Charge density influences C1 domain ligand affinity and membrane interactions.

The C1 domain, which represents the recognition motif on protein kinase C for the lipophilic second messenger diacylglycerol and its ultrapotent analogues, the phorbol esters, has emerged as a promising therapeutic target for cancer and other indications. Potential target selectivity is markedly enhanced both because binding reflects ternary complex formation between the ligand, C1 domain, and phospholipid, and because binding drives membrane insertion of the C1 domain, permitting aspects of the C1 domain surface outside the binding site, per se, to influence binding energetics. Here, focusing on charged residues identified in atypical C1 domains which contribute to their loss of ligand binding activity, we showed that increasing charge along the rim of the binding cleft of the protein kinase C δ C1 b domain raises the requirement for anionic phospholipids. Correspondingly, it shifts the selectivity of C1 domain translocation to the plasma membrane, which is more negatively charged than internal membranes. This change in localization is most pronounced in the case of more hydrophilic ligands, which provide weaker membrane stabilization than do the more hydrophobic ligands and thus contributes an element to the structure-activity relations for C1 domain ligands. Coexpressing pairs of C1-containing constructs with differing charges each expressing a distinct fluorescent tag provided a powerful tool to demonstrate the effect of increasing charge in the C1 domain.

Molecular basis for failure of "atypical" C1 domain of Vav1 to bind diacylglycerol/phorbol ester.

C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu(9), Glu(10), Thr(11), Thr(24), and Tyr(26)) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1.

Common congenital heart problems in acute and intensive care.

Over the past five decades, there have been multiple advances in the treatment of congenital heart defects, resulting in an increasing population of adults living with congenital heart disease (CHD). Despite improved survival, CHD patients often have residual haemodynamic sequelae and limited physiologic reserve and are at increased risk for acute decompensation with occurrence of arrhythmias, heart failure, and other medical conditions. Comorbidities occur more frequently and at an earlier age in CHD patients than in the general population. The management of the critically ill CHD patient requires an understanding of the unique aspects of congenital cardiac physiology as well as the recognition of other organ systems that may be involved. Certain patients may be candidates for mechanical circulatory support, and goals of care should be established with advanced care planning.

Right ventricular contraction patterns in healthy children using three-dimensional echocardiography.

Background: The right ventricle (RV) has complex geometry and function, with motion along three separate axes-longitudinal, radial, and anteroposterior. Quantitative assessment of RV function by two-dimension echocardiography (2DE) has been limited as a consequence of this complexity, whereas newer three dimensional (3D) analysis offers the potential for more comprehensive assessment of the contributors to RV function. The aims of this study were to quantify the longitudinal, radial and anteroposterior components of global RV function using 3D echocardiography in a cohort of healthy children and to examine maturational changes in these parameters. Methods: Three-dimensional contours of the RV were generated from a cohort of healthy pediatric patients with structurally normal hearts at two centers. Traditional 2D and 3D echo characteristics were recorded. Using offline analysis of 3D datasets, RV motion was decomposed into three components, and ejection fractions (EF) were calculated (longitudinal-LEF; radial-REF; and anteroposterior-AEF). The individual decomposed EF values were indexed against the global RVEF. Strain values were calculated as well. Results: Data from 166 subjects were included in the analysis; median age was 13.5 years (range 0 to 17.4 years). Overall, AEF was greater than REF and LEF (29.2 ± 6.2% vs. 25.1 ± 7.2% and 25.7 ± 6.0%, respectively; p < 0.001). This remained true when indexed to overall EF (49.8 ± 8.7% vs. 43.3 ± 11.6% and 44.4 ± 10%, respectively; p < 0.001). Age-related differences were present for global RVEF, REF, and all components of RV strain. Conclusions: In healthy children, anteroposterior shortening is the dominant component of RV contraction. Evaluation of 3D parameters of the RV in children is feasible and enhances the overall understanding of RV function, which may allow improvements in recognition of dysfunction and assessment of treatment effects in the future.

Statin therapy across the lifespan: evidence in major age groups.

This review provides needed perspective on statin efficacy and safety in individuals under 40, 40-75, and > 75 years of age. Starting with the 2013 ACC-AHA cholesterol guidelines extensive evidence base on randomized controlled trials (RCTs) we added references in the past 5 years that discussed statin efficacy and safety over the life span. In those under 40, statins are primarily used for treatment of severe hypercholesterolemia, often familial, and they are well tolerated. In middle-aged adults, statins have strong evidence for benefit in primary and secondary prevention trials; however, in primary prevention, a clinician-patient risk discussion should precede statin prescription in order to determine appropriate treatment. In those over 75, issues of statin intensity and net benefit loom large as associated comorbidity, polypharmacy, and potential for adverse effects impact the decision to use statins with RCT data strongest in support of use in secondary prevention. Statin drugs have been studied by RCTs in a large number of individuals. In those groups shown to benefit, statins have reduced the risk of atherosclerotic cardiovascular disease with few side effects as compared to controls. This review has detailed considerations that should occur when statins are given to individuals in different age groups.

Physicians' Lack of Adherence to National Heart, Lung, and Blood Institute Guidelines for Pediatric Lipid Screening.

OBJECTIVES: To determine adherence to the 2011 National Heart, Lung, and Blood Institute lipid screening guidelines and identify patient factors promoting screening. METHODS: Records of children who received well-child care at age 11 years and turned 12 in 2013 were reviewed. Subjects were stratified by guideline-defined dyslipidemia risk based on documented medical or family history risk factors. We defined adherence as the order of a lipid profile when age 11 years or completed lipid screening at 9 to 10 years. RESULTS: Of 298 subjects, 42% were assigned to the dyslipidemia high-risk subgroup. Records of 27.2% demonstrated adherence. Fifty-six percent of high-risk subjects versus 6% of their non-high-risk counterparts received lipid screening by age 12 (P < .001). Among screened subjects, history of obesity and parental history of dyslipidemia were significantly associated with lipid testing. CONCLUSIONS: Lipid screening rates were low. Strategies to increase lipid screening in the primary care setting are needed.

Critical role of VCP/p97 in the pathogenesis and progression of non-small cell lung carcinoma.

BACKGROUND: Valosin-containing protein (VCP)/p97 is an AAA ATPase molecular chaperone that regulates vital cellular functions and protein-processing. A recent study indicated that VCP expression levels are correlated with prognosis and progression of non-small cell lung carcinoma (NSCLC). We not only verified these findings but also identified the specific role of VCP in NSCLC pathogenesis and progression. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that VCP is significantly overexpressed in non-small cell lung carcinoma (NSCLC) as compared to normal tissues and cell lines (p<0.001). Moreover, we observed the corresponding accumulation of ubiquitinated-proteins in NSCLC cell lines and tissues as compared to the normal controls. VCP inhibition by si/shRNA or small-molecule (Eeyarestatin I, EerI) significantly (p<0.05, p<0.00007) suppressed H1299 proliferation and migration but induced (p<0.00001) apoptosis. Cell cycle analysis by flow cytometry verified this data and shows that VCP inhibition significantly (p<0.001, p<0.003) induced cell cycle arrest in the G0/G1 phases. We also found that VCP directly regulates p53 and NFκB protein levels as a potential mechanism to control tumor cell proliferation and progression. Finally, we evaluated the therapeutic potential of VCP inhibition and observed significantly reduced NSCLC tumor growth in both in vitro and xenograft murine (athymic-nude) models after EerI treatment (p<0.05). CONCLUSIONS/SIGNIFICANCE: Thus, targeting VCP in NSCLC may provide a novel strategy to restore p53 and NFκB levels and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes.