Differential protein profiling as a potential multi-marker approach for TSE diagnosis

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177This "proof of concept" study, examines the use of differential protein expression profiling using surface enhanced laser desorption and ionisationtime of flight mass spectrometry (SELDI-TOF) for the diagnosis of TSE disease. Spectral output from all proteins selectively captured from individual murine brain homogenate samples, are compared as "profiles" in groups of infected and non-infected animals. Differential protein expression between groups is thus highlighted and statistically significant protein "peaks" used to construct a panel of disease specific markers. Studies at both terminal stages of disease and throughout the time course of disease have shown a disease specific protein profile or "disease fingerprint" which could be used to distinguish between groups of TSE infected and uninfected animals at an early time point of disease. Results Our results show many differentially expressed proteins in diseased and control animals, some at early stages of disease. Three proteins identified by SELDI-TOF analysis were verified by immunohistochemistry in brain tissue sections. We demonstrate that by combining the most statistically significant changes in expression, a panel of markers can be constructed that can distinguish between TSE diseased and normal animals. Conclusion Differential protein expression profiling has the potential to be used for the detection of disease in TSE infected animals. Having established that a "training set" of potential markers can be constructed, more work would be required to further test the specificity and sensitivity of the assay in a "testing set". Based on these promising results, further studies are being performed using blood samples from infected sheep to assess the potential use of SELDI-TOF as a pre-mortem blood based diagnostic.https://doi.org/10.1186/1471-2334-9-1889pubpub

The Human Urinary Proteome Fingerprint Database UPdb

The use of human urine as a diagnostic tool has many advantages, such as ease of sample acquisition and noninvasiveness. However, the discovery of novel biomarkers, as well as biomarker patterns, in urine is hindered mainly by a lack of comparable datasets. To fill this gap, we assembled a new urinary fingerprint database. Here, we report the establishment of a human urinary proteomic fingerprint database using urine from 200 individuals analysed by SELDI-TOF (surface enhanced laser desorption ionisation-time of flight) mass spectrometry (MS) on several chip surfaces (SEND, HP50, NP20, Q10, CM10, and IMAC30). The database currently lists 2490 unique peaks/ion species from 1172 nonredundant SELDI analyses in the mass range of 1500 to 150000. All unprocessed mass spectrometric scans are available as “.xml” data files. Additionally, 1384 peaks were included from external studies using CE (capillary electrophoresis)-MS, MALDI (matrix assisted laser desorption/ionisation), and CE-MALDI hybrids. We propose to use this platform as a global resource to share and exchange primary data derived from MS analyses in urinary research

The Human Urinary Proteome Fingerprint Database UPdb

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177The use of human urine as a diagnostic tool has many advantages, such as ease of sample acquisition and noninvasiveness. However, the discovery of novel biomarkers, as well as biomarker patterns, in urine is hindered mainly by a lack of comparable datasets. To fill this gap, we assembled a new urinary fingerprint database. Here, we report the establishment of a human urinary proteomic fingerprint database using urine from 200 individuals analysed by SELDI-TOF (surface enhanced laser desorption ionisation-time of flight) mass spectrometry (MS) on several chip surfaces (SEND, HP50, NP20, Q10, CM10, and IMAC30). The database currently lists 2490 unique peaks/ion species from 1172 nonredundant SELDI analyses in the mass range of 1500 to 150000. All unprocessed mass spectrometric scans are available as “.xml” data files. Additionally, 1384 peaks were included from external studies using CE (capillary electrophoresis)-MS, MALDI (matrix assisted laser desorption/ionisation), and CE-MALDI hybrids. We propose to use this platform as a global resource to share and exchange primary data derived from MS analyses in urinary research.https://doi.org/10.1155/2013/7602082013pubpub76020

Differential protein profiling as a potential multi-marker approach for TSE diagnosis

