SRM targeted proteomics in search for biomarkers of HCV ‐induced progression of fibrosis to cirrhosis in HALT ‐ C patients

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92042/1/pmic7038.pd

Scatter modelling and compensation in emission tomography

In nuclear medicine, clinical assessment and diagnosis are generally based on qualitative assessment of the distribution pattern of radiotracers used. In addition, emission tomography (SPECT and PET) imaging methods offer the possibility of quantitative assessment of tracer concentration in vivo to quantify relevant parameters in clinical and research settings, provided accurate correction for the physical degrading factors (e.g. attenuation, scatter, partial volume effects) hampering their quantitative accuracy are applied. This review addresses the problem of Compton scattering as the dominant photon interaction phenomenon in emission tomography and discusses its impact on both the quality of reconstructed clinical images and the accuracy of quantitative analysis. After a general introduction, there is a section in which scatter modelling in uniform and non-uniform media is described in detail. This is followed by an overview of scatter compensation techniques and evaluation strategies used for the assessment of these correction methods. In the process, emphasis is placed on the clinical impact of image degradation due to Compton scattering. This, in turn, stresses the need for implementation of more accurate algorithms in software supplied by scanner manufacturers, although the choice of a general-purpose algorithm or algorithms may be difficul

Differential metabolic responses of clam Ruditapes philippinarum to Vibrio anguillarum and Vibrio splendidus challenges

Clam Ruditapes philippinarum is one of the important marine aquaculture species in North China. However, pathogens can often cause diseases and lead to massive mortalities and economic losses of clam. In this work, we compared the metabolic responses induced by Vibrio anguillarum and Vibrio splendidus challenges towards hepatopancreas of clam using NMR-based metabolomics. Metabolic responses suggested that both V anguillarum and V splendidus induced disturbances in energy metabolism and osmotic regulation, oxidative and immune stresses with different mechanisms, as indicated by correspondingly differential metabolic biomarkers (e.g., amino acids, ATP, glucose, glycogen, taurine, betaine, choline and hypotaurine) and altered mRNA expression levels of related genes including ATP synthase, ATPase, glutathione peroxidase, heat shock protein 90, defensin and lysozyme. However, V. anguillarum caused more severe oxidative and immune stresses in clam hepatopancreas than V splendidus. Our results indicated that metabolomics could be used to elucidate the biological effects of pathogens to the marine clam R. philippinarum. (C) 2013 Elsevier Ltd. All rights reserved.Clam Ruditapes philippinarum is one of the important marine aquaculture species in North China. However, pathogens can often cause diseases and lead to massive mortalities and economic losses of clam. In this work, we compared the metabolic responses induced by Vibrio anguillarum and Vibrio splendidus challenges towards hepatopancreas of clam using NMR-based metabolomics. Metabolic responses suggested that both V anguillarum and V splendidus induced disturbances in energy metabolism and osmotic regulation, oxidative and immune stresses with different mechanisms, as indicated by correspondingly differential metabolic biomarkers (e.g., amino acids, ATP, glucose, glycogen, taurine, betaine, choline and hypotaurine) and altered mRNA expression levels of related genes including ATP synthase, ATPase, glutathione peroxidase, heat shock protein 90, defensin and lysozyme. However, V. anguillarum caused more severe oxidative and immune stresses in clam hepatopancreas than V splendidus. Our results indicated that metabolomics could be used to elucidate the biological effects of pathogens to the marine clam R. philippinarum. (C) 2013 Elsevier Ltd. All rights reserved

Recent Advances and the Potential for Clinical Use of Autofluorescence Detection of Extra-Ophthalmic Tissues

The autofluorescence (AF) characteristics of endogenous fluorophores allow the label-free assessment and visualization of cells and tissues of the human body. While AF imaging (AFI) is well-established in ophthalmology, its clinical applications are steadily expanding to other disciplines. This review summarizes clinical advances of AF techniques published during the past decade. A systematic search of the MEDLINE database and Cochrane Library databases was performed to identify clinical AF studies in extra-ophthalmic tissues. In total, 1097 articles were identified, of which 113 from internal medicine, surgery, oral medicine, and dermatology were reviewed. While comparable technological standards exist in diabetology and cardiology, in all other disciplines, comparability between studies is limited due to the number of differing AF techniques and non-standardized imaging and data analysis. Clear evidence was found for skin AF as a surrogate for blood glucose homeostasis or cardiovascular risk grading. In thyroid surgery, foremost, less experienced surgeons may benefit from the AF-guided intraoperative separation of parathyroid from thyroid tissue. There is a growing interest in AF techniques in clinical disciplines, and promising advances have been made during the past decade. However, further research and development are mandatory to overcome the existing limitations and to maximize the clinical benefits

