Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria

DNA fingerprinting has attracted considerable interest as means for identifying, tracing and preventing the dissemination of infectious agents. Various methods have been developed for typing of pathogenic bacteria, which differ in discriminative power, reproducibility and ease of interpretation. During recent years a typing method, which uses the information provided by whole genome sequencing of bacterial species, has gained increased attention. Short sequence repeat (SSR) motifs are known to undergo frequent variation in the number of repeated units through cellular mechanisms most commonly active during chromosome replication. A class of SSRs, named variable number of tandem repeats (VNTRs), has proven to be a suitable target for assessing genetic polymorphisms within bacterial species. This review attempts to give an overview of bacterial agents where VNTR-based typing, or multiple-locus variant-repeat analysis (MLVA) has been developed for typing purposes, together with addressing advantages and drawbacks associated with the use of tandem repeated DNA motifs as targets for bacterial typing and identification.     

三维目标与粗糙面复合散射的广义稀疏矩阵平面迭代及规范网格算法

提出了快速计算二维导体粗糙面与面上金属目标复合散射的广义稀疏矩阵平面迭代及规范网格法(G-SMFSIA/CAG).推导了二维导体粗糙面与面上目标相互作用的耦合积分方程,用稀疏矩阵平面迭代及规范网格法(SMFSIA/CAG)求解粗糙面部分的表面积分方程,而用基于RWG基函数的矩量法(MOM)计算目标部分的表面积分方程,并通过更新方程的激励项迭代求解目标与粗糙面的相互耦合作用.结合Monte-Carlo方法产生具有PM(Pierson-Moskowitz)海浪谱的随机海洋粗糙面,数值分析了海面上不同形状导体目 关键词： 复合散射 广义稀疏矩阵平面迭代及规范网格法 随机海洋粗糙面 双站散射系数     

9号染色体上海洛因依赖易感基因的定位

目的:筛查和定位9号染色体上海洛因依赖易感基因的位点.方法:在10例海洛因依赖者和其正常同胞对照者中用覆盖第9号染色体的20个短串联重复序列(Short Tandem Repeats,STR)进行荧光标记的基因组扫描.结果:D9S286, D9S167和D9S164位点等位基因在海洛因依赖组和其正常同胞对照组之间分布有显著性差异(x2检验,P＜0.05).其它17个STR位点D9S288,D9S285,D9S157, D9S171,D9S161,D9S1817, D9S273,D9S175,D9S283,D9S287,D9S1690,D9S1677,D9S1766, D9S1682, D9S290,D9S1826,D9S158的等位基因在海洛因依赖组和其正常同胞对照组之间分布没有显著性差异(x2检验,P＞0.05).结论:D9S286,D9S167和D9S164位点附近可能存在海洛因依赖的易感基因,多巴胺β-羟化酶基因可以作为候选基因.     

Functional Bacterial Amyloids: Understanding Fibrillation,Regulating Biofilm Fibril Formation and Organizing Surface Assemblies

Functional amyloid is produced by many organisms but is particularly well understood in bacteria, where proteins such as CsgA (E. coli) and FapC (Pseudomonas) are assembled as functional bacterial amyloid (FuBA) on the cell surface in a carefully optimized process. Besides a host of helper proteins, FuBA formation is aided by multiple imperfect repeats which stabilize amyloid and streamline the aggregation mechanism to a fast-track assembly dominated by primary nucleation. These repeats, which are found in variable numbers in Pseudomonas, are most likely the structural core of the fibrils, though we still lack experimental data to determine whether the repeats give rise to β-helix structures via stacked β-hairpins (highly likely for CsgA) or more complicated arrangements (possibly the case for FapC). The response of FuBA fibrillation to denaturants suggests that nucleation and elongation involve equal amounts of folding, but protein chaperones preferentially target nucleation for effective inhibition. Smart peptides can be designed based on these imperfect repeats and modified with various flanking sequences to divert aggregation to less stable structures, leading to a reduction in biofilm formation. Small molecules such as EGCG can also divert FuBA to less organized structures, such as partially-folded oligomeric species, with the same detrimental effect on biofilm. Finally, the strong tendency of FuBA to self-assemble can lead to the formation of very regular two-dimensional amyloid films on structured surfaces such as graphite, which strongly implies future use in biosensors or other nanobiomaterials. In summary, the properties of functional amyloid are a much-needed corrective to the unfortunate association of amyloid with neurodegenerative disease and a testimony to nature’s ability to get the best out of a protein fold.     

