0578: Valvular atrial fibrillation and the risk of stroke and deaths: additional prognostic value of the CHA2DS2-VASc score

PurposeThe CHA2DS2-VASc score has been validated and is widely used to stratify the risk of thromboembolism in patients with non-valvular atrial fibrillation (AF). We sought to investigate whether this score could also be useful to predict the risk of stroke and death in patients with valvular AF.MethodsBetween 1998 and 2011, 1,592 consecutive patients, hospitalised for AF, 300 with valvular AF (mitral and/or aortic valve disease) and 1,292 with non-valvular AF were enrolled in the cohort. All patients were followed-up at least 6 months and cardiovascular events recorded. The end-point was defined as the first occurrence of stroke or death. The Cox analysis was adjusted on warfarin, antiplatelet and antiarrhythmic treatments at discharge.ResultsMean age was 73±14 years in valvular AF and 68±15 in non-valvular AF (p=0.0001). At baseline, in the valvular AF group CHA2DS2-VASc score were = 0 for 14 (5%) patients, = 1 for 28 (9%), ? 2 for 258 (86%). Non-valvular AF CHA2DS2-VASc scores were = 0 for 158 (12%), = 1 for 189 (15), ?2 for 945 (73%). The difference was statistically significant (p<0.0001). During a mean follow-up of 4.6±3.5 years, the patients with valvular AF experienced 154 (51%) and the patients with non-valvular AF experienced 409 (32%) strokes or deaths. The Kaplan-Meier curves (figure) show that patients with a CHA2DS2-VASc score ?2 were at higher risk of stroke or death. The adjusted Cox model, showed that valvular AF (HR, 1.57, 95%CI 1.30-1.89, p<0.0001) and a CHA2DS2-VASc score ?2 (HR, 5.30, 95%CI 3.77-7.45, p<0.0001) were predictors of risk of stroke or death.ConclusionThese results suggest that a CHA2DS2-VASc score ?2 is associated with a higher risk of stroke and deaths, at mid-term follow-up, in patients with valvular AF (figure next page).Abstract 0578 - Figure: Kaplan-Meier survival curve

Correction to: Macromolecular interactions in vitro, comparing classical and novel approaches

Correction to: European Biophysics Journal https://doi.org/10.1007/s00249-021-01517-5Peer reviewe

076: Left atrial enlargement in sickle cell disease patients: remodelling associated with haematological parameters or index of left ventricular filling pressures

PuposeDiastolic Left ventricular (LV) dysfunction is a common finding in sickle cell disease. Furthermore, left atrial (LA) size usually reflects left ventricular filling pressures.The aim of our study was to determine if LA size is an expression of left ventricular filling pressures or reflects remodelling associated with anemia and/or haemolysis in sickle cell disease.MethodsWe evaluated 127 patients with sickle cell disease in stable condition (mean age 28,6±8,5 years, 83 women) and 38 age and sex-matched healthy controls. LA size was measured with Simpson's method in apical 4-chamber view. LV filling pressures were assessed using ratio between pulsed Doppler peak E velocity and peak Ea velocity obtained with tissue Doppler imaging of the lateral annulus (E/Ea ratio). Clinical and biologic data were collected from clinical records.ResultsCompared with the normal group, patients with sickle cell disease had a LA volume and E/Ea ratio significatively increased (48,4±11,2ml/m2; and 5,9±1,7; 30,5±7,6ml/m&sup2; and 4,5±1, respectively, p<0.0001).In multivariate analysis, LA enlargement in patients is only influenced by age and haematological parameters (haemoglobin and reticulocyte levels).No correlation was found between LA volume and E/Ea ratio (figure).ConclusionSubjects with sickle cell disease have LA enlargement. However, in this population, LA dilatation is not an index of left ventricular filling pressures.Figur

Measurements of Protein-DNA Complexes Interactions by Isothermal Titration Calorimetry (ITC) and Microscale Thermophoresis (MST)

