FLASH: ultra-fast protocol to identify RNA-protein interactions in cells

Determination of the in vivo binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding sites has not been described to date. Here we report FLASH (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein-RNA interactions in living cells. The entire FLASH protocol, starting from cells on plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions in vivo

Ecumenical modal logic

The discussion about how to put together Gentzen's systems for classical and intuitionistic logic in a single unified system is back in fashion. Indeed, recently Prawitz and others have been discussing the so called Ecumenical Systems, where connectives from these logics can co-exist in peace. In Prawitz' system, the classical logician and the intuitionistic logician would share the universal quantifier, conjunction, negation, and the constant for the absurd, but they would each have their own existential quantifier, disjunction, and implication, with different meanings. Prawitz' main idea is that these different meanings are given by a semantical framework that can be accepted by both parties. In a recent work, Ecumenical sequent calculi and a nested system were presented, and some very interesting proof theoretical properties of the systems were established. In this work we extend Prawitz' Ecumenical idea to alethic K-modalities

On Subexponentials, Synthetic Connectives, and Multi-level Delimited Control

International audienceWe construct a partially-ordered hierarchy of delimited control operators similar to those of the CPS hierarchy of Danvy and Filinski. However, instead of relying on nested CPS translations, these operators are directly interpreted in linear logic extended with subexponentials (i.e., multiple pairs of ! and ?). We construct an independent proof theory for a fragment of this logic based on the principle of focusing. It is then shown that the new constraints placed on the permutation of cuts correspond to multiple levels of delimited control

Is authorship sufficient for today’s collaborative research? A call for contributor roles

Assigning authorship and recognizing contributions to scholarly works is challenging on many levels. Here we discuss ethical, social, and technical challenges to the concept of authorship that may impede the recognition of contributions to a scholarly work. Recent work in the field of authorship shows that shifting to a more inclusive contributorship approach may address these challenges. Recent efforts to enable better recognition of contributions to scholarship include the development of the Contributor Role Ontology (CRO), which extends the CRediT taxonomy and can be used in information systems for structuring contributions. We also introduce the Contributor Attribution Model (CAM), which provides a simple data model that relates the contributor to research objects via the role that they played, as well as the provenance of the information. Finally, requirements for the adoption of a contributorship-based approach are discussed

Dynamic antagonism between key repressive pathways maintains the placental epigenome

DNA and Histone 3 Lysine 27 methylation typically function as repressive modifications and operate within distinct genomic compartments. In mammals, the majority of the genome is kept in a DNA methylated state, whereas the Polycomb repressive complexes regulate the unmethylated CpG-rich promoters of developmental genes. In contrast to this general framework, the extra-embryonic lineages display non-canonical, globally intermediate DNA methylation levels, including disruption of local Polycomb domains. Here, to better understand this unusual landscape's molecular properties, we genetically and chemically perturbed major epigenetic pathways in mouse trophoblast stem cells. We find that the extra-embryonic epigenome reflects ongoing and dynamic de novo methyltransferase recruitment, which is continuously antagonized by Polycomb to maintain intermediate, locally disordered methylation. Despite its disorganized molecular appearance, our data point to a highly controlled equilibrium between counteracting repressors within extra-embryonic cells, one that can seemingly persist indefinitely without bistable features typically seen for embryonic forms of epigenetic regulation

Destruction of chemical warfare surrogates using a portable atmospheric pressure plasma jet

Today’s reality is connected with mitigation of threats from the new chemical and biological warfare agents. A novel investigation of cold plasmas in contact with liquids presented in this paper demonstrated that the chemically reactive environment produced by atmospheric pressure plasma jet (APPJ) is potentially capable of rapid destruction of chemical warfare agents in a broad spectrum. The decontamination of three different chemical warfare agent surrogates dissolved in liquid is investigated by using an easily transportable APPJ. The jet is powered by a kHz signal source connected to a low-voltage DC source and with He as working gas. The detailed investigation of electrical properties is performed for various plasmas at different distances from the sample. The measurements of plasma properties in situ are supported by the optical spectrometry measurements, whereas the high performance liquid chromatography measurements before and after the treatment of aqueous solutions of Malathion, Fenitrothion and Dimethyl Methylphosphonate. These solutions are used to evaluate destruction and its efficiency for specific neural agent simulants. The particular removal rates are found to be from 56% up to 96% during 10 min treatment. The data obtained provide basis to evaluate APPJ’s efficiency at different operating conditions. The presented results are promising and could be improved with different operating conditions and optimization of the decontamination process

Advancing our understanding of functional genome organisation through studies in the fission yeast

Significant progress has been made in understanding the functional organisation of the cell nucleus. Still many questions remain to be answered about the relationship between the spatial organisation of the nucleus and the regulation of the genome function. There are many conflicting data in the field making it very difficult to merge published results on mammalian cells into one model on subnuclear chromatin organisation. The fission yeast, Schizosaccharomyces pombe, over the last decades has emerged as a valuable model organism in understanding basic biological mechanisms, especially the cell cycle and chromosome biology. In this review we describe and compare the nuclear organisation in mammalian and fission yeast cells. We believe that fission yeast is a good tool to resolve at least some of the contradictions and unanswered questions concerning functional nuclear architecture, since S. pombe has chromosomes structurally similar to that of human. S. pombe also has the advantage over higher eukaryotes in that the genome can easily be manipulated via homologous recombination making it possible to integrate the tools needed for visualisation of chromosomes using live-cell microscopy. Classical genetic experiments can be used to elucidate what factors are involved in a certain mechanism. The knowledge we have gained during the last few years indicates similarities between the genome organisation in fission yeast and mammalian cells. We therefore propose the use of fission yeast for further advancement of our understanding of functional nuclear organisation

A simple procedure for directly obtaining haplotype sequences of diploid genomes

Background Almost all genome sequencing projects neglect the fact that diploid organisms contain two genome copies and consequently what is published is a composite of the two. This means that the relationship between alternate alleles at two or more linked loci is lost. We have developed a simplified method of directly obtaining the haploid sequences of each genome copy from an individual organism. Results The diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Samples were: 1) lymphocytes from a single Angus steer; 2) sperm cells from an Angus bull; 3) lymphocytes from East African Zebu (EAZ) cattle collected and processed in a field laboratory in Eastern Kenya. Haploid sequence from a fosmid library prepared from lymphocytes of an EAZ cow was used for comparison. Cells were serially diluted to a concentration of one cell per microlitre by counting with a haemocytometer at each dilution. One microlitre samples, each potentially containing a single cell, were lysed and divided into six aliquots (except for the sperm samples which were not divided into aliquots). Each aliquot was amplified with phi29 polymerase and sequenced. Contigs were obtained by mapping to the bovine UMD3.1 reference genome assembly and scaffolds were assembled by joining adjacent contigs that were within a threshold distance of each other. Scaffolds that appeared to contain artefacts of CNV or repeats were filtered out leaving scaffolds with an N50 length of 27–133 kb and a 88–98 % genome coverage. SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 97–201 kb but only ~27–68 % genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing. A substantial body of software for analysis and workflow management was written and is available as supplementary data. Conclusions We have developed a set of laboratory protocols and software tools that will enable any laboratory to obtain haplotype sequences at only modestly greater cost than traditional mixed diploid sequences