Fourteen-genome comparison identifies DNA markers for severe-disease-associated strains of Clostridium difficile

Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ～ 3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.     

周围神经组织工程生物材料的生物相容性评价

目的:总结周围神经组织工程生物支架材料生物相容性评价方法的进展。资料来源:应用计算机检索CNKI1990-01/2006-12有关周围神经组织工程生物材料生物相容性评价方面的文章,检索词“神经,组织工程,生物材料,生物相容性”,限定文献语言种类为中文。同时计算机检索ElsevierScienceE-journals1990-01/2006-12有关周围神经组织工程生物材料生物相容性评价方面的文章,检索词“nerve,tissueengineering,biomaterial,biocompatibility”,限定文献语言种类为English。资料选择:对资料进行初审,选取周围神经组织工程生物材料方面的相关文献,开始查找全文。排除中枢神经系统文献。对剩余的文献进行分析,总结研究内容。资料提炼:共收集到相关文献168篇,72篇符合纳入标准,其中30篇有关周围神经组织工程生物材料生物相容性评价方面的文献用于综述。资料综合:周围神经组织工程生物材料生物相容性评价可分为体外和体内两个方面。体外评价即利用生物材料与细胞共培养,通过观察细胞的形态,测定细胞的活力和凋亡等反映细胞的细胞毒性。体内评价最常用大鼠坐骨神经模型,通过普通和免疫组化染色、血液指标检测、器官病理切片等反映生物材料体内局部和整体的相容性。目前,生物相容性评价以短期为主,而其长期评价也是必须的。结论:在常用方法的基础上,一些新的方法逐渐被运用到周围神经组织工程支架材料的生物相容性评价上。对于各种评价方法的“性价比”还需要进一步的分析,以建立一套基本评价体系。     

Bone matrix degradation by the plasminogen activation system. Possible mechanism of bone destruction in arthritis

The observed increase in urokinase-type plasminogen activator (u-PA) and its receptor (u-PAR) in synovial tissue of patients with rheumatoid arthritis (RA) suggests pathophysiological involvement of the plasminogen activation (PA) system in inflammatory joint disease. In the present study, we investigated the capacity of the PA system to degrade non-mineralized and mineralized bone-like matrix in vitro as a model for bone destruction. Transfected mouse LB6 cell lines, that expressed either human u-PA or u-PAR, were cultured separately and simultaneously on radiolabelled bone matrix in the presence of plasminogen. Osteoblast-like murine calvarial MC3T3-E1 cells were used to produce a well-characterized, highly organized bone-like matrix, that could be mineralized in the presence of beta-glycerol phosphate. Bone matrix degradation was followed by the release of radioactivity in the culture medium. u-PA-producing cells, in contrast to u-PAR- producing cells, degraded both non-mineralized and mineralized bone matrix. This effect could be inhibited by anti-u-PA antibodies, as well as by tranexamic acid and by aprotinin, indicating that the degrading activity is u-PA mediated and plasmin dependent. Co-cultivation of a small portion of u-PA-producing cells with u-PAR-expressing cells resulted in a marked increase in degradation activity. Reduction of this potentiating effect by suramin or the amino-terminal fragment of u- PA, both competitive inhibitors of u-PA receptor binding, shows that this synergistic effect is due to binding of u-PA to u-PAR. u-PAR must be cell associated, as binding of u-PA to a soluble u-PAR prevented this enhancement. The capability of the PA system to degrade bone matrix in vitro, and the previously demonstrated increased expression of u-PA and u-PAR in synovial tissue of patients with RA, further support a role for the PA system in the development of bone erosions.      

