Evidence for a quantitative trait locus affecting low levels of apolipoprotein B and low density lipoprotein on chromosome 10 in Caucasian families

High plasma apolipoprotein B (apoB) and LDL cholesterol levels increase cardiovascular disease risk. These highly correlated measures may be partially controlled by common genetic polymorphisms. To identify chromosomal regions that contain genes causing low plasma levels of one or both parameters in Caucasian families ascertained for familial hypobetalipoproteinemia (FHBL), we conducted a whole-genome scan using 443 microsatellite markers typed in nine multigenerational families with at least two members with FHBL. Both variance components and regression-based linkage methods were used to identify regions of interest. Common linkage regions were identified for both measures on chromosomes 10q25.1-10q26.11 [maximum log of the odds (LOD) = 4.2 for LDL and 3.5 for apoB] and 6q24.3 (maximum LOD = 1.46 for LDL and 1.84 for apoB). There was also evidence for linkage to apoB on chromosome 13q13.2 (LOD = 1.97) and to LDL on chromosome 3p14.1 at 94 centimorgan (LOD = 1.52). Bivariate linkage analysis provided further evidence for loci contributing to both traits (6q24.3, LOD = 1.43; 10q25.1, LOD = 1.74). We evaluated single nucleotide polymorphisms (SNPs) in genes within our linkage regions to identify variants associated with apoB or LDL levels. The most significant finding was for rs2277205 in the 5' untranslated region of acyl-coenzyme A dehydrogenase short/branched chain and LDL (P = 10(-7)). Three additional SNPs were associated with apoB and/or LDL (P < 0.01). Although only the linkage signal on chromosome 10 reached genome-wide statistical significance, there are likely multiple chromosomal regions with variants that contribute to low levels of apoB and LDL and that may protect against coronary heart disease.     

A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts

Chloroplast genomes in land plants harbor approximately 20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1- and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein-protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity.     

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization

Overexpressed recombinant proteins in bacteria often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or of chaperones. This can be accomplished by growing the bacterial cells in the presence of high salt, sorbitol, and betaine as well as exposing the cells to a heat shock step. Our results suggest that by growing the cells under varied conditions one may be able to express targets as soluble proteins (from previously insoluble targets) and to improve the chances of their crystallization.     

Response of seaward-migrating European eel (Anguilla anguilla) to manipulated flow fields

Anthropogenic structures (e.g. weirs and dams) fragment river networks and restrict the movement of migratory fish. Poor understanding of behavioural response to hydrodynamic cues at structures currently limits the development of effective barrier mitigation measures. This study aimed to assess the effect of flow constriction and associated flow patterns on eel behaviour during downstream migration. In a field experiment, we tracked the movements of 40 tagged adult European eels (Anguilla anguilla) through the forebay of a redundant hydropower intake under two manipulated hydrodynamic treatments. Interrogation of fish trajectories in relation to measured and modelled water velocities provided new insights into behaviour, fundamental for developing passage technologies for this endangered species. Eels rarely followed direct routes through the site. Initially, fish aligned with streamlines near the channel banks and approached the intake semi-passively. A switch to more energetically costly avoidance behaviours occurred on encountering constricted flow, prior to physical contact with structures. Under high water velocity gradients, fish then tended to escape rapidly back upstream, whereas exploratory ‘search’ behaviour was common when acceleration was low. This study highlights the importance of hydrodynamics in informing eel behaviour. This offers potential to develop behavioural guidance, improve fish passage solutions and enhance traditional physical screening.     

A Kinetic Study of Ovalbumin Fibril Formation: The Importance of Fragmentation and End-Joining

The ability to control the morphologies of biomolecular aggregates is a central objective in the study of self-assembly processes. The development of predictive models offers the surest route for gaining such control. Under the right conditions, proteins will self-assemble into fibers that may rearrange themselves even further to form diverse structures, including the formation of closed loops. In this study, chicken egg white ovalbumin is used as a model for the study of fibril loops. By monitoring the kinetics of self-assembly, we demonstrate that loop formation is a consequence of end-to-end association between protein fibrils. A model of fibril formation kinetics, including end-joining, is developed and solved, showing that end-joining has a distinct effect on the growth of fibrillar mass density (which can be measured experimentally), establishing a link between self-assembly kinetics and the underlying growth mechanism. These results will enable experimentalists to infer fibrillar morphologies from an appropriate analysis of self-assembly kinetic data.     

Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, P _meta = 6.6×10⁻⁸, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, P _meta = 2.9×10⁻⁷, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ∼146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (P _meta = 3.2×10⁻⁷, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (P _meta = 3.5×10⁻⁴, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.     

Communication about Children's Clinical Trials as Observed and Experienced: Qualitative Study of Parents and Practitioners

Background

Recruiting children to clinical trials is perceived to be challenging. To identify ways to optimise recruitment and its conduct, we compared how parents and practitioners described their experiences of recruitment to clinical trials.

Methods and Findings

This qualitative study ran alongside four children''s clinical trials in 11 UK research sites. It compared analyses of semi-structured interviews with analyses of audio-recordings of practitioner-family dialogue during trial recruitment discussions. Parents from 59 families were interviewed; 41 had participated in audio-recorded recruitment discussions. 31 practitioners were interviewed. Parents said little in the recruitment discussions contributing a median 16% of the total dialogue and asking a median of one question. Despite this, parents reported a positive experience of the trial approach describing a sense of comfort and safety. Even if they declined or if the discussion took place at a difficult time, parents understood the need to approach them and spoke of the value of research. Some parents viewed participation as an ‘exciting’ opportunity. By contrast, practitioners often worried that approaching families about research burdened families. Some practitioners implied that recruiting to clinical trials was something which they found aversive. Many were also concerned about the amount of information they had to provide and believed this overwhelmed families. Whilst some practitioners thought the trial information leaflets were of little use to families, parents reported that they used and valued the leaflets. However, both parties agreed that the leaflets were too long and wanted them to be more reader-friendly.

Conclusions

Parents were more positive about being approached to enter their child into a clinical trial than practitioners anticipated. The concerns of some practitioners, that parents would be overburdened, were unfounded. Educating practitioners about how families perceive clinical trials and providing them with ‘moral’ support in approaching families may benefit paediatric research and, ultimately, patients.     

Development and inter-rater reliability of the Liverpool adverse drug reaction causality assessment tool

Aim

To develop and test a new adverse drug reaction (ADR) causality assessment tool (CAT).

Methods

A comparison between seven assessors of a new CAT, formulated by an expert focus group, compared with the Naranjo CAT in 80 cases from a prospective observational study and 37 published ADR case reports (819 causality assessments in total).

Main Outcome Measures

Utilisation of causality categories, measure of disagreements, inter-rater reliability (IRR).

Results

The Liverpool ADR CAT, using 40 cases from an observational study, showed causality categories of 1 unlikely, 62 possible, 92 probable and 125 definite (1, 62, 92, 125) and ‘moderate’ IRR (kappa 0.48), compared to Naranjo (0, 100, 172, 8) with ‘moderate’ IRR (kappa 0.45). In a further 40 cases, the Liverpool tool (0, 66, 81, 133) showed ‘good’ IRR (kappa 0.6) while Naranjo (1, 90, 185, 4) remained ‘moderate’.

Conclusion

The Liverpool tool assigns the full range of causality categories and shows good IRR. Further assessment by different investigators in different settings is needed to fully assess the utility of this tool.