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Precision Medicine is driven by the idea that the rapidly increasing range of relatively cheap and efficient self-tracking devices make it feasible to collect multiple kinds of phenotypic data. Advocates ofN = 1 research emphasize the countless opportunities personal data provide for optimizing individual health. At the same time, using biomarker data for lifestyle interventions has shown to entail complex challenges. In this paper, we argue that researchers in the field of precision medicine need to address the performative dimension of collecting data. We propose the fun-house mirror as a metaphor for the use of personal health data; each health data source yields a particular type of image that can be regarded as a ‘data mirror’ that is by definition specific and skewed. This requires competence on the part of individuals to adequately interpret the images thus provided.

Background Minimal residual disease (MRD) status assessed on bone marrow aspirates is a major prognostic biomarker in multiple myeloma (MM). In this study we evaluated blood-based targeted mass spectrometry (MS-MRD) as a sensitive, minimally invasive alternative to measure MM disease activity. Methods Therapy response of 41 MM patients in the IFM-2009 clinical trial (NCT01191060) was assessed with MS-MRD on frozen sera and compared to routine state-of-the-art monoclonal protein (M-protein) diagnostics and next-generation sequencing (NGS-MRD) at 2 time points. Results In all 41 patients we were able to identify clonotypic M-protein-specific peptides and perform serum-based MS-MRD measurements. MS-MRD is significantly more sensitive to detect M-protein compared to either electrophoretic M-protein diagnostics or serum free light chain analysis. The concordance between NGS-MRD and MS-MRD status in 81 paired bone marrow/sera samples was 79%. The 50% progression-free survival (PFS) was identical (49 months) for patients who were either NGS-positive or MS-positive directly after maintenance treatment. The 50% PFS was 69 and 89 months for NGS-negative and MS-negative patients, respectively. The longest 50% PFS (96 months) was observed in patients who were MRD-negative for both methods. MS-MRD relapse during maintenance treatment was significantly correlated to poor PFS (P Conclusions Our data indicate proof-of-principle that MS-MRD evaluation in blood is a feasible, patient friendly alternative to NGS-MRD assessed on bone marrow. Clinical validation of the prognostic value of MS-MRD and its complementary value in MRD-evaluation of patients with MM is warranted in an independent larger cohort.

Research consortia in Europe often compete with each other for skills, human and technical resources and, eventually, recognition of the scientific impact of their work. In response to the same EU Horizon2020 call, we received funding for our research project proposals to identify and validate novel drug targets for cardiovascular disease treatment. Each consortium followed a unique and independent research strategy. However, as coordinators of these consortia we envisioned we could increase impact, outcomes and efficiency by intensifying our interaction. At an agreed stage during our projects we chose to share our knowledge, vision and ideas. In this paper we present what we learned, in the hope that future consortia will see the benefits of this approach.

Because positron emission tomography (PET) and optical imaging are very complementary, the combination of these two imaging modalities is very enticing in the oncology field. Such bimodal imaging generally relies on imaging agents bearing two different imaging reporters. In the bioconjugation field, this is mainly performed by successive random conjugations of the two reporters on the protein vector, but these random conjugations can alter the vector properties. In this study, we aimed at abrogating the heterogeneity of the bimodal imaging immunoconjugate and mitigating the impact of multiple random conjugations. A trivalent platform bearing a DFO chelator for

Background Due to improved treatment, more patients with multiple myeloma (MM) reach a state of minimal residual disease (MRD). Different strategies for MM MRD monitoring include flow cytometry, allele-specific oligonucleotide–quantitative PCR, next-generation sequencing, and mass spectrometry (MS). The last 3 methods rely on the presence and the stability of a unique immunoglobulin fingerprint derived from the clonal plasma cell population. For MS-MRD monitoring it is imperative that MS-compatible clonotypic M-protein peptides are identified. To support implementation of molecular MRD techniques, we studied the presence and stability of these clonotypic features in the CoMMpass database. Methods An analysis pipeline based on MiXCR and HIGH-VQUEST was constructed to identify clonal molecular fingerprints and their clonotypic peptides based on transcriptomic datasets. To determine the stability of the clonal fingerprints, we compared the clonal fingerprints during disease progression for each patient. Results The analysis pipeline to establish the clonal fingerprint and MS-suitable clonotypic peptides was successfully validated in MM cell lines. In a cohort of 609 patients with MM, we demonstrated that the most abundant clone harbored a unique clonal molecular fingerprint and that multiple unique clonotypic peptides compatible with MS measurements could be identified for all patients. Furthermore, the clonal immunoglobulin gene fingerprints of both the light and heavy chain remained stable during MM disease progression. Conclusions Our data support the use of the clonal immunoglobulin gene fingerprints in patients with MM as a suitable MRD target for MS-MRD analyses.