A Ligand That Targets CUG Trinucleotide Repeats

The development of small molecules that can recognize specific RNA secondary and tertiary structures is currently an important research topic for developing tools to modulate gene expression and therapeutic drugs. Expanded CUG trinucleotide repeats, known as toxic RNA, capture the splicing factor MBNL1 and are causative of neurological disorder myotonic dystrophy type 1 (DM1). Herein, the rational molecular design, synthesis, and binding analysis of 2,9‐diaminoalkyl‐substituted 1,10‐phenanthroline (DAP), which bound to CUG trinucleotide repeats, is described. The results of melting temperature (T_m) analyses, surface plasmon resonance (SPR) assay, and electrospray spray ionization time‐of‐flight (ESI‐TOF) mass spectrometry showed that DAP bound to r(CUG)₉ but not to r(CAG)₉ and r(CGG)₉. The dual luciferase assay clearly indicated DAP bound to the r(CUG)_n repeat by affecting the translation in vitro.     

A novel diagnostic strategy for trisomy 21 using short tandem repeats

Molecular technique with STRs can rapidly diagnose aneuploidy. In order to improve its fidelity, we developed a novel STR-based strategy for fast diagnosis of trisomy 21 and constructed a multimarker diagnostic system according to it. The system is based on nine STRs, of which two were previously known and seven were newly identified from the genomic sequence of the long arm of chromosome 21. They were confirmed to be highly polymorphic in the Chinese population by PCR amplification and gel electrophoresis. The combination of nine STR markers, when applied to DNA from 102 Chinese individuals with normal karyotype, did not yield any false-positives, and clearly revealed three different alleles in DNA from 15 out of 18 trisomy 21 patients. The results show that our new strategy can provide an alternative molecular technique for the rapid detection of aneuploidy.     

Aggregation mechanism of polyglutamine diseases revealed using quantum chemical calculations, fragment molecular orbital calculations, molecular dynamics simulations, and binding free energy calculations

Polyglutamine (polyQ) diseases, including Huntington’s disease (HD), are caused by expansion of polyQ-encoding repeats within otherwise unrelated gene products. The aggregation mechanism of polyQ diseases, the inhibition mechanism of Congo red, and the alleviation mechanism of trehalose were proposed here based on quantum chemical calculations and molecular dynamics simulations. The calculations and simulations revealed the following. The effective molecular bonding is between glutamine (Gln) and Gln (Gln + Gln), between Gln and Congo red (Gln + Congo red), and between Gln and trehalose (Gln + trehalose). The bonding strength is −13.1 kcal/mol for Gln + Gln, −24.4 kcal/mol for Gln + Congo red, and −12.0 kcal/mol for Gln + trehalose. In the polyQ region, both the number of intermolecular Gln + Gln formations and the total calories generated by the Gln + Gln formation are proportional to the number of repetitions of Gln. We propose an aggregation mechanism whose heat generated by the intermolecular Gln + Gln formation causes the pathogeny of polyQ disease. In our aggregation mechanism, this generated heat collapses the host protein and promotes fibrillogenesis. Without contradiction, our mechanism can explain all the experimental results reported to date. Our mechanism can also explain the inhibition mechanism by Congo red as an inhibitor of polyglutamine-induced protein aggregation and the alleviation mechanism by trehalose as an alleviator of that aggregation. The inhibition mechanism by Congo red is explained by the strong interaction with Gln and by the characteristic structure of Congo red.