Nanoscale spatially resolved infrared spectra from single microdroplets

Droplet microfluidics has emerged as a powerful platform allowing a large number of individual reactions to be carried out in spatially distinct microcompartments. Due to their small size, however, the spectroscopic characterisation of species encapsulated in such systems remains challenging. In this paper, we demonstrate the acquisition of infrared spectra from single microdroplets containing aggregation-prone proteins. To this effect, droplets are generated in a microfluidic flow-focussing device and subsequently deposited in a square array onto a ZnSe prism using a micro stamp. After drying, the solutes present in the droplets are illuminated locally by an infrared laser through the prism, and their thermal expansion upon absorption of infrared radiation is measured with an atomic force microscopy tip, granting nanoscale resolution. Using this approach, we resolve structural differences in the amide bands of the spectra of monomeric and aggregated lysozyme from single microdroplets with picolitre volume.Comment: 5 pages, 3 Figure

Fibrogenic fibroblasts increase intercellular adhesion strength by reinforcing individual OB-cadherin bonds

We have previously shown that the switch from N-cadherin to OB-cadherin expression increases intercellular adhesion between fibroblasts during their transition from a migratory to a fibrogenic phenotype. Using atomic force microscopy we here show that part of this stronger adhesion is accomplished because OB-cadherin bonds resist similar to twofold higher forces compared with N-cadherin junctions. By assessing the adhesion force between recombinant cadherin dimers and between native cadherins in the membrane of spread fibroblasts, we demonstrate that cadherin bonds are reinforced over time with two distinct force increments. By modulating the degree of lateral cadherin diffusion and F-actin organization we can attribute the resulting three force states to the single-molecule bond rather than to cadherin cluster formation. Notably, association with actin filaments enhances cadherin adhesion strength on the single-molecule level up to threefold; actin depolymerization reduces single-bond strength to the level of cadherin constructs missing the cytoplasmic domain. Hence, fibroblasts reinforce intercellular contacts by: (1) switching from N- to OB-cadherin expression; (2) increasing the strength of single-molecule bonds in three distinct steps; and (3) actin-promoted intrinsic activation of cadherin extracellular binding. We propose that this plasticity adapts fibroblast adhesions to the changing mechanical microenvironment of tissue under remodeling

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

Photocatalytic and phototoxic properties of TiO2-based nanofilaments: ESR and AFM assays

There is uncertainty in understanding of the relationship between physico-chemical parameters of nanosized titanium dioxide (nano-TiO2) and its toxicity when brought into contact with living cells. This study provides a multidisciplinary experimental insight into the toxicity and phototoxicity of the custom-made TiO2-based nanowires (TiO2-NWs). We employed electron spin resonance (ESR) to detect reactive oxygen species (ROS) generated in aqueous suspensions of TiO2-NWs and combined these results with atomic force microscopy (AFM) to trace the onset of toxic effects towards human melanoma cells. The cells were treated with low concentrations (similar to 2.5 mu g/ml) of TiO2-NWs and Degussa P25. High-resolution AFM surface topography and cell elasticity measurements revealed toxic effects both in cells incubated with TiO2-NWs in the dark and exposed to the photo-oxidative stress under UVA radiation. In contrast to ROS generation efficacy in the absence of cells in vitro, no direct correlation was found between the physical parameters of nano-TiO2 and cell toxicity

Hydrogel Microspheres: Influence of Chemical Composition on Surface Morphology, Local Elastic Properties, and Bulk Mechanical Characteristics

Hydrogel microspheres, beads, and capsules of uniform size, differing in their chem. compn., have been prepd. by electrostatic complex formation of sodium alginate with divalent cations and polycations. These have served as model spheres to study the influence of the chem. compn. on both surface characteristics and bulk mech. properties. Resistance to compression expts. yielding the compression work clearly identified differences as a function of the compn., with forces at maximal compression in the range of 34-455 mN. The suitability and informative value of at. force microscopy have been confirmed for the case where surface characterization is performed in a liq. environment equiv. to physiol. conditions. Surface imaging and mech. response to indentation revealed different av. surface roughness and Young's moduli for all hydrogel types ranging from 0.9 to 14.4 nm and from 0.4 to 440 kPa, resp. The hydrogels exhibited pure elastic behavior. Despite a relatively high std. deviation, resulting from both surface and batch heterogeneity, nonoverlapping ranges of Young's moduli were reproducibly identified for the selected model spheres. The findings indicate the reliability of contact mode at. force microscopy to quantify local surface properties, which may have an impact on the biocompatibility of alginate-based hydrogel materials of different compn. and conditions of prepn. Moreover, it seems that local elastic properties and bulk mech. characteristics are subject to analogous compn. influences. [on SciFinder (R)

MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation.

BACKGROUND: The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models. RESULTS: In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression. CONCLUSIONS: This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo

Probing fibronectin-antibody interactions using AFM force spectroscopy and lateral force microscopy

The first experiment showing the effects of specific interaction forces using lateral force microscopy (LFM) was demonstrated for lectin-carbohydrate interactions some years ago. Such measurements are possible under the assumption that specific forces strongly dominate over the non-specific ones. However, obtaining quantitative results requires the complex and tedious calibration of a torsional force. Here, a new and relatively simple method for the calibration of the torsional force is presented. The proposed calibration method is validated through the measurement of the interaction forces between human fibronectin and its monoclonal antibody. The results obtained using LFM and AFM-based classical force spectroscopies showed similar unbinding forces recorded at similar loading rates. Our studies verify that the proposed lateral force calibration method can be applied to study single molecule interactions

Motion of a colloidal particle in an optical trap

Thermal position fluctuations of a colloidal particle in an optical trap are measured with microsecond resolution using back-focal-plane interferometry. The mean-square displacement MSD(t) and power spectral density are in excellent agreement with the theory for a Brownian particle in a harmonic potential that accounts for hydrodynamic memory effects. The motion of a particle is dominated at short times by memory effects and at longer times by the potential. We identify the time below which the particle's motion is not influenced by the potential, and find it to be approximately tau_k/20, where tau_k is the relaxation time of the restoring force of the potential. This allows us to exclude the existence of free diffusive motion (MSD(t) proprtional t) even for a sphere with a radius as small as 0.27 µm in a potential as weak as 1.5 µN/m. As the physics of Brownian motion can be used to calibrate an optical trap, we show that neglecting memory effects leads to an underestimation of more than 10% in the detector sensitivity and the trap stiffness for an experiment with a micrometer-sized particle and a sampling frequency above 200 kHz. Furthermore, these calibration errors increase in a nontrivial fashion with particle size, trap stiffness, and sampling frequency. Finally, we present a method to evaluate calibration errors caused by memory effects for typical optical trapping experiments