Intrinsic protein disorder and interaction promiscuity are widely associated with dosage sensitivity.

Why are genes harmful when they are overexpressed? By testing possible causes of overexpression phenotypes in yeast, we identify intrinsic protein disorder as an important determinant of dosage sensitivity. Disordered regions are prone to make promiscuous molecular interactions when their concentration is increased, and we demonstrate that this is the likely cause of pathology when genes are overexpressed. We validate our findings in two animals, Drosophila melanogaster and Caenorhabditis elegans. In mice and humans the same properties are strongly associated with dosage-sensitive oncogenes, such that mass-action-driven molecular interactions may be a frequent cause of cancer. Dosage-sensitive genes are tightly regulated at the transcriptional, RNA, and protein levels, which may serve to prevent harmful increases in protein concentration under physiological conditions. Mass-action-driven interaction promiscuity is a single theoretical framework that can be used to understand, predict, and possibly treat the effects of increased gene expression in evolution and disease.

Rapid selection of transgenic C. elegans using antibiotic resistance.

Caenorhabditis elegans is an important model organism in biology, but until now no antibiotic selection markers have been successfully demonstrated for this species. We have developed a selection system using puromycin that allows the rapid and easy isolation of large populations of transgenic worms. This approach is sufficiently powerful to select single-copy transgenes, does not require any particular genetic background and also works in C. briggsae.

RGS10 restricts upregulation by chemokines of T cell adhesion mediated by α4ß1 and αLß2 integrins.

Chemokines rapidly and transiently upregulate α4ß1 and αLß2 integrin-mediated adhesion during T lymphocyte extravasation by activating Gα-dependent inside-out signaling. To limit and terminate Gα-mediated signaling, cells can use several mechanisms, including the action of regulator of G protein signaling (RGS) proteins, which accelerate the GTPase activity of Gα subunits. Using human T cells silenced for or overexpressing RGS10, we show in this article that RGS10 functions as an inhibitor of Gα(i)-dependent, chemokine-upregulated T cell adhesion mediated by α4ß1 and αLß2. Shear stress-dependent detachment and cell spreading analyses revealed that RGS10 action mainly targets the adhesion strengthening and spreading phases of α4ß1-mediated cell attachment. Associated with these observations, chemokine-stimulated Vav1-Rac1 activation was longer sustained and of higher intensity in RGS10-silenced T cells, or inhibited in cells overexpressing RGS10. Of importance, expression of constitutively activated Rac1 forms in cells overexpressing RGS10 led to the rescue of CXCL12-stimulated adhesion to VCAM-1 to levels similar to those in control transfectants. Instead, adhesion under flow conditions, soluble binding experiment, flow cytometry, and biochemical analyses revealed that the earlier chemokine-triggered integrin activation step was mostly independent of RGS10 actions. The data strongly suggest that RGS10 opposes activation by chemokines of the Vav1-Rac1 pathway in T cells, leading to repression of adhesion strengthening mediated by α4ß1. In addition to control chemokine-upregulated T cell attachment, RGS10 also limited adhesion-independent cell chemotaxis and activation of cdc42. These results identify RGS10 as a key molecule that contributes to the termination of Gα-dependent signaling during chemokine-activated α4ß1- and αLß2-dependent T cell adhesion.

Intracellular signaling required for CCL25-stimulated T cell adhesion mediated by the integrin alpha4beta1.

The alpha4beta1 integrin is expressed on thymocytes and mediates cell attachment to its ligands CS-1/fibronectin (CS-1/FN) and VCAM-1 in the thymus. The chemokine CCL25 is highly expressed in the thymus, where it binds to its receptor CCR9 on thymocytes promoting migration and activation. We show here that alpha4beta1 and CCR9 are coexpressed mainly on double- and single-positive thymocytes and that CCL25 strongly stimulates CD4(+)CD8(+) and CD4(+)CD8(-) adhesion to CS-1/FN and VCAM-1. CCL25 rapidly activated the GTPases Rac and Rap1 on thymocytes, and this activation was required for stimulation of adhesion, as detected using the CCR9(+)/alpha4beta1(+) human T cell line Molt-4. To study the role on CCL25-stimulated adhesion of the Rac downstream effector Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2) as well as of Rap1-GTP-interacting proteins, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP-interacting adapter molecule (RIAM), we knocked down their expression and tested transfectant attachment to alpha4beta1 ligands. We found that WAVE2 and RAPL but not RIAM were required for efficient triggering by CCL25 of T cell adhesion to CS-1/FN and VCAM-1. Although Rac and Rap1 activation was required during early steps of T cell adhesion stimulated by CCL25, WAVE2 was needed for the development of actin-dependent T cell spreading subsequent to adhesion strengthening but not during initial alpha4beta1-ligand interactions. These results suggest that regulation by CCL25 of adhesion of thymocyte subpopulations mediated by alpha4beta1 could contribute to control their trafficking in the thymus during maturation, and identify Rac-WAVE2 and Rap1-RAPL as pathways whose activation is required in inside-out signaling, leading to stimulated adhesion.

