Management and analysis of high-throughput sequence data for infectious animal diseases.

Advances in technology and decreasing costs have accelerated the use of high-throughput sequencing (HTS) for both diagnosis and characterisation of infectious animal diseases. High-throughput sequencing offers several advantages over previous techniques, including rapid turnaround times and the ability to resolve single nucleotide changes among samples, both of which are important for epidemiological investigations of outbreaks. However, due to the plethora of genetic data being routinely generated, the storage and analysis of these data are proving challenging in their own right. In this article, the authors provide insight into the aspects of data management and analysis that should be considered before adopting HTS for routine animal health diagnostics. These elements fall largely into three interrelated categories: data storage, data analysis and quality assurance. Each has numerous complexities and may need to be adapted as HTS evolves. Making appropriate strategic decisions about bioinformatic sequence analysis early on in project development will help to avert major issues in the long term.

Les avancées technologiques dans le domaine du séquençage à haut débit (SHD) et la diminution des coûts liés à cette technique en ont accéléré l'utilisation à des fins de diagnostic et de caractérisation des maladies animales infectieuses. Le séquençage à haut débit offre plusieurs avantages par rapport aux techniques antérieures, en particulier la rapidité de son exécution et une résolution de l'ordre d'un seul changement de nucléotide parmi plusieurs échantillons, ce qui présente un grand intérêt lors des enquêtes épidémiologiques sur les foyers. Néanmoins, la pléthore de données génétiques générées en routine par le SHD devient un véritable problème en termes de stockage et d'analyse de ces données. Les auteurs apportent un éclairage sur les aspects de la gestion et de l'analyse des données qu'il convient de prendre en compte avant d'adopter le SHD pour le diagnostic de routine en santé animale. Ces éléments relèvent de trois catégories étroitement reliées : le stockage de données, l'analyse de données et l'assurance qualité. Chacun de ces aspects présente de nombreuses complexités et nécessitera sans doute d'être adapté à mesure que le SHD évolue. Lorsqu'elles sont prises dès la phase initiale d'un projet, des décisions stratégiques appropriées en matière d'analyse bio-informatique de séquences peuvent contribuer à éviter des problèmes majeurs sur le long terme.

Los avances tecnológicos y la reducción de los costos han acelerado el uso de la secuenciación de alto rendimiento (SAR) con fines de diagnóstico y caracterización de enfermedades animales infecciosas. La secuenciación de alto rendimiento presenta varias ventajas en comparación con otras técnicas anteriores, en particular ciclos más rápidos y una resolución que permite detectar diferencias de un solo nucleótido entre las muestras, aspectos ambos de gran importancia para el estudio epidemiológico de brotes infecciosos. Sin embargo, debido al sinnúmero de datos genéticos que constantemente se generan, no es de extrañar que esté resultando problemático almacenar y analizar los datos obtenidos. Los autores arrojan luz sobre los aspectos de la gestión y el análisis de datos que conviene tener en cuenta antes de aplicar la SAR a las labores sistemáticas de diagnóstico en sanidad animal. Estos elementos corresponden a grandes líneas a tres categorías relacionadas entre sí: el almacenamiento de datos; el análisis de datos; y la garantía de calidad. Cada una de ellas presenta multitud de complicaciones y exige un proceso permanente de adaptación a medida que la técnica de secuenciación va evolucionando. El hecho de adoptar las buenas decisiones estratégicas sobre el análisis bioinformático de secuencias en los primeros momentos de la concepción de un proyecto ayudará a evitar importantes problemas a largo plazo.

Longitudinal study on the occurrence in pigs of colistin-resistant Escherichia coli carrying mcr-1 following the cessation of use of colistin.

AIMS: In 2015, colistin-resistant Escherichia coli and Salmonella with the mcr-1 gene were isolated from a pig farm in Great Britain. Pigs were subsequently monitored over a ~20-month period for the occurrence of mcr-1-mediated colistin resistance and the risk of mcr-1 E. coli entering the food chain was assessed. METHODS AND RESULTS: Pig faeces and slurry were cultured for colistin-resistant E. coli and Salmonella, tested for the mcr-1 gene by PCR and selected isolates were further analysed. Seventy-eight per cent of faecal samples (n = 275) from pigs yielded mcr-1 E. coli after selective culture, but in positive samples only 0·2-1·3% of the total E. coli carried mcr-1. Twenty months after the initial sampling, faecal samples (n = 59) were negative for E. coli carrying mcr-1. CONCLUSIONS: The risk to public health from porcine E. coli carrying mcr-1 was assessed as very low. Twenty months after cessation of colistin use, E. coli carrying mcr-1 was not detected in pig faeces on a farm where it was previously present. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that cessation of colistin use may help over time to reduce or possibly eliminate mcr-1 E. coli on pig farms where it occurs.

