Use of the Roche LightCycler instrument in a real-time PCR for Trichomonas vaginalis in urine samples from females and males

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted infection (STI) that can result in vaginitis, urethritis, and preterm birth. Traditional methods of diagnosis, including wet preparation, can be unreliable. In this study, we describe the adaptation of an existing PCR method for specific detection of T. vaginalis DNA into a rapid real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe chemistry. The FRET-based assay described demonstrated high sensitivity with a detection limit of 1.06 organisms, as well as a high specificity. A total of 253 urine samples collected prospectively from both men and women were tested for T. vaginalis DNA with both the FRET-based assay and a previously validated PCR assay. When the validated PCR assay was used as the "gold standard" and after discrepancies had been resolved, our FRET-based assay demonstrated an analytical sensitivity and specificity of 90.1 and 100%, respectively. Overall results suggest that FRET-based assays can provide rapid, accurate, and high-throughput detection of T. vaginalis and may prove useful in clinical settings and for large-scale screening programs.     

Age-dependent and iron-independent expression of two mRNA isoforms of divalent metal transporter 1 in rat brain

The DMT1(Nramp2/DCT1) is a newly discovered proton-coupled metal-ion transport protein. The cellular localization and functional characterization of DMT1 suggest that it might play a role in physiological iron transport in the brain. In the study, we evaluated effects of dietary iron and age on iron content and DMT1 expression in four brain regions: cortex, hippocampus, striatum, substantia nigra. Total iron content in all regions was significantly lower in the low-iron diet rats and higher in the high-iron diet rats than that in the control animals, showing that dietary iron treatment for 6-weeks can alter brain iron levels. Contrary to our expectation, there was no significant alternation in DMT1(+IRE) and (-IRE) mRNA expression and protein content in all brain regions examined in spite of the existence of the altered iron levels in these regions after 6-weeks' diet treatment although TfR mRNA expression and protein level were affected significantly, as was expected. The data demonstrates that expression of DMT1(+IRE) and (-IRE) was not regulated by iron in these regions of adult rats. The lack of response of DMT1 to iron status in the brain suggests that the IRE of brain DMT1 mRNA might be not really iron-responsive and that DMT1-mediated iron transport might be not the rate-limiting step in brain iron uptake in adult rats. Our findings also showed that development can significantly affect brain iron and DMT1(+IRE) and (-IRE) expression but the effect varies in different brain regions, indicating a regionally specific regulation in the brain.     

Phylogenomics of the dog and fox family (Canidae,Carnivora) revealed by chromosome painting

Canid species (dogs and foxes) have highly rearranged karyotypes and thus represent a challenge for conventional comparative cytogenetic studies. Among them, the domestic dog is one of the best-mapped species in mammals, constituting an ideal reference genome for comparative genomic study. Here we report the results of genome-wide comparative mapping of dog chromosome-specific probes onto chromosomes of the dhole, fennec fox, and gray fox, as well as the mapping of red fox chromosome-specific probes onto chromosomes of the corsac fox. We also present an integrated comparative chromosome map between the species studied here and all canids studied previously. The integrated map demonstrates an extensive conservation of whole chromosome arms across different canid species. In addition, we have generated a comprehensive genome phylogeny for the Canidae on the basis of the chromosome rearrangements revealed by comparative painting. This genome phylogeny has provided new insights into the karyotypic relationships among the canids. Our results, together with published data, allow the formulation of a likely Canidae ancestral karyotype (CAK, 2n = 82), and reveal that at least 6–24 chromosomal fission/fusion events are needed to convert the CAK karyotype to that of the modern canids. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rapid identification and susceptibility testing using the VITEK(R) 2 system using culture fluids from positive BacT/ALERT(R) blood cultures

