Rapid detection and identification of human adenovirus species by adenoplex, a multiplex PCR-enzyme hybridization assay

Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10(-2), 10(-1), 10(-1), 10(-2), 10(-1), and 10(-2), respectively. Similarly, the LOD for the six DNA controls ranged from 10(2) to 10(3) copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.     

Number of C-bands of human isochromosome Xqi and relation to 45,X mosaicism

With combination of C and G banding techniques, three morphologically different types of isochromosome for the long arm of X (Xqi) have been identified, i.e. those with one C-band and symmetrical banding patterns of both arms, those with two C-bands and symmetrical banding patterns of two arms, and those with two C-bands but with asymmetrical banding patterns of two arms. The last type is a heterogeneous group with various different asymmetrical patterns. We have studied 6 cases of Xqi with C, G, and Q banding: 3 showed one C-band and symmetrical arms and all these were without 45,X mosaicism; the other 3 cases showed two C-bands, 2 of the cases having symmetrical arms and being mosaic for 45X/46,XXqi/47,XXqiXqi (those 2 were a pair of identical twins). One other had asymmetrical arms and was mosaic for 45,X/46,XXqi. Including our 6 cases, there have been a total of 30 reported cases of Xqi with C and G banding studies. Two-thirds of Xqi's were found to have 2 C-bands and one-third to have 1 C-band. Mosaicism was found in 85% of Xqi's with 2 C-bands and in only 44% of Xqi's with 1 C-band. Apparently, Xqi's with 2 C-bands have a greater tendency for anaphase lag and mitotic nondisjunction. Several possible mechanisms for the formation of the different types of Xqi's are discussed.     

A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension

Kinetochores play an essential role in chromosome segregation by forming dynamic connections with spindle microtubules. Here, we identify a set of 10 copurifying kinetochore proteins from Caenorhabditis elegans, seven of which were previously uncharacterized. Using in vivo assays to monitor chromosome segregation, kinetochore assembly, and the mechanical stability of chromosome-microtubule attachments, we show that this copurifying protein network plays a central role at the kinetochore-microtubule interface. In addition, our analysis suggests that the network is comprised of three groups of proteins that contribute in distinct ways to this interface: KNL proteins act after the assembly of centromeric chromatin to generate the core of the microtubule-binding interface, MIS proteins control the rate and extent of formation of this interface, and NDC proteins are necessary to sustain tension during interactions with spindle microtubules. We also purify a similar set of associated proteins from human cells that includes four novel proteins and has recognizable homologs from each functional class. Thus, this protein network is a conserved constituent of the outer kinetochore, and the functions defined by our analysis in C. elegans are likely to be widely relevant.     

Pattern and predictors of weight gain during pregnancy among HIV-1-infected women from Tanzania

Progression of HIV disease is often accompanied by weight loss and wasting. Gestational weight gain is a strong determinant of maternal and neonatal outcomes; however, the pattern and predictors of weight gain during pregnancy among HIV-positive women are unknown. We obtained monthly anthropometric measurements in a cohort of 957 pregnant women from Tanzania who were HIV infected. We estimated the weekly rate of weight gain at various points during the second and third trimesters of pregnancy and computed rate differences between levels of sociodemographic, nutritional, immunologic, and parasitic variables at the first prenatal visit. The change in mid-upper arm circumference (MUAC) from baseline to delivery was also examined. The rate of weight gain decreased progressively during pregnancy. There was an average decline of 1 cm in MUAC between weeks 12 and 38. Lower level of education and helminthic infections at first visit were associated with decreased adjusted rates of weight gain during the third trimester. High baseline MUAC, not contributing to household income, lower serum retinol and selenium concentrations, advanced clinical stage of HIV disease, and malaria infection were related to decreased rates of weight gain during the second trimester. Low baseline CD4 T-cell counts were related to a poorer pattern of weight gain throughout pregnancy. Prevention and treatment of parasitic infections and improvement of nutritional status are likely to enhance the pattern of gestational weight gain among HIV-infected women.     

