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1.  Effects of perhexiline-induced fuel switch on the cardiac proteome and metabolome 
Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms of heart disease. It provides an oxygen sparing effect in the myocardium by creating a switch from fatty acid to glucose metabolism through partial inhibition of carnitine palmitoyltransferase 1 and 2. However, the precise molecular mechanisms underlying the cardioprotective effects elicited by perhexiline are not fully understood. The present study employed a combined proteomics, metabolomics and computational approach to characterise changes in murine hearts upon treatment with perhexiline. According to results based on difference in-gel electrophoresis, the most profound change in the cardiac proteome related to the activation of the pyruvate dehydrogenase complex. Metabolomic analysis by high-resolution nuclear magnetic resonance spectroscopy showed lower levels of total creatine and taurine in hearts of perhexiline-treated mice. Creatine and taurine levels were also significantly correlated in a cross-correlation analysis of all metabolites. Computational modelling suggested that far from inducing a simple shift from fatty acid to glucose oxidation, perhexiline may cause complex rebalancing of carbon and nucleotide phosphate fluxes, fuelled by increased lactate and amino acid uptake, to increase metabolic flexibility and to maintain cardiac output. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".
Graphical abstract
► Mice were fed perhexiline to achieve steady state concentrations. ► Hearts were analysed using a combined proteomic and metabolomic approach. ► Computer modelling was used to cross-validate the findings. ► Perhexiline has more wide-ranging and complex metabolic effects than previously thought.
PMCID: PMC3573230  PMID: 23277191
CPT, carnitine palmitoyltransferase; DIGE, difference in-gel electrophoresis; FCS, foetal calf serum; FDR, false discovery rate; GO, Gene ontology; 1H NMR, proton nuclear magnetic resonance spectroscopy; LC-MS/MS, liquid chromatography tandem mass spectrometry; TCA, tricarboxylic acid; Metabolomics; Proteomics; Cardioprotection; Metabolism; Heart failure
2.  Novel Role of ADAMTS-5 Protein in Proteoglycan Turnover and Lipoprotein Retention in Atherosclerosis* 
The Journal of Biological Chemistry  2012;287(23):19341-19345.
Background: In atherosclerosis, proteoglycan accumulation results in increased lipoprotein retention.
Results: ADAMTS-5 is reduced in aortas of apolipoprotein E-null mice. ADAMTS-5 deficiency impairs processing of vascular proteoglycans, and ADAMTS-5 activity affects proteoglycan-mediated lipoprotein retention.
Conclusion: ADAMTS-5 regulates vascular proteoglycan catabolism and alters lipoprotein retention.
Significance: This is the first study implicating ADAMTS-5 proteolytic activity in atherosclerosis.
Atherosclerosis is initiated by the retention of lipoproteins on proteoglycans in the arterial intima. However, the mechanisms leading to proteoglycan accumulation and lipoprotein retention are poorly understood. In this study, we set out to investigate the role of ADAMTS-5 (a disintegrin and metalloprotease with thrombospondin motifs-5) in the vasculature. ADAMTS-5 was markedly reduced in atherosclerotic aortas of apolipoprotein E-null (apoE−/−) mice. The reduction of ADAMTS-5 was accompanied by accumulation of biglycan and versican, the major lipoprotein-binding proteoglycans, in atherosclerosis. ADAMTS-5 activity induced the release of ADAMTS-specific versican (DPEAAE441) and aggrecan (374ALGS) fragments as well as biglycan and link protein from the aortic wall. Fibroblast growth factor 2 (FGF-2) inhibited ADAMTS-5 expression in isolated aortic smooth muscle cells and blocked the spontaneous release of ADAMTS-generated versican and aggrecan fragments from aortic explants. In aortas of ADAMTS-5-deficient mice, DPEAAE441 versican neoepitopes were not detectable. Instead, biglycan levels were increased, highlighting the role of ADAMTS-5 in the catabolism of vascular proteoglycans. Importantly, ADAMTS-5 proteolytic activity reduced the LDL binding ability of biglycan and released LDL from human aortic lesions. This study provides the first evidence implicating ADAMTS-5 in the regulation of proteoglycan turnover and lipoprotein retention in atherosclerosis.