Host cell protein quantitation by LC-MS. Experimental demonstration, qualification, and comparison of methods in USP 1132.1

The development of biologics necessitates reliable assays to characterize and control Host Cell Protein (HCP) impurities. Liquid Chromatography-Mass Spectrometry (LC-MS)-based HCP assays have emerged as a powerful orthogonal method to HCP ELISA, providing detailed information on individual HCPs. In response to a growing need, the U.S Pharmacopeia (USP) introduced General Chapter &lt; 1132.1 &gt;, which provides the best practices and outlines three quantitative LC-MS methods. This study explores the practical application and validation readiness of the following strategies: A – Relative to Product Protein, B – Relative to Spiked-in Protein, and C – Relative to Spiked-in Peptide. Two common HCPs—Clusterin and Lipoprotein Lipase—were quantified using LC-MS in a purified mAb drug substance spiked with a CHO cell culture harvest to simulate in-process HCP levels. All three methods were demonstrated in the same samples and dilutions, enabling direct comparison of the three methods from a single dataset. Method performance was assessed according to ICH Q2(R2) guidelines for analytical method validation, focusing on linearity, accuracy, precision, and specificity. Results include a comparative assessment and discussion of the advantages and disadvantages, and application of each aforementioned HCP quantification method. This study provides practical insights into the implementation of USP &lt; 1132.1 &gt; supporting the growing role of LC-MS in HCP analysis for biologics development.</p

Quantitative comparative analysis of human erythrocyte surface proteins between individuals from two genetically distinct populations

Abstract: Red blood cells (RBCs) play a critical role in oxygen transport, and are the focus of important diseases including malaria and the haemoglobinopathies. Proteins at the RBC surface can determine susceptibility to disease, however previous studies classifying the RBC proteome have not used specific strategies directed at enriching cell surface proteins. Furthermore, there has been no systematic analysis of variation in abundance of RBC surface proteins between genetically disparate human populations. These questions are important to inform not only basic RBC biology but additionally to identify novel candidate receptors for malarial parasites. Here, we use ‘plasma membrane profiling’ and tandem mass tag-based mass spectrometry to enrich and quantify primary RBC cell surface proteins from two sets of nine donors from the UK or Senegal. We define a RBC surface proteome and identify potential Plasmodium receptors based on either diminished protein abundance, or increased variation in RBCs from West African individuals

Simplified quantification of insulin, its synthetic analogs and C-peptide in human plasma by means of LC-HRMS.

The quantification of peptide hormones by means of liquid chromatography (LC) coupled to mass spectrometry (MS) or other techniques (e.g. immunoassays) has been a challenging task in modern analytical chemistry. Especially for insulin, its synthetic analogs, and C-peptide, reliable determinations are urgently needed due to their diagnostic value in the management of diabetes and insulin resistance and because of the illicit use of insulin as a performance-enhancing agent in professional sports or as an effective toxin in forensic toxicology. The concomitant measurement of C-peptide and insulin offers an established tool for the diagnostic workup of hypoglycemia (endogenous vs. exogenous hyperinsulinemia), characterizing hepatic insulin clearance, and the assessment of beta-cell function (insulin secretion). Thus, the present approach offers the possibility to determine human insulin and its synthetic analogs (lispro, glulisine, aspart, glargine metabolite, degludec, detemir, porcine, and bovine) and C-peptide simultaneously after sample preparation utilizing protein precipitation and a mixed-mode cation-exchange solid-phase extraction, and subsequent detection by LC-high resolution MS. The method was fully validated regarding the following parameters: specificity, limit of detection (0.2 ng/mL), limit of quantification (0.6 ng/mL), recovery (40-90%), accuracy (78-128%), linearity, precision (< 21%), carry over, robustness, and matrix effects. The proof-of-concept was shown by analyzing authentic plasma samples from adults with class II obesity and prediabetes collected in the course of an oral glucose tolerance test. All sample preparation steps were controlled by two stable isotope-labeled internal standards, namely [[2 H10 ] Leu B6, B11, B15, B17 ]-insulin, and [[13 C6 ] Leu 26, 30 ] C-peptide

