HO-1 was higher in MCI group than in control group both in CSF [(631.38±32.17)vs(480.75±17.98)ng/ L,P＜0.05],and in serum [(612.52±111.48)vs.(384.16±56.86)ng/ L,P＜0.05].The MCI and normal people HO-1 level had no significant difference between CSF group and serum group (P＞0.05).In MCI group,the levels of serum and CSF HO-1 had a positive correlation with MMSE scores (P＜0.05),but had no obvious correlation with MoCA scores (P＞0.05).In the normal group,the level of HO-1 was negatively related with MMSE scores in serum and CSF,and with MoCA scores in CSF (P＜0.05),but no obvious correlation in serum (P＞0.05).The levels of serum and CSF HO-1 had no obvious correlation with age in both groups (P＞0.05).Conclusions HO-1 concentration in both CSF and serum is significantly higher in MCI group than in normal group,and positively related with MMSE score.Thus the increase of HO-1concentration in both CSF and/or serum might be a new marker for the diagnosis of MCI.

Objective To observe the influence of Huiyang Shengji Ointment and its modified formulae on interleukin-1 (IL-1α) and thromboxane B2 (TXB2) in diabetic rats with chronic skin ulcers, and explore the mechanism for promoting the healing of ulcer.Methods Six out of 160 rats were randomly selected as a blank group, without any further processing. The remaining rats were made diabetic model and randomly divided into five groups after 2 weeks:1 d, 3 d, 5 d, 7 d and 14 d groups. Then, these groups were further divided into normal group (Vaseline ointments), model group (Vaseline ointments), Huiyang Shengji Ointment group (whole formula Ointment), Wenyang Yiqi group (Yiqi group, modified Wenyang Yiqi formula ointments) and Huoxue Shengji group (Huoxue group, modified Huoxue Shengji formula ointments). Normal group and model group were given Vaseline ointments;whole formula group, Yiqi group and Huoxue group were given corresponding ointment. Normal group used the method of skin excision, while other groups used STZ injection-hydrocortisone interference-skin excision-foreign body embedded preparation of composite factors for chronic skin ulcer model. After the appropriate treatment period, the rats were executed and tested for the contents of IL-1α and TXB2 in serum by enzyme-linked immunosorbent assay of five time points.Results In treatment 3 d, the contents of IL-1α in Yiqi group were significantly higher than the blank group, model group, whole formula group and Huoxue group (P<0.05). In treatment 5 d, the contents of IL-1α in whole formula group were significantly higher than the blank group and model group (P<0.05). In treatment 7 d, the contents of IL-1α in each treatment group were significantly higher than blank group and model group (P<0.05), and the whole formula group was higher than the Yiqi group and Huoxue group. In treatment 14 d, the contents of IL-1α in model group and Huoxue group were lower than the blank group (P<0.05). In treatment 3 d, the contents of TXB2 in normal group and the whole formula group were higher than the blank group (P<0.05). In treatment 5 d, the contents of TXB2 in whole formula group were higher than the blank group and the normal group (P<0.05). In treatment 7 d, the contents of TXB2 in Yiqi group were higher than the blank, the model, the whole formula and Huoxue groups (P<0.05). In treatment 14 d, the contents of TXB2 in Huoxue group were higher than the blank and model group (P<0.05), and the contents of TXB2 in the blank group and normal group was lower than those treatment groups (P<0.05).Conclusion Huiyang Shengji Ointment and its modified formulae could promote inflammation, stimulate secretion of inflammatory cytokines, while Yiqi Wenyang ointments played a more active role in promoting inflammation of the early phase of wound surface.

Through the analysis of the current status and problems of innovative medical devices evaluation, tnis paper discussed the strategies of evaluation, and ultimately raises the frame of evaluation, so as to provide reference for scientific evaluation of medical devices in China.
China ; Equipment and Supplies ; standards ; Evaluation Studies as Topic

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2.METHODS:RT-PCR and Western blot was used to determine the expres-sion of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29.HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay.ELISPOT as-say was used to investigate the levels of IFN-γ.The cytotoxicity assay in vitro was also used to determine the ability of indu-cing T cell response by the peptides.RESULTS:The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule.ELISPOT assay showed P485 and P965 induced CTLs of IFN-γrelease form CTLs.The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION:The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.