PMCID: PMC3365970  PMID: 22493487
Atherosclerosis; Cardiovascular Disease; Extracellular Matrix; Lipoprotein; Protease; Proteoglycan; ADAMTS-5
3.  Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution* 
Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.
PMCID: PMC2849712  PMID: 20037178
4.  Antifactor Xa activity in critically ill patients receiving antithrombotic prophylaxis with standard dosages of certoparin: a prospective, clinical study 
Critical Care  2005;9(5):R541-R548.
Deep venous thrombosis with subsequent pulmonary embolism or post-thrombotic syndrome is a feared complication in the intensive care unit. Therefore, routine prophylactic anticoagulation is widely recommended. Aside from unfractionated heparin, low molecular weight heparins, such as certoparin, have become increasingly used for prophylactic anticoagulation in critically ill patients. In this prospective study, we evaluated the potency of 3,000 IU certoparin administered once daily to reach antithrombotic antifactor Xa (aFXa) levels of 0.1 to 0.3 IU/ml in 62 critically ill patients.
AFXa levels were determined 4, 12 and 24 h after injection of certoparin. Prothrombin time, activated partial thromboplastin time, antithrombin, fibrinogen, hemoglobin, platelet count, serum urea and creatinine concentrations were documented before and 12 and 24 h after injection of certoparin.
Four hours after certoparin injection (n = 32), 28% of patients were within the antithrombotic aFXa range. After 12 and 24 h, 6% achieved antithrombotic aFXa levels. Because of a severe pulmonary embolism in one study patient, an interim analysis was performed, and the dosage of certoparin was increased to 3,000 IU twice daily. This regime attained recommended antithrombotic aFXa levels in 47%, 27%, 40% and 30% of patients at 4, 12, 16 and 24 h, respectively, after twice daily certoparin injection (n = 30). Antithrombin and fibrinogen concentrations slightly increased during the observation period. Low antithrombin concentrations before certoparin were independently correlated with underdosing of certoparin. Patients with aFXa levels <0.1 IU/ml 4 h after certoparin injection required vasopressors more often and had lower serum concentrations of creatinine and urea than patients with antithrombotic aFXa levels.
Standard dosages of certoparin of 3,000 IU given once or twice daily are ineffective for attaining the recommended aFXa levels of 0.1 to 0.3 IU/ml in critically ill patients. Low antithrombin levels before certoparin administration were independently associated with low aFXa levels. Renal function and vasopressor therapy may further influence the effectiveness of certoparin in ensuring adequate antithrombotic prophylaxis.
PMCID: PMC1297619  PMID: 16277716
5.  Exacerbated vein graft arteriosclerosis in protein kinase Cδ–null mice 
Journal of Clinical Investigation  2001;108(10):1505-1512.
Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCδ knockout mice and performed vein bypass grafts on these animals. PKCδ–/– mice developed normally and were fertile. Vein segments from PKCδ–/– mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCδ+/+ vein grafts. Arteriosclerotic lesions in PKCδ–/– mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCδ–/– mice. SMCs derived from PKCδ–/– aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCδ–/– relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCδ maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.
PMCID: PMC209416  PMID: 11714742
6.  Freebies 
PMCID: PMC2306017  PMID: 21289744
7.  Competitiveness across the Life Span: The Feisty Fifties 
Psychology and aging  2011;27(2):278-285.
Existing theories on life-span changes in confidence or motivation suggest that individuals’ preferences to enter competitive situations should gradually decline with age. We examined competitive preferences in a field experiment using real financial stakes in 25 to 75 year olds (N=543). The critical dependent variable was whether participants chose to perform a simple mental arithmetic task either under a piece-rate payment schedule (i.e., $.25 per solved item) or a competitive payment schedule ($.50 per solved item if the overall score is better than that of a randomly selected opponent, $0 otherwise). Results revealed that competitive preferences increased across the life span until they peaked around age 50, and dropped thereafter. We also found that throughout, men had a substantially larger preference for competing than women—extending previous findings on college-aged participants. The age/gender differences in preferences were neither accounted for by actual differences in performance nor individuals’ subjective confidence. This first systematic attempt to characterize age differences in competitive behavior suggests that a simple decline conception of competitiveness needs to be reconsidered.
PMCID: PMC3969030  PMID: 22059714
8.  From data gathering to systems medicine 
Cardiovascular Research  2013;97(4):599-600.
