Neuropathological Staging of Spinocerebellar Ataxia Type 2 by Semiquantitative 1C2‐Positive Neuron Typing. Nuclear Translocation of Cytoplasmic 1C2 Underlies Disease Progression of Spinocerebellar Ataxia Type 2

Spinocerebellar ataxia type 2 (SCA2) is a hereditary neurodegenerative disorder caused by the expansion of the trinucleotide CAG repeats encoding elongated polyglutamine tract in ataxin‐2, the SCA2 gene product. Polyglutamine diseases comprise nine genetic entities, including seven different forms of spinocerebellar ataxias, Huntington's disease, and spinal and bulbar muscular atrophy. These are pathologically characterized by neuronal loss and intranuclear aggregates or inclusions of mutant proteins including expanded polyglutamine in selected neuronal groups. Previously, we examined immunolocalization of ubiquitin, expanded polyglutamine (probed by 1C2 antibody), and ataxin‐2 in genetically confirmed SCA2 patients. In the present study, we expanded this approach by distinguishing different patterns of subcellular 1C2 immunoreactivity (“granular cytoplasmic,” “cytoplasmic and nuclear” and “nuclear with inclusions.”) and by quantifying their regional frequencies in three autopsied SCA2 brains at different stage of the disease. Comparison with neuronal loss and gliosis revealed that overall 1C2 immunoreactivity was paralleled with their severity. Furthermore, appearance of granular cytoplasmic pattern corresponded to early stage, cytoplasmic and nuclear pattern to active stage, and nuclear with inclusions pattern to final stage. We conclude that this 1C2‐immunoreactive typing may be useful for evaluating the overall severity and extent of affected regions and estimating the neuropathological stage of SCA2.     

Diversity among strains of Pseudomonas aeruginosa from manure and soil,evaluated by multiple locus variable number tandem repeat analysis and antibiotic resistance profiles

The results of a multiple locus variable number of tandem repeat (VNTR) analysis (MLVA)-based study designed to understand the genetic diversity of soil and manure-borne Pseudomonas aeruginosa isolates, and the relationship between these isolates and a set of clinical and environmental isolates, are hereby reported. Fifteen described VNTR markers were first selected, and 62 isolates recovered from agricultural and industrial soils in France and Burkina Faso, and from cattle and horse manure, along with 26 snake-related isolates and 17 environmental and clinical isolates from international collections, were genotyped. Following a comparison with previously published 9-marker MLVA schemes, an optimal 13-marker MLVA scheme (MLVA₁₃-Lyon) was identified that was found to be the most efficient, as it showed high typability (90%) and high discriminatory power (0.987). A comparison of MLVA with PFGE for typing of the snake-related isolates confirmed the MLVA₁₃-Lyon scheme to be a robust method for quickly discriminating and inferring genetic relatedness among environmental isolates. The 62 isolates displayed wide diversity, since 41 MLVA types (i.e. MTs) were observed, with 26 MTs clustered in 10 MLVA clonal complexes (MCs). Three and eight MCs were found among soil and manure isolates, respectively. Only one MC contained both soil and manure-borne isolates. No common MC was observed between soil and manure-borne isolates and the snake-related or environmental and clinical isolates. Antibiotic resistance profiles were performed to determine a potential link between resistance properties and the selective pressure that might be present in the various habitats. Except for four soil and manure isolates resistant to ticarcillin and ticarcillin/clavulanic acid and one isolate from a hydrocarbon-contaminated soil resistant to imipenem, all environmental isolates showed wild-type antibiotic profiles.     

Extended-spectrum β-lactamase producing Klebsiella spp. in chicken meat and humans: a comparison of typing methods

Recently, chicken meat was identified as a plausible source of extended-spectrum β-lactamase (ESBL) -producing Escherichia coli in humans. We investigated the relatedness of ESBL-producing Klebsiella spp. in chicken meat and humans. Furthermore, we tested the performance of SpectraCell RA^® (River Diagnostics), a new typing method based on Raman spectroscopy, in comparison with multilocus sequence typing (MLST) for Klebsiella pneumoniae. Twenty-seven phenotypically and genotypically confirmed ESBL-producing Klebsiella spp. isolates were typed with MLST and SpectraCell RA. The isolates derived from chicken meat, human rectal swabs and clinical blood cultures. In the 22 ESBL-producing K. pneumoniae isolates, CTX-M15 was the predominant genotype, found in five isolates of human origin and in one chicken meat isolate. With MLST, 16 different STs were found, including five new STs. Comparing the results of SpectraCell RA with MLST, we found a sensitivity of 70.0% and a specificity of 81.8% for the new SpectraCell RA typing method. Therefore, we conclude that SpectraCell RA is not a suitable typing method when evaluating relationships of ESBL-producing Klebsiella spp. at the population level. Although no clustering was found with isolates of chicken meat and human origin containing the same ESBL genes, MLST showed no clustering into distinctive clones of isolates from chicken meat and human origin. More studies are needed to elucidate the role of chicken meat in the rise of ESBL-producing Klebsiella spp. in humans.     

