The future of association studies: gene-based analysis and replication

Historically, association tests were limited to single variants, so that the allele was considered the basic unit for association testing. As marker density increases and indirect approaches are used to assess association through linkage disequilibrium, association is now frequently considered at the haplotypic level. We suggest that there are difficulties in replicating association findings at the single-nucleotide-polymorphism (SNP) or the haplotype level, and we propose a shift toward a gene-based approach in which all common variation within a candidate gene is considered jointly. Inconsistencies arising from population differences are more readily resolved by use of a gene-based approach rather than either a SNP-based or a haplotype-based approach. A gene-based approach captures all of the potential risk-conferring variations; thus, negative findings are subject only to the issue of power. In addition, chance findings due to multiple testing can be readily accounted for by use of a genewide-significance level. Meta-analysis procedures can be formalized for gene-based methods through the combination of P values. It is only a matter of time before all variation within genes is mapped, at which point the gene-based approach will become the natural end point for association analysis and will inform our search for functional variants relevant to disease etiology.     

MAGP-2 has multiple binding regions on fibrillins and has covalent periodic association with fibrillin-containing microfibrils

The interactions of microfibril-associated glycoprotein (MAGP)-2 have been investigated with fibrillins and fibrillin-containing microfibrils. Solid phase binding assays were conducted with recombinant fragments covering fibrillin-1 and most of fibrillin-2. MAGP-2, and its structure relative MAGP-1, were found to bind two fragments spanning the N-terminal half of fibrillin-1 and an N-terminal fragment of fibrillin-2. Blocking experiments indicated that MAGP-2 had a binding site(s) close to the N terminus of the fibrillin-1 molecule that was distinct from that for MAGP-1 and an additional, more central binding site(s) that may be shared by the two MAGPs. Immunogold labeling of developing nuchal ligament tissue showed that MAGP-2 had regular covalent and periodic (about 56 nm) association with fibrillin-containing microfibrils of elastic fibers in this tissue. Further analysis of isolated microfibrils indicated that MAGP-2 was attached at two points along the microfibril substructure, "site 1" on the "beads" and "site 2" at the "shoulder" of the interbead region close to where the two "arms" fuse. In contrast, MAGP-1 was located only on the beads. Comparison of the MAGP-2 binding data with known fibrillin epitope maps of the microfibrils showed that site 1 correlated with the N-terminal MAGP-2 binding region, and site 2 correlated with the second, more central, MAGP-2 binding region on the fibrillin-1 molecule. Of particular note, immunolabeling at site 2 was markedly decreased, relative to that at site 1, on extended microfibrils with bead-to-bead periods over 90 nm, suggesting that site 2 may move toward the beads when the microfibril is stretched. The study points to MAGP-2 being an integral component of some populations of fibrillin-containing microfibrils. Moreover, the identification of multiple MAGP-binding sequences on fibrillins supports the concept that MAGPs may function as molecular cross-linkers, stabilizing fibrillin monomers in folded conformation within or between the microfibrils, and thus MAGPs may be implicated in the modulation of the elasticity of these structures.     

Misexpression screen for genes altering the olfactory map in Drosophila

Despite the identification of a number of guidance molecules, a comprehensive picture has yet to emerge to explain the precise anatomy of the olfactory map. From a misexpression screen of 1,515 P{GS} lines, we identified 23 genes that, when forcibly expressed in the olfactory receptor neurons, disrupted the stereotyped anatomy of the Drosophila antennal lobes. These genes, which have not been shown previously to control olfactory map development, encode novel proteins as well as proteins with known roles in axonal outgrowth and cytoskeletal remodeling. We analyzed Akap200, which encodes a Protein Kinase A-binding protein. Overexpression of Akap200 resulted in fusion of the glomeruli, while its loss resulted in misshapen and ectopic glomeruli. The requirement of Akap200 validates our screen as an effective approach for recovering genes controlling glomerular map patterning. Our finding of diverse classes of genes reveals the complexity of the mechanisms that underlie olfactory map development.     

