Impact of Hapten Presentation on Antibody Binding at Lipid Membrane Interfaces

AbstractWe report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (KD) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, KD(bulk)=1.7±0.2 nM vs. 2.9±0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, KD=3.6±1.1 nM vs. 2.0±0.2 μM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands’ relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the KD value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4±1.1μM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (KD=21±10nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands

Dual mechanism of brain injury and novel treatment strategy in maple syrup urine disease

Maple syrup urine disease (MSUD) is an inherited disorder of branched-chain amino acid metabolism presenting with lifethreatening cerebral oedema and dysmyelination in affected individuals. Treatment requires life-long dietary restriction and monitoring of branched-chain amino acids to avoid brain injury. Despite careful management, children commonly suffer metabolic decompensation in the context of catabolic stress associated with non-specific illness. The mechanisms underlying this decompensation and brain injury are poorly understood. Using recently developed mouse models of classic and intermediate maple syrup urine disease, we assessed biochemical, behavioural and neuropathological changes that occurred during encephalopathy in these mice. Here, we show that rapid brain leucine accumulation displaces other essential amino acids resulting in neurotransmitter depletion and disruption of normal brain growth and development. A novel approach of administering norleucine to heterozygous mothers of classic maple syrup urine disease pups reduced branched-chain amino acid accumulation in milk as well as blood and brain of these pups to enhance survival. Similarly, norleucine substantially delayed encephalopathy in intermediate maple syrup urine disease mice placed on a high protein diet that mimics the catabolic stress shown to cause encephalopathy in human maple syrup urine disease. Current findings suggest two converging mechanisms of brain injury in maple syrup urine disease including: (i) neurotransmitter deficiencies and growth restriction associated with branchedchain amino acid accumulation and (ii) energy deprivation through Krebs cycle disruption associated with branched-chain ketoacid accumulation. Both classic and intermediate models appear to be useful to study the mechanism of brain injury and potential treatment strategies for maple syrup urine disease. Norleucine should be further tested as a potential treatment to prevent encephalopathy in children with maple syrup urine disease during catabolic stress

Use of Sex-Specific Clinical and Exercise Risk Scores to Identify Patients at Increased Risk for All-Cause Mortality

Importance Risk assessment tools for exercise treadmill testing may have limited external validity. Cardiovascular mortality has decreased in recent decades, and women have been underrepresented in prior cohorts. Objectives To determine whether exercise and clinical variables are associated with differential mortality outcomes in men and women and to assess whether sex-specific risk scores better estimate all-cause mortality. Design, Setting, and Participants This retrospective cohort study included 59 877 patients seen at the Cleveland Clinic Foundation (CCF cohort) from January 1, 2000, through December 31, 2010, and 49 278 patients seen at the Henry Ford Hospital (FIT cohort) from January 1, 1991, through December 31, 2009. All patients were 18 years or older and underwent exercise treadmill testing. Data were analyzed from January 1, 2000, to October 27, 2011, in the CCF cohort and from January 1, 1991, to April 1, 2013, in the FIT cohort. Main Outcomes and Measurements The CCF cohort was divided randomly into derivation and validation samples, and separate risk scores were developed for men and women. Net reclassification, C statistics, and integrated discrimination improvement were used to compare the sex-specific risk scores with other tools that have all-cause mortality as the outcome. Discrimination and calibration were also evaluated with these sex-specific risk scores in the FIT cohort. Results The CCF cohort included 59 877 patients (59.4% men; 40.5% women) with a median (interquartile range [IQR]) age of 54 (45-63) years and 2521 deaths (4.2%) during a median follow-up of 7 (IQR, 4.1-9.6) years. The FIT cohort included 49 278 patients (52.5% men; 47.4% women) with a median (IQR) age of 54 (46-64) years and 6643 deaths (13.5%) during a median (IQR) follow-up of 10.2 (7-13.4) years. C statistics for the sex-specific risk scores in the CCF validation sample were higher (0.79 in women and 0.81 in men) than C statistics using other tools in women (0.70 for Duke Treadmill Score; 0.74 for Lauer nomogram) and men (0.72 for Duke Treadmill Score; 0.75 for Lauer nomogram). Net reclassification and integrated discrimination improvement were superior with the sex-specific risk scores, mostly owing to correct reclassification of events. The sex-specific risk scores in the FIT cohort demonstrated similar discrimination (C statistic, 0.78 for women and 0.79 for men), and calibration was reasonable. Conclusions and Relevance Sex-specific risk scores better estimate mortality in patients undergoing exercise treadmill testing. In particular, these sex-specific risk scores help to identify patients at the highest residual risk in the present era

Dynamic plasticity of large-scale chromatin structure revealed by self-assembly of engineered chromosome regions