PMCID: PMC3583261  PMID: 23386274
9.  Executive Dysfunction Assessed with a Task-Switching Task following Concussion 
PLoS ONE  2014;9(3):e91379.
Concussion frequently results in executive function deficits that can be specifically probed using task-switching tasks. The current study examined in detail the influence of concussion on task switching performance using both spatial and numerical stimuli. Individuals with concussion (n = 16) were tested within 48 hours of injury and 7, 14, and 28 days later. Healthy sex-, age-, height-, weight- and activity-matched controls (n = 16) were also tested at the same intervals. Switch costs were significantly greater in the participants with concussion than in the controls for both types of stimuli. By contrast, the global costs on non-switching trials were unaffected by concussion. We conclude that concussion has pronounced negative effects on the ability to switch task sets that generalize across task combinations (spatial or numerical) and that persist across at least a month after injury.
PMCID: PMC3950211  PMID: 24618717
10.  Relation of plasma adiponectin levels and aortic stiffness after acute ST-segment elevation myocardial infarction 
Pulse wave velocity is a measure of aortic stiffness and an independent predictor of cardiovascular morbidity and mortality. Adiponectin is involved in atherosclerosis and inflammation. In the present study we aimed to explore the association between plasma adiponectin concentrations and pulse wave velocity in the acute phase after ST-segment elevation myocardial infarction (STEMI).
Forty-six consecutive STEMI patients (mean age 57±11 years) treated with primary percutaneous coronary intervention (PCI) were enrolled in this cross-sectional study. Plasma adiponectin was measured 2 days after index event by enzyme-linked immunosorbent assay. Aortic pulse wave velocity (PWV) was calculated by the transit-time method with the use of a velocity-encoded, phase-contrast cardiac magnetic resonance protocol.
Median plasma adiponectin concentration was 2385 ng/ml (interquartile range 1735–5403). Males had lower plasma adiponectin values than females and current smokers had lower values than non-smokers (all p<0.02). Adiponectin was significantly associated with PWV (r=0.505, p<0.001), age (r=0.437, p=0.002), and total cholesterol (r=0.468, p=0.001). Multiple linear regression analysis revealed adiponectin as a predictor of PWV independently of age, sex, smoking status, total cholesterol, and N-terminal pro-B-type natriuretic peptide (p=0.027).
Plasma adiponectin concentrations are strongly associated with aortic stiffness in patients after acute STEMI treated with primary PCI. Our data support a possible role for adiponectin as an independent risk marker for increased aortic stiffness in STEMI patients.
PMCID: PMC3932779  PMID: 24337918
Adiponectin; aortic stiffness; cardiac magnetic resonance; pulse wave velocity; ST-segment elevation myocardial infarction
11.  Infection Rate and Acute Organ Dysfunction Risk as Explanations for Racial Differences in Severe Sepsis 
Severe sepsis, defined as infection complicated by acute organ dysfunction, occurs more frequently and leads to more deaths in black than in white individuals. The optimal approach to minimize these disparities is unclear.
To determine the extent to which higher severe sepsis rates in black than in white patients are due to higher infection rates or to a higher risk of acute organ dysfunction.
Design, Setting, and Participants
Analysis of infection-related hospitalizations from the 2005 hospital discharge data of 7 US states and infection-related emergency department visits from the 2003-2007 National Hospital Ambulatory Care Survey.
Main Outcome Measure
Age- and sex-standardized severe sepsis and infection hospitalization rates and the risk of acute organ dysfunction.
