The rockefeller university

Prokaryotic organisms are threatened by a large array of viruses and have developed numerous defence strategies. Among these, only clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity against foreign elements. Upon viral injection, a small sequence of the viral genome, known as a spacer, is integrated into the CRISPR locus to immunize the host cell. Spacers are transcribed into small RNA guides that direct the cleavage of the viral DNA by Cas nucleases. Immunization through spacer acquisition enables a unique form of evolution whereby a population not only rapidly acquires resistance to its predators but also passes this resistance mechanism vertically to its progeny.

Huntington's disease (HD) is a neurodegenerative disorder characterized by massive loss of medium spiny neurons in the striatum. However, the mechanisms by which mutant huntingtin leads to this selective neuronal death remain incompletely understood. Brain-derived neurotrophic factor (BDNF) has been shown to be neuroprotective on HD striatal neurons both in vitro and in vivo. ProBDNF, the precursor of mature BDNF (mBDNF), also can be secreted but promotes apoptosis of neurons expressing p75(NTR) and sortilin receptors. Although a reduction of total striatal BDNF protein has been reported in HD patients and mouse models, it remains unclear whether conversion of proBDNF to mBDNF is altered in HD, and whether the proBDNF receptors, p75(NTR) and sortilin are dysregulated, leading to impaired striatal neuron survival. To test these hypotheses, we generated bdnf-HA knock-in (MI) mice on the zQ175 HD background to accurately quantitate the levels of both proBDNF and mBDNF in the HD striatum. In aged zQ175 HD mice, we observed a significant loss of mBDNF and decreased TrkB activation, but no increase of proBDNF or p75(NTR) levels either in the sensorimotor cortex or the striatum cortilin receptor are both increased in immature striatal oligodenHD striatum. Taken together, the present study through the TrkB receptor, rather than an induction factor to the vulnerability of striatal neurons in the zQ175 HD mouse model. (C) 2015 Elsevier Inc All rights reserved.

Transposable elements (TEs) and repetitive sequences are ubiquitously present in eukaryotic genomes which are in general epigenetically silenced by DNA methylation and/or histone 3 lysine 9 methylation (H3K9me). RNA-directed DNA methylation (RdDM) is the major pathway that initiates de novo DNA methylation in Arabidopsis and sets up a self-reinforcing silencing loop between DNA methylation and H3K9me. However, a key issue is the requirement of a basal level transcript from the target loci to initiate the RNA-based silencing. How the heterochromatic silenced loci are transcribed remains largely unknown. Here, we show that JMJ24, a JmjC domain-containing protein counteracts H3K9me to promote basal level transcription of endogenous silenced loci in Arabidopsis. JMJ24 functionally resembles the fission yeast JmjC protein Epe1. The transcript promoted by JMJ24 is, at least in part, processed to small RNA to initiate the RdDM. Genome-wide transcriptome profiling indicates that transcript levels of TEs are more likely regulated by JMJ24, compared with protein-coding genes. Our data suggest that JMJ24 plays a conserved role in promoting basal level transcription of endogenous silenced loci to reinforce the silencing. We also provide evidence of a physical association between JMJ24 and RNA-dependent RNA polymerase 2 (RDR2), which represents an evolved property of the RNA silencing pathway.

