Gotowa bibliografia na temat „Virtual ribosome”

Chloroethylagelastatin A (CEAA) is an analogue of agelastatin A (AA), a natural alkaloid derived from a marine sponge. It is under development for therapeutic use against brain tumors as it has excellent central nervous system (CNS) penetration and pre-clinical therapeutic activity against brain tumors. Recently, AA was shown to inhibit protein synthesis by binding to the ribosomal A-site. In this study, we developed a novel virtual screening platform to perform a comprehensive screening of various AA analogues showing that AA analogues with proven therapeutic activity including CEAA have significant ribosomal binding capacity whereas therapeutically inactive analogues show poor ribosomal binding and revealing structural fingerprint features essential for drug-ribosome interactions. In particular, CEAA was found to have greater ribosomal binding capacity than AA. Biological tests showed that CEAA binds the ribosome and contributes to protein synthesis inhibition. Our findings suggest that CEAA may possess ribosomal inhibitor activity and that our virtual screening platform may be a useful tool in discovery and development of novel ribosomal inhibitors.

Oxazolidinones are a broad-spectrum class of synthetic antibiotics that bind to the 50S ribosomal subunit of Gram-positive and Gram-negative bacteria. Many crystal structures of the ribosomes with oxazolidinone ligands have been reported in the literature, facilitating structure-based design using methods such as molecular docking. It would be of great interest to know in advance how well docking methods can reproduce the correct ligand binding modes and rank these correctly. We examined the performance of five molecular docking programs (AutoDock 4, AutoDock Vina, DOCK 6, rDock, and RLDock) for their ability to model ribosomal–ligand interactions with oxazolidinones. Eleven ribosomal crystal structures with oxazolidinones as the ligands were docked. The accuracy was evaluated by calculating the docked complexes’ root-mean-square deviation (RMSD) and the program’s internal scoring function. The rankings for each program based on the median RMSD between the native and predicted were DOCK 6 > AD4 > Vina > RDOCK >> RLDOCK. Results demonstrate that the top-performing program, DOCK 6, could accurately replicate the ligand binding in only four of the eleven ribosomes due to the poor electron density of said ribosomal structures. In this study, we have further benchmarked the performance of the DOCK 6 docking algorithm and scoring in improving virtual screening (VS) enrichment using the dataset of 285 oxazolidinone derivatives against oxazolidinone binding sites in the S. aureus ribosome. However, there was no clear trend between the structure and activity of the oxazolidinones in VS. Overall, the docking performance indicates that the RNA pocket’s high flexibility does not allow for accurate docking prediction, highlighting the need to validate VS. protocols for ligand-RNA before future use. Later, we developed a re-scoring method incorporating absolute docking scores and molecular descriptors, and the results indicate that the descriptors greatly improve the correlation of docking scores and pMIC values. Morgan fingerprint analysis was also used, suggesting that DOCK 6 underpredicted molecules with tail modifications with acetamide, n-methylacetamide, or n-ethylacetamide and over-predicted molecule derivatives with methylamino bits. Alternatively, a ligand-based approach similar to a field template was taken, indicating that each derivative’s tail groups have strong positive and negative electrostatic potential contributing to microbial activity. These results indicate that one should perform VS. campaigns of ribosomal antibiotics with care and that more comprehensive strategies, including molecular dynamics simulations and relative free energy calculations, might be necessary in conjunction with VS. and docking.

Ribosomal profiling is an emerging experimental technology to measure protein synthesis by sequencing short mRNA fragments undergoing translation in ribosomes. Applied on the genome wide scale, this is a powerful tool to profile global protein synthesis within cell populations of interest. Such information can be utilized for biomarker discovery and detection of treatment-responsive genes. However, analysis of ribosomal profiling data requires careful preprocessing to reduce the impact of artifacts and dedicated statistical methods for visualizing and modeling the high-dimensional discrete read count data. Here we present Ribosomal Profiling Reports (RP-REP), a new open-source cloud-enabled software that allows users to execute start-to-end gene-level ribosomal profiling and RNA-Seq analysis on a pre-configured Amazon Virtual Machine Image (AMI) hosted on AWS or on the user’s own Ubuntu Linux server. The software works with FASTQ files stored locally, on AWS S3, or at the Sequence Read Archive (SRA). RP-REP automatically executes a series of customizable steps including filtering of contaminant RNA, enrichment of true ribosomal footprints, reference alignment and gene translation quantification, gene body coverage, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially translated genes, and generation of heatmaps, co-translated gene clusters, enriched pathways, and other custom visualizations. RP-REP provides functionality to contrast RNA-SEQ and ribosomal profiling results, and calculates translational efficiency per gene. The software outputs a PDF report and publication-ready table and figure files. As a use case, we provide RP-REP results for a dengue virus study that tested cytosol and endoplasmic reticulum cellular fractions of human Huh7 cells pre-infection and at 6 h, 12 h, 24 h, and 40 h post-infection. Case study results, Ubuntu installation scripts, and the most recent RP-REP source code are accessible at GitHub. The cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3)).