Abundance and ecological significance of the clam Rangia cuneata (Sowerby, 1831) in the upper Barataria Estuary (Louisiana,USA)

We proposed that Rangia cuneata (Sowerby, 1831) is an important estuarine bivalve with ecological significance in three coastal lakes in Barataria Bay, Gulf of Mexico—Lake Cataouatche, Lake Salvador and Lac des Allemands. Our goals were to determine the abundance and distribution of Rangia in these lakes and to measure clearance times to elucidate its potential impacts on phytoplankton communities. The estimated average densities of R. cuneata in Lake Cataouatche, Lake Salvador, and Lac des Allemands were 63, 157, and 107 individuals m⁻², respectively, which is 30% lower than that observed in nearby Lake Pontchartrain. The size of clams in Lake Salvador was between 4 and 50 mm, while individuals in Lake Cataouatche and Lac des Allemands were mostly >20 mm. We postulate that a relatively infrequent large tropical storm transported the larvae from Lake Salvador to the other two lakes 1 year before our sampling to create this size difference. The clams were up to 99.9% of the total benthic biomass in Lake Salvador, 15.9% in Lake Cataouatche, and 40.0% in Lac des Allemands. The R. cuneata biomass values were between 16.2 and 27.6 g m⁻² and the clearance times were 1.0–1.5 days. The clearance times are among the highest previously reported for coastal bivalve communities, which were from cooler climates. The results demonstrate that Rangia can be a critical part of the ecological processes in shallow water systems of the Gulf of Mexico.     

Visualization of p53(264-272)/HLA-A*0201 complexes naturally presented on tumor cell surface by a multimeric soluble single-chain T cell receptor

Intracellular Ags are processed into small peptides that are presented on cell surfaces in the context of HLA class I molecules. These peptides are recognized by TCRs displayed by CD8+ T lymphocytes (T cells). To date, direct identification and quantitation of these peptides has relied primarily on mass spectrometry analysis, which is expensive and requires large quantities of diseased tissues to obtain useful results. Here we demonstrate that multimerization of a soluble single-chain TCR (scTCR), recognizing a peptide from p53 presented in the context of HLA-A2.1, could be used to directly visualize and quantitate peptide/MHC complexes on unmanipulated human tumor cells. Tumor cells displaying as few as 500 peptide/MHC complexes were readily detectable by flow cytometry. The scTCR/multimers exhibited exquisite recognition capability and could distinguish peptides differing in as little as a single amino acid. We also demonstrate that scTCR/multimers could specifically stain human tumors generated in mice, as well as tumors obtained from patient biopsies. Thus, scTCR/multimers represent a novel class of immunostaining reagents that could be used to validate, quantitate, or monitor epitope presentation by cancer cells.     

Confirmation of linkage to chromosome 1q for spine bone mineral density in southern Chinese

Chromosome 1q has previously been linked to bone mineral density (BMD) variation in the general population in several genome-wide linkage studies in both humans and mouse model. The aim of present study is to replicate and fine map the QTL influencing BMD in chromosome 1q in southern Chinese. Twelve microsatellite markers were genotyped for a 57 cΜ region in the chromosome 1q in 306 southern Chinese families with 1,459 subjects. Each of these families was ascertained through a proband with BMD Z-scores less than −1.3 at the hip or spine. BMD (g/cm²) at the L1-4 lumbar spine, femoral neck (FN), trochanter and total hip was measured by dual-energy X-ray absortiometry. Linkage analyses were performed using the variance component linkage analysis method implemented in Merlin software. Four markers (D1S2878, D1S196, D1S452, and D1S218) achieved a LOD score greater than 1.0 with spine BMD, with the maximum multipoint LOD score of 2.36 at the marker D1S196. We did not detect a LOD score greater than 1.0 for BMD at the FN, trochanter, or total hip in multipoint linkage analyses. Our results present the first evidence for the presence of an osteoporosis susceptibility gene on chromosome 1q in non-Caucasian subjects. Further analyses of candidate genes are warranted to identify QTL genes and variants underlying the variations of BMD in this region.     

Enhanced store-operated Ca²+ entry and TRPC channel expression in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats

Pulmonary hypertension (PH) is associated with profound vascular remodeling and alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Previous studies show that canonical transient receptor potential (TRPC) genes are upregulated and store-operated Ca(2+) entry (SOCE) is augmented in PASMCs of chronic hypoxic rats and patients of pulmonary arterial hypertension (PAH). Here we further examine the involvement of TRPC and SOCE in PH with a widely used rat model of monocrotaline (MCT)-induced PAH. Rats developed severe PAH, right ventricular hypertrophy, and significant increase in store-operated TRPC1 and TRPC4 mRNA and protein in endothelium-denuded pulmonary arteries (PAs) 3 wk after MCT injection. Contraction of PA and Ca(2+) influx in PASMC evoked by store depletion using cyclopiazonic acid (CPA) were enhanced dramatically, consistent with augmented SOCE in the MCT-treated group. The time course of increase in CPA-induced contraction corresponded to that of TRPC1 expression. Endothelin-1 (ET-1)-induced vasoconstriction was also potentiated in PAs of MCT-treated rats. The response was partially inhibited by SOCE blockers, including Gd(3+), La(3+), and SKF-96365, as well as the general TRPC inhibitor BTP-2, suggesting that TRPC-dependent SOCE was involved. Moreover, the ET-1-induced contraction and Ca(2+) response in the MCT group were more susceptible to the inhibition caused by the various SOCE blockers. Hence, our study shows that MCT-induced PAH is associated with increased TRPC expression and SOCE, which are involved in the enhanced vascular reactivity to ET-1, and support the hypothesis that TRPC-dependent SOCE is an important pathway for the development of PH.     

Mechanisms of FGFR-mediated carcinogenesis

In this review, the evidence for a role of fibroblast growth factor receptor (FGFR) mediated signalling in carcinogenesis are considered and relevant underlying mechanisms highlighted. FGF signalling mediated by FGFR follows a classic receptor tyrosine kinase signalling pathway and its deregulation at various points of its cascade could result in malignancy. Here we review the accumulating reports that revealed the association of FGF/FGFRs to various types of cancer at a genetic level, along with in vitro and in vivo evidences available so far, which indicates the functional involvement of FGF signalling in tumour formation and progression. An increasing number of drugs against the FGF pathways is currently in clinical testing. We will discuss the strategies for future FGF research in cancer and translational approaches.     

Antibody-dependent cellular cytotoxicity against primary HIV-infected CD4+ T cells is directly associated with the magnitude of surface IgG binding

Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is thought to potentially play a role in vaccine-induced protection from HIV-1. The characteristics of such antibodies remain incompletely understood. Furthermore, correlates between ADCC and HIV-1 immune status are not clearly defined. We screened the sera of 20 HIV-1-positive (HIV-1(+)) patients for ADCC. Normal human peripheral blood mononuclear cells were used to derive HIV-infected CD4(+) T cell targets and autologous, freshly isolated, natural killer (NK) cells in a novel assay that measures granzyme B (GrB) and HIV-1-infected CD4(+) T cell elimination (ICE) by flow cytometry. We observed that complex sera mediated greater levels of ADCC than anti-HIV-1 envelope glycoprotein (Env)-specific monoclonal antibodies and serum-mediated ADCC correlated with the amount of IgG and IgG1 bound to HIV-1-infected CD4(+) T cells. No correlation between ADCC and viral load, CD4(+) T cell count, or neutralization of HIV-1(SF162) or other primary viral isolates was detected. Sera pooled from clade B HIV-1(+) individuals exhibited breadth in killing targets infected with HIV-1 from clades A/E, B, and C. Taken together, these data suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of ADCC and that polyvalent antigen-specific Abs are required for a robust ADCC response. In addition, Abs elicited by a vaccine formulated with immunogens from a single clade may generate a protective ADCC response in vivo against a variety of HIV-1 species. Increased understanding of the parameters that dictate ADCC against HIV-1-infected cells will inform efforts to stimulate ADCC activity and improve its potency in vaccinees.     

A novel property of spider silk: chemical defence against ants

Spider webs are made of silk, the properties of which ensure remarkable efficiency at capturing prey. However, remaining on, or near, the web exposes the resident spiders to many potential predators, such as ants. Surprisingly, ants are rarely reported foraging on the webs of orb-weaving spiders, despite the formidable capacity of ants to subdue prey and repel enemies, the diversity and abundance of orb-web spiders, and the nutritional value of the web and resident spider. We explain this paradox by reporting a novel property of the silk produced by the orb-web spider Nephila antipodiana (Walckenaer). These spiders deposit on the silk a pyrrolidine alkaloid (2-pyrrolidinone) that provides protection from ant invasion. Furthermore, the ontogenetic change in the production of 2-pyrrolidinone suggests that this compound represents an adaptive response to the threat of natural enemies, rather than a simple by-product of silk synthesis: while 2-pyrrolidinone occurs on the silk threads produced by adult and large juvenile spiders, it is absent on threads produced by small juvenile spiders, whose threads are sufficiently thin to be inaccessible to ants.