Characterization of Thy-1 (CDw90) expression in CD34+ acute leukemia

Thy-1 (CDw90) is a phosphatidylinositol-anchored cell surface molecule which, when coexpressed with CD34 in normal human bone marrow, identifies a population of immature cells that includes putative hematopoietic stem cells. To date, the characterization of Thy-1 expression has been confined largely to normal tissues and cell lines. In this study, we evaluated the frequency and intensity of Thy-1 expression as defined by reactivity with the anti-Thy-1 antibody 5E10 in 38 cases of CD34+ acute leukemia (21 acute myelogenous leukemia [AML], 8 chronic myelogenous leukemia [CML] in blast crisis, and 9 acute lymphoblastic leukemia [ALL]). In 34 of 38 cases (89%) the CD34+ cells lacked expression of the Thy-1 antigen. High-density Thy-1 expression was found in 1 case of CML in lymphoid blast crisis, and low- density Thy-1 expression was identified on a portion of the leukemic cells in 2 cases of AML with myelodysplastic features, and 1 case of CML in myeloid blast crisis, suggesting a possible correlation between Thy-1 expression and certain instances of stem cell disorders such as CML and AML with dysplastic features. In contrast, the dissociation of Thy-1 and CD34 expression in the majority of acute leukemias studied suggests that the development of these leukemias occurs at a later stage than the hematopoietic stem cell. Characterization of Thy-1 expression in acute leukemia may eventually provide insights into the origin of the disease. In addition, separation of leukemic blasts from normal stem cells based on Thy-1 expression may prove useful in assessing residual disease, as well as in excluding leukemic blasts from stem cell preparations destined for autologous bone marrow or peripheral stem cell transplantation.     

Effect of precordial electrocardiographic electrode placement on st-segment measurement during exercise

Research protocols often utilize serial exercise testing to examine the efficacy of anti-ischemic therapies. These tests, however, are prone to multiple sources of bias. This investigation sought to determine the influence of varying precordial electrocardiographic (ECG) electrode placement on the detection of exercise-induced ST-segment shifts. Fifteen coronary artery disease patients with abnormal exercise tests were studied. Based on the previous exercise test, the precordial electrode position exhibiting the greatest ST-segment shift was selected as the reference electrode. Four additional electrodes were placed around this reference electrode and exercise testing was performed. ECG strips were recorded every minute. The time-to-onset and -offset of ischemic-type ST-segment depression was recorded. ST-segment depression was recorded during exercise from the reference electrode in 12 of 15 patients. Ischemic-type ST-depression was also recorded in each of these 12 patients with the surrounding electrodes; however, the time-to-onset detected by all four surrounding electrodes concurred in only 5 of 12 (42%) patients. The time-to-offset of the ST-segment depression concurred in 9 of 12 (75%) patients. Serial ECGs recorded from similar but not exactly the same precordial ECG electrode position should yield similar results for the detection of ischemia, but time-to-onset or -offset of ischemia may differ by 60 s or more. Small changes in the time-to-onset and -offset of ischemia should not be considered reliable indicators of anti-ischemia efficacy.     

Platelet adhesion to cyanogen-bromide fragments of collagen alpha 1(I) under flow conditions

The aim of this investigation was to identify domains of collagen type I that can support platelet adhesion under flow conditions. Four cyanogen bromide (CB) fragments composing 87% of the collagen alpha 1(I)-chain were studied under static and flow conditions. Under static conditions, bovine and human collagen fragment alpha 1(I)CB3 induced aggregate formation, whereas alpha 1(I)CB7 and alpha 1(I)CB8 supported adhesion of dendritic and contact platelets. Bovine alpha 1(I)CB6 weakly supported platelet adhesion. At shear rate 300/s, collagen fragment alpha 1(I)CB3 strongly supported platelet adhesion, whereas lower platelet adhesion was observed to alpha 1(I)CB7 and alpha 1(I)CB8. The fragment alpha 1(I)CB6 did not support platelet adhesion under flow conditions. Adhesion to alpha 1(I)CB3 was completely inhibited by a low concentration (0.6 IgG microgram/mL) of anti-GPIa monoclonal antibody (MoAb), whereas this concentration of antibody partially inhibited adhesion to alpha 1(I)CB7 and alpha 1(I)CB8. At higher concentrations (3 micrograms/mL) the anti-glycoprotein Ia (GPIa) antibody completely inhibited adhesion to alpha 1(I)CB8 and further reduced adhesion to alpha 1(I)CB7. Platelet adhesion to alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 was strongly inhibited by an anti-GPIb MoAb. A MoAb against the GPIb-binding site of von Willebrand factor (vWF) strongly inhibited platelet adhesion to alpha 1(I)CB7 and alpha 1(I)CB8, whereas platelet adhesion to alpha 1(I)CB3 was not inhibited. We conclude that under flow conditions alpha 1(I)CB3, alpha 1(I)CB7, and alpha 1(I)CB8 support GPIa/IIa-dependent platelet adhesion. The GPIb-vWF interaction is important under flow conditions for adhesion to alpha 1(I)CB7 and alpha 1(I)CB8 and probably also to alpha 1(I)CB3.