Longitudinal NT-proBNP: Associations With Echocardiographic Changes and Outcomes in Heart Failure.

BACKGROUND: The relationship of serial NT-proBNP (N-terminal pro-B-type natriuretic peptide) measurements with changes in cardiac features and outcomes in heart failure (HF) remains incompletely understood. We determined whether common clinical covariates impact these relationships. METHODS AND RESULTS: In 2 nationwide observational populations with HF, the relationship of serial NT-proBNP measurements with serial echocardiographic parameters and outcomes was analyzed, further stratified by HF with reduced versus preserved left ventricular ejection fraction, inpatient versus outpatient enrollment, age, obesity, chronic kidney disease, atrial fibrillation, and attainment of ≥50% guideline-recommended doses of renin-angiotensin system inhibitors and ß-blockers. Among 1911 patients (mean±SD age, 65.1±13.4 years; 26.6% women; 62% inpatient and 38% outpatient), NT-proBNP declined overall, with more rapid declines among inpatients, those with obesity, those with atrial fibrillation, and those attaining ≥50% guideline-recommended doses. Each doubling of NT-proBNP was associated with increases in left ventricular volume (by 6.1 mL), E/e' (transmitral to mitral annular early diastolic velocity ratio) (by 1.4 points), left atrial volume (by 3.6 mL), and reduced left ventricular ejection fraction (by -2.1%). The effect sizes of these associations were lower among patients with HF with preserved ejection fraction, atrial fibrillation, or advanced age (Pinteraction<0.001). A landmark analysis identified that an SD increase in NT-proBNP over 6 months was associated with a 27% increase in the risk of the composite event of HF hospitalization or all-cause death between 6 months and 2 years (adjusted hazard ratio, 1.27 [95% CI, 1.15-1.40]; P<0.001). CONCLUSIONS: The relationships between NT-proBNP and structural/functional remodeling differed by age, presence of atrial fibrillation, and HF phenotypes. The association of increased NT-proBNP with increased risk of adverse outcomes was consistent in all subgroups.

Global longitudinal strain and plasma biomarkers for prognosis in heart failure complicated by diabetes: a prospective observational study.

BACKGROUND: Heart failure (HF) and diabetes are associated with increased incidence and worse prognosis of each other. The prognostic value of global longitudinal strain (GLS) measured by cardiovascular magnetic resonance (CMR) has not been established in HF patients with diabetes. METHODS: In this prospective, observational study, consecutive patients (n = 315) with HF underwent CMR at 3T, including GLS, late gadolinium enhancement (LGE), native T1, and extracellular volume fraction (ECV) mapping. Plasma biomarker concentrations were measured including: N-terminal pro B-type natriuretic peptide(NT-proBNP), high-sensitivity troponin T(hs-TnT), growth differentiation factor 15(GDF-15), soluble ST2(sST2), and galectin 3(Gal-3). The primary outcome was a composite of all-cause mortality or HF hospitalisation. RESULTS: Compared to those without diabetes (n = 156), the diabetes group (n = 159) had a higher LGE prevalence (76 vs. 60%, p < 0.05), higher T1 (1285±42 vs. 1269±42ms, p < 0.001), and higher ECV (30.5±3.5 vs. 28.8±4.1%, p < 0.001). The diabetes group had higher NT-pro-BNP, hs-TnT, GDF-15, sST2, and Gal-3. Diabetes conferred worse prognosis (hazard ratio (HR) 2.33 [95% confidence interval (CI) 1.43-3.79], p < 0.001). In multivariable Cox regression analysis including clinical markers and plasma biomarkers, sST2 alone remained independently associated with the primary outcome (HR per 1 ng/mL 1.04 [95% CI 1.02-1.07], p = 0.001). In multivariable Cox regression models in the diabetes group, both GLS and sST2 remained prognostic (GLS: HR 1.12 [95% CI 1.03-1.21], p = 0.01; sST2: HR per 1 ng/mL 1.03 [95% CI 1.00-1.06], p = 0.02). CONCLUSIONS: Compared to HF patients without diabetes, those with diabetes have worse plasma and CMR markers of fibrosis and a more adverse prognosis. GLS by CMR is a powerful and independent prognostic marker in HF patients with diabetes.

Experiences of using the MINDSET Self-Management mobile health app among Hispanic Patients:Results of a qualitative study.

