Near-complete genome sequencing of swine vesicular disease virus using the Roche GS FLX sequencing platform

Swine vesicular disease virus (SVDV) is an enterovirus that is both genetically and antigenically closely related to human coxsackievirus B5 within the Picornaviridae family. SVDV is the causative agent of a highly contagious (though rarely fatal) vesicular disease in pigs. We report a rapid method that is suitable for sequencing the complete protein-encoding sequences of SVDV isolates in which the RNA is relatively intact. The approach couples a single PCR amplification reaction, using only a single PCR primer set to amplify the near-complete SVDV genome, with deep-sequencing using a small fraction of the capacity of a Roche GS FLX sequencing platform. Sequences were initially verified through one of two criteria; either a match between a de novo assembly and a reference mapping, or a match between all of five different reference mappings performed against a fixed set of starting reference genomes with significant genetic distances within the same species of viruses. All reference mappings used an iterative method to avoid bias. Further verification was achieved through phylogenetic analysis against published SVDV genomes and additional Enterovirus B sequences. This approach allows high confidence in the obtained consensus sequences, as well as provides sufficiently high and evenly dispersed sequence coverage to allow future studies of intra-host variation

Isolation of Proteinase K-Sensitive Prions Using Pronase E and Phosphotungstic Acid

Disease-related prion protein, PrPSc, is classically distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrPSc using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrPC, while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrPC

Childhood socioeconomic position and adult mental wellbeing:Evidence from four British birth cohort studies

There is much evidence showing that childhood socioeconomic position is associated with physical health in adulthood; however existing evidence on how early life disadvantage is associated with adult mental wellbeing is inconsistent. This paper investigated whether childhood socioeconomic position (SEP) is associated with adult mental wellbeing and to what extent any association is explained by adult SEP using harmonised data from four British birth cohort studies.The sample comprised 20,717 participants with mental wellbeing data in the Hertfordshire Cohort Study (HCS), the MRC National Survey of Health and Development (NSHD), the National Child Development Study (NCDS), and the British Cohort Study (BCS70). Warwick Edinburgh Mental Wellbeing Scale (WEMWBS) scores at age 73 (HCS), 60-64 (NSHD), 50 (NCDS), or 42 (BCS70) were used. Harmonised socioeconomic position (Registrar General's Social Classification) was ascertained in childhood (age 10/11) and adulthood (age 42/43). Associations between childhood SEP, adult SEP, and wellbeing were tested using linear regression and multi-group structural equation models.More advantaged father's social class was associated with better adult mental wellbeing in the BCS70 and the NCDS. This association was independent of adult SEP in the BCS70 but fully mediated by adult SEP in the NCDS. There was no evidence of an association between father's social class and adult mental wellbeing in the HCS or the NSHD.Socioeconomic conditions in childhood are directly and indirectly, through adult socioeconomic pathways, associated with adult mental wellbeing, but findings from these harmonised data suggest this association may depend on cohort or age

PrP(Sc)-specific antibodies with the ability to immunodetect prion oligomers.

The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs) derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p(0/0) cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aβ and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids

Does a SLAP lesion affect shoulder muscle recruitment as measured by EMG activity during a rugby tackle?

Background: The study objective was to assess the influence of a SLAP lesion on onset of EMG activity in shoulder muscles during a front on rugby football tackle within professional rugby players. Methods: Mixed cross-sectional study evaluating between and within group differences in EMG onset times. Testing was carried out within the physiotherapy department of a university sports medicine clinic. The test group consisted of 7 players with clinically diagnosed SLAP lesions, later verified on arthroscopy. The reference group consisted of 15 uninjured and full time professional rugby players from within the same playing squad. Controlled tackles were performed against a tackle dummy. Onset of EMG activity was assessed from surface EMG of Pectorialis Major, Biceps Brachii, Latissimus Dorsi, Serratus Anterior and Infraspinatus muscles relative to time of impact. Analysis of differences in activation timing between muscles and limbs (injured versus non-injured side and non injured side versus matched reference group). Results: Serratus Anterior was activated prior to all other muscles in all (P = 0.001-0.03) subjects. In the SLAP injured shoulder Biceps was activated later than in the non-injured side. Onset times of all muscles of the noninjured shoulder in the injured player were consistently earlier compared with the reference group. Whereas, within the injured shoulder, all muscle activation timings were later than in the reference group. Conclusions: This study shows that in shoulders with a SLAP lesion there is a trend towards delay in activation time of Biceps and other muscles with the exception of an associated earlier onset of activation of Serratus anterior, possibly due to a coping strategy to protect glenohumeral stability and thoraco-scapular stability. This trend was not statistically significant in all cases