Mammalian hibernation involves virtual cessation of energetically consumptive processes normally vital to homeostasis, including gene transcription and protein synthesis. As animals enter torpor, the bulk of initiation of translation is blocked at a body temperature of 18°C in golden-mantled ground squirrels [ Spermophilus (Callospermophilus) lateralis ]. Previous data demonstrated regulation of cap-dependent initiation of translation during torpor. We asked what happens to cap-independent, specifically, internal ribosome entry site (IRES)-mediated initiation of translation during hibernation. We analyzed polysome fractions for mRNAs that are known to contain or not to contain IRES elements. Here, we show that mRNAs harboring IRES elements preferentially associate with ribosomes as a torpor bout progresses. Squirrels allowed to naturally complete a torpor cycle have a higher IRES preference index than those animals that are prematurely aroused from torpor. Data indicate that this change in preference is not associated with gene expression, i.e., change is due to change in mRNA association with ribosomes as opposed to mRNA abundance. Thus, although processes like transcription and translation are virtually arrested during torpor, ribosomes are preferentially loaded with IRES-containing transcripts when squirrels arouse from torpor and translation resumes. Differential translation of preexisting mRNAs may allow for the preferential production of key stress proteins critical for survival of physiological insults that are lethal to other mammals.

New RNA-binding small-molecule scaffolds are needed to unleash the pharmacological potential of RNA targets. Here we have applied a pharmacophore-based virtual screening approach, seldom used in the RNA recognition field, to identify novel conformational inhibitors of the hepatitis C virus internal ribosome entry site. The conformational effect of the screening hits was assessed with a fluorescence resonance energy transfer assay, and the affinity, specificity, and binding site of the ligands were determined using a combination of fluorescence intensity and NMR spectroscopy experiments. The results indicate that this strategy can be successfully applied to discover RNA conformational inhibitors bearing substantially less positive charge than the reference ligands. This methodology can potentially be accommodated to other RNA motifs of pharmacological interest, facilitating the discovery of novel RNA-targeted molecules.

ABSTRACTThe mono-ADP-ribosyltransferase toxins are bacterial virulence factors that contribute to many disease states in plants, animals, and humans. These toxins function as enzymes that target various host proteins and covalently attach an ADP-ribose moiety that alters target protein function. We tested compounds from a virtual screen of commercially available compounds combined with a directed poly(ADP-ribose) polymerase (PARP) inhibitor library and found several compounds that bind tightly and inhibit toxins fromPseudomonas aeruginosaandVibrio cholerae. The most efficacious compounds completely protected human lung epithelial cells against the cytotoxicity of these bacterial virulence factors. Moreover, we determined high-resolution crystal structures of the best inhibitors in complex with cholix toxin to reveal important criteria for inhibitor binding and mechanism of action. These results provide new insight into development of antivirulence compounds for treating many bacterial diseases.