Aims: Patients with diabetes have a higher incidence of infections with Candida albicans, Staphylococcus aureus and Mycobacterium tuberculosis, yet factors contributing to this increased risk are largely unknown. We hypothesize that altered innate and adaptive immune responses during diabetes contribute to an increased susceptibility to infections. Materials and methods: We studied cytokine responses to ex vivo pathogenic stimulations in a cohort with type 1 diabetes (n = 243) and non-diabetic healthy control subjects (n = 56) using isolated peripheral blood mononuclear cells (PBMCs). Clinical phenotypical data including BMI, duration of diabetes, and HbA1c levels were collected and related to the cytokine production capacity. Results: Adjusted for age, sex and BMI, the presence of diabetes was associated with significantly lower IL-1β, IL-6, TNF-α, and IL-17 production upon ex vivo stimulation of PBMCs with C. albicans and S. aureus (all, p < 0.05). In response to stimulation with M. tuberculosis only IL-17 (p < 0.001) was lower in patients with diabetes. Patients with the shortest diabetes duration had a significant lower IL-1β, IL-6 and TNF-α production (all, p < 0.01) after M. tuberculosis stimulation. Older patients had a significant lower IFN-γ (p < 0.05) production after stimulation with all three pathogens. HbA1c levels and BMI had no significant impact on cytokine production. Conclusions: PBMCs of patients with type 1 diabetes demonstrate significantly lower cytokine production in response to stimulation with several pathogens, which likely explain, at least in part, the increased susceptibility for these infections.

An effective response to the coronavirus disease 2019 (COVID-19) pandemic requires a better understanding of the biology of the infection and the identification of validated biomarker profiles that would increase the availability, accuracy, and speed of COVID-19 testing. Here, we describe the strategic objectives and action lines of the European Alliance of Medical Research Infrastructures (AMRI), established to improve the research process and tackle challenges related to diagnostic tests and biomarker development. Recommendations include: the creation of a European taskforce for validation of novel diagnostic products, the definition and promotion of criteria for COVID-19 samples biobanking, the identification and validation of biomarkers as clinical endpoints for clinical trials, and the definition of immune biomarker signatures at different stages of the disease. An effective management of the COVID-19 pandemic is possible only if there is a high level of knowledge and coordination between the public and private sectors within a robust quality framework., Oldoni et al debate relevant issues related to coronavirus disease 2019 testing and biomarker development. European Medical Research Infrastructures call for a high level of knowledge and coordination between public and private sectors, likewise for quality and reproducibility along the research pipeline.

Background: Overweight and obesity can lead to adipose tissue inflammation, which causes insulin resistance and on the long-term type 2 diabetes mellitus (T2D). The inflammatory changes of obese-adipose tissue are characterized by macrophage infiltration and activation, but validated circulating biomarkers for adipose tissue inflammation for clinical use are still lacking. One of the most secreted enzymes by activated macrophages is chitotriosidase (CHIT1). Objective: To test whether circulating CHIT1 enzymatic activity levels reflect adipose tissue inflammation. Methods: Plasma and adipose tissue samples of 105 subjects (35 lean, 37 overweight, and 33 T2D patients) were investigated. CHIT1 mRNA levels were determined in adipose tissue-resident innate immune cells. CHIT1 mRNA levels, protein abundance, and plasma enzymatic activity were subsequently measured in adipose tissue biopsies and plasma of control subjects with varying levels of obesity and adipose tissue inflammation as well as in T2D patients. Results: In adipose tissue, CHIT1 mRNA levels were higher in stromal vascular cells compared to adipocytes, and higher in adipose tissue-residing macrophages compared to circulating monocytes (p < 0.001). CHIT1 mRNA levels in adipose tissue were enhanced in overweightcompared to lean subjects and even more in T2D patients (p < 0.05). In contrast, plasma CHIT1 enzymatic activity did not differ between lean, overweight subjects and T2D patients. A mutation of the CHIT1 gene decreases plasma CHIT1 activity. Conclusions: CHIT1 is expressed by adipose tissue macrophages and expression is higher in overweight subjects and T2D patients, indicating its potential as tissue biomarker for adipose tissue inflammation. However, these differences do not translate into different plasma CHIT1 activity levels. Moreover, a common CHIT1 gene mutation causing loss of plasma CHIT1 activity interferes with its use as a biomarker of adipose tissue inflammation. These results indicate that plasma CHIT1 activity is of limited value as a circulating biomarker for adipose tissue inflammation in human subjects.