Relapsing polychondritis complicated by cognitive dysfunction: two distinct clinical phenotypes?

Relapsing polychondritis (RPC) is a rare, immune-mediated condition affecting approximately 3.5 per million population per year. Neurological involvement in RPC is still rarer and is presumed to be the result of a vasculitic process, although this is seldom confirmed in the literature. We present two cases of RPC complicated by cognitive dysfunction with contrasting clinical trajectories. Our findings suggest that there are two clinical phenotypes of cognitive dysfunction in RPC. The first is a fulminant, multisystem presentation with sub-acute cognitive decline mimicking central nervous system vasculitis, and we provide histopathological evidence of this process occurring. The other is an insidious cognitive decline without associated constitutional or systemic symptoms.

A new highly penetrant form of obesity due to deletions on chromosome 16p11.2.

Obesity has become a major worldwide challenge to public health, owing to an interaction between the Western 'obesogenic' environment and a strong genetic contribution. Recent extensive genome-wide association studies (GWASs) have identified numerous single nucleotide polymorphisms associated with obesity, but these loci together account for only a small fraction of the known heritable component. Thus, the 'common disease, common variant' hypothesis is increasingly coming under challenge. Here we report a highly penetrant form of obesity, initially observed in 31 subjects who were heterozygous for deletions of at least 593 kilobases at 16p11.2 and whose ascertainment included cognitive deficits. Nineteen similar deletions were identified from GWAS data in 16,053 individuals from eight European cohorts. These deletions were absent from healthy non-obese controls and accounted for 0.7% of our morbid obesity cases (body mass index (BMI) >or= 40 kg m(-2) or BMI standard deviation score >or= 4; P = 6.4 x 10(-8), odds ratio 43.0), demonstrating the potential importance in common disease of rare variants with strong effects. This highlights a promising strategy for identifying missing heritability in obesity and other complex traits: cohorts with extreme phenotypes are likely to be enriched for rare variants, thereby improving power for their discovery. Subsequent analysis of the loci so identified may well reveal additional rare variants that further contribute to the missing heritability, as recently reported for SIM1 (ref. 3). The most productive approach may therefore be to combine the 'power of the extreme' in small, well-phenotyped cohorts, with targeted follow-up in case-control and population cohorts.

Brain morphometric correlates of metabolic variables in HIV: the CHARTER study.

Obesity and other metabolic variables are associated with abnormal brain structural volumes and cognitive dysfunction in HIV-uninfected populations. Since individuals with HIV infection on combined antiretroviral therapy (CART) often have systemic metabolic abnormalities and changes in brain morphology and function, we examined associations among brain volumes and metabolic factors in the multisite CNS HIV AntiRetroviral Therapy Effects Research (CHARTER) cohort, cross-sectional study of 222 HIV-infected individuals. Metabolic variables included body mass index (BMI), total blood cholesterol (C), low- and high-density lipoprotein C (LDL-C and HDL-C), blood pressure, random blood glucose, and diabetes. MRI measured volumes of cerebral white matter, abnormal white matter, cortical and subcortical gray matter, and ventricular and sulcal CSF. Multiple linear regression models allowed us to examine metabolic variables separately and in combination to predict each regional volume. Greater BMI was associated with smaller cortical gray and larger white matter volumes. Higher total cholesterol (C) levels were associated with smaller cortex volumes; higher LDL-C was associated with larger cerebral white matter volumes, while higher HDL-C levels were associated with larger sulci. Higher blood glucose levels and diabetes were associated with more abnormal white matter. Multiple atherogenic metabolic factors contribute to regional brain volumes in HIV-infected, CART-treated patients, reflecting associations similar to those found in HIV-uninfected individuals. These risk factors may accelerate cerebral atherosclerosis and consequent brain alterations and cognitive dysfunction.

Apolipoprotein E4 genotype does not increase risk of HIV-associated neurocognitive disorders.