Many studies have shown the trend that students’ conceptualisations of scientific phenomena are limited to the ontological category of objects. The ontological category of processes, on the other hand, is not developed enough to build valid scientific mental models (Chi , 2005; Ferrari and Chi, 1998; Vosniadou, 1991). A correct conceptualisation of any scientific concept results from a proper understanding of object properties and processes involving these objects. Moreover, Pata and Sarapuu (2003) have raised the problem that the traditional way to teach science is usually focused on the ontological category of objects. The active-learning idea, coming from constructivism, predicts that virtual manipulations improve deeper learning (Evans and Gibbons, 2007). With interactive activities, learners are the main actors of their own construction of scientific concepts. According to this approach, deeper conceptualisation will be enhanced when manipulations are possible. In the context of web-based learning, some manipulatives, which are defined as movable representations or movable objects on the computer screen (Moyer et al., 2002), can be used to create interactivity. This study investigates the students’ ontological understanding in a virtual learning environment and the effect of manipulatives on the students’ construction of ontological conceptions. More precisely, this work attempts to answer the following research questions: (1) What is the learners’ understanding about ontological category of objects comparing to ontological category of processes? (2) How do manipulatives and non-manipulatives help learners to conceptualise through ontological categories? A process-based virtual learning environment, “Cell World” (http://bio.edu.ee/models/fr/), was applied by 59 French students of junior year of high school in sciences (aged 16-17). The sample consisted of students from three average level French schools. The results presented in this paper were drawn from a pilot survey using the model translation of “Cell World”. In this model, students can move each manipulative from the store (on the left of the screen) to the area of animations where non-manipulatives are (on the right of the screen). If the selected manipulative is incorrect, learners receive a feedback which instructs them to drag the correct manipulative in order to continue the translation process. If the manipulative is correct, the animation continues so that students can observe interaction processes involving directly manipulatives (e.g. interaction between tRNA and mRNA) and/or only non-manipulatives (e.g. the fixing process of ribosome onto mRNA molecule). For leading students to use the environment correctly, a worksheet containing instructions was developed. Moreover, the worksheet also consisted of ten questions to investigate students’ understanding about ontological categories of objects (four questions) and processes (six questions). The questions about processes lead students to think in term of emergent interactions between objects. Questions about objects lead students to use factual knowledge about the properties of molecules. These questions involve also either manipulatives or non-manipulatives to investigate the role of interactivity on students’ conceptions. In order to obtain results about learners’ understanding of ontological categories, students were grouped according to their results about the two categories of questions, objects and processes: 43 students out of 59 expressed a high level of objects’ understanding – they obtained at least two-third of maximum points, whereas only 29 students expressed a high level of processes’ understanding. The Wilcoxon signed-ranks test also revealed significant differences (Z=-3.803, P<0.001) between the answers of the two categories of questions (objects and processes): 45 students demonstrated better performances concerning questions about objects than about processes, 13 answered better about objects and one was equal in both categories. Thus, the results provide evidence that the ontological category of processes is significantly less constructed and understood than the objects’ category. For studying the role of manipulatives on the student’s ontological categories of objects and processes, a Wilcoxon signed-ranks test was used. The test compared questions about objects involving manipulatives and questions about non-manipulatives: 45 students obtained better results about manipulable molecules, 10 students about non-manipulable and only 4 learners performed with non-manipulable as well as manipulable molecules (Z=-3.868, P<0.001). Another Wilcoxon signed-ranks test was used to compare students’ answers about processes when manipulatives or non-manipulatives were available in theses processes: 47 students obtained better results concerning category of processes when these processes involved manipulable molecules. Seven students performed better when processes involved non-manipulable molecule and 5 learners got the same results for manipulable and non-manipulable molecules (Z=-5.070, P<0.001). Thus, the great majority of the students showed better understanding in both ontological categories – objects and processes, when manipulatives were involved. In conclusion, these outcomes confirm precedent works: the ontological category of processes is not developed enough to build valid scientific mental models. Thus, the main challenge for teaching scientific phenomena is to lead learners to think through ontological category of processes. One possibility to help students to improve understanding in both ontological categories is to introduce manipulatives at most crucial aspects of a scientific model. -References: -Chi, M. T. H. (2005). Common sense conceptions of emergent processes: Why some misconceptions are robust. Journal of the Learning Sciences, 14, 161–199. -Evans, C., Gibbons N. J. (2007). The interactivity effect in multimedia learning. Computers & Education, 49, 1147–1160. -Ferrari, M. & Chi, M. T. H. (1998). The nature of naive explanations of natural selection. International Journal of Science Education, 20(10), 1231-1256. -Moyer, P. S., & Bolyard, J. J. (2002). Exploring Representation in the Middle Grades: Investigations in Geometry with Virtual Manipulatives. The Australian Mathematics Teacher, 58(1), 19-25. -Pata, K. & Sarapuu, T. (2003). Framework for scaffolding the development of problem representationsby collaborative design. In B. Wasson, S. Ludvigsen & U. Hoppe (Eds.), Designing for Change in Networked Learning Environments. Proceedings of CSCL’ 2003 Conference. Kluwer Academic Publishers, Dordrecht, 189-198. -Vosniadou, S. (1991). Conceptual development in astronomy. In S. M. Glynn & e. al. (Eds.), The Psychology of Learning Science. Hillsdale, N.J.: Lawrence Erlbaum Associates Publishers, 149-177.