International audience; Thanks to proteomics investigations, our vision of the role of different protein isoforms in the pathophysiology of diseases has largely evolved. The idea that protein biomarkers like tau, amyloid peptides, ApoE, cystatin, or neurogranin are represented in body fluids as single species is obviously over-simplified, as most proteins are present in different isoforms and subjected to numerous processing and post-translational modifications. Measuring the intact mass of proteins by MS has the advantage to provide information on the presence and relative amount of the different proteoforms. Such Top-Down approaches typically require a high degree of sample pre-fractionation to allow the MS system to deliver optimal performance in terms of dynamic range, mass accuracy and resolution. In clinical studies, however, the requirements for pre-analytical robustness and sample size large enough for statistical power restrict the routine use of a high degree of sample pre-fractionation. In this study, we have investigated the capacities of current-generation Ultra-High Resolution Q-Tof systems to deal with high complexity intact protein samples and have evaluated the approach on a cohort of patients suffering from neurodegenerative disease. Statistical analysis has shown that several proteoforms can be used to distinguish Alzheimer disease patients from patients suffering from other neurodegenerative disease.SIGNIFICANCE:Top-down approaches have an extremely high biological relevance, especially when it comes to biomarker discovery, but the necessary pre-fractionation constraints are not easily compatible with the robustness requirements and the size of clinical sample cohorts. We have demonstrated that intact protein profiling studies could be run on UHR-Q-ToF with limited pre-fractionation. The proteoforms that have been identified as candidate biomarkers in the-proof-of concept study are derived from proteins known to play a role in the pathophysiology process of Alzheimer disease.

Contains fulltext : 225249.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Assessing the specificity of protein binders is an essential first step in protein biomarker assay development. Affimers are novel protein binders and can potentially replace antibodies in multiple protein capture-based assays. Affimers are selected for their high specificity against the target protein and have benefits over antibodies like batch-to-batch reproducibility and are stable across a wide range of chemical conditions. Here we mimicked a typical initial screening of affimers and commercially available monoclonal antibodies against two non-related proteins, IL-37b and proinsulin, to assess the potential of affimers as alternative to antibodies. METHODS: Binding specificity of anti-IL-37b and anti-proinsulin affimers and antibodies was investigated via magnetic bead-based capture of their recombinant protein targets in human plasma. Captured proteins were analyzed using SDS-PAGE, Coomassie blue staining, Western blotting and LC-MS/MS-based proteomics. RESULTS: All affimers and antibodies were able to bind their target protein in human plasma. Gel and LC-MS/MS analysis showed that both affimer and antibody-based captures resulted in co-purified background proteins. However, affimer-based captures showed the highest relative enrichment of IL-37b and proinsulin. CONCLUSIONS: For both proteins tested, affimers show higher specificity in purifying their target proteins from human plasma compared to monoclonal antibodies. These results indicate that affimers are promising antibody-replacement tools for protein biomarker assay development.

Contains fulltext : 225915.pdf (Publisher’s version ) (Open Access) Parkinson's disease (PD) is the most common neurodegenerative disorder of movement worldwide. To date, only symptomatic treatments are available. Implantation of collagen-encapsulated human umbilical cord mesenchymal stem cells (hUC-MSCs) is being developed as a novel therapeutic approach to potentially modify PD progression. However, implanted collagen scaffolds may induce a host tissue response. To gain insight into such response, hUC-MSCs were encapsulated into collagen hydrogels and implanted into the striatum of hemi-Parkinsonian male Sprague-Dawley rats. One or 14 days after implantation, the area of interest was dissected using a cryostat. Total protein extracts were subjected to tryptic digestion and subsequent LC-MS/MS analyses for protein expression profiling. Univariate and multivariate analyses were performed to identify differentially expressed protein profiles with subsequent gene ontology and pathway analysis for biological interpretation of the data; 2,219 proteins were identified by MaxQuant at 1% false discovery rate. A high correlation of label-free quantification (LFQ) protein values between biological replicates (r = .95) was observed. No significant differences were observed between brains treated with encapsulated hUC-MSCs compared to appropriate controls. Proteomic data were highly robust and reproducible, indicating the suitability of this approach to map differential protein expression caused by the implants. The lack of differences between conditions suggests that the effects of implantation may be minimal. Alternatively, effects may only have been focal and/or could have been masked by nonrelevant high-abundant proteins. For follow-up assessment of local changes, a more accurate dissection technique, such as laser micro dissection, and analysis method are recommended.