BACKGROUND: Interventions focusing on epilepsy self-management (ESM) are vital for promoting the health of people living with epilepsy. E-technology and mobile health (mHealth) tools are becoming increasingly integrated into practice to promote self-management strategies for chronic diseases, enhance care delivery, and reduce health disparities. Management Information and Decision Support Epilepsy Tool (MINDSET), a bilingual decision support tool (available in English and Spanish), was found to be both feasible and effective in facilitating goal-based ESM in the clinic. PURPOSE: To assess the experience of using MINDSET as an ESM intervention among Hispanic patients with epilepsy to inform future interventional studies. METHODS: This study used a Qualitative Descriptive (QD) framework to provide a rich and straightforward description of patients' subjective experiences using MINDSET. Participants were enrolled in the intervention group of a larger parent study (RCT) to assess the efficacy of MINDSET among Hispanic People with Epilepsy (PWE). The purposive, convenient, criterion-based sample for this qualitative analysis comprised of 42 patients who agreed to participate in a semi-structured interview at the end of the larger RCT. This RCT was conducted between August 2017 and January 2019. Spanish and English-speaking Hispanic adult patients (n = 94) with epilepsy in Arizona (n = 53) and Texas (n = 41) were randomly assigned within 6 neurology clinics to treatment (MINDSET plus Usual Care, hereafter referred to as MINDSET; n = 46) and comparison (Usual Care Only; n = 48) conditions. RESULTS: Patient demographics, epilepsy conditions, and ESM behavioral characteristics were representative of the intervention group. Study participants were Hispanic, mainly of Mexican descent (94 %), with a mean age of 39 years, mostly female (53 %), and most of the participants reported having had one or more seizures per month (54 %). The MINDSET intervention revealed five ESM themes: (1) Awareness and Realization of Epilepsy Self-Management, (2) Communication and Partnership with Health Care Providers HCP, (3) Epilepsy Self-Management and Quality of Life, (4) Seizure Control, and (5) Optimism and Agency. CONCLUSION: The participants who used MINDSET as a self-management intervention reported an overall positive experience. Qualitative data in this study show that MINDSET is a valuable ESM tool for Hispanic patients with epilepsy. Findings from this qualitative study were consistent with results from a larger parent study that recognized MINDSET as an effective platform for improving epilepsy self-management adherence.

Distinguishing heart failure with reduced ejection fraction from heart failure with preserved ejection fraction: A phenomics approach.

AIM: Pathophysiological differences between patients with heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction (EF) remain unclear. Therefore we used a phenomics approach, integrating selected proteomics data with patient characteristics and cardiac structural and functional parameters, to get insight into differential pathophysiological mechanisms and identify potential treatment targets. METHODS AND RESULTS: We report data from a representative subcohort of the prospective Singapore Heart Failure Outcomes and Phenotypes (SHOP), including patients with HFrEF (EF <40%, n = 217), HFpEF (EF ≥50%, n = 213), and age- and sex-matched controls without HF (n = 216). We measured 92 biomarkers using a proximity extension assay and assessed cardiac structure and function in all participants using echocardiography. We used multi-block projection to latent structure analysis to integrate clinical, echocardiographic, and biomarker variables. Candidate biomarker targets were cross-referenced with small-molecule and drug databases. The total cohort had a median age of 65 years (interquartile range 60-71), and 50% were women. Protein profiles strongly discriminated patients with HFrEF (area under the curve [AUC] = 0.89) and HFpEF (AUC = 0.94) from controls. Phenomics analyses identified unique druggable inflammatory markers in HFpEF from the tumour necrosis factor receptor superfamily (TNFRSF), which were positively associated with hypertension, diabetes, and increased posterior and relative wall thickness. In HFrEF, interleukin (IL)-8 and IL-6 were possible targets related to lower EF and worsening renal function. CONCLUSION: We identified pathophysiological mechanisms related to increased cardiac wall thickness parameters and potentially druggable inflammatory markers from the TNFRSF in HFpEF.

Serum Placental Growth Factor as a Marker of Cerebrovascular Disease Burden in Alzheimer's Disease.