Clara cell adhesion and migration to extracellular matrix

<p>Abstract</p> <p>Background</p> <p>Clara cells are the epithelial progenitor cell of the small airways, a location known to be important in many lung disorders. Although migration of alveolar type II and bronchiolar ciliated epithelial cells has been examined, the migratory response of Clara cells has received little attention.</p> <p>Methods</p> <p>Using a modification of existing procedures for Clara cell isolation, we examined mouse Clara cells and a mouse Clara-like cell line (C22) for adhesion to and migration toward matrix substrate gradients, to establish the nature and integrin dependence of migration in Clara cells.</p> <p>Results</p> <p>We observed that Clara cells adhere preferentially to fibronectin (Fn) and type I collagen (Col I) similar to previous reports. Migration of Clara cells can be directed by a fixed gradient of matrix substrates (haptotaxis). Migration of the C22 cell line was similar to the Clara cells so integrin dependence of migration was evaluated with this cell line. As determined by competition with an RGD containing-peptide, migration of C22 cells toward Fn and laminin (Lm) 511 (formerly laminin 10) was significantly RGD integrin dependent, but migration toward Col I was RGD integrin independent, suggesting that Clara cells utilize different receptors for these different matrices.</p> <p>Conclusion</p> <p>Thus, Clara cells resemble alveolar type II and bronchiolar ciliated epithelial cells by showing integrin mediated pro-migratory changes to extracellular matrix components that are present in tissues after injury.</p

Transmission characteristics of heterozygous cases of Creutzfeldt-Jakob disease with variable abnormal prion protein allotypes

In the human prion disease Creutzfeldt-Jakob disease (CJD), different CJD neuropathological subtypes are defined by the presence in normal prion protein (PrPC) of a methionine or valine at residue 129, by the molecular mass of the infectious prion protein PrPSc, by the pattern of PrPSc deposition, and by the distribution of spongiform change in the brain. Heterozygous cases of CJD potentially add another layer of complexity to defining CJD subtypes since PrPSc can have either a methionine (PrPSc-M129) or valine (PrPSc-V129) at residue 129. We have recently demonstrated that the relative amount of PrPSc-M129 versus PrPSc-V129, i.e. the PrPSc allotype ratio, varies between heterozygous CJD cases. In order to determine if differences in PrPSc allotype correlated with different disease phenotypes, we have inoculated 10 cases of heterozygous CJD (7 sporadic and 3 iatrogenic) into two transgenic mouse lines overexpressing PrPC with a methionine at codon 129. In one case, brain-region specific differences in PrPSc allotype appeared to correlate with differences in prion disease transmission and phenotype. In the other 9 cases inoculated, the presence of PrPSc-V129 was associated with plaque formation but differences in PrPSc allotype did not consistently correlate with disease incubation time or neuropathology. Thus, while the PrPSc allotype ratio may contribute to diverse prion phenotypes within a single brain, it does not appear to be a primary determinative factor of disease phenotype

Impact of movement training on upper limb motor strategies in persons with shoulder impingement syndrome