Aim To investigate whether a history of severe hypoglycaemia (SH) or the associated presence of impaired awareness of hypoglycaemia (IAH) is characterized by a pro‐inflammatory profile in people with type 1 diabetes. Research design and methods We measured circulating inflammatory markers and pro‐ and anti‐inflammatory cytokine production after ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) in a well‐characterized cohort of individuals with type 1 diabetes (n = 239) and in people without diabetes (n = 56). Data were corrected for confounders by using multivariate linear regression models. Results People with type 1 diabetes had higher circulating concentrations of high‐sensitivity C‐reactive protein (hs‐CRP; 0.91 [0.36–2.25] vs. 0.52 [0.20–0.98] pg/mL, P < 0.001 and interleukin‐18‐binding protein (IL‐18BP; 1746 [1304–2112] vs. 1381 [1191–1807] pg/mL; P = 0.001) than those without diabetes. In multivariate analysis, only higher hs‐CRP concentrations persisted. Neither circulating immune cells nor ex vivo cytokine levels produced by PBMCs in response to an extensive panel of stimuli differed in groups defined by awareness state or a history of SH, apart from elevated IL‐18BP in people with, versus those without, history of SH (1524 [1227–1903] vs. 1913 [1459–2408] pg/mL; P < 0.001). Conclusions IAH or history of SH in people with type 1 diabetes was not associated with altered inflammatory profiles, arguing against chronically elevated inflammatory activity mediating the increased cardiovascular risk associated with hypoglycaemia. The finding of higher circulating concentrations of IL‐18BP in individuals with a history of SH requires further investigation.

© 2020 The Author(s)., [Introduction]: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. [Areas covered]: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. [Expert commentary]: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine., This paper was funded by the European cooperation in science and technology - COST action No. CA16113 - CliniMARK: ‘good biomarker practice’ to increase the number of clinically validated biomarkers.

ObjectivePrevious studies indicate a role for Oncostatin M (OSM) in atherosclerosis and other chronic inflammatory diseases for which inhibitory antibodies are in development. However, to date no intervention studies with OSM have been performed, and its relation to coronary heart disease (CHD) has not been studied.Approach and resultsGene expression analysis on human normal arteries (n = 10) and late stage/advanced carotid atherosclerotic arteries (n = 127) and in situ hybridization on early human plaques (n = 9) showed that OSM, and its receptors, OSM receptor (OSMR) and Leukemia Inhibitory Factor Receptor (LIFR) are expressed in normal arteries and atherosclerotic plaques. Chronic OSM administration in APOE*3Leiden.CETP mice (n = 15/group) increased plasma E-selectin levels and monocyte adhesion to the activated endothelium independently of cholesterol but reduced the amount of inflammatory Ly-6CHigh monocytes and atherosclerotic lesion size and severity. Using aptamer-based proteomics profiling assays high circulating OSM levels were shown to correlate with post incident CHD survival probability in the AGES-Reykjavik study (n = 5457).ConclusionsChronic OSM administration in APOE*3Leiden.CETP mice reduced atherosclerosis development. In line, higher serum OSM levels were correlated with improved post incident CHD survival probability in patients, suggesting a protective cardiovascular effect.