A New Optimized Percutaneous Access System for CIPII.

In recent years, continuous intraperitoneal insulin infusion (CIPII) has become a favored treatment alternative for patients with subcutaneous insulin resistance, mainly due to its ability of mimicking physiological conditions of insulin absorption. CIPII has been shown to improve glycemic control as well as to reduce hypoglycemic events and to lead to increased patient satisfaction and quality of life (QoL). Among CIPII delivery systems, Diaport stands out due to its low side effects, its demonstrated clinical efficacy and the potential for integration into closed-loop systems.

Need for Outcome Scenario Analysis of Clinical Trials in Diabetes.

Since the FDA requirement for cardiovascular safety of all new antihyperglycemic drugs to enter the market, the number and extent of phase 3 clinical trials has markedly increased. Unexpected trial results imply an enormous economic, personal and time cost and has deleterious effects over R&D. To prevent unforeseen developments in clinical trials, we recommend performing a comprehensive prospective outcome scenario analysis before launching the trial. In this commentary, we discuss the most important factors to take in consideration for prediction of clinical trial outcome scenarios and propose a theoretical model for decision making.

Impact on Diabetes Self-Management and Glycemic Control of a New Color-Based SMBG Meter.

BACKGROUND: Self-monitoring of blood glucose (SMBG) is a key pillar of personal diabetes management. The objective of this observational study was to analyze diabetes self-management (DSM) and glycemic outcomes before and during system implementation in real-life settings of a blood glucose meter system with a color-coded display of glucose levels, which helps identify out-of-range levels. METHODS: A total of 193 insulin-treated diabetes patients (11% T1DM; 55% male, age 60 ± 4 years, mean diabetes duration 14 ± 9 years, HbA1c 8.68 ± 1.2%) were enrolled into the study. Both the Diabetes Self-Management Questionnaire (DSMQ) and glycemic control were analyzed at baseline and 3 and 6 months after study initiation. RESULTS: DSMQ general perception improved significantly by the end of the study period ("Sum Scale," P < .05). Moreover, after 6 months patient's attitudes on self-care (Q16, P = .0046) and nutrition ("Dietary Control," P = .004) showed significant improvements. Use of the blood glucose meter resulted in improved glycemic control, as shown by mean HbA1c levels, which decreased from 8.68 ± 1.2% at baseline to 8.13 ± 1.02% after 3 months ( P < .0001) and to 7.9 ± 1.1% at 6 months ( P < .0001). Both patients and diabetes educators agreed in the advantages of the color-coded indicator and on its helpfulness in assisting patients on their diabetes management, as drawn from the results of the self-reported satisfaction questionnaire. CONCLUSION: This real-world study demonstrates that SMBG implemented via this new blood glucose meter not only leads to an improvement in metabolic control, but also is associated with a significant improvement in diabetes management.

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4ß1.

Stimulation by chemokines of integrin α4ß1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4ß1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4ß1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase-inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4ß1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4ß1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4ß1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76-, ADAP-, and Pyk2-regulated cell adhesion involving α4ß1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4ß1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation.

Predicting phenotypic variation in yeast from individual genome sequences.

A central challenge in genetics is to predict phenotypic variation from individual genome sequences. Here we construct and evaluate phenotypic predictions for 19 strains of Saccharomyces cerevisiae. We use conservation-based methods to predict the impact of protein-coding variation within genes on protein function. We then rank strains using a prediction score that measures the total sum of function-altering changes in different sets of genes reported to influence over 100 phenotypes in genome-wide loss-of-function screens. We evaluate our predictions by comparing them with the observed growth rate and efficiency of 15 strains tested across 20 conditions in quantitative experiments. The median predictive performance, as measured by ROC AUC, was 0.76, and predictions were more accurate when the genes reported to influence a trait were highly connected in a functional gene network.