This is a cross-sectional, observational study to evaluate the hypothesis that HIV-seropositive (HIV+) apolipoprotein E4 (APOE4) carriers are at increased risk for HIV-associated neurocognitive disorders (HAND) compared to APOE4 noncarriers with HIV in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) Group sample. APOE genotype was determined in 466 CHARTER participants with varying disease stages and histories of antiretroviral treatment who did not have severe psychiatric or medical comorbid conditions that preclude diagnosis of HAND. HAND diagnoses were based on results of comprehensive neurobehavioral evaluation and use of current neuroAIDS diagnostic criteria. HAND status consists of two levels: neuropsychologically normal status (i.e., no HAND) and any HAND diagnosis (i.e., asymptomatic neurocognitive impairment, minor neurocognitive disorder, HIV-associated dementia). Logistic regression analyses revealed no association between APOE4 carrier status and HAND, and there were no interactions between APOE4 carrier status and ethnicity, age, substance use disorders, duration of infection, or nadir CD4. Results did not differ when analysis was restricted to symptomatic HAND, and no APOE4 gene dose-dependent relationship to HAND emerged. APOE4 status was not associated with concurrent HAND in this large, well-characterized sample. This does not preclude emergence of an association between APOE4 status and HAND as this population ages. Prospective, longitudinal studies are needed to examine APOE4 as a risk factor for neurocognitive decline, incident HAND at older ages, and potential associations with cerebrospinal fluid amyloid.

PKR-like ER kinase (PERK) Haplotypes Are Associated with Depressive Symptoms in People with HIV.

Background: Depression is a debilitating and difficult-to-treat condition in people with HIV (PWH) despite viral suppression on antiretroviral therapy (ART). Depression is associated with activation of the PKR-like ER kinase (PERK) pathway, which regulates protein synthesis in response to metabolic stress. We evaluated common PERK haplotypes that influence PERK expression in relation to depressed mood in PWH. Methods: PWH from 6 research centers were enrolled in the study. Genotyping was conducted using targeted sequencing with TaqMan. The major PERK haplotypes A, B, and D were identified. Depressive symptom severity was assessed using the Beck Depression Inventory-II (BDI-II). Covariates including genetically-defined ancestry, demographics, HIV disease/treatment parameters and antidepressant treatments were assessed. Data were analyzed using multivariable regression models. Results: A total of 287 PWH with a mean (SD) age of 57.1±7.8 years were enrolled. Although the largest ethnic group was non-Hispanic white (n=129, 45.3%), African-American (n=124, 43.5%) and Hispanic (n=30, 10.5%) made up over half the sample. 20.3% were female and 96.5% were virally suppressed. Mean BDI-II was 9.6±9.5, and 28.9% scored above the cutoff for mild depression (BDI-II>13). PERK haplotype frequencies were AA57.8%, AB25.8%, AD 10.1%, and BB4.88%. PERK haplotypes were differentially represented according to genetic ancestry (p=6.84e-6). BDI-II scores were significantly higher in participants with the AB haplotype (F=4.45, p=0.0007).This finding was robust to consideration of potential confounds. Conclusion: PERK haplotypes were associated with depressed mood in PWH.Consequently, pharmacological targeting of PERK-related pathways might amelioratedepression in PWH.

Diagnosing symptomatic HIV-associated neurocognitive disorders: self-report versus performance-based assessment of everyday functioning.

Three types of HIV-associated neurocognitive disorders (HAND) exist that are distinguished by presence and severity of impairment in cognitive and everyday functioning. Although well-validated neurocognitive measures exist, determining impairment in everyday functioning remains a challenge. We aim to determine whether Self-Report measures of everyday functioning are as effective in characterizing HAND as Performance-Based measures. We assessed 674 HIV-infected participants with a comprehensive neurocognitive battery; 233 met criteria for a HAND diagnosis by having at least mild neurocognitive impairment. Functional decline was measured via Self-Report and Performance-Based measures. HAND diagnoses were determined according to published criteria using three approaches to assess functional decline: (1) Self-Report measures only, (2) Performance-Based measures only, and (3) Dual-method combining Self-Report and Performance-Based measures. The Dual-method classified the most symptomatic HAND, compared to either singular method. Singular method classifications were 76% concordant with each other. Participants classified as Performance-Based functionally impaired were more likely to be unemployed and more immunosuppressed, whereas those classified as Self-Report functionally impaired had more depressive symptoms. Multimodal methods of assessing everyday functioning facilitate detection of symptomatic HAND. Singular Performance-Based classifications were associated with objective functional and disease-related factors; reliance on Self-Report classifications may be biased by depressive symptoms.

