Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: A case study with the metastatic breast cancer cell line MDA-MB-231

Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing must effectively remove the extracellular media components and quenching should stop the metabolic activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES (−50 °C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted. Together, these findings provide for the first time a systematic study into the washing and quenching steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will improve reliability in the application of metabolomics technology to study adherent mammalian cell metabolism

Ethnic differences in atrial fibrillation among patients with heart failure in Asia

Aims We aimed to characterize ethnic differences in prevalence, clinical correlates, and outcomes of atrial fibrillation (AF) in heart failure (HF) with preserved and reduced ejection fraction (HFpEF and HFrEF) across Asia. Methods and results Among 5504 patients with HF prospectively recruited across 11 Asian regions using identical protocols in the Asian Sudden Cardiac Death in Heart Failure study (mean age 61 +/- 13 years, 27% women, 83% HFrEF), 1383 (25%) had AF defined as a history of AF and/or AF/flutter on baseline electrocardiogram. Clinical correlates of AF were similar across ethnicities and included older age, prior stroke, higher NT-proBNP, and larger left atria. Diabetes was associated with lower odds of AF in HFrEF [adjusted odds ratio (AOR) 0.79, 95% CI 0.66-0.95] and HFpEF (AOR 0.58, 95% CI 0.39-0.84) regardless of ethnicity. Compared with Chinese ethnicity, Japanese/Koreans had higher odds of AF in HFrEF (AOR 1.76, 95% CI 1.40-2.21), while Indians had lower odds in HFrEF (AOR 0.18, 95% CI 0.13-0.24) and HFpEF (AOR 0.28, 95% CI 0.16-0.49) even after adjusting for clinical covariates. Interaction between ethnicity and region was observed among Indians, with Southeast Asian Indians having higher odds of AF (AOR 3.01, 95% CI 1.60-5.67) compared with South Asian Indians. AF was associated with poorer quality of life and increased risk of 1 year all-cause mortality or HF hospitalisation (adjusted hazard ratio 1.39, 95% CI 1.18-1.63) regardless of ethnicity. Conclusions Among patients with HF across Asia, clinical correlates and adverse outcomes associated with AF are similar across ethnicities; however, there are striking ethnic variations in the prevalence of AF that are not accounted for by known risk factors

Heart failure with improved versus persistently reduced left ventricular ejection fraction:A comparison of the BIOSTAT-CHF (European) study with the ASIAN-HF registry

AimsWe investigated the prevalence, clinical characteristics, and prognosis of patients with heart failure (HF) with improved ejection fraction (HFimpEF).Methods and resultsWe used data from BIOSTAT-CHF including patients with a left ventricular ejection fraction (LVEF) ≤40% at baseline who had LVEF re-assessed at 9 months. HFimpEF was defined as a LVEF >40% and a LVEF ≥10% increase from baseline at 9 months. We validated findings in the ASIAN-HF registry. The primary outcome was a composite of time to HF rehospitalization or all-cause mortality. In BIOSTAT-CHF, about 20% of patients developed HFimpEF, that was associated with a lower primary event rate of all-cause mortality (hazard ratio [HR] 0.52, 95% confidence interval [CI] 0.28–0.97, p = 0.040) and the composite endpoint (HR 0.46, 95% CI 0.30–0.70, p < 0.001) compared with patients who remained in persistent HF with reduced ejection fraction (HFrEF). The findings were similar in the ASIAN-HF (HR 0.40, 95% CI 0.18–0.89, p = 0.024, and HR 0.29, 95% CI 0.17–0.48, p < 0.001). Five independently common predictors for HFimpEF in both BIOSTAT-CHF and ASIAN-HF were female sex, absence of ischaemic heart disease, higher LVEF, smaller left ventricular end-diastolic and end-systolic diameter at baseline. A predictive model combining only five predictors (absence of ischaemic heart disease and left bundle branch block, smaller left ventricular end-systolic and left atrial diameter, and higher platelet count) for HFimpEF in the BIOSTAT-CHF achieved an area under the curve of 0.772 and 0.688 in the ASIAN-HF (due to missing left atrial diameter and platelet count).ConclusionsApproximately 20–30% of patients with HFrEF improved to HFimpEF within 1 year with better clinical outcomes. In addition, the predictive model with clinical predictors could more accurately predict HFimpEF in patients with HFrEF

Effects of combined renin-angiotensin-aldosterone system inhibitor and beta-blocker treatment on outcomes in heart failure with reduced ejection fraction:insights from BIOSTAT-CHF and ASIAN-HF registries

Background: Angiotensin‐converting enzyme inhibitors (ACEi)/angiotensin receptor blockers (ARB) and β‐blockers are guideline‐recommended first‐line therapies in heart failure (HF) with reduced ejection fraction (HFrEF). Previous studies showed that individual drug classes were under‐dosed in many parts of Europe and Asia. In this study, we investigated the association of combined up‐titration of ACEi/ARBs and β‐blockers with all‐cause mortality and its combination with hospitalization for HF. Methods and results: A total of 6787 HFrEF patients (mean age 62.6 ± 13.2 years, 77.7% men, mean left ventricular ejection fraction 27.7 ± 7.2%) were enrolled in the prospective multinational European (BIOSTAT‐CHF; n = 2100) and Asian (ASIAN‐HF; n = 4687) studies. Outcomes were analysed according to achieved percentage of guideline‐recommended target doses (GRTD) of combination ACEi/ARB and β‐blocker therapy, adjusted for indication bias. Only 14% (n = 981) patients achieved ≥50% GRTD for both ACEi/ARB and β‐blocker. The best outcomes were observed in patients who achieved 100% GRTD of both ACEi/ARB and β‐blocker [hazard ratio (HR) 0.32, 95% confidence interval (CI) 0.26–0.39 vs. none]. Lower dose of combined therapy was associated with better outcomes than 100% GRTD of either monotherapy. Up‐titrating β‐blockers was associated with a consistent and greater reduction in hazards of all‐cause mortality (HR for 100% GRTD: 0.40, 95% CI 0.25–0.63) than corresponding ACEi/ARB up‐titration (HR 0.75, 95% CI 0.53–1.07). Conclusion: This study shows that best outcomes were observed in patients attaining GRTD for both ACEi/ARB and β‐blockers, unfortunately this was rarely achieved. Achieving >50% GRTD of both drug classes was associated with better outcome than target dose of monotherapy. Up‐titrating β‐blockers to target dose was associated with greater mortality reduction than up‐titrating ACEi/ARB

Prescribing patterns of evidence-based heart failure pharmacotherapy and outcomes in the ASIAN-HF registry:a cohort study

Background Angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs), beta blockers, and mineralocorticoid receptor antagonists (MRAs) are of proven benefit and are recommended by guidelines for management of patients with heart failure and reduced ejection fraction (HFrEF). We aimed to examine the first prospective multinational data from Asia on prescribing patterns of guideline-directed medical therapies and analyse its effect on outcomes. Methods In the prospective multinational ASIAN-HF registry (with enrolment from 46 centres in 11 countries in Asia), we enrolled patients aged 18 years or older, with symptomatic heart failure (stage C, with at least one episode of decompensated heart failure in the past 6 months that resulted in admission to hospital or was treated in an outpatient clinic) and left ventricular systolic dysfunction (ejection fraction Findings Between Oct 1, 2012, and Dec 31, 2015, we enrolled 5276 patients with HFrEF (mean age 59.6 years [SD 13.2], 77% men, body-mass index 24.9 kg/m(2) [5.1], 33% New York Heart Association class III or IV). Followup data were available for 4544 (90%) of 5061 eligible patients taking medication for heart failure, with median follow-up of 417 days (IQR 214-735). ACE inhibitors or ARBs were prescribed to 3868 (77%) of 5005 patients, beta blockers to 3975 (79%) of 5061, and MRAs to 2998 (58%) of 5205, with substantial regional variation. Guideline-recommended dose was achieved in only 17% of cases for ACE inhibitors or ARB, 13% for beta blockers, and 29% for MRAs. Country (all three drug classes), increasing body-mass index (ACE inhibitors or ARBs and MRAs), and in-patient recruitment (ACE inhibitors or ARBs and beta blockers) were associated with attainment of guideline-recommended dose (all p= 100%]). Interpretation Guideline-directed medical therapies at recommended doses are underutilised in patients with HFrEF. Improved uptake and uptitration of guideline-directed medical therapies are needed for better patient outcomes. Copyright (C) 2018 The Author(s). Published by Elsevier Ltd