Capsular polysaccharide gene diversity of pneumococcal serotypes 6A, 6B, 6C,and 6D

This study was performed to better understand the genetic diversity and evolutionary relatedness of pneumococcal serotypes 6A, 6B, 6C, and 6D. Multi-locus sequence typing (MLST) was performed for 160 serogroup 6 isolates from clinical specimens collected from children between 1991 and 2010. We identified 38 sequence types (STs) comprising five clonal complexes with 12 singletons. Although most STs were confined to a single serotype, some STs were shared by two serotypes, and one ST was shared by three serotypes. Many STs of serotype 6A showed genetic relatedness with those of serotype 6C or 6D in eBURST analysis. Five capsular polysaccharide (cps) genes – wchA, wciO, wciP, wzy, and wzx – were analysed in 74 isolates from our clinical samples and in 36 isolates from GenBank. There were several profiles and clades in each serotype on the analysis of the concatenated sequences of the five cps genes. Small genetic distances between serotypes 6A and 6B and between serotypes 6C and 6D were observed while serotype 6B with an indel sequence formed a distinct clade. When comparing the individual cps genes between the serotypes, there was also a high level of similarity in the wchA and wciO gene sequences between serotype 6C and serotype 6D. On the other hand, serotypes 6A and 6D had the most highly similar wzy and wzx gene sequences. The wzy sequences of serotype 6C were nearly identical (99.6%) to those of serotype 6A clade II strains. In conclusion, we revealed the diversity of the genetic background and cps sequences in each pneumococcal serotype of serogroup 6. Pneumococcal serotype diversity might be attributable to complex serial mutation and recombination events.     

Resolution of Ambiguous HLA Genotyping in Korean by Multi-Group-Specific Sequence-Based Typing

Purpose

To evaluate a multi-group-specific sequence-based typing (SBT) method for resolving ambiguous results from human leukocyte antigen (HLA) genotyping.

Materials and Methods

A total of 50 samples that showed ambiguous genotypes for at least two HLA loci from HLA-A, -B, -C and -DRB1 by the conventional SBT assay were evaluated using a new SBT test, the AVITA plus assay. The most likely HLA genotypes for the respective samples considering allele frequencies in Korean were concordant between the AVITA and conventional SBT assays.

Results

An average of 3.3 loci among the HLA-A, -B, -C and -DRB1 loci per sample gave results with two or more possible allele combinations with the conventional SBT, and 48 (96.0%) out of 50 showed reduced numbers of possible genotypes for at least one HLA locus with the AVITA. A total of 41, 43, 42, and 38 cases among the 50 samples showed ambiguous results for HLA-A, -B, -C, and -DRB1 typing by the conventional SBT, respectively. The average numbers of possible allele combinations for the respective four HLA loci were 8.2, 6.7, 5.9, and 3.2, and they were reduced to 1.5, 2.2, 4.4, and 1.8, respectively, by the AVITA. Ambiguity was resolved by the AVITA in 33 (80.5%), 31 (72.1%), 17 (40.5%) and 28 (73.7%) samples among the ambiguous cases from the conventional SBT for HLA-A, -B, -C, and -DRB1 typing, respectively.

Conclusion

The multi-group-specific SBT method considerably reduced the number of ambiguous results, and thus may be useful for accurate HLA typing in clinical laboratories.     

利用基因芯片技术筛选早期乳腺癌的差异表达基因

目的 比较相同病理类型和临床分期预后不同的早期(Ⅰ、Ⅱ期)乳腺癌样本的基因表达差异,寻找有显著差异的基因,探索与早期乳腺癌预后有关的基因分型.方法 用Agilent4×44K人全基因组Oligo芯片对47例早期乳腺癌患者的组织样本,结合其预后好、差数据,根据其临床预后的不同分为对照组(预后好)24例和实验组(预后差)23例,进行差异基因表达分析;采用Real-time PCR技术,对两组乳腺癌样本中差异表达的基因进行验证.结果 基因芯片检测分析发现,实验组样本比对照组样本有差异表达基因126个,其中60个基因显著上调,66个基因显著下调(差异均在2倍以上).结论 不同预后的早期乳腺癌样本中,基因表达存在显著差异,早期乳腺癌的预后与这些基因的表达有关.     

