Factors affecting the accuracy of urine-based biomarkers of BSE

<p>Abstract</p> <p>Background</p> <p>Transmissible spongiform encephalopathy diseases are untreatable, uniformly fatal degenerative syndromes of the central nervous system that can be transmitted both within as well as between species. The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob disease (vCJD), have profoundly influenced beef production processes as well as blood donation and surgical procedures. Simple, robust and cost effective diagnostic screening and surveillance tools are needed for both the preclinical and clinical stages of TSE disease in order to minimize both the economic costs and zoonotic risk of BSE and to further reduce the risk of secondary vCJD.</p> <p>Objective</p> <p>Urine is well suited as the matrix for an ante-mortem test for TSE diseases because it would permit non-invasive and repeated sampling. In this study urine samples collected from BSE infected and age matched control cattle were screened for the presence of individual proteins that exhibited disease specific changes in abundance in response to BSE infection that might form the basis of such an ante-mortem test.</p> <p>Results</p> <p>Two-dimensional differential gel electrophoresis (2D-DIGE) was used to identify proteins exhibiting differential abundance in two sets of cattle. The known set consisted of BSE infected steers and age matched controls throughout the course of the disease. The blinded unknown set was composed of BSE infected and control samples of both genders, a wide range of ages and two different breeds. Multivariate analyses of individual protein abundance data generated classifiers comprised of the proteins best able to discriminate between the samples based on disease state, breed, age and gender.</p> <p>Conclusion</p> <p>Despite the presence of confounding factors, the disease specific changes in abundance exhibited by a panel of urine proteins permitted the creation of classifiers able to discriminate between control and infected cattle with a high degree of accuracy.</p

The identification of disease-induced biomarkers in the urine of BSE infected cattle

<p>Abstract</p> <p>Background</p> <p>The bovine spongiform encephalopathy (BSE) epidemic and the emergence of a new human variant of Creutzfeldt-Jakob Disease (vCJD) have led to profound changes in the production and trade of agricultural goods. The rapid tests currently approved for BSE monitoring in slaughtered cattle are all based on the detection of the disease related isoform of the prion protein, PrP^d, in brain tissue and consequently are only suitable for post-mortem diagnosis. Objectives: In instances such as assessing the health of breeding stock for export purposes where post-mortem testing is not an option, there is a demand for an ante-mortem test based on a matrix or body fluid that would permit easy access and repeated sampling. Urine and urine based analyses would meet these requirements.</p> <p>Results</p> <p>Two dimensional differential gel eletrophoresis (2D-DIGE) and mass spectrometry analyses were used to identify proteins exhibiting differential abundance in the urine of BSE infected cattle and age matched controls over the course of the disease. Multivariate analyses of protein expression data identified a single protein able to discriminate, with 100% accuracy, control from infected samples. In addition, a subset of proteins were able to predict with 85% ± 13.2 accuracy the time post infection that the samples were collected.</p> <p>Conclusion</p> <p>These results suggest that in principle it is possible to identify biomarkers in urine useful in the diagnosis, prognosis and monitoring of disease progression of transmissible spongiform encephalopathy diseases (TSEs).</p

Non-Productive Infection of Glial Cells with SARS-CoV-2 in Hamster Organotypic Cerebellar Slice Cultures

The numerous neurological syndromes associated with COVID-19 implicate an effect of viral pathogenesis on neuronal function, yet reports of direct SARS-CoV-2 infection in the brain are conflicting. We used a well-established organotypic brain slice culture to determine the permissivity of hamster brain tissues to SARS-CoV-2 infection. We found levels of live virus waned after inoculation and observed no evidence of cell-to-cell spread, indicating that SARS-CoV-2 infection was non-productive. Nonetheless, we identified a small number of infected cells with glial phenotypes; however, no evidence of viral infection or replication was observed in neurons. Our data corroborate several clinical studies that have assessed patients with COVID-19 and their association with neurological involvement

Urine Proteins Identified by Two-Dimensional Differential Gel Electrophoresis Facilitate the Differential Diagnoses of Scrapie

<div><p>The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry.</p></div

Principle component analysis of the 11 Alzheimer's disease model (red) and the 12 control samples (green) collected from 4 diseased and 4 control mice at 3 time points: 32, 36, and 40 weeks of age.

<p>A sample from one of the diseased mice was not obtained at 32 weeks of age. A classifier based on 84 features was able to correctly classify the 23 samples of the training set with 100% accuracy. (PC1 = 62.0, PC2 = 11.9).</p

Principle component analysis of 7 scrapie infected samples (red) at 15 and 17 weeks of age, 11 Alzheimer's disease samples (green) at 32, 36 and 40 weeks of age, and the 20 corresponding control samples (blue) when analyzed by the 20 protein Disease Discriminant Classifier.

<p><b>Samples were produced by 4 scrapie infected mice, 4 Alzheimer diseased mice and the 8 corresponding control mice.</b> Thirty seven of the 38 samples were correctly classified. The 32 week old control sample, misclassified as having Alzheimer's disease, is identified by an arrow.</p

Flow chart depicting the development of the Disease Discriminant Classifier based upon 20 identified proteins from the 169 proteins that were present in 80% of the 38 gel images representing the clinical stage Alzheimer and scrapie sample sets exhibiting significant differential abundance (ANOVA p≤.01).

<p>Flow chart depicting the development of the Disease Discriminant Classifier based upon 20 identified proteins from the 169 proteins that were present in 80% of the 38 gel images representing the clinical stage Alzheimer and scrapie sample sets exhibiting significant differential abundance (ANOVA p≤.01).</p