Thalamic versus midbrain tremor; two distinct types of Holmes' Tremor: a review of 17 cases.

INTRODUCTION: Holmes Tremor (HT) is a unique and debilitating movement disorder. It usually results from lesions of the midbrain and its connection but can also result from posterior thalamic injury. Clinical examination can help lesion localization between these two areas. We studied the clinical features and their radiological correlations to distinguish midbrain HT (HT-m) from thalamic HT (HT-t). METHODS: Retrospective review of 17 patients with a HT-type presentation was conducted. Tremor characteristics, associated clinical signs and radiological findings were studied. RESULTS: Eleven patients had a myorythmic rest tremor, large amplitude proximal tremor with goal-directed worsening, with or without mild distal dystonic posturing, representing HT-m. Six patients had slow, large amplitude proximal tremors and distal choreathetoid movements, significant proximal/distal dystonic posturing, associated with proprioceptive sensory loss, representing HT-t. Haemorrhagic lesions were the predominant cause of HT-m; whereas, ischaemia was more commonly associated with HT-t. CONCLUSION: When assessing patients with HT, attentiveness to the presence of associated signs in the affected limb, such as a proprioceptive sensory deficits and additional movement disorders, can aid lesion localisation, which can have implications for management.

Molecular chaperones: the plant connection.

Molecular chaperones are a family of unrelated proteins found in all types of cell. They mediate the correct assembly of other polypeptides, but are not components of the mature assembled structures. Chaperones function by binding specifically to interactive protein surfaces that are exposed transiently during many cellular processes and so prevent them from undergoing incorrect interactions that might produce nonfunctional structures. The concept of molecular chaperones originated largely from studies of the chloroplast enzyme rubisco, which fixes carbon dioxide in plant photosynthesis; the function of chaperones forces a rethinking of the principle of protein self-assembly.

[Chloroplast ribosomes: stereospecificity of inhibition by chloramphenicol].

Chloroplast ribosomes from tobacco leaves show the same stereospecificity of inhibition by chloramphenicol as bacterial ribosomes do. Cytoplasmic ribosomes from the same leaves are unaffected by chloramphenicol. These results remove doubts raised by nonstereospecific effects of chloramphenicol on higher plant cells and support the concept that chloroplasts have evolved from prokaryotes.

Neutron imaging of laser fusion targets.

Along-term goal of inertial-confinement fusion research is the generation of energy by imploding capsules containing deuterium-tritium fuel. Progress in designing the capsules is aided by accurate imaging of the fusion burn. Penumbral coded-aperture techniques have been used to obtain neutron images that are a direct measurement of the fusion burn region in the capsules.

Macromolecular crowding: obvious but underappreciated.

Biological macromolecules evolve and function within intracellular environments that are crowded with other macromolecules. Crowding results in surprisingly large quantitative effects on both the rates and the equilibria of interactions involving macromolecules, but such interactions are commonly studied outside the cell in uncrowded buffers. The addition of high concentrations of natural and synthetic macromolecules to such buffers enables crowding to be mimicked in vitro, and should be encouraged as a routine variable to study. The stimulation of protein aggregation by crowding might account for the existence of molecular chaperones that combat this effect. Positive results of crowding include enhancing the collapse of polypeptide chains into functional proteins, the assembly of oligomeric structures and the efficiency of action of some molecular chaperones and metabolic pathways.

Molecular chaperones: proteins essential for the biogenesis of some macromolecular structures.

Many polypeptides can self-assemble into functional structures while others assemble only in the presence of additional proteins (molecular chaperones) which are not components of the final structure. We discuss here the effect that the recognition of the essential roles played by these proteins in assembly processes may have on the principle of spontaneous self-assembly.

Single photon emission computerized tomography with capromab pendetide plus computerized tomography image set co-registration independently predicts biochemical failure.