BACKGROUND: Concomitant cerebrovascular diseases (CeVD) have been identified as an important determinant of Alzheimer's disease (AD) progression. Development of robust blood-based biomarkers will provide critical tools to evaluate prognosis and potential interventional strategies for AD with CeVD. OBJECTIVE: This study investigated circulating placental growth factor (PlGF), a potent pro-angiogenic factor related to endothelial dysfunction and vascular inflammation, in an Asian memory clinic cohort of non-demented individuals as well as AD, including its associations with neuroimaging markers of CeVD. METHODS: 109 patients with AD, 76 cognitively impaired with no dementia (CIND), and 56 non-cognitively impaired (NCI) were included in this cross-sectional study. All subjects underwent 3T brain magnetic resonance imaging to assess white matter hyperintensities (WMH), lacunes, cortical infarcts, and cerebral microbleeds (CMBs). Serum PlGF concentrations were measured by electrochemiluminescence immunoassays. RESULTS: Serum PlGF was elevated in AD, but not CIND, compared to the NCI controls. Adjusted concentrations of PlGF were associated with AD only in the presence of significant CeVD. Elevated PlGF was significantly associated with higher burden of WMH and with CMBs in AD patients. CONCLUSIONS: Serum PlGF has potential utility as a biomarker for the presence of CeVD, specifically WMH and CMBs, in AD. Further studies are needed to elucidate the underlying pathophysiological mechanisms linking PlGF to CeVD, as well as to further assess PlGF's clinical utility.

Elf1 promotes Rad26's interaction with lesion-arrested Pol II for transcription-coupled repair.

Transcription-coupled nucleotide excision repair (TC-NER) is a highly conserved DNA repair pathway that removes bulky lesions in the transcribed genome. Cockayne syndrome B protein (CSB), or its yeast ortholog Rad26, has been known for decades to play important roles in the lesion-recognition steps of TC-NER. Another conserved protein ELOF1, or its yeast ortholog Elf1, was recently identified as a core transcription-coupled repair factor. How Rad26 distinguishes between RNA polymerase II (Pol II) stalled at a DNA lesion or other obstacles and what role Elf1 plays in this process remains unknown. Here, we present cryo-EM structures of Pol II-Rad26 complexes stalled at different obstacles that show that Rad26 uses a common mechanism to recognize a stalled Pol II, with additional interactions when Pol II is arrested at a lesion. A cryo-EM structure of lesion-arrested Pol II-Rad26 bound to Elf1 revealed that Elf1 induces further interactions between Rad26 and a lesion-arrested Pol II. Biochemical and genetic data support the importance of the interplay between Elf1 and Rad26 in TC-NER initiation. Together, our results provide important mechanistic insights into how two conserved transcription-coupled repair factors, Rad26/CSB and Elf1/ELOF1, work together at the initial lesion recognition steps of transcription-coupled repair.

Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies.

Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1-8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57-60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.

Differential Associations of A-/B-Type Natriuretic Peptides With Cardiac Structure, Function, and Prognosis in Heart Failure.

BACKGROUND: Natriuretic peptide (NP) elevations are prognostic in heart failure (HF), but relative atrial NP deficiency in acute HF has been suggested. OBJECTIVES: The authors compared plasma concentrations and relative strength of associations of A- and B-type NPs with cardiac structure/function and clinical outcomes in HF. METHODS: Midregional pro-atrial natriuretic peptide (MR-proANP), B-type natriuretic peptide (BNP), and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were measured in patients with compensated HF in a prospective, multicenter study. The primary outcome was a composite of HF-hospitalization or all-cause mortality. Secondary outcomes included individual primary outcome components and cardiovascular admission. RESULTS: Among 1,278 patients (age 60.1 ± 12.1 years, 82% men, left ventricular ejection fraction [LVEF] 34% ± 14%), median concentrations of MR-proANP were 990 pg/mL (Q1-Q3: 557-1,563 pg/mL), NT-proBNP 1,648 pg/mL (Q1-Q3: 652-3,960 pg/mL), and BNP 291 pg/mL (Q1-Q3: 103-777 pg/mL). No subpopulation with inappropriately low MR-proANP (relative to BNP/NT-proBNP) was observed. Clinical event rates were similar for biomarker tertiles. Increments in MR-proANP exhibited steeper associations with concurrent shifts in left ventricular size, diastolic indexes and LVEF than BNP/NT-proBNP at baseline and serially (P < 0.05), and lower odds of beneficial left ventricular reverse remodeling: OR: 0.35 (95% CI: 0.18-0.70). In single-biomarker models, MR-proANP(log10) was associated with the highest hazard (4 to 6 times) for each outcome. In multimarker models, independent associations were observed for the primary outcome (MR-proANP and NT-proBNP), HF-hospitalization and cardiovascular admission (MR-proANP only), and all-cause mortality (NT-proBNP only) (P < 0.05). The discriminative value of MR-proANP was superior to BNP/NT-proBNP (HF-hospitalization) and BNP (primary outcome) (P < 0.05). CONCLUSIONS: MR-proANP was not inappropriately low relative to concurrent BNP/NT-proBNP values. Proportional increments in MR-proANP were more pronounced than for B-peptides for given decrements in cardiac structure/function. MR-proANP offered greater independent predictive power overall.