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177This "proof of concept" study, examines the use of differential protein expression profiling using surface enhanced laser desorption and ionisationtime of flight mass spectrometry (SELDI-TOF) for the diagnosis of TSE disease. Spectral output from all proteins selectively captured from individual murine brain homogenate samples, are compared as "profiles" in groups of infected and non-infected animals. Differential protein expression between groups is thus highlighted and statistically significant protein "peaks" used to construct a panel of disease specific markers. Studies at both terminal stages of disease and throughout the time course of disease have shown a disease specific protein profile or "disease fingerprint" which could be used to distinguish between groups of TSE infected and uninfected animals at an early time point of disease. Results Our results show many differentially expressed proteins in diseased and control animals, some at early stages of disease. Three proteins identified by SELDI-TOF analysis were verified by immunohistochemistry in brain tissue sections. We demonstrate that by combining the most statistically significant changes in expression, a panel of markers can be constructed that can distinguish between TSE diseased and normal animals. Conclusion Differential protein expression profiling has the potential to be used for the detection of disease in TSE infected animals. Having established that a "training set" of potential markers can be constructed, more work would be required to further test the specificity and sensitivity of the assay in a "testing set". Based on these promising results, further studies are being performed using blood samples from infected sheep to assess the potential use of SELDI-TOF as a pre-mortem blood based diagnostic.https://doi.org/10.1186/1471-2334-9-1889pubpub

Microdissection: A method developed to investigate mechanisms involved in transmissible spongiform encephalopathy pathogenesis

BACKGROUND: The transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting both human and animals. The neuroanatomical changes which occur in the central nervous system (CNS) of TSE infected animals include vacuolation, gliosis, neuronal loss and the deposition of a disease specific protein, PrP(Sc). Experimental murine models of scrapie, a TSE of sheep, have revealed that pathology may be confined to specific brain areas with targeting of particular neuronal subsets depending on route of injection and scrapie isolate. To assess the biochemical changes which are taking place in these targeted areas it was necessary to develop a reliable sampling procedure (microdissection) which could be used for a variety of tests such as western blotting and magnetic resonance spectroscopy. METHODS: The method described is for the microdissection of murine brains. To assess the usefulness of this dissection technique for producing similar sample types for analysis by various down-stream biochemical techniques, the areas dissected were analysed for PrP(Sc )by western blotting and compared to immunocytochemical (ICC) techniques. RESULTS: Results show that the method generates samples yielding a consistent protein content which can be analysed for PrP(Sc). The areas in which PrP(Sc )is found by western blotting compares well with localisation visualised by immunocytochemistry. CONCLUSION: The microdisssection method described can be used to generate samples suitable for a range of biochemical techniques. Using these samples a range of assays can be carried out which will help to elucidate the molecular and cellular mechanisms underlying TSE pathogenesis. The method would also be useful for any study requiring the investigation of discrete areas within the murine brain

