Structural characterization of cephaeline binding to the eukaryotic ribosome using Cryo-Electron Microscopy

The eukaryotic ribosome is emerging as a promising target against human pathogens, includ- ing amoeba, protozoans, and fungi. Among the eukaryotic-specific families of inhibitors, al- kaloids are known to bind to the eukaryotic ribosome and inhibit translocation. However, these inhibitors have varying medical indications and toxicity to humans. Structural information is available for only two of them, cryptopleurine and emetine. Aim. In our work, we aimed to elucidate the binding mechanism of another alkaloid, cephaeline, to the eukaryotic ribosome. Methods. We used cryogenic electron microscopy and cell-free assays to reveal its mechanism of action. Results. Our results indicate that cephaeline binds to the E-tRNA binding site on the small subunit of the eukaryotic ribosome. Similar to emetine, cephaeline forms a stacking interaction with G889 of 18S rRNA and L132 of the protein uS11. We propose the hypothesis of cephaeline specificity to eukaryotes by comparing the interaction pattern of cephaeline with other inhibitors binding to the E-site of the mRNA tunnel. Conclusions. The high-resolution structure of ribosome-bound cephaeline (2.45 Å) allowed us to precisely determine the in- hibitor’s position in the binding site, which holds potential for the development of the next generation of drugs targeting the mRNA tunnel of the ribosome

Электропривод приёмного устройства линии производства гибких нагревательных элементов

Целью работы является разработка и исследование системы векторного управления асинхронным двигателем с короткозамкнутым ротором для приёмного устройства линии производства гибких нагревательных элементов с инвертором с синусоидальной ШИМ с предмодуляцией третьей гармоникой, а также исследование свойств и выявление достоинств и недостатков различных способов управления автономных инверторов напряжения по принципу широтно-импульсной модуляции.The aim of the work is to develop and analyze the system of vector control of induction motor with squirrel-cage rotor for take-up machine of the cable production line with the voltage source inverter with third harmonic injection sinusoidal PWM, as well as to analyze the properties and identify the advantages and disadvantages of various methods of control of voltage source inverter on the principle of pulse width modulation

E-site drug specificity of the human pathogen Candida albicans ribosome

International audienceCandida albicans is a widespread commensal fungus with substantial pathogenic potential and steadily increasing resistance to current antifungal drugs. It is known to be resistant to cycloheximide (CHX) that binds to the E–transfer RNA binding site of the ribosome. Because of lack of structural information, it is neither possible to understand the nature of the resistance nor to develop novel inhibitors. To overcome this issue, we determined the structure of the vacant C. albicans 80 S ribosome at 2.3 angstroms and its complexes with bound inhibitors at resolutions better than 2.9 angstroms using cryo–electron microscopy. Our structures reveal how a change in a conserved amino acid in ribosomal protein eL42 explains CHX resistance in C. albicans and forms a basis for further antifungal drug development

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Inhibition of primer extension by ribosome–mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture

Nested effects models for high-dimensional phenotyping screens

Motivation: In high-dimensional phenotyping screens, a large number of cellular features is observed after perturbing genes by knockouts or RNA interference. Comprehensive analysis of perturbation effects is one of the most powerful techniques for attributing functions to genes, but not much work has been done so far to adapt statistical and computational methodology to the specific needs of large-scale and high-dimensional phenotyping screens. Results: We introduce and compare probabilistic methods to efficiently infer a genetic hierarchy from the nested structure of observed perturbation effects. These hierarchies elucidate the structures of signaling pathways and regulatory networks. Our methods achieve two goals: (1) they reveal clusters of genes with highly similar phenotypic profiles, and (2) they order (clusters of) genes according to subset relationships between phenotypes. We evaluate our algorithms in the controlled setting of simulation studies and show their practical use in two experimental scenarios: (1) a data set investigating the response to microbial challenge in Drosophila melanogaster, and (2) a compendium of expression profiles of Saccharomyces cerevisiae knockout strains. We show that our methods identify biologically justified genetic hierarchies of perturbation effects. Availability: The software used in our analysis is freely available in the R package ‘nem’ from www.bioconductor.or