Mapping the Sensitive Volume of an Ion-Counting Nanodosimeter

We present two methods of independently mapping the dimensions of the sensitive volume in an ion-counting nanodosimeter. The first method is based on a calculational approach simulating the extraction of ions from the sensitive volume, and the second method on probing the sensitive volume with 250 MeV protons. Sensitive-volume maps obtained with both methods are compared and systematic errors inherent in both methods are quantified.Comment: 27 pages, 8 figures. Submitted to JINST, Jan. 16 200

Molecular recording of mammalian embryogenesis.

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes

R&D on co-working transport schemes in Geant4

A research and development (R&D) project related to the extension of the Geant4 toolkit has been recently launched to address fundamental methods in radiation transport simulation. The project focuses on simulation at different scales in the same experimental environment; this problem requires new methods across the current boundaries of condensed-random-walk and discrete transport schemes. The new developments have been motivated by experimental requirements in various domains, including nanodosimetry, astronomy and detector developments for high energy physics applications.Comment: To be published in the Proceedings of the CHEP (Computing in High Energy Physics) 2009 conferenc

A novel class of microRNA-recognition elements that function only within open reading frames.

MicroRNAs (miRNAs) are well known to target 3' untranslated regions (3' UTRs) in mRNAs, thereby silencing gene expression at the post-transcriptional level. Multiple reports have also indicated the ability of miRNAs to target protein-coding sequences (CDS); however, miRNAs have been generally believed to function through similar mechanisms regardless of the locations of their sites of action. Here, we report a class of miRNA-recognition elements (MREs) that function exclusively in CDS regions. Through functional and mechanistic characterization of these 'unusual' MREs, we demonstrate that CDS-targeted miRNAs require extensive base-pairing at the 3' side rather than the 5' seed; cause gene silencing in an Argonaute-dependent but GW182-independent manner; and repress translation by inducing transient ribosome stalling instead of mRNA destabilization. These findings reveal distinct mechanisms and functional consequences of miRNAs that target CDS versus the 3' UTR and suggest that CDS-targeted miRNAs may use a translational quality-control-related mechanism to regulate translation in mammalian cells

Using hiCLIP to identify RNA duplexes that interact with a specific RNA-binding protein

The structure of RNA molecules has a critical role in regulating gene expression, largely through influencing their interactions with RNA-binding proteins (RBPs). RNA hybrid and individual-nucleotide resolution UV cross-linking and immunoprecipitation (hiCLIP) is a transcriptome-wide method of monitoring these interactions by identifying RNA duplexes bound by a specific RBP. The hiCLIP protocol consists of the following steps: in vivo cross-linking of RBPs to their bound RNAs; partial RNA digestion and purification of RNA duplexes interacting with the specific RBP using immunoprecipitation; ligation of the two arms of RNA duplexes via a linker; reverse transcription; cDNA library amplification; and finally high-throughput DNA sequencing. Mapping of the sequenced arms to a reference transcriptome identifies the exact locations of duplexes. hiCLIP data can directly identify all types of RNA duplexes bound by RBPs, including those that are challenging to predict computationally, such as intermolecular and long-range intramolecular duplexes. Moreover, the use of an adaptor that links the two arms of the RNA duplex permits hiCLIP to unambiguously identify the duplexes. Here we describe in detail the procedure for a hiCLIP experiment and the subsequent streamlined data analysis with an R package, 'hiclipr' (https://github.com/luslab/hiclipr/). Preparation of the library for high-throughput DNA sequencing takes ∼7 d and the basic bioinformatic pipeline takes 1 d

Essentials of miRNA-dependent control of mRNA translation and decay, miRNA targeting principles, and methods for target identification

MicroRNAs appear to be involved in nearly all biological processes, as the large majority of protein coding genes are assumed to be regulated by one or several miRNAs. To exert their repressive function on translation and transcript stability, miRNAs guide Argonaute (AGO) proteins to partially complementary sites in target RNAs. A short hexamer of the miRNAs, called the seed, is especially involved in target binding and complementarity to the seed can be used to predict targets of miRNAs, albeit with considerable number of false predictions. Several biochemical methods have been developed to identify miRNA targets, amongst others crosslinking and immunoprecipitation approaches. These identify target sites at nucleotide resolution and can also directly tell which miRNA is binding, when the data is analyzed for miRNA:target site ligation products. This chapter will give an overview on the mechanism by which miRNAs exert their repressive function and on the experimental methods that have been used to identify their targets. We further discuss the potential of unambiguous identification of miRNA interactions by ligations in the brain, where an extraordinary high number of miRNAs are expressed and huma- or primate-specific miRNAs are of special interest

Bayesian reconstruction of nanodosimetric cluster distributions at 100% detection efficiency

Ionisation spectra in nanometric volumes at a given distance from a charged particle track are obtained by using electron (or ion) gas detectors, having non-uniformly distributed detection efficiency. Therefore, such spectra should be properly processed in order to reconstruct the frequency distribution of clusters really produced in the detector gas. A Bayesian unfolding has been applied to ionisation distributions due to 5.4 MeV alpha particles in a 20-mn site obtained by Monte Carlo simulations, taking into account different detection efficiency conditions. It will be shown that Bayesian analysis provides a valid tool for reconstructing the true ionisation distributions, well beyond the maximum measured cluster size