Of 8 661 227 non–childbirth-related discharges, 2 261 857 were associated with an infection, and of these, 381 787 (16.8%) had severe sepsis. Black patients had a 67% higher age- and sex-standardized severe sepsis rate than did white patients (9.4; 95% confidence interval [CI], 9.3-9.5 vs 5.6; 95% CI, 5.6-5.6 per 1000 population; P<.001) and 80% higher standardized mortality (1.8, 95% CI, 1.8-1.9 vs 1.0, 95% CI, 1.0-1.1 per 1000 population; P<.001). The higher severe sepsis rate was explained by both a higher infection rate in black patients (47.3; 95% CI, 47.1-47.4 vs 34.0; 95% CI, 33.9-34.0 per 1000 population; incidence rate ratio, 1.39; P<.001) and a higher risk of developing acute organ dysfunction (age- and sex-adjusted odds ratio [OR],1.29; 95% CI, 1.27-1.30; P<.001). Differences in infection presented broadly across different sites and etiology of infection and for community- and hospital-acquired infections and occurred despite a lower likelihood of being admitted for infection from the emergency department (adjusted OR, 0.70; 95% CI, 0.64-0.76; P<.001). The higher risk of organ dysfunction persisted but was attenuated after adjusting for age, sex, comorbid conditions, poverty, and hospital effect (OR, 1.14; 95% CI, 1.13-1.16; P<.001). Racial disparities in infection and severe sepsis incidence and mortality rates were largest among younger adults (eg, the proportion of invasive pneumococcal disease occurring in adults <65 years was 73.9% among black patients vs 44.5% among white patients, P<.001).
Racial differences in severe sepsis are explained by both a higher infection rate and a higher risk of acute organ dysfunction in black than in white individuals.
PMCID: PMC3910506  PMID: 20571016
12.  Adolescent Self-Organization Predicts Midlife Memory in a Prospective Birth Cohort Study 
Psychology and Aging  2013;28(4):958-968.
Childhood and adolescent mental health have a lasting impact on adult life chances, with strong implications for subsequent health, including cognitive aging. Using the British 1946 birth cohort, the authors tested associations between adolescent conduct problems, emotional problems and aspects of self-organization, and verbal memory at 43 years and rate of decline in verbal memory from 43 to 60–64 years. After controlling for childhood intelligence, adolescent self-organization was positively associated with verbal memory at 43 years, mainly through educational attainment, although not with rate of memory decline. Associations between adolescent conduct and emotional problems and future memory were of negligible magnitude. It has been suggested that interventions to improve self-organization may save a wide range of societal costs; this study also suggests that this might also benefit cognitive function in later life.
PMCID: PMC3906799  PMID: 24364401
self-organization; memory; life course
13.  Rapidly Progressive Lupus Nephritis with Extremely High Levels of Antineutrophil Cytoplasmic Antibodies 
A 43-year-old woman, with a 3-month history of fatigue, anaemia and swollen lymph nodes, underwent biopsy of a lymph node, which revealed reactive lymphadenopathy. Due to an increased serum creatinine concentration and severe proteinuria, a kidney biopsy was performed, which revealed diffuse, segmental, proliferative, immune-complex glomerulonephritis with crescents. Electron microscopy showed tubulo-reticular structures within one endothelial cell. These were a typical clinical presentation and compatible histopathological findings for systemic lupus erythematosus; however, the anti-myeloperoxidase antineutrophil cytoplasmic antibody (MPO-ANCA) level was extraordinarily high. In spite of treatment with intravenous cyclophosphamide and methylprednisolone pulse therapy, the patient's kidney function declined. Starting plasma exchange improved her renal function and removed MPO-ANCAs, which were suspected to play the major role in the pathogenesis of glomerulonephritis. These findings indicate that in addition to lupus nephritis, MPO-ANCAs may be involved in the pathogenesis of glomerulonephritis and that the coincidence of systemic lupus erythematosus and ANCA may be responsible for the severe clinical course in our patient.
PMCID: PMC3934786  PMID: 24575116
Lupus nephritis; MPO-ANCA; Plasma exchange
14.  Effects of paracentesis on hemodynamic parameters and respiratory function in critically ill patients 
BMC Gastroenterology  2014;14:18.
Ascites is a major and common complication of liver cirrhosis. Large or refractory ascites frequently necessitates paracentesis. The aim of our study was to investigate the effects of paracentesis on hemodynamic and respiratory parameters in critically ill patients.
Observational study comparing hemodynamic and respiratory parameters before and after paracentesis in 50 critically ill patients with advanced hemodynamic monitoring. 28/50 (56%) required mechanical ventilation.
Descriptive statistics are presented as mean ± standard deviation for normally distributed data and median, range, and interquartile range (IQR) for non-normally distributed data. Comparisons of hemodynamic and respiratory parameters before and after paracentesis were performed by Wilcoxon signed-rank tests. Bivariate relations were assessed by Spearman’s correlation coefficient and univariate regression analyses.
