The mechanism of non-blocking inhibition of sodium channels revealed by conformation-selective photolabeling

Sodium channel inhibitors can be used to treat hyperexcitability-related diseases, including epilepsies, pain syndromes, neuromuscular disorders, and cardiac arrhythmias. The applicability of these drugs is limited by their nonspecific effect on physiological function. They act mainly by sodium channel block and in addition by modulation of channel kinetics. While channel block inhibits healthy and pathological tissue equally, modulation can preferentially inhibit pathological activity. An ideal drug designed to target the sodium channels of pathological tissue would act predominantly by modulation. Thus far, no such drug has been described.Patch-clamp experiments with ultra-fast solution exchange and photolabeling-coupled electrophysiology were applied to describe the unique mechanism of riluzole on Nav1.4 sodium channels. In silico docking experiments were used to study the molecular details of binding.We present evidence that riluzole acts predominantly by non-blocking modulation. We propose that, being a relatively small molecule, riluzole is able to stay bound to the binding site, but nonetheless stay off the conduction pathway, by residing in one of the fenestrations. We demonstrate how this mechanism can be recognized.Our results identify riluzole as the prototype of this new class of sodium channel inhibitors. Drugs of this class are expected to selectively prevent hyperexcitability, while having minimal effect on cells firing at a normal rate from a normal resting potential

Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803

Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. strain PCC6803 the two metabolic pathways are linked by coregulated genes that are involved in arsenic transport, sulfide oxidation, and probably in sulfide-based alternative photosynthesis. Although Synechocystis sp. strain PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis, we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. suoS (sll5036) encodes a light-dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200-fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as an arsenite importer at permissive concentrations. We propose that the proteins encoded by the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and that the operon was acquired by the cyanobacterium via horizontal gene transfer

Limnological changes in South Carpathian glacier-formed lakes (Retezat Mountains, Romania) during the Late Glacial and the Holocene: A synthesis

Remains of aquatic biota preserved in mountain lake sediments provide an excellent tool to study lake ecosystem responses to past climate change. In the PROLONG project a multi-proxy study was performed on sediments of glacier-formed lakes from the Retezat Mountains, Southern Carpathians (Romania). The studied lakes (Lake Brazi and Gales) are situated on the northern slope of the mountain at different altitudes (1740 m and 1990 m a.s.l.). Our main objectives were 1) to describe the main limnological changes in these lakes during the last ca. 15,000 years and 2) to summarize the environmental history of the studied lakes based on taxonomical and functional patterns of the biological proxies. For this synthesis we used the results of diatom and chironomid analyses, and indirect biotic and abiotic parameters, including sediment organic matter (LOI) content, geochemical element concentrations (Al, Ca, S, Sr) and biogenic silica content. Using multivariate numerical approaches we analysed changes in the assemblage structure of siliceous algae and chironomids, compared temporal patterns among proxies, examined the relationship between potential driving factors, chironomid and diatom assemblage changes and identified paleolimnological phases of the lake successions. Changes in assemblage composition and aquatic ecosystem state apparently followed summer insolation, local climatic conditions and local productivity changes driven by these. Diatom and chironomid assemblages generally changed in a similar direction and at a similar time within a lake, but differed to some extent between Lake Brazi and Gales. At both lakes the strongest variations were observed in the Late Glacial and the first half of the Holocene. The strongest Holocene assemblage changes took place in the earliest Holocene in Lake Brazi, but extended into the mid-Holocene in Lake Gales, following long-term insolation changes and climatic changes. In addition, three common zone boundaries were identified: at ca. 14,200 and at ca. 6500 cal yr BP for every records and at ca. 3100 cal yr BP for diatom records in both of the lakes and for the chironomid record of Lake Brazi. This multi-proxy synthesis provides comprehensive data that increase our understanding of the past variability of lake ecosystem functioning and biodiversity in East-Central Europe. Keyword

Constraints on the physical properties of Type Ia supernovae from photometry

We present a photometric study of 17 Type Ia supernovae (SNe) based on multi-color (Bessell BVRI) data taken at Piszkesteto mountain station of Konkoly Observatory, Hungary between 2016 and 2018. We analyze the light curves (LCs) using the publicly available LC-fitter SNooPy2 to derive distance and reddening information. The bolometric LCs are fit with a radiation-diffusion Arnett-model to get constraints on the physical parameters of the ejecta: the optical opacity, the ejected mass and the expansion velocity in particular. We also study the pre-maximum (B-V) color evolution by comparing our data with standard delayed detonation and pulsational delayed detonation models, and show that the Ni56 masses of the models that fit the (B-V) colors are consistent with those derived from the bolometric LC fitting. We find similar correlations between the ejecta parameters (e.g. ejecta mass, or Ni56 mass vs decline rate) as published recently by Scalzo et al. (2019

Catalytic mechanism of alpha-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling.

Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason-Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the alpha-phosphate site. Phosphorus-31 NMR spectroscopy (31P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue alpha,beta-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme-product complex structure

Policy instruments for managing road safety on EU-roads

Directive 2008/96/EC on road infrastructure safety management requires the establishment and implementation of procedures relating to road safety impact assessments (RSIA), road safety audits (RSA), ranking of high accident concentration sections and network safety ranking (NSR) and road safety inspections (RSI). The aim of this article is to present the outputs of BALTRIS project. The goal of the international project BALTRIS is to elaborate the road and street infrastructure safety management procedures and teaching material consistently explaining the above mentioned infrastructure management procedures. Four Baltic Sea region countries (Sweden, Estonia, Latvia, and Lithuania), represented by universities and national road administrations participate in the elaboration of these procedures and teaching material. This article describes the scope of NSR, RSA and RSI procedures prepared in the frame of BALTRIS project, also article provides detailed implementation and execution of procedures for the EU Member States. NSR means a method for identifying, analysing and classifying parts of the existing road network according to their potential for safety development and accident cost savings. Ranking of high accident concentration sectionsmeans a method to identify, analyse and rank sections of the road network which have been in operation for 3÷5 years and upon which a large number of fatal/injury accidents in proportion to the traffic flow or compared to respective conditions have occurred. RSI is a strategic comparative analysis of the impact of the new road or a substantial modification to the existing network on the safety performance of the road network. RSA is a formal safety performance examination of the existing or future road or intersection by an independent audit team

Angiotensin type 1A receptor regulates β-arrestin binding of the β2-adrenergic receptor via heterodimerization

Heterodimerization between angiotensin type 1A receptor (AT1R) and β2-adrenergic receptor (β2AR) has been shown to modulate G protein-mediated effects of these receptors. Activation of G protein-coupled receptors (GPCRs) leads to β-arrestin binding, desensitization, internalization and G protein-independent signaling of GPCRs. Our aim was to study the effect of heterodimerization on β-arrestin coupling. We found that β-arrestin binding of β2AR is affected by activation of AT1Rs. Costimulation with angiotensin II and isoproterenol markedly enhanced the interaction between β2AR and β-arrestins, by prolonging the lifespan of β2AR-induced β-arrestin2 clusters at the plasma membrane. While candesartan, a conventional AT1R antagonist, had no effect on the β-arrestin2 binding to β2AR, TRV120023, a β-arrestin biased agonist, enhanced the interaction. These findings reveal a new crosstalk mechanism between AT1R and β2AR, and suggest that enhanced β-arrestin2 binding to β2AR can contribute to the pharmacological effects of biased AT1R agonists. © 201

Cadaverine, a metabolite of the microbiome, reduces breast cancer aggressiveness through trace amino acid receptors

Recent studies showed that changes to the gut microbiome alters the microbiome-derived metabolome, potentially promoting carcinogenesis in organs that are distal to the gut. In this study, we assessed the relationship between breast cancer and cadaverine biosynthesis. Cadaverine treatment of Balb/c female mice (500 nmol/kg p.o.q.d.) grafted with 4T1 breast cancer cells ameliorated the disease (lower mass and infiltration of the primary tumor, fewer metastases, and lower grade tumors). Cadaverine treatment of breast cancer cell lines corresponding to its serum reference range (100-800 nM) reverted endothelial-to-mesenchymal transition, inhibited cellular movement and invasion, moreover, rendered cells less stem cell-like through reducing mitochondrial oxidation. Trace amino acid receptors (TAARs), namely, TAAR1, TAAR8 and TAAR9 were instrumental in provoking the cadaverine-evoked effects. Early stage breast cancer patients, versus control women, had reduced abundance of the CadA and LdcC genes in fecal DNA, both responsible for bacterial cadaverine production. Moreover, we found low protein expression of E. coli LdcC in the feces of stage 1 breast cancer patients. In addition, higher expression of lysine decarboxylase resulted in a prolonged survival among early-stage breast cancer patients. Taken together, cadaverine production seems to be a regulator of early breast cancer

Detection and isolation of cell-derived microparticles are compromised by protein complexes due to shared biophysical parameters

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (e.g. ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light scattering analysis and flow cytometry, for the first time we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematological disorders, infections and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs, and contribute to correct the clinical laboratory assessment of the presence and biological functions of MPs in health and disease