Determination of hepatitis C virus genotype by Pyrosequencing

A simple sequencing-based assay is described for genotyping of hepatitis C virus (HCV). RT-PCR was employed to amplify a 237-nucleotide-long fragment from the 5' untranslated region (UTR) of the genome using one biotinylated and one normal primer. Subsequent to capture of the PCR products on streptavidin-coated beads, single-stranded DNA separation, and hybridization of sequencing primer, Pyrosequencing was performed. The genotype of 98 samples out of which 77 samples were from American veterans and 21 samples were from Iran was determined. The samples from the American veterans contained six different subtypes, while five subtypes were found in Iranian samples. For rapid population-specific HCV subtyping, a multiplex assay was developed. This study demonstrates the suitability of this technology for low-cost, high throughput and accurate microbial genotyping.     

Lactobacillus crispatus strain SJ‐3C‐US induces human dendritic cells (DCs) maturation and confers an anti‐inflammatory phenotype to DCs

Lactobacillus crispatus is one of the most predominant species in the healthy vagina microbiota. Nevertheless, the interactions between this commensal bacterium and the immune system are largely unknown. Given the importance of the dendritic cells (DCs) in the regulation of the immunity, this study was performed to elucidate the influence of vaginal isolated L. crispatus SJ‐3C‐US from healthy Iranian women on DCs, either directly by exposure of DCs to ultraviolet‐inactivated (UVI) and heat‐killed (HK) L. crispatus SJ‐3C‐US or indirectly to its cell‐free supernatant (CFS), and the outcomes of immune response. In this work we showed that L. crispatus SJ‐3C‐US induced strong dose‐dependent activation of dendritic cells and production of high levels of IL‐10, whereas IL‐12p70 production was induced at low level in an inverse dose‐dependent manner. This stimulation skewed T cells polarization toward CD4⁺ CD25⁺ FOXP3⁺ Treg cells and production of IL‐10 in a dose‐dependent manner in mixed leukocyte reaction (MLR) test. The mode of bacterial inactivation did not affect the DCs activation pattern, upon encounter with L. crispatus SJ‐3C‐US. Moreover, while DCs stimulated with CFS showed moderate phenotypic maturation and IL‐10 production, it failed to skew T cells polarization toward CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg) and production of IL‐10. This study showed that L. crispatus SJ‐3C‐US confers an anti‐inflammatory phenotype to DCs through up‐regulation of anti‐inflammatory/regulatory IL‐10 cytokine production and induction of CD4⁺ CD25⁺ FOXP3⁺ T cells at optimal dosage. Our findings suggest that L. crispatus SJ‐3C‐US could be a potent candidate as protective probiotic against human immune‐mediated pathologies, such as chronic inflammation, vaginitis or pelvic inflammatory disease (PID).     

Distress tolerance and special alcohol metacognitions behave differently in the association of negative affect with alcohol-related patterns in men with problematic alcohol use in the abstinence phase

Negative affect may be related to alcohol-related patterns (e.g., craving and problematic alcohol use). Distress intolerance and positive and negative alcohol-related metacognitions may be underlying mechanisms in this link. This study aimed to evaluate the effect of negative affect including depressive, anxious, and stress symptoms on alcohol craving and problematic alcohol use via the paths of distress tolerance and both positive and negative alcohol-related metacognitions. Three hundred men with problematic alcohol use during the abstinence phase completed psychological and clinical measures. Results showed that craving and negative alcohol metacognitions mediated the relationship between negative affect and problematic alcohol use. Negative affect had a direct and positive effect on craving and indirect effect via distress intolerance and positive alcohol metacognitions. In turn, distress intolerance and positive alcohol metacognitions indirectly and positively affected problematic alcohol use via craving. The study indicates that distress tolerance and distinct alcohol metacognitions may be differently related to various patterns of alcohol-related problems, such that alcohol drinkers with high levels of negative affect, distress intolerance, and positive alcohol metacognitions show higher levels of craving, while high negative affect in relation to high negative alcohol metacognitions and alcohol craving is related to the perpetuation of alcohol use or problematic alcohol use.     

L-statistics of absolute differences for quantifying the agreement between two variables

In many clinical studies, Lin’s (1989) concordance correlation coefficient (CCC) is a popular measure of agreement for continuous outcomes. Most commonly, it is used under the assumption that data are normally distributed. However, in many practical applications, data are often skewed and/or thick-tailed. King and Chinchilli (2001) proposed robust estimation methods of alternative CCC indices, and we propose an approach that extends the existing methods of robust estimators by focusing on functionals that yield robust L-statistics. We provide two data examples to illustrate the methodology, and we discuss the results of computer simulation studies that evaluate statistical performance.     

Mitochondrial genotype and risk for Alzheimer’s disease: cross-sectional data from the Vienna-Transdanube-Aging “VITA” study

Summary. The Vienna Transdanube Aging (VITA) study searches for early markers of Alzheimers disease (AD) by examining the mental status in a community-based cohort of 606, 75-years old volunteers that are then related to various clinical and genetic analyses. To determine whether mutations in mtDNA are involved in expression of AD, the mtDNA of 79 control participants is screened for alterations by sequencing of hot-spot-regions. This study on mtDNA mutations has eliminated the influence of aging on the occurrence of mtDNA alterations by sequencing samples from persons at the age of exactly 75 years. Thus, our cohort reveals a snap-shot of mitochondrial sequences of elderly persons.So far, a high percentage (56%) of persons with known or unknown mutations in the fragments analyzed were found. These data will be compared in due time to a cohort of participants with proven late-onset AD.