Median amount of ascites removed was 5.99 L (IQR, 3.33-7.68 L). There were no statistically significant changes in hemodynamic parameters except a decrease in mean arterial pressure (-7 mm Hg; p = 0.041) and in systemic vascular resistance index (-116 dyne·sec/cm5/m2; p = 0.016) when measured 2 hours after paracentesis. In all patients, oxygenation ratio (PaO2/FiO2; median, 220 mmHg; IQR, 161–329 mmHg) increased significantly when measured immediately (+58 mmHg; p = 0.001), 2 hours (+9 mmHg; p = 0.004), and 6 hours (+6 mmHg); p = 0.050) after paracentesis. In mechanically ventilated patients, lung injury score (cumulative points without x-ray; median, 6; IQR, 4–7) significantly improved immediately (5; IQR, 4–6; p < 0.001), 2 hours (5; IQR, 4–7; p = 0.003), and 6 hours (6; IQR 4–6; p = 0.012) after paracentesis.
Paracentesis in critically ill patients is safe regarding circulatory function and is related to immediate and sustained improvement of respiratory function.
PMCID: PMC3906760  PMID: 24467993
Ascites; Dynamic respiratory system compliance; Hemodynamics; Transpulmonary thermodilution; Hemodynamic monitoring
15.  Boosting the Concordance Index for Survival Data – A Unified Framework To Derive and Evaluate Biomarker Combinations 
PLoS ONE  2014;9(1):e84483.
The development of molecular signatures for the prediction of time-to-event outcomes is a methodologically challenging task in bioinformatics and biostatistics. Although there are numerous approaches for the derivation of marker combinations and their evaluation, the underlying methodology often suffers from the problem that different optimization criteria are mixed during the feature selection, estimation and evaluation steps. This might result in marker combinations that are suboptimal regarding the evaluation criterion of interest. To address this issue, we propose a unified framework to derive and evaluate biomarker combinations. Our approach is based on the concordance index for time-to-event data, which is a non-parametric measure to quantify the discriminatory power of a prediction rule. Specifically, we propose a gradient boosting algorithm that results in linear biomarker combinations that are optimal with respect to a smoothed version of the concordance index. We investigate the performance of our algorithm in a large-scale simulation study and in two molecular data sets for the prediction of survival in breast cancer patients. Our numerical results show that the new approach is not only methodologically sound but can also lead to a higher discriminatory power than traditional approaches for the derivation of gene signatures.
PMCID: PMC3882229  PMID: 24400093
16.  Immunosuppressive Glycodelin A is an independent marker for poor prognosis in endometrial cancer 
BMC Cancer  2013;13:616.
Knowledge on immunosuppressive factors in the pathogenesis of endometrial cancer is scarce. The aim of this study was to assess Glycodelin (Gd) and its immunosuppressive isoform Glycodelin A (GdA) in endometrial cancer tissue and to analyze its impact on clinical and pathological features and patient outcome.
292 patients diagnosed and treated for endometrial cancer were included. Patient characteristics, histology and follow-up data were available. Gd and GdA was determined by immunohistochemistry and in situ hybridization was performed for Gd mRNA.
Endometrial cancer shows intermediate (52.2%) or high (20.6%) expression for Gd in 72.8%, and GdA in 71.6% (intermediate 62.6%, high 9.0%) of all cases. The glycosylation dependent staining of GdA is tumour specific and correlates with the peptide-specific Gd staining though neither of the two is associated with estrogen receptor, progesterone receptor or clinic-pathological features. Also Gd protein positively correlates with Gd mRNA as quantified by in situ hybridization. Gd positive cases have a favourable prognosis (p = 0.039), while GdA positive patients have a poor outcome (p = 0.003). Cox-regression analysis proofed GdA to be an independent prognostic marker for patient survival (p = 0.002), besides tumour stage, grade and the concomitant diagnosis of hypertension.
Gd and GdA are commonly expressed in endometrial cancer tissue and seem to be of relevance in tumourigenesis. They differ not only in glycosylation but also in their biological activity, since only GdA holds prognostic significance for a poor overall survival in endometrial cancer patients. This finding might be explained by GdAs immunosuppressive capacity.
PMCID: PMC3898404  PMID: 24377825
Endometrial cancer; Glycodelin; Glycodelin A; Immunohistochemistry; In situ hybridization; Prognosis
17.  Epidemiology of severe sepsis 
Virulence  2013;5(1):4-11.