Aptamers are an emerging class of highly specific targeting ligands. They can be selected in vitro for a large variety of targets, ranging from small molecules to whole cells. Most aptamers selected are nucleic acid-based, allowing chemical synthesis and easy modification. Although their properties make them interesting drug candidates for a broad spectrum of applications and an interesting alternative to antibodies or fusion proteins, they are not yet broadly used. One major drawback of aptamers is their susceptibility to abundant serum nucleases, resulting in their fast degradation in biological fluids. Using modified nucleic acids has become a common strategy to overcome these disadvantages, greatly increasing their half-life under cell culture conditions or even in vivo. Whereas pre-selective modifications of the initial library for aptamer selection are relatively easy to obtain, post-selective modifications of already selected aptamers are still generally very labor-intensive and often compromise the aptamers ability to bind its target molecule.Here we report the selection, characterization and post-selective modification of a 34 nucleotide (nt) RNA aptamer for a non-dominant, novel target site (domain 3) of the interleukin-6 receptor (IL-6R). We performed structural analyses and investigated the affinity of the aptamer to the membrane-bound and soluble forms (sIL-6R) of the IL-6R. Further, we performed structural analyses of the aptamer in solution using small-angle X-ray scattering and determined its overall shape and oligomeric state. Post-selective exchange of all pyrimidines against their 2-fluoro analogs increased the aptamers stability significantly without compromising its affinity for the target protein. The resulting modified aptamer could be shortened to its minimal binding motif without loss of affinity.

A search for pair production of neutral color-octet weak-triplet scalar particles (Theta(0)) is performed in processes where one Theta(0) decays to a pair of b quark jets and the other to a Z boson plus a jet, with the Z boson decaying to a pair of electrons or muons. The search is performed with data collected by the CMS experiment at the CERN LHC corresponding to an integrated luminosity of 19.7 fb(-1) of proton-proton collisions at root s = 8TeV. The number of observed events is found to be in agreement with the standard model predictions. The 95% confidence level upper limit on the product of the cross section and branching fraction is obtained as a function of the Theta(0) mass. The 95% confidence level lower bounds on the Theta(0) mass are found to be 623 and 426 GeV, for two different octo-triplet theoretical scenarios. These are the first direct experimental bounds on particles predicted by the octo-triplet model.

Motivation: We have created an R package named phylogeo that provides a set of geographic utilities for sequencing-based microbial ecology studies. Although the geographic location of samples is an important aspect of environmental microbiology, none of the major software packages used in processing microbiome data include utilities that allow users to map and explore the spatial dimension of their data. phylogeo solves this problem by providing a set of plotting and mapping functions that can be used to visualize the geographic distribution of samples, to look at the relatedness of microbiomes using ecological distance, and to map the geographic distribution of particular sequences. By extending the popular phyloseq package and using the same data structures and command formats, phylogeo allows users to easily map and explore the geographic dimensions of their data from the R programming language.

For degenerative disorders of the CNS, the main obstacle to therapeutic advancement has been the challenge of identifying the key molecular mechanisms underlying neuronal loss. We developed a combinatorial approach including translational profiling and brain regulatory network analysis to search for key determinants of neuronal survival or death. Following the generation of transgenic mice for cell type-specific profiling of midbrain dopaminergic neurons, we established and compared translatome libraries reflecting the molecular signature of these cells at baseline or under degenerative stress. Analysis of these libraries by interrogating a context-specific brain regulatory network led to the identification of a repertoire of intrinsic upstream regulators that drive the dopaminergic stress response. The altered activity of these regulators was not associated with changes in their expression levels. This strategy can be generalized for the identification of molecular determinants involved in the degeneration of other classes of neurons.

Pancreatic cancer is one of the leading causes of cancer-related death in the United States. Gemcitabine is a common treatment, but response rates are low, perhaps due in part to tumor hypoxia. We utilized C-14-labeled gemcitabine to map distribution of the drug with respect to perfused and hypoxic regions of the tumor microenvironment in two orthotopic xenograft models of pancreatic cancer. There was only a slight reduction in gemcitabine in hypoxic areas, with similar to 78% of the drug present in hypoxic compared to perfused areas. In addition, only a 4% reduction in gemcitabine was measured at >100 mu m from perfused blood vessels. Thus, despite significant areas of hypoxia in these tumors, gemcitabine distribution is relatively homogenous. Ours is the first study to directly measure gemcitabine distribution within tumor tissue, demonstrating that in these models, tumor tissue does not represent a barrier to gemcitabine penetration.

Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin-proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IB degradation assay, Thsp is a slow inhibitor and 4hrs of treatment achieves the same effects as MG-132 at 30min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes invitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.