Introduction : Obesity is associated with adipose tissue inflammation which in turn drives insulin resistance and the development of type 2 diabetes. Oxylipins are a collection of lipid metabolites, subdivided in different classes, which are involved in inflammatory cascades. They play important roles in regulating adipose tissue homeostasis and inflammation and are therefore putative biomarkers for obesity-associated adipose tissue inflammation and the subsequent risk of type 2 diabetes onset. The objective for this study is to design an assay for a specific oxylipin class and evaluate these as potential prognostic biomarker for obesity-associated adipose tissue inflammation and type 2 diabetes. Methods : An optimized workflow was developed to extract oxylipins from plasma using solid-phase extraction followed by analysis using ultra-high performance liquid chromatography coupled to a triple quadrupole mass spectrometer in multiple reaction monitoring mode. This workflow was applied to clinical plasma samples obtained from obese-type 2 diabetes patients and from lean and obese control subjects. Results : The assay was analytically validated and enabled reproducible analyses of oxylipins extracted from plasma with acceptable sensitivities. Analysis of clinical samples revealed discriminative values for four oxylipins between the type 2 diabetes patients and the lean and obese control subjects, viz. PGF2α, PGE2, 15-keto-PGE2 and 13,14-dihydro-15-keto-PGE2. The combination of PGF2α and 15-keto-PGE2 had the most predictive value to discriminate type 2 diabetic patients from lean and obese controls. Conclusions : This proof-of-principle study demonstrates the potential value of oxylipins as biomarkers to discriminate obese individuals from obese-type 2 diabetes patients.

Background Choosing which biomarker tests to select for further research and development is not only a matter of diagnostic accuracy, but also of the clinical and monetary benefits downstream. Early health economic modeling provides tools to assess the potential effects of biomarker innovation and support decision-making. Methods We applied early health economic modeling to the case of diagnosing primary aldosteronism in patients with resistant hypertension. We simulated a cohort of patients using a Markov cohort state-transition model. Using the headroom method, we compared the currently used aldosterone-to-renin ratio to a hypothetical new test with perfect diagnostic properties to determine the headroom based on quality-adjusted life-years (QALYs) and costs, followed by threshold analyses to determine the minimal diagnostic accuracy for a cost-effective product. Results Our model indicated that a perfect diagnostic test would yield 0.027 QALYs and increase costs by €43 per patient. At a cost-effectiveness threshold of €20,000 per QALY, the maximum price for this perfect test to be cost-effective is €498 (95% confidence interval [CI]: €275–€808). The value of the perfect test was most strongly influenced by the sensitivity of the current biomarker test. Threshold analysis showed the novel test needs a sensitivity of at least 0.9 and a specificity of at least 0.7 to be cost-effective. Conclusions Our model-based approach evaluated the added value of a clinical biomarker innovation, prior to extensive investment in development, clinical studies and implementation. We conclude that early health economic modeling can be a valuable tool when prioritizing biomarker innovations in the laboratory.

Background The complement system is a central component of the innate immune system. Constitutive biosynthesis of complement proteins is essential for homeostasis. Dysregulation as a consequence of genetic or environmental cues can lead to inflammatory syndromes or increased susceptibility to infection. However, very little is known about steady state levels in children or its kinetics during infection. Methods With a newly developed multiplex mass spectrometry-based method we analyzed the levels of 32 complement proteins in healthy individuals and in a group of pediatric patients infected with bacterial or viral pathogens. Findings In plasma from young infants we found reduced levels of C4BP, ficolin-3, factor B, classical pathway components C1QA, C1QB, C1QC, C1R, and terminal pathway components C5, C8, C9, as compared to healthy adults; whereas the majority of complement regulating (inhibitory) proteins reach adult levels at very young age. Both viral and bacterial infections in children generally lead to a slight overall increase in complement levels, with some exceptions. The kinetics of complement levels during invasive bacterial infections only showed minor changes, except for a significant increase and decrease of CRP and clusterin, respectively. Interpretation The combination of lower levels of activating and higher levels of regulating complement proteins, would potentially raise the threshold of activation, which might lead to suppressed complement activation in the first phase of life. There is hardly any measurable complement consumption during bacterial or viral infection. Altogether, expression of the complement proteins appears surprisingly stable, which suggests that the system is continuously replenished. Fund European Union's Horizon 2020, project PERFORM, grant agreement No. 668303., Graphical abstract Unlabelled Image

Introduction: The onset of type 2 diabetes mellitus (T2DM) is strongly associated with obesity and subsequent perturbations in immuno-metabolic responses. To understand the complexity of these systemic changes and better monitor the health status of people at risk, validated clinical biomarkers are needed. Omics technologies are increasingly applied to measure the interplay of genes, proteins and metabolites in biological systems, which is imperative in understanding molecular mechanisms of disease and selecting the best possible molecular biomarkers for clinical use. Areas covered: This review describes the complex onset of T2DM, the contribution of obesity and adipose tissue inflammation to the T2DM disease mechanism, and the output of current biomarker strategies. A new biomarker approach is described that combines published and new self-generated data to merge multiple -omes (i.e. genome, proteome, metabolome etc.) toward understanding of mechanism of disease on the individual level and design multiparameter biomarker panels that drive significant impacts on personalized healthcare. Expert commentary: We here propose an approach to use cross-omics analyses to contextualize published biomarker data and better understand molecular mechanisms of health and disease. This will improve the current and future innovation gaps in translation of discovered putative biomarkers to clinically applicable biomarker tests. © 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group.

