Quantitative protein microarrays for time-resolved measurements of protein phosphorylation

The quantitative analysis of signaling networks requires highly sensitive methods for the time-resolved determination of protein phosphorylation. For this reason, we developed a quantitative protein microarray that monitors the activation of multiple signaling pathways in parallel, and at high temporal resolution. A label-free sandwich approach was combined with near infrared detection, thus permitting the accurate quantification of low-level phosphoproteins in limited biological samples corresponding to less than 50,000 cells, and with a very low standard deviation of approximately 5%. The identification of suitable antibody pairs was facilitated by determining their accuracy and dynamic range using our customized software package Quantpro. Thus, we are providing an important tool to generate quantitative data for systems biology approaches, and to drive innovative diagnostic applications.     

Submembraneous microtubule cytoskeleton: biochemical and functional interplay of TRP channels with the cytoskeleton

Much work has focused on the electrophysiological properties of transient receptor potential channels. Recently, a novel aspect of importance emerged: the interplay of transient receptor potential channels with the cytoskeleton. Recent data suggest a direct interaction and functional repercussion for both binding partners. The bi-directionality of physical and functional interaction renders therefore, the cytoskeleton a potent integration point of complex biological signalling events, from both the cytoplasm and the extracellular space. In this minireview, we focus mostly on the interaction of the cytoskeleton with transient receptor potential vanilloid channels. Thereby, we point out the functional importance of cytoskeleton components both as modulator and as modulated downstream effector. The resulting implications for patho-biological situations are discussed.     

Age-variability in body shape associated with excess weight: the UK National Sizing Survey

Background: The health risks of obesity are disproportionately due to central abdominal adiposity; however, the extent to which age is associated with the body shape of obese adults is not known. Objective: Three‐dimensional (3D) data on body shape from the UK National Sizing Survey were analyzed to investigate age‐associated changes in body shape within the BMI bands <20, 20–24.99, 25–29.99 and ≥30 kg/m². Methods: Measurements of anthropometry (weight and height) and a 3D body scan were obtained in 4,344 men and 5,266 women recruited from eight British cities. Results: The body shape of men showed high consistency within BMI bands between early adulthood and old age. In contrast, the body shape of women altered within each BMI band with increasing age. In obese, overweight, and normal weight women, age was associated with decreased thigh girth, increased waist, and bust girth. Whereas young obese women maintained an hourglass shape, in old age the body shape of obese women converged on that of obese men. Discussion: The association of age with body shape is markedly different between the sexes, with the impact of obesity on shape strongly age‐dependent in women but not in men. The age delay in the association between obesity and high waist girth in women may contribute to the sex‐difference in life expectancy. The relationship between body shape change and cardiovascular risk merits longitudinal investigation within individuals.     

C-ME: a 3D community-based, real-time collaboration tool for scientific research and training

The need for effective collaboration tools is growing as multidisciplinary proteome-wide projects and distributed research teams become more common. The resulting data is often quite disparate, stored in separate locations, and not contextually related. Collaborative Molecular Modeling Environment (C-ME) is an interactive community-based collaboration system that allows researchers to organize information, visualize data on a two-dimensional (2-D) or three-dimensional (3-D) basis, and share and manage that information with collaborators in real time. C-ME stores the information in industry-standard databases that are immediately accessible by appropriate permission within the computer network directory service or anonymously across the internet through the C-ME application or through a web browser. The system addresses two important aspects of collaboration: context and information management. C-ME allows a researcher to use a 3-D atomic structure model or a 2-D image as a contextual basis on which to attach and share annotations to specific atoms or molecules or to specific regions of a 2-D image. These annotations provide additional information about the atomic structure or image data that can then be evaluated, amended or added to by other project members.     

Identification of PLCL1 gene for hip bone size variation in females in a genome-wide association study

Osteoporosis, the most prevalent metabolic bone disease among older people, increases risk for low trauma hip fractures (HF) that are associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of the key measurable risk factors for HF. Although hip BS is highly genetically determined, genetic factors underlying the trait are still poorly defined. Here, we performed the first genome-wide association study (GWAS) of hip BS interrogating approximately 380,000 SNPs on the Affymetrix platform in 1,000 homogeneous unrelated Caucasian subjects, including 501 females and 499 males. We identified a gene, PLCL1 (phospholipase c-like 1), that had four SNPs associated with hip BS at, or approaching, a genome-wide significance level in our female subjects; the most significant SNP, rs7595412, achieved a p value of 3.72x10(-7). The gene's importance to hip BS was replicated using the Illumina genotyping platform in an independent UK cohort containing 1,216 Caucasian females. Two SNPs of the PLCL1 gene, rs892515 and rs9789480, surrounded by the four SNPs identified in our GWAS, achieved p values of 8.62x10(-3) and 2.44x10(-3), respectively, for association with hip BS. Imputation analyses on our GWAS and the UK samples further confirmed the replication signals; eight SNPs of the gene achieved combined imputed p values<10(-5) in the two samples. The PLCL1 gene's relevance to HF was also observed in a Chinese sample containing 403 females, including 266 with HF and 177 control subjects. A SNP of the PLCL1 gene, rs3771362 that is only approximately 0.6 kb apart from the most significant SNP detected in our GWAS (rs7595412), achieved a p value of 7.66x10(-3) (odds ratio = 0.26) for association with HF. Additional biological support for the role of PLCL1 in BS comes from previous demonstrations that the PLCL1 protein inhibits IP3 (inositol 1,4,5-trisphosphate)-mediated calcium signaling, an important pathway regulating mechanical sensing of bone cells. Our findings suggest that PLCL1 is a novel gene associated with variation in hip BS, and provide new insights into the pathogenesis of HF.