Sequence-Level Analysis of the Major European Huntington Disease Haplotype

Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.     

Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations

This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible.     

Structural analysis of human and macaque mAbs 2909 and 2.5B: implications for the configuration of the quaternary neutralizing epitope of HIV-1 gp120

The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 ? in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.     

Design and initial characterization of the SC-200 proteomics standard mixture

High-throughput (HTP) proteomics studies generate large amounts of data. Interpretation of these data requires effective approaches to distinguish noise from biological signal, particularly as instrument and computational capacity increase and studies become more complex. Resolving this issue requires validated and reproducible methods and models, which in turn requires complex experimental and computational standards. The absence of appropriate standards and data sets for validating experimental and computational workflows hinders the development of HTP proteomics methods. Most protein standards are simple mixtures of proteins or peptides, or undercharacterized reference standards in which the identity and concentration of the constituent proteins is unknown. The Seattle Children's 200 (SC-200) proposed proteomics standard mixture is the next step toward developing realistic, fully characterized HTP proteomics standards. The SC-200 exhibits a unique modular design to extend its functionality, and consists of 200 proteins of known identities and molar concentrations from 6 microbial genomes, distributed into 10 molar concentration tiers spanning a 1,000-fold range. We describe the SC-200's design, potential uses, and initial characterization. We identified 84% of SC-200 proteins with an LTQ-Orbitrap and 65% with an LTQ-Velos (false discovery rate?=?1% for both). There were obvious trends in success rate, sequence coverage, and spectral counts with protein concentration; however, protein identification, sequence coverage, and spectral counts vary greatly within concentration levels.     

Effects of different elastic cord assistance levels on vertical jump

Tran, TT, Brown, LE, Coburn, JW, Lynn, SK, Dabbs, NC, Schick, MK, Schick, EE, Khamoui, AV, Uribe, BP, and Noffal, GJ. Effects of different elastic cord assistance levels on vertical jump. J Strength Cond Res 25(12): 3472-3478, 2011-Currently, little research has been conducted using body weight reduction (BWR) as a means to enhance vertical jump. The purpose of this study was to determine the effects of different elastic cord assistance levels on vertical jump height (JH), takeoff velocity (TOV), relative ground reaction force (rGRF), relative impact force (RIF), and descent velocity (DV). Thirty recreationally trained college men and women (M = 15, W = 15) completed 3 testing sessions consisting of 5 conditions: 0, 10, 20, 30, and 40% BWR. In all BWR conditions, the subjects wore a full body harness while being attached to 2 elastic cords suspended from the ceiling and a linear velocity transducer. They then performed 3 maximal countermovement jumps with arm swing on a force plate. The results indicated no interaction of condition by sex for any variable; however, there was a significant (p < 0.05) main effect for condition for each variable. The JH significantly increased across all conditions (0%: 43.73 ± 1.62 cm, 40%: 64.77 ± 2.36 cm). The TOV at 30% (2.73 ± 0.34 m·s) was significantly greater than that at 0% (2.59 ± 0.39 m·s) and 10% (2.63 ± 0.34 m·s), whereas that at 40% (2.79 ± 0.43 m·s) was significantly greater than that at >0, 10, and 20%. The rGRF at 30% (18.62 ± 4.35 N·kg) was significantly greater than that at >0, 10, and 20%, whereas that at 40% (21.38 ± 5.21 N·kg) was significantly greater than in all conditions. The RIF at 20, 30, and 40% (40%: 61.60 ± 18.53 N·kg) was significantly greater than that at 0% (44.46 ± 9.12 N·kg). The DV at 20% (2.61 ± 0.31 m·s) was significantly greater than at 10%, whereas those at 30 and 40% (2.8 ± 0.41 m·s) were significantly greater than at 0, 10, and 20%. These results demonstrate that using different elastic cord levels to reduce body weight appears effective for increasing ascent and descent force and velocity variables. Future research should investigate greater BWR% and chronic training.