AIMS:Endothelial activation is involved in many chronic inflammatory diseases, such as atherosclerosis, and is often initiated by cytokines. Oncostatin M (OSM) is a relatively unknown cytokine that has been suggested to play a role in both endothelial activation and atherosclerosis. We comprehensively investigated the effect of OSM on endothelial cell activation from different vascular beds and in APOE*3Leiden.CETP mice. METHODS AND RESULTS:Human umbilical vein endothelial cells, human aortic endothelial cells and human microvascular endothelial cells cultured in the presence of OSM express elevated MCP-1, IL-6 and ICAM-1 mRNA levels. Human umbilical vein endothelial cells and human aortic endothelial cells additionally expressed increased VCAM-1 and E-selectin mRNA levels. Moreover, ICAM-1 membrane expression is increased as well as MCP-1, IL-6 and E-selectin protein release. A marked increase was observed in STAT1 and STAT3 phosphorylation indicating that the JAK/STAT pathway is involved in OSM signaling. OSM signals through the LIF receptor alfa (LIFR) and the OSM receptor (OSMR). siRNA knockdown of the LIFR and the OSMR revealed that simultaneous knockdown is necessary to significantly reduce MCP-1 and IL-6 secretion, VCAM-1 and E-selectin shedding and STAT1 and STAT3 phosphorylation after OSM stimulation. Moreover, OSM administration to APOE*3Leiden.CETP mice enhances plasma E-selectin levels and increases ICAM-1 expression and monocyte adhesion in the aortic root area. Furthermore, Il-6 mRNA expression was elevated in the aorta of OSM treated mice. CONCLUSION:OSM induces endothelial activation in vitro in endothelial cells from different vascular beds through activation of the JAK/STAT cascade and in vivo in APOE*3Leiden.CETP mice. Since endothelial activation is an initial step in atherosclerosis development, OSM may play a role in the initiation of atherosclerotic lesion formation.

BACKGROUND AND AIMS:With the intention to gain support for the hypothesis that incident ischemic complications of atherosclerotic disease involve a stochastic aspect, we performed a histological, qualitative evaluation of the epidemiology of coronary atherosclerotic disease in a cohort of aortic valve donors. PATIENTS AND METHODS:Donors (n = 695, median age 54, range 11-65 years) were dichotomized into a non-cardiovascular (non-CVD) and a cardiovascular disease death (CVD) group. Consecutive 5 mm proximal left coronary artery segments were Movat stained, and the atherosclerotic burden for each segment was graded (revised AHA-classification). RESULTS:Non-CVD and CVD groups showed steep increase of atherosclerosis severity beyond the age of 40, resulting in an endemic presence of advanced atherosclerosis in men over 40 and women over 50 years. In fact, only 19% of the non-CVD and 6% of the CVD donors over 40 years were classified with a normal LCA or a so called non-progressive lesion type. Fibrous calcified plaques (FCP), the consolidated remnants of earlier ruptured lesions, dominated in both non-CVD and CVD donors. Estimates of the atherosclerosis burden (i.e. average lesion grade, proportion of FCPs, and average number of FCPs per cross-section) were all higher in the CVD group (p

Few biomarkers progress from discovery to become validated tools or diagnostics. To bridge this gap, three European biomedical research infrastructures — EATRIS-ERIC (focused on translational medicine), BBMRI-ERIC (focused on biobanking) and ELIXIR (focused on data sharing) — are paving the way to developing and sharing best practices for biomarker validation. Few biomarkers progress from discovery to become validated tools or diagnostics. To bridge this gap, three European biomedical research infrastructures — EATRIS-ERIC (focused on translational medicine), BBMRI-ERIC (focused on biobanking) and ELIXIR (focused on data sharing) — are paving the way to developing and sharing best practices for biomarker validation.