BACKGROUND AND PURPOSE: In order to reduce the turnaround time for laboratory diagnosis of bacteremia, the efficacy of identification and antimicrobial susceptibility testing using samples taken directly from positive BacT/ALERT(R) standard aerobic and standard anaerobic blood culture bottles was evaluated. METHODS: 160 positive blood culture bottles were examined and incubated at 35 degrees C in 5% carbon dioxide for 4-24 h, and an aliquot of the culture fluid was Gram stained. Samples containing Gram-negative bacilli were inoculated on VITEK(R) 2 ID-GNB (identification-Gram-negative bacilli) and AST (antimicrobial susceptibility testing)-GN04 cards, and those containing Gram-positive cocci were inoculated on ID-GPC (identification-Gram-positive cocci) and AST-P526 cards. The same samples were also examined by the standard method, involving subculture from positive BacT/ALERT standard blood culture bottles. RESULTS: Eighty seven of 97 Gram-negative bacilli (89.7%) and 21 of 63 Gram-positive cocci (33.3%) were correctly identified to the species level. For antimicrobial susceptibility testing, the direct method had an overall error rate of 5.4% for Gram-negative bacilli, with 0.9% very major, 0.9% major, and 3.6% minor discrepancies compared to the standard method. The overall error rate in antimicrobial susceptibility testing for the 13 Staphylococcus spp. was 10.3%, with 6.0% very major, 2.6% major, and 1.7% minor discrepancies. CONCLUSION: These data suggest that VITEK 2 cards inoculated with samples taken directly from positive Bact/ALERT blood culture bottles would provide acceptable identification and antimicrobial susceptibility testing results for Gram-negative bacilli, but not for Gram-positive cocci. Compared to the standard method, the direct method would reduce turnaround time by at least 24 h.     

长期慢性脑外伤大鼠脑线粒体功能的变化

研究长期慢性轻度脑外伤对大鼠脑线粒体功能的影响。大鼠连续1、5、10、15、20、25、30d轻度闭合性颅脑撞击后分离脑线粒体,测定线粒体肿胀度、膜流动性、膜磷脂含量、呼吸功能、线粒体呼吸酶、超氧化物歧化酶(SOD)、丙二醛(MDA)和Ca2 等指标以显示线粒体功能、抗氧化能力的变化。结果显示,第15、20、25、30d大鼠脑线粒体明显肿胀,膜磷脂降解,膜流动性下降,呼吸功能衰减,呼吸酶、SOD活性降低,Ca2 、MDA含量升高。由此认为,经常性头部撞击可造成大鼠脑线粒体功能受损,其机制可能与脑线粒体膜损伤后继发的自由基生成增加、脑线粒体能量代谢障碍有关。     

差异筛选肝癌凋亡细胞cDNA文库的简便方法

目的差异筛选人肝癌凋亡细胞ｃＤＮＡ文库。方法消减杂交和点杂交相结合的噬菌斑原位杂交法筛选ｃＤＮＡ文库，首先采用（－）ｃＤＮＡ探针杂交，挑取（－）的噬菌斑克隆，再用（－）和（＋）两种ｃＤＮＡ探针与初筛出的噬菌斑克隆杂交的差异筛选方法。结果得到４个充分孤立的噬菌斑克隆，其插入片段长度为１．５ｋｂ左右。结论该方法简便、快速，是差异筛选ｃＤＮＡ文库的一种较为可行的简便方法。     

Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching

Switching of Saccharomyces mating type by replacement of sequences at the MAT locus involves a choice between two donors, HML and HMR. MATα cells inhibit recombination along the entire left arm of chromosome III, including HML, whereas MATa cells activate this same region. MATa-dependent activation of HML depends on a small, cis-acting DNA sequence designated the recombination enhancer (RE), located 17 kb centromere-proximal to HML. A comparison of RE sequences interchangeable between Saccharomyces cerevisiae and Saccharomyces carlsbergensis defines a minimum RE of 244 bp. RE activity is repressed in MATα cells by binding of the Matα2–Mcm1 corepressor to a site within the RE. Mutation of the two Matα2 binding sites removes most, but not all, of this repression, and RE chromatin structure in MATα cells becomes indistinguishable from that seen in MATa. Surprisingly, a 2-bp mutation in the Mcm1 binding site completely abolishes RE activity in MATa cells; moreover, RE chromatin structure in the MATa mutant becomes very similar to that seen in MATα cells with a normal RE, displaying highly ordered nucleosomes despite the absence of Matα2. Further, a mutation that alters the ability of Mcm1 to act with Matα2 in repressing a-specific genes also alters donor preference in either mating type. Thus, Mcm1 is critically responsible for the activation as well as the Matα2-Mcm1-mediated repression of RE activity.     

