Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Focused clamping of a single neuronal SNARE complex by complexin under high mechanical tension

The SNARE complex enables the fusion of synaptic vesicles with presynaptic membrane via a zippering process that is modulated by the protein complexin, though the precise mechanism remains unclear. Here, the authors used magnetic tweezers to show how complexin prepares a SNARE complex for fusion under mechanical tension

Submicrometer elasticity of double-stranded DNA revealed by precision force-extension measurements with magnetic tweezers

Submicrometer elasticity of double-stranded DNA (dsDNA) governs nanoscale bending of DNA segments and their interactions with proteins. Single-molecule force spectroscopy, including magnetic tweezers (MTs), is an important tool for studying DNA mechanics. However, its application to short DNAs under 1 mm is limited. We developed an MT-based method for precise force-extension measurements in the 100-nm regime that enables in situ correction of the error in DNA extension measurement, and normalizes the force variability across beads by exploiting DNA hairpins. The method reduces the lower limit of tractable dsDNA length down to 198 base pairs (bp) (67 nm), an order-of-magnitude improvement compared to conventional tweezing experiments. Applying this method and the finite worm-like chain model we observed an essentially constant persistence length across the chain lengths studied (198 bp to 10 kbp), which steeply depended on GC content and methylation. This finding suggests a potential sequence-dependent mechanism for short-DNA elasticity.11Nsciescopu

Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy

Extremely weak protein-protein interactions (PPIs), signified by micromolar or even millimolar dissociation constants, are one of the keys to understanding the rapid responses of cellular systems. Although single-molecule methods are particularly useful in determining kinetics of biological processes, their application is largely limited to rather strong interactions because of the diffraction-limited observation volume. In this study, we report a single-molecule method that allows the characterization of PPIs using a prey concentration 4 orders of magnitude lower than the dissociation constant. Instead of increasing the concentration of diffusing molecules, which is inevitably limited by the optical diffraction limit, we employed an increased density of surface bait protein. The low occupancy of the surface baits permitted determination of the kinetics with single-molecule resolution. We used this approach to study a PPI network consisting of Ras and its downstream proteins including full-length Rafs and catalytic subunits of phosphoinositide 3-kinase. © 2016 American Chemical Society1541sciescopu

Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy

Extremely weak protein-protein interactions (PPIs), signified by micromolar or even millimolar dissociation constants, are one of the keys to understanding the rapid responses of cellular systems. Although single-molecule methods are particularly useful in determining kinetics of biological processes, their application is largely limited to rather strong interactions because of the diffraction-limited observation volume. In this study, we report a single-molecule method that allows the characterization of PPIs using a prey concentration 4 orders of magnitude lower than the dissociation constant. Instead of increasing the concentration of diffusing molecules, which is inevitably limited by the optical diffraction limit, we employed an increased density of surface bait protein. The low occupancy of the surface baits permitted determination of the kinetics with single molecule resolution. We used this approach to study a PPI network consisting of Ras and its downstream proteins including full-length Rafs and catalytic subunits of phosphoinositide 3-kinase.11Nsciescopu

Tension exerted on cells by magnetic nanoparticles regulates differentiation of human mesenchymal stem cells

Cells can ???sense??? physical cues in the surrounding microenvironment and ???react??? by changing their function. Previous studies have focused on regulating the physical properties of the matrix, such as stiffness and topog-raphy, thus changing the tension ???felt??? by the cell as a result. In this study, by directly applying a quantified magnetic force to the cell, a correlation between differentiation and tension was shown. The magnetic force, quantified by magnetic tweezers, was applied by incorporating magnetotactic bacteria-isolated magnetic nanoparticles (MNPs) in human mesenchymal stem cells. As the applied tension increased, the expression levels of osteogenic differentiation marker genes and proteins were proportionally upregulated. Additionally, the translocation of YAP and RUNX2, deformation of nucleus, and activation of the MAPK signaling pathway were observed in tension-based osteogenic differentiation. Our findings provide a platform for the quantitative control of tension, a key factor in stem cell differentiation, between cells and the matrix using MNPs. Furthermore, these findings improve the understanding of osteogenic differentiation by mechanotransduction.11Nsciescopu

Tension exerted on cells by magnetic nanoparticles regulates differentiation of human mesenchymal stem cells

Cells can ‘sense’ physical cues in the surrounding microenvironment and ‘react’ by changing their function. Previous studies have focused on regulating the physical properties of the matrix, such as stiffness and topography, thus changing the tension ‘felt’ by the cell as a result. In this study, by directly applying a quantified magnetic force to the cell, a correlation between differentiation and tension was shown. The magnetic force, quantified by magnetic tweezers, was applied by incorporating magnetotactic bacteria-isolated magnetic nanoparticles (MNPs) in human mesenchymal stem cells. As the applied tension increased, the expression levels of osteogenic differentiation marker genes and proteins were proportionally upregulated. Additionally, the translocation of YAP and RUNX2, deformation of nucleus, and activation of the MAPK signaling pathway were observed in tension-based osteogenic differentiation. Our findings provide a platform for the quantitative control of tension, a key factor in stem cell differentiation, between cells and the matrix using MNPs. Furthermore, these findings improve the understanding of osteogenic differentiation by mechanotransduction. © 2022 Elsevier B.V.11Nsciescopu

Profiling protein-protein interactions of single cancer cells with in situ lysis and co-immunoprecipitation

Heterogeneity in a tumor allows a small portion of cancer cells to survive and regrow upon targeted cancer therapy, eventually leading to cancer relapse. Such drug-resistant cells often exhibit dynamic adaptation of their signaling pathways at the level of protein- protein interactions (PPIs). To probe the rewiring of signaling pathways and the heterogeneity across individual cancer cells, we developed a single-cell version of the co-immunoprecipitation (co-IP) analysis that examines the amount and PPIs of target proteins immunoprecipitated from individual cells. The method captures cancer cells at predefined locations using a microfluidic chip, pulls down target proteins on the surface using antibodies, and lyses the captured cells in situ. Then, subsequent addition of eGFP-labeled downstream proteins enables the determination of the corresponding PPIs for the minimal amount of target proteins sampled from a single cell. We applied the technique to probe epidermal growth factor receptors (EGFRs) in PC9 lung adenocarcinoma cells. The results reveal that the strength of EGFR PPIs can be largely uncorrelated with the expression level of EGFRs in single cells. In addition, the individual PC9 cells showed markedly different patterns of PPIs, indicating a high heterogeneity in EGFR signaling within a genetically homogeneous population.11Nsciescopu