Severe sepsis is a leading cause of death in the United States and the most common cause of death among critically ill patients in non-coronary intensive care units (ICU). Respiratory tract infections, particularly pneumonia, are the most common site of infection, and associated with the highest mortality. The type of organism causing severe sepsis is an important determinant of outcome, and gram-positive organisms as a cause of sepsis have increased in frequency over time and are now more common than gram-negative infections.
Recent studies suggest that acute infections worsen pre-existing chronic diseases or result in new chronic diseases, leading to poor long-term outcomes in acute illness survivors. People of older age, male gender, black race, and preexisting chronic health conditions are particularly prone to develop severe sepsis; hence prevention strategies should be targeted at these vulnerable populations in future studies.
PMCID: PMC3916382  PMID: 24335434
severe sepsis; epidemiology; risk factors; infection; organ dysfunction
18.  The EndoPredict score provides prognostic information on late distant metastases in ER+/HER2− breast cancer patients 
British Journal of Cancer  2013;109(12):2959-2964.
ER+/HER2− breast cancers have a proclivity for late recurrence. A personalised estimate of relapse risk after 5 years of endocrine treatment can improve patient selection for extended hormonal therapy.
A total of 1702 postmenopausal ER+/HER2− breast cancer patients from two adjuvant phase III trials (ABCSG6, ABCSG8) treated with 5 years of endocrine therapy participated in this study. The multigene test EndoPredict (EP) and the EPclin score (which combines EP with tumour size and nodal status) were predefined in independent training cohorts. All patients were retrospectively assigned to risk categories based on gene expression and on clinical parameters. The primary end point was distant metastasis (DM). Kaplan–Meier method and Cox regression analysis were used in an early (0–5 years) and late time interval (>5 years post diagnosis).
EP is a significant, independent, prognostic parameter in the early and late time interval. The expression levels of proliferative and ER signalling genes contribute differentially to the underlying biology of early and late DM. The EPclin stratified 64% of patients at risk after 5 years into a low-risk subgroup with an absolute 1.8% of late DM at 10 years of follow-up.
The EP test provides additional prognostic information for the identification of early and late DM beyond what can be achieved by combining the commonly used clinical parameters. The EPclin reliably identified a subgroup of patients who have an excellent long-term prognosis after 5 years of endocrine therapy. The side effects of extended therapy should be weighed against this projected outcome.
PMCID: PMC3859949  PMID: 24157828
EndoPredict; endocrine therapy; late relapse
19.  Development and Validation of Sensitive Liquid Chromatography/Tandem Mass Spectrometry Methods for Quantification of Bendamustine in Mouse Brain Tissue 
A liquid chromatography-tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue was developed and fully validated. Methanol was used to precipitate proteins in brain tissue. Bendamustine and internal standard (chlorambucil) were separated with reverse phase chromatography on a C-18 column with a gradient of water and 95% methanol in 0.1% formic acid. Positive mode electrospray ionization was applied with selected reaction monitoring to achieve 5 ng/ml lower limits of quantitation in mouse brain tissue. The calibration curve for bendamustine in mouse brain was linear between 5 and 2000 ng/ml. The within- and between- batch accuracy and precision of the assay were within 15% at 10, 100 and 1000 ng/ml. The recovery and matrix effect of bendamustine in mouse brain tissue ranged from 41.1% to 51.6% and 107.4% to 110.3%, respectively. Validated the method was then applied to quantitate bendamustine in an animal study. Results indicate the assay can be applied to evaluate bendamustine disposition in mouse brain tissue. This assay will be applied in the future to detect and quantify bendamustine in human brain tissue samples.