Poly(D,L-lactic acid) surfaces modified by silk fibroin: effects on the culture of osteoblast in vitro.

The objective of this study was to modify the surface of poly(D,L-lactic acid) (PDLLA) with different molecular weight of silk fibroins, and assess the effects of the modified surfaces on the functions of rat osteoblasts cultured in vitro. The properties of the modified PDLLA surface and the control one were investigated by contact angle and electron spectroscopy for chemical analysis (ESCA). The former indicated the variation of hydrophilicity and the latter suggested that the modified PDLLA film using silk fibroin is enriched with nitrogen atoms. The biocompatibility of the PDLLA film may be altered and in turn affects the seeded cell functions. Therefore, attachment and proliferation of osteoblasts seeded on the modified PDLLA films and the control one were examined. Cell morphologies on these films were studied by scanning electron microscopy (SEM) and cell viability was evaluated by MTT assay. In addition, differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. These results suggest that the silk fibroin-modified PDLLA surface can improve the interaction between osteoblasts and the PDLLA films.     

STAT-3 overexpression and p21 up-regulation accompany impaired regeneration of fatty livers

Fatty liver is an important cause of morbidity in humans and is linked to impaired liver regeneration after liver injury, but the mechanisms for impaired liver regeneration remain unknown. In the normal liver, the interleukin (IL)-6/STAT-3 pathway is thought to play a central role in regeneration because this pathway is disrupted in IL-6-deficient mice that exhibit impaired liver regeneration after 70% partial hepatectomy (PH). To determine whether inhibition of STAT-3 is involved in fatty liver-related mitoinhibition, regenerative induction of STAT-3 was compared in normal mice and leptin-deficient ob/ob mice that have fatty livers and markedly impaired liver regeneration after PH. In both groups, two waves of STAT-3 activation were observed, the first in endothelia and the second in hepatocytes. Before PH, a significantly higher percentage of ob/ob endothelial and hepatocyte nuclei expressed phosphorylated (activated) STAT-3. After PH, phospho-STAT-3 accumulated in liver nuclei of lean mice and this response was markedly exaggerated in ob/ob mice. Moreover, a striking inverse correlation was noted between hepatocyte nuclear accumulation of phospho-STAT-3 and DNA synthesis (as assessed by bromodeoxyuridine labeling), as well as cyclin D1 mRNA induction and protein expression. In contrast, STAT-3 activation was positively correlated with p21 protein expression in both groups of mice. Because these results link exaggerated STAT-3 activation with impaired hepatocyte proliferation, STAT-3 inhibition cannot be a growth-arrest mechanism in ob/ob fatty livers. Rather, hyperinduction of this factor may promote mitoinhibition by up-regulating mechanisms that impede cell cycle progression.     

珊瑚涂层人工骨的制备及溶血实验研究

目的根据生物工程及化工等原理,制备珊瑚人工骨,并检测此珊瑚(Coral)复合人工骨的血液相容性。方法将制备的珊瑚复合人工骨、按照国际标准化组织和我国国家标准规定要求,采用将珊瑚复合人工骨及其浸提液与抗凝稀释兔血直接接触,测定红细胞释放的血红蛋白量(OD值),将测得的OD值换算为溶血率。结果珊瑚复合人工骨材料浸提液组的溶血率为1．81％,材料与血液直接接触组溶血率为1．41％,阴性对照组和阳性对照组溶血率分别为0％、100％。结论珊瑚复合人工骨材料无溶血作用,血液相容性良好。