BACKGROUND Because cerebrospinal fluid (CSF) is in close contact with diseased areas in neurological disorders, it is an important source of material in the search for molecular biomarkers. However, sample handling for CSF collected from patients in a clinical setting might not always be adequate for use in proteomics and metabolomics studies. METHODS We left CSF for 0, 30, and 120 min at room temperature immediately after sample collection and centrifugation/removal of cells. At 2 laboratories CSF proteomes were subjected to tryptic digestion and analyzed by use of nano-liquid chromatography (LC) Orbitrap mass spectrometry (MS) and chipLC quadrupole TOF-MS. Metabolome analysis was performed at 3 laboratories by NMR, GC-MS, and LC-MS. Targeted analyses of cystatin C and albumin were performed by LC–tandem MS in the selected reaction monitoring mode. RESULTS We did not find significant changes in the measured proteome and metabolome of CSF stored at room temperature after centrifugation, except for 2 peptides and 1 metabolite, 2,3,4-trihydroxybutanoic (threonic) acid, of 5780 identified peptides and 93 identified metabolites. A sensitive protein stability marker, cystatin C, was not affected. CONCLUSIONS The measured proteome and metabolome of centrifuged human CSF is stable at room temperature for up to 2 hours. We cannot exclude, however, that changes undetectable with our current methodology, such as denaturation or proteolysis, might occur because of sample handling conditions. The stability we observed gives laboratory personnel at the collection site sufficient time to aliquot samples before freezing and storage at −80 °C.

The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

We introduce a matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FT-ICR) method for quantitative peptide profiling, using peak height as a measure for abundance. Relative standard deviations in peak height of peptides spiked over 3 orders of magnitude in concentration were below 10% and allowed for accurate comparisons between multiple sclerosis and controls. Application on a set of 163 cerebrospinal fluid (CSF) samples showed significantly differential abundant peptides, which were subsequently identified into proteins (e.g., chromogranin A, clusterin, and complement C3).

To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples, The factors assessed were a 30 or 120 min time delay at room temperature before storage at -80 degrees C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 degrees C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze-thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 degrees C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at -80 degrees C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.

International audience; Cockayne syndrome is a multisystem sun-sensitive genetic disorder associated with a specific defect in the ability to perform transcription-coupled repair of active genes after UV irradiation. Two complementation groups (CS-A and CS-B) have been identified, and 80% of patients have been assigned to the CS-B complementation group. We have analyzed the sites of the mutations in the CSB gene in 16 patients, to determine the spectrum of mutations in this gene and to see whether the nature of the mutation correlates with the type and severity of the clinical symptoms. In nine of the patients, the mutations resulted in truncated products in both alleles, whereas, in the other seven, at least one allele contained a single amino acid change. The latter mutations were confined to the C-terminal two-thirds of the protein and were shown to be inactivating by their failure to restore UV-irradiation resistance to hamster UV61 cells, which are known to be defective in the CSB gene. Neither the site nor the nature of the mutation correlated with the severity of the clinical features. Severe truncations were found in different patients with either classical or early-onset forms of the disease.

Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His6-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine. As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts. These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions.

In the past years, it has become increasingly evident that basal metabolic processes within the cell are intimately linked and influenced by one another. One such link that recently has attracted much attention is the close interplay between nucleotide excision DNA repair and transcription. This is illustrated both by the preferential repair of the transcribed strand of active genes (a phenomenon known as transcription-coupled repair, TCR) as well as by the distinct dual involvement of proteins in both processes. The mechanism of TCR in eukaryotes is still largely unknown. It was first discovered in mammals by the pioneering studies of Hanawalt and colleagues, and subsequently identified in yeast and Escherichia coli. In the latter case, one protein, the transcription repair-coupling factor, was found to accomplish this function in vitro, and a plausible model for its activity was proposed. While the E. coli model still functions as a paradigm for TCR in eukaryotes, recent observations prompt us to believe that the situation in eukaryotes is much more complex, involving dual functionality of multiple proteins.

International audience; Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription.

Contains fulltext : 128506.pdf (Publisher’s version ) (Open Access) Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification-mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP.