PMCID: PMC3856370  PMID: 22925718
Bendamustine; Liquid Chromatography-Mass Spectrometry; LC-MS/MS; Brain
20.  Dense genotyping of immune-related disease regions identifies nine new risk loci for primary sclerosing cholangitis 
Liu, Jimmy Z. | Hov, Johannes Roksund | Folseraas, Trine | Ellinghaus, Eva | Rushbrook, Simon M. | Doncheva, Nadezhda T. | Andreassen, Ole A. | Weersma, Rinse K. | Weismüller, Tobias J. | Eksteen, Bertus | Invernizzi, Pietro | Hirschfield, Gideon M. | Gotthardt, Daniel Nils | Pares, Albert | Ellinghaus, David | Shah, Tejas | Juran, Brian D. | Milkiewicz, Piotr | Rust, Christian | Schramm, Christoph | Müller, Tobias | Srivastava, Brijesh | Dalekos, Georgios | Nöthen, Markus M. | Herms, Stefan | Winkelmann, Juliane | Mitrovic, Mitja | Braun, Felix | Ponsioen, Cyriel Y. | Croucher, Peter J. P. | Sterneck, Martina | Teufel, Andreas | Mason, Andrew L. | Saarela, Janna | Leppa, Virpi | Dorfman, Ruslan | Alvaro, Domenico | Floreani, Annarosa | Onengut-Gumuscu, Suna | Rich, Stephen S. | Thompson, Wesley K. | Schork, Andrew J. | Næss, Sigrid | Thomsen, Ingo | Mayr, Gabriele | König, Inke R. | Hveem, Kristian | Cleynen, Isabelle | Gutierrez-Achury, Javier | Ricaño-Ponce, Isis | van Heel, David | Björnsson, Einar | Sandford, Richard N. | Durie, Peter R. | Melum, Espen | Vatn, Morten H | Silverberg, Mark S. | Duerr, Richard H. | Padyukov, Leonid | Brand, Stephan | Sans, Miquel | Annese, Vito | Achkar, Jean-Paul | Boberg, Kirsten Muri | Marschall, Hanns-Ulrich | Chazouillères, Olivier | Bowlus, Christopher L. | Wijmenga, Cisca | Schrumpf, Erik | Vermeire, Severine | Albrecht, Mario | Rioux, John D. | Alexander, Graeme | Bergquist, Annika | Cho, Judy | Schreiber, Stefan | Manns, Michael P. | Färkkilä, Martti | Dale, Anders M. | Chapman, Roger W. | Lazaridis, Konstantinos N. | Franke, Andre | Anderson, Carl A. | Karlsen, Tom H.
Nature genetics  2013;45(6):670-675.
PMCID: PMC3667736  PMID: 23603763
genetic association study; disease genetics; immunogenetics; liver
21.  Infantile peripheral neuropathy, deafness, and proximal tubulopathy associated with a novel mutation of the RRM2B gene 
Croatian Medical Journal  2013;54(6):579-584.
Mitochondrial DNA depletion syndromes are a group of autosomal recessive hereditary disorders characterized by reduction of the amount of mitochondrial DNA in the affected tissue (muscle, liver, brain, or kidneys). We report a case of an infant with myopathy, deafness, peripheral neuropathy, nephrocalcinosis, proximal renal tubulopathy, moderate lactic acidosis, and a novel mutation of the RRM2B gene.
PMCID: PMC3893987  PMID: 24382854
22.  Phosphoregulation of the Titin-cap Protein Telethonin in Cardiac Myocytes* 
The Journal of Biological Chemistry  2013;289(3):1282-1293.
Background: Telethonin mutations are associated with cardiomyopathy through unknown mechanisms.
Results: Telethonin is a substrate for CaMK family kinases and exists in a bis-phosphorylated state in cardiomyocytes, in which non-phosphorylated telethonin disrupts transverse tubule organization and intracellular calcium transients.
Conclusion: Telethonin phosphorylation is critical for the maintenance of normal cardiomyocyte morphology and calcium handling.
Significance: Disruption of phospho-telethonin functions may contribute to pathogenesis in cardiomyopathy.
Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca2+/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca2+ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca2+ transients.
PMCID: PMC3894314  PMID: 24280220
CaMKII; Cardiac Muscle; Cardiomyopathy; Excitation-Contraction Coupling; Protein Kinase D (PKD); Protein Phosphorylation
23.  Does Minimally Invasive Total Knee Arthroplasty Improve Isokinetic Torque? 
Less invasive dissection of the extensor apparatus, one of the aspects of minimally invasive surgical (MIS) TKA, might result in less reduction of postoperative quadriceps strength. However, it is unclear whether MIS is associated with less strength reduction.
We asked whether knee strength (extensor or flexor torque) would be greater in patients having a MIS TKA compared with patients having a standard TKA and whether knee scores, alignment, ROM, tourniquet and operating times, and hospital length of stay were improved.