A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase.

Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition.

Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase.

Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds-Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.

Prognostic Value of Left Atrial Strain in Aortic Stenosis: A Competing Risk Analysis.

BACKGROUND: The role of left atrial (LA) strain as an imaging biomarker in aortic stenosis is not well established. The aim of this study was to investigate the prognostic performance of phasic LA strain in relation to clinical and echocardiographic variables and N-terminal pro-B-type natriuretic peptide in asymptomatic and minimally symptomatic patients with moderate to severe aortic stenosis and left ventricular ejection fraction > 50%. METHODS: LA reservoir strain (LASr), LA conduit strain (LAScd), and LA contractile strain (LASct) were measured using speckle-tracking echocardiography. The primary outcome was a composite of all-cause mortality, heart failure hospitalization, progression to New York Heart Association functional class III or IV, acute coronary syndrome, or syncope. Secondary outcomes 1 and 2 comprised the same end points but excluded acute coronary syndrome and additionally syncope, respectively. The prognostic performance of phasic LA strain cutoffs was evaluated in competing risk analyses, aortic valve replacement being the competing risk. RESULTS: Among 173 patients (mean age, 69 ± 11 years; mean peak transaortic velocity, 4.0 ± 0.8 m/sec), median LASr, LAScd, and LASct were 27% (interquartile range [IQR], 22%-32%), 12% (IQR, 8%-15%), and 16% (IQR, 13%-18%), respectively. Over a median of 2.7 years (IQR, 1.4-4.6 years), the primary outcome and secondary outcomes 1 and 2 occurred in 66 (38%), 62 (36%), and 59 (34%) patients, respectively. LASr < 20%, LAScd < 6%, and LASct < 12% were identified as optimal cutoffs of the primary outcome. In competing risk analyses, progressing from echocardiographic to echocardiographic-clinical and combined models incorporating N-terminal pro-B-type natriuretic peptide, LA strain parameters outperformed other key echocardiographic variables and significantly predicted clinical outcomes. LASr < 20% was associated with the primary outcome and secondary outcome 1, LAScd < 6% with all clinical outcomes, and LASct < 12% with secondary outcome 2. LAScd < 6% had the highest specificity (95%) and positive predictive value (82%) for the primary outcome, and competing risk models incorporating LAScd < 6% had the best discriminative value. CONCLUSIONS: In well-compensated patients with moderate to severe aortic stenosis and preserved left ventricular ejection fractions, LA strain was superior to other echocardiographic indices and incremental to N-terminal pro-B-type natriuretic peptide for risk stratification. LAScd < 6%, LASr < 20%, and LASct < 12% identified patients at higher risk for adverse outcomes.

One-Shot Generation of Epitope-Directed Monoclonal Antibodies to Multiple Nonoverlapping Targets: Peptide Selection, Antigen Preparation, and Epitope Mapping.

This chapter describes an epitope-directed approach to generate antipeptide monoclonal antibodies to multiple nonoverlapping protein sites using a cocktail of fusion peptides as immunogen. It provides a step-by-step protocol on how antigenic peptides on a target protein can be identified by in silico prediction and discusses considerations for final peptide selection. Each antigenic peptide (10-20 amino acids long) is displayed as three-copy inserts on the surface exposed loop of a thioredoxin scaffold protein. The corresponding DNA coding sequence specifying the tripeptide insert flanked by Gly-Ser-Gly-Ser-Gly linkers is cloned in-frame into the Rsr II site of the thioredoxin gene in the pET-32a vector. The presence of a C-terminal polyhistidine tag (His6-tag) allows the soluble fusion proteins to be purified by one-step native immobilized metal affinity chromatography (IMAC) to greater than 95% purity. Multiple thioredoxin fusion proteins are mixed in equimolar concentrations and used as an immunogen cocktail for animal immunization. The use of short antigenic peptides of known sequence facilitates direct epitope mapping requiring only small mutagenesis scan peptide libraries in the multipin peptide format.