Metabolomics using anion-exchange chromatography mass spectrometry for the analysis of cells, tissues and biofluids

The direct coupling of ion-exchange chromatography with mass spectrometry using electrochemical ion suppression creates a hyphenated technique with selectivity and specificity for the analysis of highly polar and ionic compounds. The technique has enabled new applications in environmental chemistry, food chemistry, forensics, cell biology and, more recently, metabolomics. Robust, reproducible and quantitative methods for the analysis of highly polar and ionic metabolites help meet a longstanding analytical need in metabolomics. Here, we provide step-by-step instructions for both untargeted and semi-targeted metabolite analysis from cell, tissue or biofluid samples by using anion-exchange chromatography–high-resolution tandem mass spectrometry (AEC-MS/MS). The method requires minimal sample preparation and is robust, sensitive and selective. It provides comprehensive coverage of hundreds of metabolites found in primary and secondary metabolic pathways, including glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, purine and pyrimidine metabolism, amino acid degradation and redox metabolism. An inline electrolytic ion suppressor is used to quantitatively neutralize OH− ions in the eluent stream, after chromatographic separation, enabling AEC to be directly coupled with MS. Counter ions are also removed during this process, creating a neutral pH, aqueous eluent with a simplified matrix optimal for negative ion MS analysis. Sample preparation through to data analysis and interpretation is described in the protocol, including a guide to which metabolites and metabolic pathways are suitable for analysis by using AEC-MS/MS

Evidence for models of diagnostic service provision in the community: literature mapping exercise and focused rapid reviews

Background Current NHS policy favours the expansion of diagnostic testing services in community and primary care settings. Objectives Our objectives were to identify current models of community diagnostic services in the UK and internationally and to assess the evidence for quality, safety and clinical effectiveness of such services. We were also interested in whether or not there is any evidence to support a broader range of diagnostic tests being provided in the community. Review methods We performed an initial broad literature mapping exercise to assess the quantity and nature of the published research evidence. The results were used to inform selection of three areas for investigation in more detail. We chose to perform focused reviews on logistics of diagnostic modalities in primary care (because the relevant issues differ widely between different types of test); diagnostic ultrasound (a key diagnostic technology affected by developments in equipment); and a diagnostic pathway (assessment of breathlessness) typically delivered wholly or partly in primary care/community settings. Databases and other sources searched, and search dates, were decided individually for each review. Quantitative and qualitative systematic reviews and primary studies of any design were eligible for inclusion. Results We identified seven main models of service that are delivered in primary care/community settings and in most cases with the possible involvement of community/primary care staff. Not all of these models are relevant to all types of diagnostic test. Overall, the evidence base for community- and primary care-based diagnostic services was limited, with very few controlled studies comparing different models of service. We found evidence from different settings that these services can reduce referrals to secondary care and allow more patients to be managed in primary care, but the quality of the research was generally poor. Evidence on the quality (including diagnostic accuracy and appropriateness of test ordering) and safety of such services was mixed. Conclusions In the absence of clear evidence of superior clinical effectiveness and cost-effectiveness, the expansion of community-based services appears to be driven by other factors. These include policies to encourage moving services out of hospitals; the promise of reduced waiting times for diagnosis; the availability of a wider range of suitable tests and/or cheaper, more user-friendly equipment; and the ability of commercial providers to bid for NHS contracts. However, service development also faces a number of barriers, including issues related to staffing, training, governance and quality control. Limitations We have not attempted to cover all types of diagnostic technology in equal depth. Time and staff resources constrained our ability to carry out review processes in duplicate. Research in this field is limited by the difficulty of obtaining, from publicly available sources, up-to-date information about what models of service are commissioned, where and from which providers. Future work There is a need for research to compare the outcomes of different service models using robust study designs. Comparisons of ‘true’ community-based services with secondary care-based open-access services and rapid access clinics would be particularly valuable. There are specific needs for economic evaluations and for studies that incorporate effects on the wider health system. There appears to be no easy way of identifying what services are being commissioned from whom and keeping up with local evaluations of new services, suggesting a need to improve the availability of information in this area. Funding The National Institute for Health Research Health Services and Delivery Research programme