Objective Establish an objective and fair evaluation index system for the implementation effectiveness of the National Major New Drug Development Program of China.Methods Based on literature review and Delphi method,the framework of the evaluation index system was built and analytic hierarchy process was applied to determine the priority of each indicator of the system.The objectiveness and validness of the results were assured by determining expert activeness coefficient,authority coefficient,indicator rationality,and by applying consistency check.Results A targeted and systematic evaluation index system was constructed for the National Major New Drug Development Program,and through expert consultation and analytic hierarchy process,the factor of product result was considered of the highest weight of 39.03％,followed by technical result,theoretical result and organization and management result.The evaluation index system suggested that product result and technical result are two relatively more significant factors to be considered during evaluation.Conclu sion After consultation on expert opinions,the evaluation index system for the National Major New Drug Development Program was constructed with each factor been assigned with a scientific and rational weight,and this will provide a guideline for the decision making of program administrators and evaluation practice.

Objective To indentify the relationship between hepatitis B virus covalently closed circular DNA (HBV cccDNA) and postoperative recurrence of HBV in HBcAb positive liver donors by detecting HBV cccDNA in liver from HBcAb positive liver donors.Method Eighty-five of 1200 patients underwent liver transplantation for hepatitis B-related end-stage liver disease in our hospital from January 2007 to January 2010 were retrospectively analyzed.According to the situation of HBV infection in donor liver,the recipients were divided into 3 groups:(1) the experimental group (livers positive for HBcAb,and negative for HBsAg,n =40),(2) control group (livers positive for HBsAg,n =15),and (3) normal group (donor livers without HBV infection,n =30).HBV cccDNA of donors was detected by fluorescence quantitative PCR.Serum HBV and HBV DNA were regularly tested,and biopsy was done in those positive for HBsAg or HBV DNA to confirm HBV recurrence.The relationship between HBV cccDNA and postoperative recurrence of HBV in HBcAb positive liver donors was analyzed.Result The positive rate of cccDNA was 30％ in experimental group (12、40),73.3％ in control group (11/50),and 0 (normal group).The rate of HBV recurrence in experimental,control and normal groups was 10％ (4/40),80％ (12/15) and 3.3％ (1/30) respectively.The rate of HBV recurrence in the experimental group of cccDNA (+) and cccDNA (-) was 7.5％ (3/40),and 2.5％ (1/40).Conclusion The cccDNA in HBcAb positive donors may be one of the risk factors in HBV recurrence of hepatitis B-related liver transplantation patients.The screening of HBV cccDNA in the donor livers positive for HBcAb before liver transplantation is recommended to reduce the positive HBV recurrence and expand the pool of liver donors for patients with HBV-related liver disease.

OBJECTIVETo identify potential mutations of PKD1 gene in a family affected with autosomal dominant polycystic kidney disease (ADPKD).

METHODSThe coding regions of the PKD1 gene were subjected to PCR and Sanger sequencing. Reverse transcription-PCR (RT-PCR) was used to determine the relative mRNA expression in the patient.

RESULTSA splicing site mutation, c.8791+1_8791+5delGTGCG (IVS23+1_+5delGTGCG), was detected in the PKD1 gene in all 5 patients from the pedigree but not in 6 phenotypically normal relatives and 40 healthy controls. Sequencing of RNA has confirmed that there were 8 bases inserted in the 3' end of exon 23 of the PKD1 gene.

CONCLUSIONThe novel c.8791+1_8791+5delGTGCG mutation has created a new splice site and led to a frameshift, which probably underlies the ADPKD in the family. Above finding has enriched the mutation spectrum of the PKD1 gene.

Adult ; Female ; Humans ; Male ; Mutation ; genetics ; Pedigree ; Polycystic Kidney, Autosomal Dominant ; genetics ; RNA Splicing ; genetics ; TRPP Cation Channels ; genetics ; Young Adult

OBJECTIVETo detect the disease-causing mutation in a pedigree affected with autosomal dominant congenital cataract.

METHODSGenomic DNA was extracted and purified from peripheral blood samples from members of the pedigree and 100 healthy controls. Coding regions of 18 candidate genes were screened with PCR and Sanger sequencing. Identified mutations were verified among 100 healthy individuals to exclude single nucleotide polymorphisms.

RESULTSA heterozygous nonsense mutation c.471G>A of the CRYGD gene, which resulted in p.Trp157Term, was identified in all three patients. The same mutation was not found in the two normal individuals from the family and 100 healthy controls. The nonsense mutation was predicted to be "disease causing" by Mutation t@sting program.

CONCLUSIONThe nonsense mutation c.471G>A of the CRYGD gene probably underlies the congenital cataract in the pedigree.

Cataract ; etiology ; genetics ; Child ; Codon, Nonsense ; Humans ; Male ; Sequence Analysis, DNA ; gamma-Crystallins ; genetics