PURPOSE: We evaluate the usefulness of pretreatment (111)Indium capromab pendetide (ProstaScint) planar imaging (immunoscintigraphy) plus single photon emission tomography co-registration with computerized tomography scans to detect occult metastatic disease and predict for biochemical failure, in a cohort of patients with a clinical diagnosis of localized adenocarcinoma of the prostate referred for primary radiotherapy. MATERIALS AND METHODS: Patients were followed after radiotherapy for evidence of biochemical failure using 2 criteria of prostate specific antigen clinical nadir +2 ng/ml and American Society for Therapeutic Radiology and Oncology Consensus definitions. Median followup was 58.8 months (mean 64.8). Clinical risk factors defined 3 risk groups of high (51), intermediate (72) and low (116). RESULTS: Overall biochemical failure was 18.3% vs 11.8% by the 2-BFC at 8-year actuarial analysis with 58.8 months median followup. By the CN +2 definition the control date for the cohort is 34.8 months. Pretreatment SPECT/CT suggested prostate cancer metastasis (22), seminal vesicle extension (20) and organ confined disease (197). Biochemical failure in patients having extra-periprostatic metastatic prostate cancer, seminal vesicle extension and organ confined disease uptake on SPECT/CT was 43.2%, 16.0% vs 14.7% (p = 0.0006); and 33.3%, 15.0% vs 8.7% (p = 0.0017) by the 2-BFC, respectively. Cox multiple regression analysis demonstrated that a finding of extra-periprostatic metastatic prostate on SPECT/CT significantly predicted a 4.2-fold greater risk (p = 0.0012) and a 4.5-fold greater risk (p = 0.0011) of failure by the 2-BFC than organ confined disease adjusting for treatment and risk group. CONCLUSIONS: Unconfirmed findings of extra-periprostatic metastatic prostate cancer on SPECT/CT immunoscintigraphy independently and significantly predicted an increased risk of biochemical failure in patients presenting for radiotherapy with a clinical diagnosis of localized prostate cancer.

Impact of long-term treatment with neurotoxic dideoxynucleoside antiretrovirals: implications for clinical care in resource-limited settings.

OBJECTIVES: A minority of HIV-infected patients taking an antiretroviral (ARV) regimen containing dideoxynucleosides (d-drugs) such as stavudine (d4T) and didanosine (DDI) experiences dose-limiting neuropathic pain and paraesthesias, usually within weeks of starting these drugs. Because d-drugs are among the few affordable options available in developing countries, continuing d-drug therapy would be a desirable strategy for many HIV-infected individuals. Therefore, we evaluated the safety of continuing d-drug therapy. METHODS: In a US cohort, we compared the rates of worsening neuropathic symptoms and signs in HIV-infected individuals on stable ARV regimens that did (n=252) or did not (n=250) include d-drugs. Rates of worsening were compared using proportional hazards model and the log-rank test. RESULTS: The risk ratios (RR) were not significantly larger for worsening neuropathy signs [0.94; 95% confidence interval (CI) 0.84-1.07] or symptoms (0.99; 95% CI 0.88-1.14) in patients taking d-drugs continuously compared to those not taking d-drugs. CONCLUSIONS: Continued d-drug exposure among patients tolerating an initial trial did not increase the risk of worsening neuropathy compared to non-d-drug-containing regimens. If applicable in developing countries, these findings suggest that in most patients d-drugs can be continued safely in the long term without increasing the risk of worsening neuropathy.

Molecular chaperones. Opening and closing the Anfinsen cage.

Dynamic interactions between chaperonins allow newly synthesized polypeptides to begin correct folding inside a transiently closed cage. Specialized chaperonins may be used to deal with recalcitrant proteins.

Molecular chaperones: inside and outside the Anfinsen cage.

The GroEL/GroES chaperonin system acts as a passive anti-aggregation cage for refolding rubisco and rhodanese, and not as an active unfolding device. Refolding aconitase is too large to enter the cage but reversible binding to GroEL reduces its aggregration. Unexpectedly, confinement in the cage increases the rate of refolding of rubisco, but not rhodanese.

Molecular chaperones: avoiding the crowd.

The involvement of two types of molecular chaperone in folding newly synthesized proteins can be rationalized in terms of the crowded nature of the intracellular environment. Recent work sheds light on how these chaperones recognise their substrates and protect them from the problems of macromolecular crowding.

Molecular chaperones: pathways and networks.

Some proteins synthesized by growing eukaryotic cells are transferred along unidirectional pathways of molecular chaperones until the risk of aggregation has decreased and they can be released safely. Mature proteins denatured by stress may instead be handled by chaperones acting in branched, reversible networks.