Mutational Patterns in RNA Secondary Structure Evolution Examined in Three RNA Families

The goal of this work was to study mutational patterns in the evolution of RNA secondary structure. We analyzed bacterial tmRNA, RNaseP and eukaryotic telomerase RNA secondary structures, mapping structural variability onto phylogenetic trees constructed primarily from rRNA sequences. We found that secondary structures evolve both by whole stem insertion/deletion, and by mutations that create or disrupt stem base pairing. We analyzed the evolution of stem lengths and constructed substitution matrices describing the changes responsible for the variation in the RNA stem length. In addition, we used principal component analysis of the stem length data to determine the most variable stems in different families of RNA. This data provides new insights into the evolution of RNA secondary structures and patterns of variation in the lengths of double helical regions of RNA molecules. Our findings will facilitate design of improved mutational models for RNA structure evolution

AUG_hairpin: prediction of a downstream secondary structure influencing the recognition of a translation start site

<p>Abstract</p> <p>Background</p> <p>The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. The recognition of the start AUG codon by eukaryotic ribosomes is considered to depend on its nucleotide context. However, the fraction of eukaryotic mRNAs with the start codon in a suboptimal context is relatively large. It may be expected that mRNA should possess some features providing efficient translation, including the proper recognition of a translation start site. It has been experimentally shown that a downstream hairpin located in certain positions with respect to start codon can compensate in part for the suboptimal AUG context and also increases translation from non-AUG initiation codons. Prediction of such a compensatory hairpin may be useful in the evaluation of eukaryotic mRNA translation properties.</p> <p>Results</p> <p>We evaluated interdependency between the start codon context and mRNA secondary structure at the CDS beginning: it was found that a suboptimal start codon context significantly correlated with higher base pairing probabilities at positions 13 – 17 of CDS of human and mouse mRNAs. It is likely that the downstream hairpins are used to enhance translation of some mammalian mRNAs <it>in vivo</it>. Thus, we have developed a tool, <it>AUG_hairpin</it>, to predict local stem-loop structures located within the defined region at the beginning of mRNA coding part. The implemented algorithm is based on the available published experimental data on the CDS-located stem-loop structures influencing the recognition of upstream start codons.</p> <p>Conclusion</p> <p>An occurrence of a potential secondary structure downstream of start AUG codon in a suboptimal context (or downstream of a potential non-AUG start codon) may provide researchers with a testable assumption on the presence of additional regulatory signal influencing mRNA translation initiation rate and the start codon choice. <it>AUG_hairpin</it>, which has a convenient Web-interface with adjustable parameters, will make such an evaluation easy and efficient.</p

In vitro and in vivo delivery of a sustained release nanocarrier-based formulation of an MRTF/SRF inhibitor in conjunctival fibrosis

Abstract Background Sustained drug delivery is a large unmet clinical need in glaucoma. Here, we incorporated a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor, CCG-222740, into slow release large unilamellar vesicles derived from the liposomes DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and tested their effects in vitro and in vivo. Results The vesicles were spherical particles of around 130 nm and were strongly cationic. A large amount of inhibitor could be incorporated into the vesicles. We showed that the nanocarrier CCG-222740 formulation gradually released the inhibitor over 14 days using high performance liquid chromatography. Nanocarrier CCG-222740 significantly decreased ACTA2 gene expression and was not cytotoxic in human conjunctival fibroblasts. In vivo, nanocarrier CCG-222740 doubled the bleb survival from 11.0 ± 0.6 days to 22.0 ± 1.3 days (p = 0.001), decreased conjunctival scarring and did not have any local or systemic adverse effects in a rabbit model of glaucoma filtration surgery. Conclusions Our study demonstrates proof-of-concept that a nanocarrier-based formulation efficiently achieves a sustained release of a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor and prevents conjunctival fibrosis in an established rabbit model of glaucoma filtration surgery.https://deepblue.lib.umich.edu/bitstream/2027.42/146540/1/12951_2018_Article_425.pd

Utz Wachil: Findings from an International Study of Indigenous Perspectives on Health and Environment

This article reports previously unpublished results of a collaborative study undertaken in 2003 by health workers of the UK-based organisation Health Unlimited, and by researchers of the London School of Hygiene and Tropical Medicine. This study marked the first of a series of collaborative activities aimed at highlighting the situation of Indigenous peoples, some in the most isolated ecosystems of the planet. While many researchers focus on quantitative analysis of the health and environmental conditions of Indigenous peoples, our 2003 study aimed at exploring the views of Indigenous peoples in isolated communities in five countries on their environment and their health. In this article we look closely at the web of knowledge and belief that underpins Indigenous peoples' concepts of health and well-being, and their relationship to land and the environment. Although many Indigenous people have been forced off their traditional lands and live in rural settlements, towns, and cities, there are still a large number of people living in very small Indigenous communities in remote areas. This article focuses on 20 such communities in six countries. We explore traditional knowledge and practice and its relationship to Western medicine and services. The research findings highlight the importance of Indigenous knowledge systems for the emerging ecohealth community and suggest that we have much to learn from Indigenous peoples in our pursuit of a more holistic science

Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis

SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures