Heterogeneous and rate-dependent streptavidin-biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations

Receptor-ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, detailed information of the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spectroscopy on the streptavidin-biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and rate-dependent induced-fit motions for intermediates, which might be relevant for other receptor-ligand bonds.Comment: 21 pages, 4 figure

Entanglement detection with trace polynomials

We provide a systematic method for nonlinear entanglement detection based on trace polynomial inequalities. In particular, this allows to employ multi-partite witnesses for the detection of bipartite states, and vice versa. We identify witnesses for which linear detection of an entangled state fails, but for which nonlinear detection succeeds. With the trace polynomial formulation a great variety of witnesses arise from immamant inequalities, which can be implemented in the laboratory through randomized measurements

Results of MAGIC on Galactic sources

MAGIC is a single-dish Cherenkov telescope located on La Palma (Spain), hence with an optimal view on the Northern sky. Sensitive in the 30 GeV-30 TeV energy band, it is nowadays the only ground-based instrument being able to measure high-energy gamma-rays below 100 GeV. We review the most recent experimental results on Galactic sources obtained using MAGIC. These include pulsars, binary systems, supernova remnants and unidentified sources.Comment: 4 pages, 8 figures, to appear in the proceedings of "4th Heidelberg International Symposium on High Energy Gamma-Ray Astronomy 2008

Results of MAGIC on Galactic sources

MAGIC is a single-dish Cherenkov telescope located on La Palma (Spain), hence with an optimal view on the Northern sky. Sensitive in the 30 GeV-30 TeV energy band, it is nowadays the only ground-based instrument being able to measure high-energy gamma-rays below 100 GeV. We review the most recent experimental results on Galactic sources obtained using MAGIC. These include pulsars, binary systems, supernova remnants and unidentified sources.Comment: 4 pages, 8 figures, to appear in the proceedings of "4th Heidelberg International Symposium on High Energy Gamma-Ray Astronomy 2008

A microfluidic method generating monodispersed microparticles with controllable sizes and mechanical properties

International audienceSeeking to produce microparticles that mimic red blood cells (RBCs), we present a microfluidic method of generating monodispersed hydrogel microparticles of Na-/Ca-alginate with controllable sizes (micrometer range) and mechanical properties. No surfactant is used. Transformation from Na-alginate to Ca-alginate microparticles is realized via ex situ gelation, which proves essential to obtaining desired microparticle properties, such as insolubility in water and RBC-like mechanical properties. For both Na-alginate and Ca-alginate microparticles, a smooth surface and a porous inner structure are observed under a scanning electron microscope. A platform of microgrippers is successfully developed to manipulate the microparticles. The Young’s modulus measured using an atomic force microscope on the surface of Ca-alginate microparticles is of the same order as that of RBCs

Occurrence and ecological risks of pharmaceuticals in a Mediterranean river in Eastern Spain

Pharmaceuticals are biologically active molecules that may exert toxic effects to a wide range of aquatic organisms. They are considered contaminants of emerging concern due to their common presence in wastewaters and in the receiving surface waters, and the lack of specific regulations to monitor their environmental occurrence and risks. In this work, the environmental exposure and risks of pharmaceuticals have been studied in the Mijares River, Eastern Mediterranean coast (Spain). A total of 57 surface water samples from 19 sampling points were collected in three monitoring campaigns between June 2018 and February 2019. A list of 40 compounds was investigated using a quantitative target UHPLC-MS/MS method. In order to complement the data obtained, a wide-scope screening of pharmaceuticals and metabolites was also performed by UHPLC-HRMS. The ecological risks posed by the pharmaceutical mixtures were evaluated using species sensitivity distributions built with chronic toxicity data for aquatic organisms. In this study, up to 69 pharmaceuticals and 9 metabolites were identified, out of which 35 compounds were assessed using the quantitative method. The highest concentrations in water corresponded to acetaminophen, gabapentin, venlafaxine, valsartan, ciprofloxacin and diclofenac. The compounds that were found to exert the highest toxic pressure on the aquatic ecosystems were principally analgesic/anti-inflammatory drugs and antibiotics. These were: phenazone > azithromycin > diclofenac, and to a lower extent norfloxacin > ciprofloxacin > clarithromycin. The monitored pharmaceutical mixtures are expected to exert severe ecological risks in areas downstream of WWTP discharges, with the percentage of aquatic species affected ranging between 65% and 82% in 3 out of the 19 evaluated sites. In addition, five antibiotics were found to exceed antibiotic resistance thresholds, thus potentially contributing to resistance gene enrichment in environmental bacteria. This work illustrates the wide use and impact of pharmaceuticals in the area under study, and the vulnerability of surface waters if only conventional wastewater treatments are applied. Several compounds included in this study should be incorporated in future water monitoring programs to help in the development of future regulations, due to their potential risk to the aquatic environment

Experimental test of Sinai's model in DNA unzipping

The experimental measurement of correlation functions and critical exponents in disordered systems is key to testing renormalization group (RG) predictions. We mechanically unzip single DNA hairpins with optical tweezers, an experimental realization of the diffusive motion of a particle in a one-dimensional random force field, known as the Sinai model. We measure the unzipping forces $F_w$ as a function of the trap position $w$ in equilibrium and calculate the force-force correlator $\Delta_m(w)$, its amplitude, and correlation length, finding agreement with theoretical predictions. We study the universal scaling properties since the effective trap stiffness $m^2$ decreases upon unzipping. Fluctuations of the position of the base pair at the unzipping junction $u$ scales as $u \sim m^{-\zeta}$, with a roughness exponent $\zeta=1.34\pm0.06$, in agreement with the analytical prediction $\zeta = \frac{4}{3}$. Our study provides a single-molecule test of the functional RG approach for disordered elastic systems in equilibrium.Comment: 5 main + 6 supplementary pages, 8 figure

A Combination of screening and computational approaches for the identification of novel compounds that decrease mast cell degranulation

High-content screening of compound libraries poses various challenges in the early steps in drug discovery such as gaining insights into the mode of action of the selected compounds. Here, we addressed these challenges by integrating two biological screens through bioinformatics and computational analysis. We screened a small-molecule library enriched in amphiphilic compounds in a degranulation assay in rat basophilic leukemia 2H3 (RBL-2H3) cells. The same library was rescreened in a high-content image-based endocytosis assay in HeLa cells. This assay was previously applied to a genome-wide RNAi screen that produced quantitative multiparametric phenotypic profiles for genes that directly or indirectly affect endocytosis. By correlating the endocytic profiles of the compounds with the genome-wide siRNA profiles, we identified candidate pathways that may be inhibited by the compounds. Among these, we focused on the Akt pathway and validated its inhibition in HeLa and RBL-2H3 cells. We further showed that the compounds inhibited the translocation of the Akt-PH domain to the plasma membrane. The approach performed here can be used to integrate chemical and functional genomics screens for investigating the mechanism of action of compounds

Wide-scope screening of pharmaceuticals, illicit drugs and their metabolites in the Amazon River

Only a limited number of households in the Amazon are served by sewage collection or treatment facili- ties, suggesting that there might be a significant emission of pharmaceuticals and other wastewater contaminants into freshwater ecosystems. In this work, we performed a wide-scope screening to assess the occurrence of pharmaceuticals, illicit drugs and their metabolites in freshwater ecosystems of the Brazilian Amazon. Our study included 40 samples taken along the Amazon River, in three of its major tributaries, and in small tributaries crossing four important urban areas (Manaus, Santarém, Macapá, Belém). More than 900 compounds were investigated making use of target and suspect screening approaches, based on liquid chromatography coupled to high-resolution mass spectrometry with ion mobility separation. Empirical collision-cross section (CCS) values were used to help and confirm identifications in target screening, while in the suspect screening approach CCS values were predicted using Artificial Neural Networks to increase the confidence of the tentative identification. In this way, 51 compounds and metabolites were identified. The highest prevalence was found in streams crossing the urban areas of Manaus, Macapáand Belém, with some samples containing up to 30 - 40 compounds, while samples taken in Santarém showed a lower number (8 - 11), and the samples taken in the main course of the Amazon River and its tributaries contained between 1 and 7 compounds. Most compounds identified in areas with significant urban impact belonged to the analgesics and antihypertensive categories, followed by stimulants and antibiotics. Compounds such as caffeine, cocaine and its metabolite benzoylecgonine, and cotinine (the metabolite of nicotine), were also detected in areas with relatively low anthropogenic impact and showed the highest total prevalence. This study supports the need to improve the sanitation system of urban areas in the Brazilian Amazon and the development of follow-up studies aimed at quantifying exposure levels and risks for Amazonian freshwater biodiversity

Information-to-work conversion in single molecule experiments: from discrete to continuous feedback

We theoretically investigate the extractable work in single molecule unfolding-folding experiments with applied feedback. Using a simple two-state model, we obtain a description of the full work distribution, from discrete to continuous feedback. The effect of the feedback is captured by a detailed fluctuation theorem, accounting for the information aquired. We find analytical expressions for the average work extraction as well as an experimentally measurable bound thereof, which becomes tight in the continuous feedback limit. We further determine the parameters for maximal power, or rate of work extraction. While our two-state model only depends on a single, effective transition rate, we find quantitative agreement with Monte Carlo simulations of DNA hairpin unfolding-folding dynamics.Comment: 5 pages, 4 figures, 5 pages of supplementary informatio