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

Analysis of shared heritability in common disorders of the brain

ience, this issue p. eaap8757 Structured Abstract INTRODUCTION Brain disorders may exhibit shared symptoms and substantial epidemiological comorbidity, inciting debate about their etiologic overlap. However, detailed study of phenotypes with different ages of onset, severity, and presentation poses a considerable challenge. Recently developed heritability methods allow us to accurately measure correlation of genome-wide common variant risk between two phenotypes from pools of different individuals and assess how connected they, or at least their genetic risks, are on the genomic level. We used genome-wide association data for 265,218 patients and 784,643 control participants, as well as 17 phenotypes from a total of 1,191,588 individuals, to quantify the degree of overlap for genetic risk factors of 25 common brain disorders. RATIONALE Over the past century, the classification of brain disorders has evolved to reflect the medical and scientific communities' assessments of the presumed root causes of clinical phenomena such as behavioral change, loss of motor function, or alterations of consciousness. Directly observable phenomena (such as the presence of emboli, protein tangles, or unusual electrical activity patterns) generally define and separate neurological disorders from psychiatric disorders. Understanding the genetic underpinnings and categorical distinctions for brain disorders and related phenotypes may inform the search for their biological mechanisms. RESULTS Common variant risk for psychiatric disorders was shown to correlate significantly, especially among attention deficit hyperactivity disorder (ADHD), bipolar disorder, major depressive disorder (MDD), and schizophrenia. By contrast, neurological disorders appear more distinct from one another and from the psychiatric disorders, except for migraine, which was significantly correlated to ADHD, MDD, and Tourette syndrome. We demonstrate that, in the general population, the personality trait neuroticism is significantly correlated with almost every psychiatric disorder and migraine. We also identify significant genetic sharing between disorders and early life cognitive measures (e.g., years of education and college attainment) in the general population, demonstrating positive correlation with several psychiatric disorders (e.g., anorexia nervosa and bipolar disorder) and negative correlation with several neurological phenotypes (e.g., Alzheimer's disease and ischemic stroke), even though the latter are considered to result from specific processes that occur later in life. Extensive simulations were also performed to inform how statistical power, diagnostic misclassification, and phenotypic heterogeneity influence genetic correlations. CONCLUSION The high degree of genetic correlation among many of the psychiatric disorders adds further evidence that their current clinical boundaries do not reflect distinct underlying pathogenic processes, at least on the genetic level. This suggests a deeply interconnected nature for psychiatric disorders, in contrast to neurological disorders, and underscores the need to refine psychiatric diagnostics. Genetically informed analyses may provide important "scaffolding" to support such restructuring of psychiatric nosology, which likely requires incorporating many levels of information. By contrast, we find limited evidence for widespread common genetic risk sharing among neurological disorders or across neurological and psychiatric disorders. We show that both psychiatric and neurological disorders have robust correlations with cognitive and personality measures. Further study is needed to evaluate whether overlapping genetic contributions to psychiatric pathology may influence treatment choices. Ultimately, such developments may pave the way toward reduced heterogeneity and improved diagnosis and treatment of psychiatric disorders

Differential protein expression profiling in BSE disease

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177Item not available in this repository.Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disease affecting cattle. Current tests for the detection of BSE are based solely on the only definitive marker of the disease, an abnormal conformer (PrPd), of the host encoded prion protein (PrPc). Recent evidence that other transmissible spongiform encephalopathy diseases can be present in the absence of PrPd, coupled with the need to establish pre-mortem diagnostic assays have led to a search for alternative diagnostic approaches. In this study we apply differential protein expression profiling for the prediction of BSE disease in post-mortem bovine brain tissue. The protein profiles of groups of 27 BSE diseased cattle were compared with 28 control animals. Analysis using statistical learning (and linear discriminant analysis) techniques established protein markers of disease with good predictive power (sensitivity 85% and specificity 71%). Further work will be required to test the predictive markers in a wider range of diseases, particularly other neurological conditions.https://doi.org/10.3109/13813455.2010.504211116pubpub4-

Protocols and Hospital Mortality in Critically Ill Patients: The United States Critical Illness and Injury Trials Group Critical Illness Outcomes Study

Objective: Clinical protocols may decrease unnecessary variation in care and improve compliance with desirable therapies. We evaluated whether highly protocolized ICUs have superior patient outcomes compared with less highly protocolized ICUs. Design: Observational study in which participating ICUs completed a general assessment and enrolled new patients 1 day each week. Patients: A total of 6,179 critically ill patients. Setting: Fifty-nine ICUs in the United States Critical Illness and Injury Trials Group Critical Illness Outcomes Study. Interventions: None. Measurements and Main Results: The primary exposure was the number of ICU protocols; the primary outcome was hospital mortality. A total of 5,809 participants were followed prospectively, and 5,454 patients in 57 ICUs had complete outcome data. The median number of protocols per ICU was 19 (interquartile range, 15-21.5). In single-variable analyses, there were no differences in ICU and hospital mortality, length of stay, use of mechanical ventilation, vasopressors, or continuous sedation among individuals in ICUs with a high versus low number of protocols. The lack of association was confirmed in adjusted multivariable analysis (p = 0.70). Protocol compliance with two ventilator management protocols was moderate and did not differ between ICUs with high versus low numbers of protocols for lung protective ventilation in acute respiratory distress syndrome (47% vs 52%; p = 0.28) and for spontaneous breathing trials (55% vs 51%; p = 0.27). Conclusions: Clinical protocols are highly prevalent in U.S. ICUs. The presence of a greater number of protocols was not associated with protocol compliance or patient mortality