Circulating erythroferrone has diagnostic utility for acute decompensated heart failure in patients presenting with acute or worsening dyspnea.

Objectives: In dyspneic patients with atrial fibrillation (AF) or obesity, the diagnostic performance of NT-proBNP for acute heart failure is reduced. We evaluated the erythroblast derived protein erythroferrone (ERFE) as an ancillary biomarker for the diagnosis of acute decompensated heart failure (ADHF) in these comorbid subgroups in both Western and Asian populations. Methods: The diagnostic performance of ERFE (Intrinsic Lifesciences) and NT-proBNP (Roche Cobas e411) for ADHF was assessed in 479 New Zealand (NZ) and 475 Singapore (SG) patients presenting with breathlessness. Results: Plasma ERFE was higher in ADHF, compared with breathlessness from other causes, in both countries (NZ; 4.9 vs. 1.4 ng/ml, p < 0.001) and (SG; 4.2 vs. 0.4 ng/ml, p = 0.021). The receiver operating characteristic (ROC) areas under the curve (AUCs) for discrimination of ADHF were reduced in the NZ cohort compared to SG for ERFE (0.75 and 0.84, p = 0.007) and NT-proBNP (0.86 and 0.92, p = 0.004). Optimal cut-off points for ERFE yielded comparable sensitivity and positive predictive values in both cohorts, but slightly better specificity, negative predictive values and accuracy in SG compared with NZ. In patients with AF, the AUC decreased for ERFE in each cohort (NZ: 0.71, n = 105, SG: 0.61, n = 44) but increased in patients with obesity (NZ: 0.79, n = 150, SG: 0.87, n = 164). Conclusions: Circulating ERFE is higher in patients with ADHF than in other causes of new onset breathlessness with fair diagnostic utility, performing better in Asian than in Western patients. The diagnostic performance of ERFE is impaired in patients with AF but not patients with obesity.

Immunoassay-Compatible Inactivation of SARS-CoV-2 in Plasma Samples for Enhanced Handling Safety.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) inactivation is an important step toward enhanced biosafety in testing facilities and affords a reduction in the biocontainment level necessary for handling virus-positive biological specimens. Virus inactivation methods commonly employ heat, detergents, or combinations thereof. In this work, we address the dearth of information on the efficacy of SARS-CoV-2 inactivation procedures in plasma and their downstream impact on immunoassays. We evaluated the effects of heat (56 °C for 30 min), detergent (1-5% Triton X-100), and solvent-detergent (SD) combinations [0.3-1% tri-n-butyl phosphate (TNBP) and 1-2% Triton X-100] on 19 immunoassays across different assay formats. Treatments are deemed immunoassay-compatible when the average and range of percentage recovery (treated concentration relative to untreated concentration) lie between 90-110 and 80-120%, respectively. We show that SD treatment (0.3% TNBP/1% Triton-X100) is compatible with more than half of the downstream immunoassays tested and is effective in reducing SARS-CoV-2 infectivity in plasma to below detectable levels in plaque assays. This facile method offers enhanced safety for laboratory workers handling biological specimens in clinical and research settings.

Circulating cardiac biomarkers improve risk stratification for incident cardiovascular disease in community dwelling populations.