The Immature Heart: The Roles of Bone Marrow Stromal Stem Cells in Growth and Myocardial Repair

Studies have shown that adult bone marrow derived stem cells (MSCs) can participate in repair of myocardial injury in adult hearts, as well as in cardiac growth during fetal development in utero. Yet, no studies have evaluated the role of MSCs with respect to normal growth or tissue repair in immature hearts after birth. The present study examines whether MSCs may participate in the myocardial growth and injury in the post-natal immature hearts. MSCs were isolated from adult Lewis rats and labeled with Lac-Z gene using retroviral vectors. These MSCs were injected systemically into groups of neonatal (NB=2days-old), immature (B=30days-old) and adult (A=>3months-old) isogeneic Lewis rats. Additionally, left coronary artery ligation was carried out in subgroups of immature (BL) and adult (AL) rats one week after MSCs injection. The hearts were harvested serially from 2-days to 6-weeks, stained with X-Gal for labeled MSCs. Cardiomyocyte phenotypic expression was evaluated by immunohistological staining for Troponin I-C and Connexin-43. Labeled MSCs were found to home into the bone marrow in all rats of different developmental stages. They could be recruited from bone marrow into the infarcted site of myocardium only in groups AL and BL. They were also capable of differentiating into cardiomyocyte phenotype after myocardial injury. In contrast to that reported in the developing fetus, MSCs did not appear to contribute to the growth of non-injured hearts after birth. However, they can be recruited from the bone marrow and regenerate damaged myocardium both in the adult and in the immature hearts

Identification and Characterization of Nucleolin as a COUP-TFII Coactivator of Retinoic Acid Receptor β Transcription in Breast Cancer Cells

The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine-resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade.Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII

Rheumatoid arthritis - clinical aspects: 134. Predictors of Joint Damage in South Africans with Rheumatoid Arthritis

Background: Rheumatoid arthritis (RA) causes progressive joint damage and functional disability. Studies on factors affecting joint damage as clinical outcome are lacking in Africa. The aim of the present study was to identify predictors of joint damage in adult South Africans with established RA. Methods: A cross-sectional study of 100 black patients with RA of >5 years were assessed for joint damage using a validated clinical method, the RA articular damage (RAAD) score. Potential predictors of joint damage that were documented included socio-demographics, smoking, body mass index (BMI), disease duration, delay in disease modifying antirheumatic drug (DMARD) initiation, global disease activity as measured by the disease activity score (DAS28), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and autoantibody status. The predictive value of variables was assessed by univariate and stepwise multivariate regression analyses. A p value <0.05 was considered significant. Results: The mean (SD) age was 56 (9.8) years, disease duration 17.5 (8.5) years, educational level 7.5 (3.5) years and DMARD lag was 9 (8.8) years. Female to male ratio was 10:1. The mean (SD) DAS28 was 4.9 (1.5) and total RAAD score was 28.3 (12.8). The mean (SD) BMI was 27.2 kg/m2 (6.2) and 93% of patients were rheumatoid factor (RF) positive. More than 90% of patients received between 2 to 3 DMARDs. Significant univariate predictors of a poor RAAD score were increasing age (p = 0.001), lower education level (p = 0.019), longer disease duration (p < 0.001), longer DMARD lag (p = 0.014), lower BMI (p = 0.025), high RF titre (p < 0.001) and high ESR (p = 0.008). The multivariate regression analysis showed that the only independent significant predictors of a higher mean RAAD score were older age at disease onset (p = 0.04), disease duration (p < 0.001) and RF titre (p < 0.001). There was also a negative association between BMI and the mean total RAAD score (p = 0.049). Conclusions: Patients with longstanding established RA have more severe irreversible joint damage as measured by the clinical RAAD score, contrary to other studies in Africa. This is largely reflected by a delay in the initiation of early effective treatment. Independent of disease duration, older age at disease onset and a higher RF titre are strongly associated with more joint damage. The inverse association between BMI and articular damage in RA has been observed in several studies using radiographic damage scores. The mechanisms underlying this paradoxical association are still widely unknown but adipokines have recently been suggested to play a role. Disclosure statement: C.I. has received a research grant from the Connective Tissue Diseases Research Fund, University of the Witwatersrand. All other authors have declared no conflicts of interes