High-throughput, high-fidelity HLA genotyping with deep sequencing

Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes.     

金黄色葡萄球菌多位点熔解曲线分型可行性研究

目的建立多位点熔解曲线体系(Mutiple locus melting curve system,MLMC)对金黄色葡萄球菌(金葡菌)进行分子分型,并评价其可行性。方法建立并应用多位点熔解曲线体系检测59株金葡菌,以脉冲场凝胶电泳技术(pulsed field gel electrophoresis,PFGE)作为金标准,评价MLMC方法应用与金葡菌食物中毒快速溯源的可行性。结果MLMC将59株金葡菌分为19个型别,包括11个流行克隆和8个散发克隆。PFGE将其分为15个型别,包括8组流行克隆和7个散发克隆。两者有13株金葡菌呈现不一致的分型结果,两种方法分型一致性为77.97%(46/59)。MLMC和PFGE的Simposn相关系数依次是0.92和0.87。结论结合流行病资料分析,认为MLMC方法具有良好的分型能力、分辨力和合理性,同时操作简单快速,分辨率高于PFGE分型技术,可应用于金葡菌食物中毒的快速溯源和筛查。     

携带NDM-1基因合并膜孔蛋白OmpK36缺失肺炎克雷伯菌耐药性分析

目的对产新德里金属β-内酰胺酶(NDM-1)肺炎克雷伯菌耐药情况、分子分型特点,携带耐药基因、传播方式,及其膜孔蛋白表达情况进行分析,以探讨产NDM-1酶肺炎克雷伯菌耐药机制。方法实验菌分离自前列腺增生患者尿液的标本,对其进行细菌鉴定和药敏试验、改良Hodge试验、EDTA协同试验和多位点序列分型(MLST);采用聚合酶链技术检测β-内酰胺酶相关耐药基因和膜孔蛋白OmpK35、OmpK36基因,并对NDM-1和膜孔蛋白OmpK35、OmpK36基因进行序列分析;利用SDS-PAGE电泳检测膜孔蛋白OmpK35、OmpK36;菌株质粒接合实验分析传播方式,对受体菌和接合子进行药敏结果比较。结果实验菌改良Hodge试验阴性、EDTA协同试验阳性,鉴定为耐碳青霉烯类肺炎克雷伯菌(CRKP)株,对13种常见抗生素耐药,MLST基因分子分型为ST15;PCR检测其携带NDM-1基因,经序列测定比对确认,同时该菌株还携带KPC基因及SHV基因,未检测到VIM、IMP、OXA-48、GES、GIM基因;OmpK35、OmpK36基因存正点突变及片段缺失的现象,SDS-PAGE分析发现膜孔蛋白OmpK36缺失;质粒接合实验阳性,接合子E.coliJ53对3种碳青霉烯类药物的抑菌圈明显减小。结论了解NDM-1肺炎克雷伯菌携带耐药基因类型及传播方式、膜孔蛋白是否缺失以及分子分型,为指导临床正确使用抗菌药物及流行病学调查具有重要意义。     

胎儿脐动脉超声血流分型对单绒毛膜双胎妊娠sIUGR预后的影响

目的探讨胎儿脐动脉超声血流分型对单绒毛膜双胎妊娠选择性宫内生长受限预后的影响.方法将91例羊绒毛膜双胎妊娠选择性宫内生长受限孕妇设为试验组,选取同期来我院就诊的正常双胎妊娠产妇31例设为对照组.两组均予以彩色多普勒超声诊断仪进行超声检查,记录两组胎儿预后和妊娠结局,比较不同预后单绒毛膜双胎妊娠选择性宫内生长受限胎儿的超声指标.结果试验组小胎儿宫内死亡率显著高于对照组(P<0.01);大、小胎儿出生体质量均显著低于对照组(P<0.01).试验组不同选择性宫内生长受限分型两胎儿、小胎儿宫内死亡率比较差异无统计学意义(P>0.05);Ⅱ型小胎儿出生体质量显著低于Ⅰ型和Ⅱ型小胎儿(P<0.05),Ⅰ型与Ⅲ型小胎儿出生体质量比较差异无统计学意义(P>0.05).实验组死亡小胎儿羊水过少、选择性宫内生长受限分型Ⅱ型检出率显著高于存活胎儿(P<0.05或0.01),帆状脐带、双胎体质量差异>25%检出率与存活胎儿比较差异无统计学意义(P>0.05).结论羊水过少及选择性宫内生长受限分型是影响单绒毛膜双胎妊娠选择性宫内生长受限胎儿预后的重要原因,对该类孕妇采用超声检查能够提供较为可靠的临床评价依据.