BACKGROUND: Plasma cardiac markers may assist in prediction of incident cardiovascular disease. METHODS: The incremental value of cardiac Troponins (T and I) and NT-proBNP added to risk factors in the PREDICT score for incident cardiovascular disease (CVD) in primary care, was assessed in 4102 asymptomatic participants in a randomised controlled trial of Vitamin D (ViDA). Findings were corroborated in 2528 participants in a separate community-based observational registry of CVD-free volunteers (HVOLS). FINDINGS: Hazard ratios for first cardiovascular events adjusted for PREDICT risk factors, comparing fifth to first quintiles of marker plasma concentrations, were 2.57 (95% CI 1.47-4.49); 3.01 (1.66-5.48) and 3.38 (2.04-5.60) for hs-cTnI, hs-cTnT and NT-proBNP respectively. The C statistic for discrimination of the primary endpoint increased from 0.755 to 0.771 (+0.016, p = 0.01). Cardiac marker data correctly reclassified risk upwards in 6.7% of patients and downwards in 3.3%. These findings were corroborated by results from HVOLS. INTERPRETATION: Increments in plasma cardiac biomarkers robustly and reproducibly predicted increased hazard of incident CVD, independent of established risk factors, in two community-dwelling populations. Cardiac markers may augment risk assessment for onset of CVD in primary care. FUNDING: ViDA was funded by the Health Research Council of New Zealand (grant 10/400) and the Accident Compensation Corporation. HVOLS was funded by the Health Research Council of NZ Programme Grants (grants 02/152 and 08/070) and by grants from the Heart Foundation of NZ and the Christchurch Heart Institute Trust. Roche Diagnostics provided in-kind support for NT-proBNP and hs-cTnT assays and Abbott Laboratories for hs-cTnI assays.

Finding a reliable assay for soluble neprilysin.

BACKGROUND: Lack of validation and standardization of research-use-only (RUO) immunoassays brings with it inherent threats to authenticity and functional quality. Poor correlation between different commercial neprilysin RUO immunoassays is concerning and discordant findings need to be resolved. We seek to identify and validate reliable neprilysin immunoassays to strengthen the scientific rigor and reproducibility of neprilysin-related investigation and of biomarker research in general. METHODS: Soluble neprilysin (sNEP) concentrations were determined in cohorts (n = 532) from Spain (Cohort 1), New Zealand (NZ, Cohort 2) and Singapore (Cohort 3), using commercial kits from six vendors. Apparent sNEP concentrations were correlated between different assays and with plasma neprilysin activity. Assay reliability was further validated by performance verification, MS analysis and cross-reactivity tests. RESULTS: sNEP in Cohorts 1 and 2 measured concurrently in Spain and NZ showed significant inter-laboratory correlation only for the Aviscera Bioscience sNEP ELISA SK00724-01. Neprilysin concentrations obtained with the R&D systems and SK00724-01 ELISAs correlated with each other but not with neprilysin activity. In Cohort 3, sNEP concentrations from the Perkin Elmer AlphaLISA and Biotechne ELLA assays agreed (r = 0.89) and both correlated with neprilysin activity (r = 0.87, 0.77 respectively). MS analysis detected authentic neprilysin in the AlphaLISA kit calibrator and in antibody pull-down material from human plasma. The AlphaLISA assay performed within acceptable limits (spike and recovery, dilutional linearity, inter- and intra-assay CV) and showed no cross-reactivity against neprilysin substrates and closely-related analogues. CONCLUSION: AlphaLISA and ELLA assays provide reliable measures of sNEP concentrations. Reliability of other commercial neprilysin assays remains in question.

Novel predictive role for mid-regional proadrenomedullin in moderate to severe aortic stenosis.

OBJECTIVE: We investigated the prognostic significance of selected known and novel circulating biomarkers in aortic stenosis (AS). METHODS: N-terminal pro-BNP (NT-proBNP), high-sensitivity troponin-T (hsTnT), growth differentiation factor-15 (GDF-15), suppression of tumorigenicity-2 (ST2), mid-regional proadrenomedullin (MR-proADM) and mid-regional proatrial natriuretic peptide (MR-proANP) were measured in patients with moderate to severe AS, New York Heart Association (NYHA) class I-II and left ventricular ejection fraction ≥50%, recruited consecutively across five centres from 2011 to 2018. Their ability to predict both primary (all-cause mortality, heart failure hospitalisation or progression to NYHA class III-IV) and secondary (additionally incorporating syncope and acute coronary syndrome) outcomes was determined by competing risk analyses. RESULTS: Among 173 patients with AS (age 69±11 years, 55% male, peak transaortic velocity (Vmax) 4.0±0.8 m/s), the primary and secondary outcomes occurred in 59 (34%) and 66 (38%), respectively. With aortic valve replacement as a competing risk, the primary outcome was determined consistently by the comorbidity index and each selected biomarker except ST2 (p<0.05), independent of NYHA class, Vmax, LV-global longitudinal strain and serum creatinine. MR-proADM had the highest discriminative value for both primary (subdistribution HR (SHR) 11.3, 95% CI 3.9 to 32.7) and secondary outcomes (SHR 12.6, 95% CI 4.7 to 33.5). Prognostic assessment of dual-biomarker combinations identified MR-proADM plus either hsTnT or NT-proBNP as the best predictive model for both clinical outcomes. Paired biomarker models were not superior to those including MR-proADM as the sole circulating biomarker. CONCLUSION: MR-proADM most powerfully portended worse prognosis and should be further assessed as possibly the biomarker of choice for risk stratification in AS.