<p>Abstract</p> <p>Background</p> <p>Movement deficits, such as changes in the magnitude of scapulohumeral and scapulathoracic muscle activations or perturbations in the kinematics of the glenohumeral, sternoclavicular and scapulothoracic joints, have been observed in people with shoulder impingement syndrome. Movement training has been suggested as a mean to contribute to the improvement of the motor performance in persons with musculoskeletal impairments. However, the impact of movement training on the movement deficits of persons with shoulder impingement syndrome is still unknown. The aim of this study was to evaluate the short-term effects of supervised movement training with feedback on the motor strategies of persons with shoulder impingement syndrome.</p> <p>Methods</p> <p>Thirty-three subjects with shoulder impingement were recruited. They were involved in two visits, one day apart. During the first visit, supervised movement training with feedback was performed. The upper limb motor strategies were evaluated before, during, immediately after and 24 hours after movement training. They were characterized during reaching movements in the frontal plane by EMG activity of seven shoulder muscles and total excursion and final position of the wrist, elbow, shoulder, clavicle and trunk. Movement training consisted of reaching movements performed under the supervision of a physiotherapist who gave feedback aimed at restoring shoulder movements. One-way repeated measures ANOVAs were run to analyze the effect of movement training.</p> <p>Results</p> <p>During, immediately after and 24 hours after movement training with feedback, the EMG activity was significantly decreased compared to the baseline level. For the kinematics, total joint excursion of the trunk and final joint position of the trunk, shoulder and clavicle were significantly improved during and immediately after training compared to baseline. Twenty-four hours after supervised movement training, the kinematics of trunk, shoulder and clavicle were back to the baseline level.</p> <p>Conclusion</p> <p>Movement training with feedback brought changes in motor strategies and improved temporarily some aspects of the kinematics. However, one training session was not enough to bring permanent improvement in the kinematic patterns. These results demonstrate the potential of movement training in the rehabilitation of movement deficits associated with shoulder impingement syndrome.</p

Bias and heteroscedastic memory error in self-reported health behavior: an investigation using covariance structure analysis

BACKGROUND: Frequent use of self-reports for investigating recent and past behavior in medical research requires statistical techniques capable of analyzing complex sources of bias associated with this methodology. In particular, although decreasing accuracy of recalling more distant past events is commonplace, the bias due to differential in memory errors resulting from it has rarely been modeled statistically. METHODS: Covariance structure analysis was used to estimate the recall error of self-reported number of sexual partners for past periods of varying duration and its implication for the bias. RESULTS: Results indicated increasing levels of inaccuracy for reports about more distant past. Considerable positive bias was found for a small fraction of respondents who reported ten or more partners in the last year, last two years and last five years. This is consistent with the effect of heteroscedastic random error where the majority of partners had been acquired in the more distant past and therefore were recalled less accurately than the partners acquired more recently to the time of interviewing. CONCLUSIONS: Memory errors of this type depend on the salience of the events recalled and are likely to be present in many areas of health research based on self-reported behavior

Protease-Sensitive Conformers in Broad Spectrum of Distinct PrPSc Structures in Sporadic Creutzfeldt-Jakob Disease Are Indicator of Progression Rate

The origin, range, and structure of prions causing the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD), are largely unknown. To investigate the molecular mechanism responsible for the broad phenotypic variability of sCJD, we analyzed the conformational characteristics of protease-sensitive and protease-resistant fractions of the pathogenic prion protein (PrPSc) using novel conformational methods derived from a conformation-dependent immunoassay (CDI). In 46 brains of patients homozygous for polymorphisms in the PRNP gene and exhibiting either Type 1 or Type 2 western blot pattern of the PrPSc, we identified an extensive array of PrPSc structures that differ in protease sensitivity, display of critical domains, and conformational stability. Surprisingly, in sCJD cases homozygous for methionine or valine at codon 129 of the PRNP gene, the concentration and stability of protease-sensitive conformers of PrPSc correlated with progression rate of the disease. These data indicate that sCJD brains exhibit a wide spectrum of PrPSc structural states, and accordingly argue for a broad spectrum of prion strains coding for different phenotypes. The link between disease duration, levels, and stability of protease-sensitive conformers of PrPSc suggests that these conformers play an important role in the pathogenesis of sCJD