Single-tube multiplex PCR using type-specific E6/E7 primers and capillary electrophoresis genotypes 21 human papillomaviruses in neoplasia

<p>Abstract</p> <p>Background</p> <p>Human papillomavirus (HPV) <it>E6/E7 </it>type-specific oncogenes are required for cervical carcinogenesis. Current PCR protocols for genotyping high-risk HPV in cervical screening are not standardized and usually use consensus primers targeting HPV capsid genes, which are often deleted in neoplasia. PCR fragments are detected using specialized equipment and extra steps, including probe hybridization or primer extension. In published papers, analytical sensitivity is typically compared with a different protocol on the same sample set.</p> <p>A single-tube multiplex PCR containing type-specific primers was developed to target the <it>E6/E7 </it>genes of two low-risk and 19 high-risk genotypes (HPV6, 11 and 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82) and the resulting short fragments were directly genotyped by high-resolution fluorescence capillary electrophoresis.</p> <p>Results</p> <p>The method was validated using long oligonucleotide templates, plasmid clones and 207 clinical samples of DNA from liquid-based cytology, fresh and formalin-fixed specimens and FTA Microcards^®imprinted with cut tumor surfaces, swabbed cervical cancers or ejected aspirates from nodal metastases of head and neck carcinomas. Between one and five long oligonucleotide targets per sample were detected without false calls. Each of the 21 genotypes was detected in the clinical sample set with up to five types simultaneously detected in individual specimens. All 101 significant cervical neoplasias (CIN 2 and above), except one adenocarcinoma, contained <it>E6/E7 </it>genes. The resulting genotype distribution accorded with the national pattern with HPV16 and 18 accounting for 69% of tumors. Rare HPV types 70 and 73 were present as the sole genotype in one carcinoma each. One cervical SCC contained DNA from HPV6 and 11 only. Six of twelve oropharyngeal cancer metastases and three neck metastases of unknown origin bore <it>E6/E7 </it>DNA; all but one were HPV16. One neck aspirate contained atypical squames with HPV26.</p> <p>Analytical sensitivity in dilute plasmid mixes was variable.</p> <p>Conclusions</p> <p>A primer-rich PCR readily detects the <it>E6/E7 </it>oncogenes of 21 HPV types in cellular and fixed tissue specimens. The method is straightforward, robust and reproducible and avoids post-PCR enzymatic and hybridization steps while detecting HPV with high clinical sensitivity in significant HPV-related neoplasia regardless of specimen type.</p

The role of clathrin in post-golgi trafficking in toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle

Assessing L2 vocabulary depth with word associates format tests: issues, findings, and suggestions

Word Associates Format (WAF) tests are often used to measure second language learners’ vocabulary depth with a focus on their network knowledge. Yet, there were often many variations in the specific forms of the tests and the ways they were used, which tended to have an impact on learners’ response behaviors and, more importantly, the psychometric properties of the tests. This paper reviews the general practices, key issues, and research findings that pertain to WAF tests in four major areas, including the design features of WAF tests, conditions for test administration, scoring methods, and test-taker characteristics. In each area, a set of variables is identified and described with relevant research findings also presented and discussed. Around eight topics, the General Discussion section provides some suggestions and directions for the development of WAF tests and the use of them as research tools in the future. This paper is hoped to help researchers become better aware that the results generated by a WAF test may vary depending on what specific design the test has, how it is administered and scored, and who the learners are, and consequently, make better decisions in their research that involves a WAF test

Factor structure of the Shoulder Pain and Disability Index in patients with adhesive capsulitis

<p>Abstract</p> <p>Background</p> <p>The Shoulder Pain and Disability Index (SPADI) is a self-administered questionnaire that aims to measure pain and disability associated with shoulder disease. It consists of a pain section and a disability section with 13 items being responded to on visual analogue scales. Few researchers have investigated SPADI validity in specified diagnostic groups, although the selection of an evaluative instrument should be based on evidence of validity in the target patient group. The aim of the present study was to investigate factor structure of the SPADI in a study population of patients with adhesive capsulitis.</p> <p>Methods</p> <p>The questionnaire was administered to 191 patients with adhesive capsulitis. Descriptive statistics for items and a comparison of scores for the two subscales were produced. Internal consistency was analyzed by use of the Cronbach alpha and a principal components analysis with varimax rotation was conducted. Study design was cross-sectional.</p> <p>Results</p> <p>Two factors were extracted, but the factor structure failed to support the original division of items into separate pain and disability sections.</p> <p>Conclusion</p> <p>We found minimal evidence to justify the use of separate subscales for pain and disability. It is our impression that the SPADI should be viewed as essentially unidimensional in patients with adhesive capsulitis.</p

Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world's oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations

Bose-Einstein condensation of photons in an optical microcavity

Bose-Einstein condensation, the macroscopic ground state accumulation of particles with integer spin (bosons) at low temperature and high density, has been observed in several physical systems, including cold atomic gases and solid state physics quasiparticles. However, the most omnipresent Bose gas, blackbody radiation (radiation in thermal equilibrium with the cavity walls) does not show this phase transition, because the chemical potential of photons vanishes and, when the temperature is reduced, photons disappear in the cavity walls. Theoretical works have considered photon number conserving thermalization processes, a prerequisite for Bose-Einstein condensation, using Compton scattering with a gas of thermal electrons, or using photon-photon scattering in a nonlinear resonator configuration. In a recent experiment, we have observed number conserving thermalization of a two-dimensional photon gas in a dye-filled optical microcavity, acting as a 'white-wall' box for photons. Here we report on the observation of a Bose-Einstein condensation of photons in a dye-filled optical microcavity. The cavity mirrors provide both a confining potential and a non-vanishing effective photon mass, making the system formally equivalent to a two-dimensional gas of trapped, massive bosons. By multiple scattering off the dye molecules, the photons thermalize to the temperature of the dye solution (room temperature). Upon increasing the photon density we observe the following signatures for a BEC of photons: Bose-Einstein distributed photon energies with a massively populated ground state mode on top of a broad thermal wing, the phase transition occurring both at the expected value and exhibiting the predicted cavity geometry dependence, and the ground state mode emerging even for a spatially displaced pump spot

Validity and responsiveness of the Clubfoot Assessment Protocol (CAP). A methodological study

BACKGROUND: The Clubfoot Assessment Protocol (CAP) is a multi dimensional instrument designed for longitudinal follow up of the clubfoot deformity during growth. Item reliability has shown to be sufficient. In this article the CAP's validity and responsiveness is studied using the Dimeglio classification scoring as a gold standard. METHODS: Thirty-two children with 45 congenital clubfeet were assessed prospectively and consecutively at ages of new-born, one, two, four months and two years of age. For convergent/divergent construct validity the Spearman's correlation coefficients were calculated. Discriminate validity was evaluated by studying the scores in bilateral clubfeet. The floor-ceiling effects at baseline (untreated clubfeet) and at two years of age (treated clubfeet) were evaluated. Responsiveness was evaluated by using effect sizes (ES) and by calculating if significant changes (Wilcoxons signed test) had occurred between the different measurement occasions. RESULTS: High to moderate significant correlation were found between CAP mobility I and morphology and the Dimeglio scores (r(s )= 0.77 and 0.44 respectively). Low correlation was found between CAP muscle function, mobility II and motion quality and the Dimeglio scoring system (r(s )= 0.20, 0.09 and 0.06 respectively). Of 13 children with bilateral clubfeet, 11 showed different CAP mobility I scores between right and left foot at baseline (untreated) compared with 5 with the Dimeglio score. At the other assessment occasions the CAP mobility I continued to show higher discrimination ability than the Dimeglio. No floor effects and low ceiling effects were found in the untreated clubfeet for both instruments. High ceiling effects were found in the CAP for the treated children and low for the Dimeglio. Responsiveness was good. ES from untreated to treated ranged from 0.80 to 4.35 for the CAP subgroups and was 4.68 for the Dimeglio. The first four treatment months, the CAP mobility I had generally higher ES compared with the Dimeglio. CONCLUSION: The Clubfoot Assessment Protocol shows in this study good validity and responsiveness. The CAP is more responsive when severity ranges between mild – moderate to severe, while the Dimeglio focuses more on the extremes. The ability to discriminate between different mobility status of the right and left foot in bilaterally affected children in this population was higher compared with the Dimeglio score implicating a better sensitivity for the CAP

The GABA transporter 1 (SLC6A1): a novel candidate gene for anxiety disorders

Recent evidence suggests that the GABA transporter 1 (GAT-1; SLC6A1) plays a role in the pathophysiology and treatment of anxiety disorders. In order to understand the impact of genetic variation within SLC6A1 on pathological anxiety, we performed a case–control association study with anxiety disorder patients with and without syndromal panic attacks. Using the method of sequential addition of cases, we found that polymorphisms in the 5′ flanking region of SLC6A1 are highly associated with anxiety disorders when considering the severity of syndromal panic attacks as phenotype covariate. Analysing the effect size of the association, we observed a constant increase in the odds ratio for disease susceptibility with an increase in panic severity (OR ~ 2.5 in severely affected patients). Nominally significant association effects were observed considering the entire patient sample. These data indicate a high load of genetic variance within SLC6A1 on pathological anxiety and highlight GAT-1 as a promising target for treatment of anxiety disorders with panic symptoms