Development and characterization of synthetic antibodies binding to the cystic fibrosis conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies

Trends over time in congenital malformations in live-born children conceived after assisted reproductive technology

IntroductionChildren born after assisted reproductive technology, particularly singletons, have been shown to have an increased risk of congenital malformations compared with children born after spontaneous conception. We wished to study whether there has been a change in the past 20 years in the risk of major congenital malformations in children conceived after assisted reproductive technology compared with children spontaneously conceived. Material and methodsPopulation-based cohort study including 90 201 assisted reproductive technology children and 482 552 children spontaneously conceived, born in Denmark, Finland, Norway and Sweden. Both singletons and twins born after in vitro fertilization, intracytoplasmatic sperm injection and frozen embryo transfer were included. Data on children were taken from when the national Nordic assisted reproductive technology registries were established until 2007. Multiple logistic regression analyses were used to estimate the risks and adjusted odds ratios for congenital malformations in four time periods: 1988-1992, 1993-1997, 1998-2002 and 2003-2007. Only major malformations were included. ResultsThe absolute risk for singletons of being born with a major malformation was 3.4% among assisted reproductive technology children vs. 2.9% among children spontaneously conceived during the study period. The relative risk of being born with a major congenital malformation between all assisted reproductive technology children and children spontaneously conceived remained similar through all four time periods (p = 0.39). However, we found that over time the number of children diagnosed with a major malformation increased in both groups across all four time periods. ConclusionWhen comparing children conceived after assisted reproductive technology and spontaneously conceived, the relative risk of being born with a major congenital malformation did not change during the study period.Peer reviewe

No effect of the turmeric root phenol curcumin on prednisolone-induced glucometabolic perturbations in men with overweight or obesity

Objectives: Preclinically, curcumin has been shown to protect against glucocorticoid-induced insulin resistance. We evaluated the effect of curcumin administered with prednisolone in healthy overweight or obese men. Methods: In a double-blind, parallel-group trial, 24 overweight/obese non-diabetic men were randomised to one of three intervention groups (A) prednisolone placebo+curcumin placebo, (B) prednisolone (50 mg/day)+curcumin placebo or (C) prednisolone and curcumin (400 mg/day). Curcumin or curcumin placebo treatment started 1 day prior to 10-day prednisolone or prednisolone placebo treatment. The primary endpoint was change in prednisolone-induced insulin resistance assessed by homeostatic model assessment of insulin resistance (HOMA2-IR). Other endpoints included anthropometric measurements, magnetic resonance spectroscopy-assessed hepatic fat content, blood pressure, circulating metabolic markers and continuous glucose monitoring measures. Results: Baseline characteristics (mean ± s.d): age 44.2 ± 13.7 years, BMI 30.1 ± 3.5 kg/m2, HbAlc 33.3 ± 3.2 mmol/mol, HOMA2-IR 1.10 ± 0.45 and fasting plasma glucose 5.2 ± 0.4 mmol/L. Prednisolone significantly increased HOMA2-IR (estimated treatment difference 0.36 (95% CI 0.16; 0.57)). Co-treatment with curcumin had no effect on HOMA2-IR (estimated treatment difference 0.08 (95% CI −0.13; 0.39)). Prednisolone increased HbAlc, insulin, C-peptide, glucagon, blood pressure, mean interstitial glucose, time spent in hyperglycaemia and glucose variability, but no protective effect of curcumin on any of these measures was observed. Conclusions: In this double-blind, placebo-controlled parallel-group study involving 24 overweight or obese men randomised to one of three treatment arms, curcumin treatment had no protective effect on prednisolone-induced insul in resistance or other glucometabolic perturbations

Women Born Preterm or with Inappropriate Weight for Gestational Age Are at Risk of Subsequent Gestational Diabetes and Pre-Eclampsia

Introduction: Low birthweight, which can be caused by inappropriate intrauterine growth or prematurity, is associated with development of gestational diabetes mellitus (GDM) as well as pre-eclampsia later in life, but the relative effects of prematurity and inappropriate intrauterine growth remain uncertain. Methods: Through nation-wide registries we identified all Danish mothers in the years 1989–2007. Two separate cohorts consisting mothers born 1974–1977 (n = 84219) and 1978–1981 (n = 32376) were studied, due to different methods o

Genomic analyses identify recurrent MEF2D fusions in acute lymphoblastic leukemia