We prospectively recruited 52 patients awaiting MIS or standard TKA, matched for age, weight, height, BMI, sex, and surgically treated side, but not randomized. Isokinetic tests were performed preoperatively and postoperatively to obtain peak values of extensor and flexor torque. Postoperative-to-preoperative ratios and the extensor-flexor torque ratio also were calculated.
Mean baseline extensor torques were 57 Nm and 53 Nm for MIS and standard TKAs, respectively. At 8 weeks postoperatively, absolute values of the mean postoperative knee extensor torque also were similar: 47 and 45 Nm for patients with MIS and standard TKAs, respectively. The postoperative-to-preoperative ratios were 0.8 for extensor and flexor torque in both groups. The extensor-flexor torque ratios were similar between groups.
Our data suggest a less invasive dissection of the extensor apparatus involved in MIS TKA does not enhance postoperative quadriceps strength.
Level of Evidence
Level III, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
PMCID: PMC3462860  PMID: 22903286
24.  Alignment for total knee replacement: a comparison of kinematic axis versus mechanical axis techniques. A cadaver study 
International Orthopaedics  2012;36(11):2249-2253.
Standard instrumentation tries to reproduce mechanical axes based on mechanical alignment (MA) guides. A kinematic alignment (KA) technique derives its plan from pre-operative MRI-measurements. This matched-pair cadaveric study compared the resulting postoperative alignments.
A prospective series of 12 torsos were acquired for a total of 24 limb specimens including intact pelvises, femoral heads, knees, and ankles.The cadavers received MRI scans to manufacture the kinematic alignment cutting guides. Two investigating surgeons performed total knee arthroplasties on randomly chosen sides using MA instruments. On the contralateral sides, KA cutting guides were used. A navigation system was used to measure final alignment.
The overall alignment showed no significant differences between the systems. In the MA group the differences between the planned and the final implantation regarding overall limb alignment ranged between 0.2° and 6.2°. In the KA group the differences between the planned and final implantation regarding overall limb alignment ranged between 0.3° and 9.1°. The differences of the deviation from plan for overall limb alignment showed no significant differences between the methods.
The different alignment strategies resulted in variations of the combinations of the three-dimensional component position on the femur and the tibia. However, the legs were aligned within comparable range for both chosen techniques.
PMCID: PMC3479289  PMID: 22890847
25.  Malignant H1299 tumour cells preferentially internalize iron-bound inositol hexakisphosphate 
Bioscience Reports  2013;33(5):e00075.
In colon enterocytes and in well-differentiated colon cancer CaCo-2 cells, InsP6 (inositol hexakisphosphate) inhibits iron uptake by forming extracellular insoluble iron/InsP6 complexes. In this study, we confirmed that CaCo-2 cells are not able to take up iron/InsP6 but, interestingly, found that the cells are able to internalize metal-free and Cr3+-bound InsP6. Thus, the inability of CaCo-2 cells to take up iron/InsP6 complexes seems to be due to the iron-bound state of InsP6. Since recently we demonstrated that the highly malignant bronchial carcinoma H1299 cells internalize and process InsP6, we examined whether these cells may be able to take up iron/InsP6 complexes. Indeed, we found that InsP6 dose-dependently increased uptake of iron and demonstrated that in the iron-bound state InsP6 is more effectively internalized than in the metal-free or Cr3+-bound state, indicating that H1299 cells preferentially take up iron/InsP6 complexes. Electron microscope and cell fraction assays indicate that after uptake H1299 cells mainly stored InsP6/iron in lysosomes as large aggregates, of which about 10% have been released to the cytosol. However, this InsP6-mediated iron transport had no significant effects on cell viability. This result together with our finding that the well-differentiated CaCo-2 cells did not, but the malignant H1299 cells preferentially took up iron/InsP6, may offer the possibility to selectively transport cytotoxic substances into tumour cells.
PMCID: PMC3804887  PMID: 24050387
H1299 tumour cell; inositol hexakisphosphate; multiple-inositol-polyphosphate-phosphatase (MINPP1); DCF, dichlorodihydrofluorescein; EM, electron microscopy; InsP6, inositol hexakisphosphate; InsPs, inositol phosphates; MDD, metal detection; MINPP1, multiple-inositol-polyphosphate-phosphatase; ROS, reactive oxygen species

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