Folding of gene loci organized in tandem arrays reveals variation of chromatin structure as a function of cell differentiation

A special gift we bestow on you for being representative of us: Considering leader

Two experiments tested hypotheses, derived from social identity and self-categorization theories, regarding the attribution of charisma to leaders. In Experiment I (N = 203), in-group prototypical leaders were attributed greater levels of charisma and were perceived to be more persuasive than in-group non-prototypical leaders. In Experiment 2 (N = 220), leaders described with in-group stereotypical characteristics were attributed relatively high levels of charisma regardless of their group-oriented versus exchange rhetoric. Leaders described with out-group stereotypical characteristics, however, had to employ group-oriented rhetoric to be attributed relatively high levels of charisma. We conclude that leadership emerges from being representative of 'us'; charisma may, indeed, be a special gift, but it is one bestowed on group members by group members for being representative of, rather than distinct from, the group itself. © 2006 The British Psychological Society

Differences in antigenic sites and other functional regions between genotype A and G mumps virus surface proteins

The surface proteins of the mumps virus, the fusion protein (F) and haemagglutinin-neuraminidase (HN), are key factors in mumps pathogenesis and are important targets for the immune response during mumps virus infection. We compared the predicted amino acid sequences of the F and HN genes from Dutch mumps virus samples from the pre-vaccine era (1957–1982) with mumps virus genotype G strains (from 2004 onwards). Genotype G is the most frequently detected mumps genotype in recent outbreaks in vaccinated communities, especially in Western Europe, the USA and Japan. Amino acid differences between the Jeryl Lynn vaccine strains (genotype A) and genotype G strains were predominantly located in known B-cell epitopes and in N-linked glycosylation sites on the HN protein. There were eight variable amino acid positions specific to genotype A or genotype G sequences in five known B-cell epitopes of the HN protein. These differences may account for the reported antigenic differences between Jeryl Lynn and genotype G strains. We also found amino acid differences in and near sites on the HN protein that have been reported to play a role in mumps virus pathogenesis. These differences may contribute to the occurrence of genotype G outbreaks in vaccinated communities

Bioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS

Genomic technologies such as next-generation sequencing (NGS) are revolutionizing molecular diagnostics and clinical medicine. However, these approaches have proven inefficient at identifying pathogenic repeat expansions. Here, we apply a collection of bioinformatics tools that can be utilized to identify either known or novel expanded repeat sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 families with a clinical diagnosis of cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Analysis of whole-genome sequence (WGS) data with five independent algorithms identified a recessively inherited intronic repeat expansion [(AAGGG)exp] in the gene encoding Replication Factor C1 (RFC1). This motif, not reported in the reference sequence, localized to an Alu element and replaced the reference (AAAAG)11 short tandem repeat. Genetic analyses confirmed the pathogenic expansion in 18 of 22 CANVAS-affected families and identified a core ancestral haplotype, estimated to have arisen in Europe more than twenty-five thousand years ago. WGS of the four RFC1-negative CANVAS-affected families identified plausible variants in three, with genomic re-diagnosis of SCA3, spastic ataxia of the Charlevoix-Saguenay type, and SCA45. This study identified the genetic basis of CANVAS and demonstrated that these improved bioinformatics tools increase the diagnostic utility of WGS to determine the genetic basis of a heterogeneous group of clinically overlapping neurogenetic disorders

Diffusion-Driven Looping Provides a Consistent Framework for Chromatin Organization

Chromatin folding inside the interphase nucleus of eukaryotic cells is done on multiple scales of length and time. Despite recent progress in understanding the folding motifs of chromatin, the higher-order structure still remains elusive. Various experimental studies reveal a tight connection between genome folding and function. Chromosomes fold into a confined subspace of the nucleus and form distinct territories. Chromatin looping seems to play a dominant role both in transcriptional regulation as well as in chromatin organization and has been assumed to be mediated by long-range interactions in many polymer models. However, it remains a crucial question which mechanisms are necessary to make two chromatin regions become co-located, i.e. have them in spatial proximity. We demonstrate that the formation of loops can be accomplished solely on the basis of diffusional motion. The probabilistic nature of temporary contacts mimics the effects of proteins, e.g. transcription factors, in the solvent. We establish testable quantitative predictions by deriving scale-independent measures for comparison to experimental data. In this Dynamic Loop (DL) model, the co-localization probability of distant elements is strongly increased compared to linear non-looping chains. The model correctly describes folding into a confined space as well as the experimentally observed cell-to-cell variation. Most importantly, at biological densities, model chromosomes occupy distinct territories showing less inter-chromosomal contacts than linear chains. Thus, dynamic diffusion-based looping, i.e. gene co-localization, provides a consistent framework for chromatin organization in eukaryotic interphase nuclei