Effect of monthly vitamin D supplementation on cardiac biomarkers: A post-hoc analysis of a randomized controlled trial.

The effects of vitamin D supplementation on cardiovascular diseases are controversial. Data on effects of vitamin D upon cardiac biomarkers, as surrogate endpoints of cardiovascular diseases, are limited and inconclusive. Therefore, we carried out a post-hoc analysis of sub-samples of a randomized, double-blinded, placebo-controlled trial with community-based older adults who were randomized to receive monthly 100,000-IU vitamin D or placebo, to determine effect of monthly vitamin D supplementation on high-sensitivity cardiac troponin I (hs-cTnI), troponin T (hs-cTnT) and N-terminal-pro-B-type natriuretic peptide (NT-proBNP). Adjusted relative difference (aRD) of follow-up geometric mean of biomarkers and adjusted relative risk (aRR) of elevated biomarkers between two groups were calculated. A total of 779 participants aged 50-84 y, randomized to vitamin D (n = 395) or placebo (n = 384) groups underwent sampling for measurement of plasma biomarkers at baseline and after one or two years treatment. Over a mean follow-up of 1.6 years, we did not find significant relative difference of geometric mean levels of three biomarkers at follow-up between vitamin D and placebo groups: hs-cTnI (aRD=1.03, 95%CI=0.97-1.09), hs-cTnT (aRD=0.99, 95%CI=0.95-1.04), and NT-proBNP (aRD=1.01, 95%CI=0.92-1.10). No significant differences were found in likelihood of clinically elevated biomarkers between two groups: hs-cTnI (aRR=0.92, 95%CI=0.51-1.69), hs-cTnT (aRR=1.11, 95%CI=0.86-1.42), and NT-proBNP (aRR=1.03,95%CI=0.89-1.20). However, among participants with initial low vitamin D status (<50 nmol/L, n = 200), follow-up NT-proBNP were significantly lower in the vitamin D group compared to placebo (geometric mean 75.9 vs 94.5 pg/mL, respectively; aRD=0.84, 95%CI=0.71-<1.00). The same results were observed for the NT-proBNP levels change from baseline between two groups. Overall, in older adults, monthly vitamin D supplementation did not reduce concentrations of hs-cTnI, hs-cTnT, and NT-proBNP. In those with low vitamin D status, vitamin D treatment was associated, on follow up and change from baseline, with lower plasma NT-proBNP compared with placebo. This potentially signals reduced risk of subsequent heart failure within this sub-group. However, we acknowledge that these findings need to be considered exploratory. Further research is required to replicate them.

RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder.

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest.

Strand-specific effect of Rad26 and TFIIS in rescuing transcriptional arrest by CAG trinucleotide repeat slip-outs.

Transcription induced CAG repeat instability is associated with fatal neurological disorders. Genetic approaches found transcription-coupled nucleotide excision repair (TC-NER) factor CSB protein and TFIIS play critical roles in modulating the repeat stability. Here, we took advantage of an in vitro reconstituted yeast transcription system to investigate the underlying mechanism of RNA polymerase II (Pol II) transcriptional pausing/stalling by CAG slip-out structures and the functions of TFIIS and Rad26, the yeast ortholog of CSB, in modulating transcriptional arrest. We identified length-dependent and strand-specific mechanisms that account for CAG slip-out induced transcriptional arrest. We found substantial R-loop formation for the distal transcriptional pausing induced by template strand (TS) slip-out, but not non-template strand (NTS) slip-out. In contrast, Pol II backtracking was observed at the proximal transcriptional pausing sites induced by both NTS and TS slip-out blockage. Strikingly, we revealed that Rad26 and TFIIS can stimulate bypass of NTS CAG slip-out, but not TS slip-out induced distal pausing. Our biochemical results provide new insights into understanding the mechanism of CAG slip-out induced transcriptional pausing and functions of transcription factors in modulating transcription-coupled CAG repeat instability, which may pave the way for developing potential strategies for the treatment of repeat sequence associated human diseases.