Creutzfeldt-Jakob disease (CJD) with a mutation at codon 148 of prion protein gene: relationship with sporadic CJD

Creutzfeldt-Jakob disease (CJD), the most common human prion disease, includes sporadic (s) and familial (f) forms. Regardless of etiology, both forms are thought to share the pathogenic mechanism whereby the cellular prion protein (PrP(C)) converts into its pathogenic isoform (PrP(Sc)). While PrP(C) conversion is thought to be random in sCJD, conversion in fCJD is facilitated by the congenital presence of mutated PrP. Differences in PrP genotype (PRNP) and in conversion circumstances lead to PrP(Sc) with distinct characteristics that elicit different disease phenotypes. Here, we describe a case of fCJD with a substitution of histidine (H) for arginine (R) at codon 148 (R148H) and heterozygosity of the methionine/valine (M/V) polymorphic codon 129, with the 129M allele coupled with the mutation. The disease phenotype and all major characteristics of PrP(Sc) of fCJD(R148H) were virtually indistinguishable from those of sCJDMV2, which has features different from those of any other sCJD. Therefore, despite the differences in etiology, PRNP, and conversion process, the two forms of PrP(Sc) had similar characteristics. Furthermore, comparison of fCJD(R148H) with a recently reported case carrying R148H and homozygosity at codon 129 suggests that codon 129 coupled with the mutation as well as that located on the normal allele can modify major phenotypic and PrP(Sc) features of fCJD(R148H).     

Rapid neurofibrillary tangle formation after localized gene transfer of mutated tau

Neurofibrillary pathology was produced in the brains of adult rats after localized gene transfer of human tau carrying the P301L mutation, which is associated with frontotemporal dementia with parkinsonism. Within 1 month of in situ transfection of the basal forebrain region of normal rats, tau-immunoreactive and argyrophilic neuronal lesions formed. The fibrillar lesions had features of neurofibrillary tangles and tau immunoreactivity at light and electron microscopic levels. In addition to neurofibrillary tangles, other tau pathology, including pretangles and neuropil threads, was abundant and widespread. Tau gene transfer to the hippocampal region of amyloid-depositing transgenic mice produced pretangles and threads, as well as intensely tau-immunoreactive neurites in amyloid plaques. The ability to produce neurofibrillary pathology in adult rodents makes this a useful method to study tau-related neurodegeneration.     

A prospective randomized,double-blinded,placebo-controlled trial comparing mifepristone and vaginal misoprostol to vaginal misoprostol alone for elective termination of early pregnancy

BACKGROUND: Vaginal misoprostol has been shown to be an effective single agent for medical abortion. This randomized, double-blinded, placebo-controlled trial compared a regimen of mifepristone and misoprostol with misoprostol alone for termination of early pregnancy. METHODS: 250 women with gestations < or = 56 days were randomized by a random number table to receive either 200 mg mifepristone orally or placebo followed 48 h later by 800 microg vaginal misoprostol. Administration of misoprostol was repeated every 24 h up to three doses if abortion failed to occur. Abortion success was defined as complete abortion without the use of surgical aspiration. RESULTS: Successful medical abortions occurred in 114 out of 119 subjects (95.7%) after mifepristone followed by vaginal misoprostol. In all, 110 out of 125 subjects (88.0%) successfully aborted after placebo and vaginal misoprostol. The higher success rate of complete abortion with the mifepristone and misoprostol regimen was statistically significant compared with the placebo and misoprostol regimen (P < 0.05). CONCLUSIONS: A regimen of mifepristone and misoprostol was significantly more effective for termination of pregnancies < or = 56 days than misoprostol alone. The 88% efficacy obtained with vaginal misoprostol alone may be clinically acceptable when mifepristone is not available.     

Lifestyle Risk Score: handling missingness of individual lifestyle components in meta-analysis of gene-by-lifestyle interactions

Recent studies consider lifestyle risk score (LRS), an aggregation of multiple lifestyle exposures, in identifying association of gene-lifestyle interaction with disease traits. However, not all cohorts have data on all lifestyle factors, leading to increased heterogeneity in the environmental exposure in collaborative meta-analyses. We compared and evaluated four approaches (Naïve, Safe, Complete and Moderator Approaches) to handle the missingness in LRS-stratified meta-analyses under various scenarios. Compared to “benchmark” results with all lifestyle factors available for all cohorts, the Complete Approach, which included only cohorts with all lifestyle components, was underpowered due to lower sample size, and the Naïve Approach, which utilized all available data and ignored the missingness, was slightly inflated. The Safe Approach, which used all data in LRS-exposed group and only included cohorts with all lifestyle factors available in the LRS-unexposed group, and the Moderator Approach, which handled missingness via moderator meta-regression, were both slightly conservative and yielded almost identical p values. We also evaluated the performance of the Safe Approach under different scenarios. We observed that the larger the proportion of cohorts without missingness included, the more accurate the results compared to “benchmark” results. In conclusion, we generally recommend the Safe Approach, a straightforward and non-inflated approach, to handle heterogeneity among cohorts in the LRS based genome-wide interaction meta-analyses.Subject terms: Genetics, Risk factors