Chromosomal rearrangements are initiating events in acute lymphoblastic leukaemia (ALL). Here using RNA sequencing of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2DBCL9. Examination of an extended cohort of 1,164 B-ALL cases identified 30 cases with MEF2D rearrangements, which include an additional fusion partner, FOXJ2; thus, MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitor treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukaemia, for which new therapeutic approaches should be considered.This work was supported in part by the American Lebanese Syrian Associated Charities of St. Jude Children’s Research Hospital; by a Stand Up to Cancer Innovative Research Grant and St. Baldrick’s Foundation Scholar Award (to C.G.M.); by a St. Baldrick’s Consortium Award (S.P.H.), by a Leukemia and Lymphoma Society Specialized Center of Research grant (S.P.H. and C.G.M.), by a Lady Tata Memorial Trust Award (I.I.), by a Leukemia and Lymphoma Society Special Fellow Award and Alex’s Lemonade Stand Foundation Young Investigator Awards (K.R.), by an Alex’s Lemonade Stand Foundation Award (M.L.) and by National Cancer Institute Grants CA21765 (St Jude Cancer Center Support Grant), U01 CA157937 (C.L.W. and S.P.H.), U24 CA114737 (to Dr Gastier-Foster), NCI Contract HHSN261200800001E (to Dr Gastier-Foster), U10 CA180820 (ECOG-ACRIN Operations) and CA180827 (E.P.); U10 CA180861 (C.D.B. and G.M.); U24 CA196171 (The Alliance NCTN Biorepository and Biospecimen Resource); CA145707 (C.L.W. and C.G.M.); and grants to the COG: U10 CA98543 (Chair’s grant and supplement to support the COG ALL TARGET project), U10 CA98413 (Statistical Center) and U24 CA114766 (Specimen Banking). This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract Number HHSN261200800001E

Finding Our Way in the Dark Proteome

The traditional structure-function paradigm has provided significant insights for well-folded proteins in which structures can be easily and rapidly revealed by X-ray crystallography beamlines. However approximately one third of the human proteome are comprised of intrinsically disordered proteins and regions (IDPs/IDRs) that do not adopt a dominant well-folded structure, and therefore remain “unseen” by traditional structural biology methods. This Perspective article considers the challenges raised by the “Dark Proteome”, in which determining the diverse conformational substates of IDPs in their free states, in encounter complexes of bound states, and in complexes retaining significant disorder, requires an unprecedented level of integration of multiple and complementary solution-based experiments that are analyzed with state-of-the art molecular simulation, Bayesian probabilistic models, and high throughput computation. We envision how these diverse experimental and computational tools can work together through formation of a “computational beamline” that will allow key functional features to be identified in IDP structural ensembles

A systematic exploration of differences in contextual factors related to implementing the MOVE! weight management program in VA: A mixed methods study

<p>Abstract</p> <p>Background</p> <p>In January 2006, Veterans Affairs (VA) disseminated the MOVE!^®Weight Management Program to VA medical centers to address the high prevalence of overweight/obesity. In its second year, MOVE! implementation varied widely across facilities. The objective of this study was to understand contextual factors that facilitated or impeded implementation of MOVE! in VA medical centers in the second year after its dissemination.</p> <p>Methods</p> <p>We used an embedded mixed methods cross-sectional study design. Qualitative and quantitative data were collected simultaneously with the primary purpose to explore contextual factors most likely to influence MOVE! implementation effectiveness at five purposively selected facilities. Facilities were selected to maximize variation with respect to participation in MOVE! by candidate Veterans. Semi-structured phone interviews were conducted with 24 staff across the five facilities. Quantitative responses were elicited followed by open-ended questions. The quantitative measures were adapted from a published implementation model. Qualitative analysis was conducted using rigorous content analysis methods.</p> <p>Results</p> <p>Qualitative and quantitative data converged to strengthen findings that point to several recommendations. Management support can help increase visibility of the program, commit needed resources, and communicate the importance of implementation efforts. Establishing a receptive implementation climate can be accomplished by emphasizing the important role that weight management may have in reducing incidence and severity of obesity-related chronic conditions. Coalescing highly functioning multi-disciplinary teams was an essential step for more effective implementation of MOVE!. In some situations, local champions can overcome challenging barriers in facilities that lack sufficient management support.</p> <p>Conclusions</p> <p>Key organizational factors at local VA medical centers were strongly associated with MOVE! implementation. Results pointed to recommendations that can help accelerate large-scale dissemination of complex weight management programs.</p

Information content and reward processing in the human striatum during performance of a declarative memory task

Negative feedback can signal poor performance, but it also provides information that can help learners reach the goal of task mastery. The primary aim of this study was to test the hypothesis that the amount of information provided by negative feedback during a paired-associate learning task influences feedback-related processing in the caudate nucleus. To do this, we manipulated the number of response options: With two options, positive and negative feedback provide equal amounts of information, whereas with four options, positive feedback provides more information than does negative feedback. We found that positive and negative feedback activated the caudate similarly when there were two response options. With four options, the caudate’s response to negative feedback was reduced. A secondary goal was to investigate the link between brain-based measures of feedback-related processing and behavioral indices of learning. Analysis of the posttest measures showed that trials with positive feedback were associated with higher posttest confidence ratings. Additionally, when positive feedback was delivered, caudate activity was greater for trials with high than with low posttest confidence. This experiment demonstrated the context sensitivity of feedback processing and provided evidence that feedback processing in the striatum can contribute to the strengthening of the representations available within declarative memory