A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori

A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOA^R), on the average 60% of the 5-FOA^R colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5 end of the pyrG gene with vectors containing a mutation near the 3 end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.     

乳腺癌（改良）根治术住院流程与平均住院日

参照业务流程重组的理论与方法研究了乳腺癌根治术的住院流程。结果显示,该病种住院流程存在的主要问题有：诊疗服务的随意性过大,等待诊疗服务的时间过长,患者入院标准的掌握过松,对该病种往院注程进行重组,将在不影响医疗服务质量的前提下有效缩短平均住院日。     

重组Calpain蛋白的免疫原性及其诊断上的应用研究

目的研究在大肠杆菌中表达的日本血吸虫Calpain蛋白的免疫原性及其在日本血吸虫诊断上的应用. 方法用重组Calpain蛋白免疫BALB/c小鼠,ELISA法测定特异性IgG抗体的动态变化及其IgG亚型(IgG1, IgG2a, IgG2b, IgG3)产生特征;同时用重组Calpain蛋白作为诊断抗原,粪检血吸虫病阳性患者血清作为一抗,肝吸虫阳性患者血清作为考核交叉反应血清. 结果重组Calpain抗原免疫小鼠后,产生了一个极高的抗Calpain特异性抗体,免疫4周后IgG类抗体达到高峰,与对照组鼠相比较,免疫鼠血清中IgG1, IgG2a, IgG2b特异性抗体也有显著性的上升; Calpain重组抗原血清诊断日本血吸虫和粪检的符合率为100%, 粪检阳性EPG低的个体抗Calpain抗体滴度高, EPG高的患者抗Calpain抗体滴度反而低, 与肝吸虫的交叉反应率为37%. 结论日本血吸虫Calpain是一个具有免疫原性的蛋白,能够激发宿主产生高水平的免疫球蛋白,能够敏感地测定日本血吸虫的感染, 说明Calpain可以发展成为日本血吸虫病的诊断抗原和日本血吸虫疫苗候选分子.     

EGF-PE4O重组毒素对喉鳞癌细胞株Hep-2的毒性作用

观察EGF－PE40重组毒素对喉鳞癌细胞的毒性作用,为寻找新型的治疗头颈鳞癌的生物制剂奠定基础。应用MTT比色法测定EGF－PE40重组毒素的生物学活性。并在光镜下观察Hep－2细胞形态变化。确定MTT比色法中Hep－2细胞浓度为2×104／well。选择10％SDS－0．01mol／LHCI作为MTT结晶的溶解液,当EGF－PE40重组毒素为0．53ng／ml时,50％的Hep－2细胞死亡,在一定范围内,随重组毒素增加,细胞损伤加重。     

小儿脊柱矫形应用同种异体骨移植的免疫学观察

目的研究同种异体骨移植在小儿脊柱矫形术中的应用及免疫学改变。方法选取1999～2004年123例脊柱矫形患儿,按照融合材料不同分为3组：第1组48例,为自体骨植骨患儿;第2组64例,为以自体骨为主、同种异体骨混合植骨融合的患儿,其中自体骨量大于60％;第3组21例,为以同种异体骨为主、自体骨混合植骨融合的患儿,其中异体骨量大于60％。观察和分析术后临床体征、实验室检查、免疫学检测、放射及同位素检查结果。结果第1组、第2组无明显排异反应;第3组3例出现早期排异反应（经病理学检查及免疫学检测证实）,包括高热、局部无菌性渗出、C-反应蛋白（CRP）持续升高,病理检查见局部大量淋巴细胞浸润,未见新骨形成,骨膜反应不明显,免疫学检测显示CD^4＋明显升高,CD^4＋／CD^8＋明显升高。3组骨痂形成无明显差异。结论大量同种异体骨移植可引起早期免疫排斥反应,将同种异体骨与自体骨混合应用可减少排斥反应,并能得到较牢固的融合结果。     

DNA损伤应答缺陷作为乳腺癌治疗靶点的研究进展

DNA损伤应答(DNA damage response,DDR)缺陷是近年来乳腺癌治疗研究的热门靶点之一。DDR通路负责DNA损伤后的识别、信号转导和修复,其功能异常可导致细胞的凋亡或基因组不稳定性的增加。目前进入临床研究阶段的乳腺癌DDR靶向药物主要包括多聚腺苷二磷酸核糖聚合酶[poly (ADP-ribose) polymerase,PARP]抑制剂、ATM抑制剂、CHEK1抑制剂、ATR抑制剂及WEE1抑制剂等。主要从DDR缺陷的概念、以DDR作为靶点的基本原理、DDR各类靶向药物的临床研究现状及其在临床应用中的难点与挑战等方面展开综述。     

Complete genome sequence analysis of novel human bocavirus reveals genetic recombination between human bocavirus 2 and human bocavirus 4

Epidemiological surveillance of human bocavirus (HBoV) was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. By partial sequence analysis of VP1 gene, an unusual strain of HBoV (CMH-S011-11), was initially identified as HBoV4. The complete genome sequence of CMH-S011-11 was performed and analyzed further to clarify whether it was a recombinant strain or a new HBoV variant. Analysis of complete genome sequence revealed that the coding sequence starting from NS1, NP1 to VP1/VP2 was 4795 nucleotides long. Interestingly, the nucleotide sequence of NS1 gene of CMH-S011-11 was most closely related to the HBoV2 reference strains detected in Pakistan, which contradicted to the initial genotyping result of the partial VP1 region in the previous study. In addition, comparison of NP1 nucleotide sequence of CMH-S011-11 with those of other HBoV1–4 reference strains also revealed a high level of sequence identity with HBoV2. On the other hand, nucleotide sequence of VP1/VP2 gene of CMH-S011–11 was most closely related to those of HBoV4 reference strains detected in Nigeria. The overall full-length sequence analysis revealed that this CMH-S011-11 was grouped within HBoV4 species, but located in a separate branch from other HBoV4 prototype strains. Recombination analysis revealed that CMH-S011-11 was the result of recombination between HBoV2 and HBoV4 strains with the break point located near the start codon of VP2.     

Novel human astrovirus strains showing multiple recombinations within highly conserved ORF1b detected from hospitalized acute watery diarrhea cases in Kolkata,India

Human astroviruses (HAstVs) associated with acute watery diarrhea among hospitalized infants, children and adults as sole or mixed infection, were earlier reported from Kolkata, India. Further, novel recombinations have been detected through sequencing of the highly conserved ORF1b (RdRp) region of seven human astrovirus strains in Kolkata, India. Primers were designed and the ORF1b region was amplified by RT-PCR and sequenced. To examine the evolutionary pressures influencing the evolution of human astroviruses we implemented evolutionary genetics analysis. Maximum recombination break points detected in Kolkata strain IDH1300 were 8 and a single break point location was detected at 1205nt position. Partition-wise phylogenetic analyses of the IDH1300 Kolkata strain did not show close homology to the reference strains. Further phylogenetic analyses of full length ORF1b region of the seven human astrovirus strains showed that they formed a close cluster with each other and displayed a separate lineage in comparison to reference human astrovirus strains worldwide. This study shows the emergence of novel recombinant human astrovirus strains in Kolkata, India, warranting stringent surveillance to monitor the genetic diversity of human astrovirus strains infecting different age groups.     

重组人生长激素对高龄病人术后恢复作用的Meta分析

目的:评价高龄病人手术后应用重组人生长激素(rh GH)对术后恢复的影响。方法:应用计算机检索Pubmed、Embase、中国生物医学文摘数据库(CBM)、维普中文期刊全文数据库中所有相关随机对照研究,按照一定标准对其筛选后进行质量评价。采用Rev Man 5.2软件进行统计和分析。结果:纳入10个随机对照研究,共计374例研究对象,其中术后应用rh GH的病人183例,对照组191例。分析结果表明,高龄病人术后应用rh GH能够1促进血清蛋白质合成,[WMD=5.60,95%CI(3.92,7.29),P0.01];促进清蛋白(ALB)水平恢复[WMD=3.79,95%CI(2.95,4.63),P0.01];促进转铁蛋白(TF)合成[WMD=0.52,95%CI(0.37,0.67),P0.000 01];促进前清蛋白(PA)合成[WMD=0.57,95%CI(0.09,1.06),P=0.02]。2促进血清免疫球蛋白水平恢复,结果分别为Ig A[WMD=0.48,95%CI(0.26,0.70),P0.01];Ig M[WMD=0.39,95%CI(0.26,0.52),P0.01];Ig G[WMD=2.31,95%CI(0.69,3.94),P0.01]。3减轻术后疲劳症状[WMD=-2.09,95%CI(-2.34,-1.85),P0.01]。结论:高龄病人术后应用rh GH可促进血清蛋白质合成,提高免疫功能,减轻疲劳综合征的症状,有利于病人康复。