Multiple Sclerosis (MScl) is a neurodegenerative disease of the CNS, associated with chronic neuroinflammation. Cerebrospinal fluid (CSF), being in closest interaction with CNS, was used to profile neuroinflammation to discover disease-specific markers. We used the commonly accepted animal model for the neuroinflammatory aspect of MScl: the experimental autoimmune/allergic encephalomyelitis (EAR). A combination of advanced (1)H NMR spectroscopy and pattern recognition methods was used to establish the metabolic profile of CSF of EAE-affected rats (representing neuroinflammation) and of two control groups (healthy and peripherally inflamed) to detect specific markers for early neuroinflammation. We found that the CSF metabolic profile for neuroinflammation is distinct from healthy and peripheral inflammation and characterized by changes in concentrations of metabolites such as creatine, arginine, and lysine. Using these disease-specific markers, we were able to detect early stage neuroinflammation, with high accuracy in a second independent set of animals. This confirms the predictive value of these markers. These findings from the EAE model may help to develop a molecular diagnosis for the early stage MScl in humans.

The nonhuman primates most commonly used in medical research are from the genus Macaca1. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.

The pharmaceutical industry must find ways to improve the unacceptably high attrition rate during drug development. Clearly, pharma has moved away from treat-and-see testing of new drugs in patients, with a strong current focus on generating translational biomarkers early in the research process to enable more predictive evaluation of drug action in clinical trials. Underlying such a translational medicine approach is the intensive search for and use of high-quality biomarkers indicative of successful drug target engagement, pharmacological effects, efficacy or safety. This review outlines our rational question-based drug development strategy in which biomarker data drive decisions on which drug candidates to progress to clinical testing.

Rodent ultraviolet light (UV)-sensitive mutant cells in complementation groups (CGs) 1 and 4 normally are known for their extraordinary (approximately 80-100 x) sensitivity to mitomycin C (MMC), although some CG1 mutants with reduced MMC sensitivity were previously reported (Stefanini et al. (1987) Cytotechnology 1, 91). We report here new CG1 and CG4 mutants with only 1.6-10 x wild-type MMC sensitivity despite low unscheduled DNA synthesis (UDS) levels. Mutant UV140, in UV CG4, has approximately 3.8 x the UV sensitivity of parental line AA8, approximately 1.6 x wild-type MMC sensitivity, wild-type X-ray and ethyl methanesulfonate (EMS) sensitivity, and is only slightly (approximately 1.4 x) hypermutable to 8-azaadenine resistance by UV light. It has moderately decreased incision of UV-damaged DNA, has moderately decreased removal of (6-4) photoproducts, and is profoundly deficient in UDS after UV. After UV, it shows abnormally decreased DNA synthesis and persistently decreased RNA synthesis. In addition a cell-free extract of this mutant displays strongly reduced nucleotide excision repair synthesis using DNA treated with N-acetoxy-acetyl-amino-fluorene (AAF). The extract selectively fails to complement extracts of group 1 and 4 mutants consistent with the notion that the affected proteins, ERCC1 and ERCC4, are part of the same complex and that mutations in one subunit also affect the other component. Mutant UV212 is a CG1 mutant with approximately 3.3 x wild-type UV and approximately 5-10 x wild-type MMC sensitivity, with profoundly deficient UDS and hypermutability (approximately 5.8 x) by UV. Mutant UV201, probably in CG1, is only slightly (approximately 1.5 x) UV-sensitive and has near wild-type (1.02X) UV mutability. These unusual group 1 and 4 mutants demonstrate that the unique UV and MMC sensitivity phenotypes displayed by these groups can be separated and support the idea that they are the result of distinct repair functions of the corresponding ERCC1 and ERCC4 genes: nucleotide excision repair for UV lesions and a separate repair pathway for removal of interstrand crosslinks.

Cells from patients with the UV-sensitive nucleotide excision repair disorder Cockayne's syndrome (CS) have a specific defect in preferential repair of lesions from the transcribed strand of active genes. This system permits quick resumption of transcription after UV exposure. Here we report the characterization of ERCC6, a gene involved in preferential repair in eukaryotes. ERCC6 corrects the repair defect of CS complementation group B (CS-B). It encodes a protein of 1493 amino acids, containing seven consecutive domains conserved between DNA and RNA helicases. The entire helicase region bears striking homology to segments in recently discovered proteins involved in transcription regulation, chromosome stability, and DNA repair. Mutation analysis of a CS-B patient indicates that the gene is not essential for cell viability and is specific for preferential repair of transcribed sequences.