Interactions between protein complexes and DNA are central regulators of the cell life. They control the activation and inactivation of a large set of nuclear processes including transcription, replication, recombination, repair, and chromosome structures. In the literature, protein-DNA interactions are characterized by highly complementary approaches including large-scale studies and analyses in cells. Biophysical approaches with purified materials help to evaluate if these interactions are direct or not. They provide quantitative information on the strength and specificity of the interactions between proteins or protein complexes and their DNA substrates. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) are widely used and are complementary methods to characterize nucleo-protein complexes and quantitatively measure protein-DNA interactions. We present here protocols to analyze the interactions between a DNA repair complex, Ku70-Ku80 (Ku) (154 kDa), and DNA substrates. ITC is a label-free method performed with both partners in solution. It serves to determine the dissociation constant (Kd), the enthalpy (ΔH), and the stoichiometry N of an interaction. MST is used to measure the Kd between the protein or the DNA labeled with a fluorescent probe. We report the data obtained on Ku-DNA interactions with ITC and MST and discuss advantages and drawbacks of both the methods

Subclinical left ventricular systolic impairment in steady state young adult patients with sickle-cell anemia

International audienceChronic volume overload in sickle-cell anemia (SCA) is associated with left ventricular (LV) enlargement and hypertrophy. The effect of the disease on LV systolic function remains debated. The aim of our study was to investigate LV systolic function in SCA patients using 2D speckle-tracking imaging. We compared 30 steady state asymptomatic adult SCA patients (17 women, mean age 24.7 ± 5.1 years) with 30 age and sex-matched healthy subjects (17 women, mean age 25.0 ± 4.9 years). In addition to conventional echocardiographic parameters including LV ejection fraction (LVEF) and LV mass index (LVMi), global longitudinal strain (GLS) and strain rate (GLSR) were measured. GLS (-17.9 ± 2.0 vs. -19.7 ± 2.5 %, p = 0.004) and GLSR (-0.92 ± 0.09 vs. -1.07 ± 0.17 s(-1), p < 0.0001) values were lower in SCA patients while LVEF values (60.1 ± 3.8 vs. 61.7 ± 4.7 %, p = 0.30) were not different. LVMi was increased in SCA patients (100.7 ± 23.5 vs. 72.4 ± 15.2 g/m(2), p = 0.0001) and GLSR was significantly lower in the subgroup of patients with LV hypertrophy (-0.88 ± 0.09 vs. -0.96 ± 0.08 s(-1), p = 0.02). In SCA patients LVMi was correlated to GLS (r = 0.58, p = 0.001) and GLSR (r = 0.45, p = 0.015) pleading in favor of a pathological LV remodeling. Asymptomatic SCA patients exhibited a subclinical alteration of LV systolic function. Myocardial dysfunction appears to be linked to the degree of LV hypertrophy. 2D speckle-tracking imaging might be useful for long-term follow-up and to study the natural course of LV dysfunction in SCA patients

Molecular and physiological logics of the pyruvate-induced response of a novel transporter in Bacillus subtilis

At the heart of central carbon metabolism, pyruvate is a pivotal metabolite in all living cells. Bacillus subtilis is able to excrete pyruvate as well as to use it as the sole carbon source. We herein reveal that ysbAB (renamed pftAB), the only operon specifically induced in pyruvate-grown B. subtilis cells, encodes a heterooligomeric membrane complex which operates as a facilitated transport system specific for pyruvate, thereby defining a novel class of transporter. We demonstrate that the LytST two-component system is responsible for the induction of pftAB in the presence of pyruvate by binding of the LytT response regulator to a palindromic region upstream of pftAB. We show that both glucose and malate, the preferred carbon sources for B. subtilis, trigger the binding of CcpA upstream of pftAB, which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate. Screening a genome-wide transposon mutant library, we find that an active malic enzyme replenishing the pyruvate pool is required for this repression. We next reveal that the higher the influx of pyruvate, the stronger the CcpA-independent repression of pftAB, which suggests that intracellular pyruvate retroinhibits pftAB induction via LytST. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry. Overall, we provide evidence for a complete system of sensors, feed-forward and feedback controllers that play a major role in environmental growth of B. subtilis. [br/] IMPORTANCE Pyruvate is a small-molecule metabolite ubiquitous in living cells. Several species also use it as a carbon source as well as excrete it into the environment. The bacterial systems for pyruvate import/export have yet to be discovered. Here, we identified in the model bacterium Bacillus subtilis the first import/export system specific for pyruvate, PftAB, which defines a novel class of transporter. In this bacterium, extracellular pyruvate acts as the signal molecule for the LytST two-component system (TCS), which in turn induces expression of PftAB. However, when the pyruvate influx is high, LytST activity is drastically retroinhibited. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry

Left atrial volume is not an index of left ventricular diastolic dysfunction in patients with sickle cell anaemia

SummaryBackgroundLeft ventricular diastolic dysfunction (LVDD) is common in sickle cell anaemia (SCA). Left atrial (LA) size is widely used as an index of LVDD; however, LA enlargement in SCA might also be due to chronic volume overload.AimTo investigate whether LA size can be used to diagnose LVDD in SCA.MethodsOne hundred and twenty-seven adults with stable SCA underwent echocardiographic assessment. LA volume was measured by the area–length method and indexed to body surface area (LAVi). Left ventricular (LV) filling pressures were assessed using the ratio of early peak diastolic velocities of mitral inflow and septal annular mitral plane (E/e′). Using mitral inflow profile and E/e′, LV diastolic function was classified as normal or abnormal. LAVi>28mL/m2 was used as the threshold to define LA enlargement.ResultsThe mean age was 28.6±8.5years; there were 83 women. Mean LAVi was 48.3±11.1mL/m2 and 124 (98%) patients had LA dilatation. In multivariable analysis, age, haemoglobin concentration and LV end-diastolic volume index were independent determinants of LAVi (R2=0.51; P<0.0001). E/e′ was not linked to LAVi (P=0.43). Twenty patients had LVDD; when compared with patients without LVDD, they had a similar LAVi (52.2±14.7 and 47.5±10.2mL/m2, respectively; P=0.29). Receiver operating characteristics curve analysis showed that LAVi could not be used to diagnose LVDD (area under curve=0.58; P=0.36).ConclusionLA enlargement is common in SCA but appears not to be linked to LVDD. LAVi in this population is related to age, haemoglobin concentration and LV morphology

Short tandem repeats are important contributors to silencer elements in T cells

International audienceThe action of cis-regulatory elements with either activation or repression functions underpins the precise regulation of gene expression during normal development and cell differentiation. Gene activation by the combined activities of promoters and distal enhancers has been extensively studied in normal and pathological contexts. In sharp contrast, gene repression by cis-acting silencers, defined as genetic elements that negatively regulate gene transcription in a position-independent fashion, is less well understood. Here, we repurpose the STARRseq approach as a novel high-throughput reporter strategy to quantitatively assess silencer activity in mammals. We assessed silencer activity from DNase hypersensitive I sites in a mouse T cell line. Identified silencers were associated with either repressive or active chromatin marks and enriched for binding motifs of known transcriptional repressors. CRISPRmediated genomic deletions validated the repressive function of distinct silencers involved in the repression of non-T cell genes and genes regulated during T cell differentiation. Finally, we unravel an association of silencer activity with short tandem repeats, highlighting the role of repetitive elements in silencer activity. Our results provide a general strategy for genome-wide identification and characterization of silencer elements

Integration of high-throughput reporter assays identify a critical enhancer of the Ikzf1 gene

International audienceThe Ikzf1 locus encodes the lymphoid specific transcription factor Ikaros, which plays an essential role in both T and B cell differentiation, while deregulation or mutation of IKZF1/ Ikzf1 is involved in leukemia. Tissue-specific and cell identity genes are usually associated with clusters of enhancers, also called super-enhancers, which are believed to ensure proper regulation of gene expression throughout cell development and differentiation. Several potential regulatory regions have been identified in close proximity of Ikzf1, however, the full extent of the regulatory landscape of the Ikzf1 locus is not yet established. In this study, we combined epigenomics and transcription factor binding along with high-through-put enhancer assay and 4C-seq to prioritize an enhancer element located 120 kb upstream of the Ikzf1 gene. We found that deletion of the E120 enhancer resulted in a significant reduction of Ikzf1 mRNA. However, the epigenetic landscape and 3D topology of the locus were only slightly affected, highlighting the complexity of the regulatory landscape regulating the Ikzf1 locus

Macromolecular interactions in vitro, comparing classical and novel approaches

International audienceBiophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France (https://mosbio.sciencesconf.org/). Twenty European students benefited from a week’s training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein–protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction