The rockefeller university

Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequence!

We profiled microRNAs (miRNAs) in cell-free serum and plasma samples from human volunteers using deep sequencing of barcoded small RNA cDNA libraries. By introducing calibrator synthetic oligonucleotides during library preparation, we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we detected placental-specific miRNA in both maternal and newborn circulations and quantitated the relative contribution of placental miRNAs to the circulating pool of miRNAs. Furthermore, sequence variation in the placental miRNA profiles could be traced to the specific placenta of origin. These deep sequencing profiles, which may serve as a model for tumor or disease detection, allow us to define the repertoire of miRNA abundance in the circulation and potential uses as biomarkers.

We present an updated search for the Higgs boson produced in association with a vector boson in the final state with missing transverse energy and two jets. We use the full CDF data set corresponding to an integrated luminosity of 9: 45 fb(-1) at a proton-antiproton center-of-mass energy of root s = 1.96 TeV. New to this analysis is the inclusion of a b-jet identification algorithm specifically optimized for H -> b (b) over bar searches. Across the Higgs boson mass range 90 <= m(H) <= 150 GeV/c(2), the expected 95% credibility level upper limits on the VH production cross section times the H -> b (b) over bar branching fraction are improved by an average of 14% relative to the previous analysis. At a Higgs boson mass of 125 GeV/c(2), the observed (expected) limit is 3.06 (3.33) times the standard model prediction, corresponding to one of the most sensitive searches to date in this final state. DOI: 10.1103/PhysRevD.87.052008

Post-translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM-dependent proteinprotein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak proteinprotein interactions. We have recently developed CLASPI (cross-linking-assisted and stable isotope labeling in cell culture-based protein identification), a chemical proteomics approach to examine proteinprotein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation-dependent proteinprotein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 l!

A measurement of the a production cross section in pp collisions at root s = 7 TeV is presented. The results are based on data corresponding to an integrated luminosity of 2.3 fb(-1) collected by the CMS detector at the LHC. Selected events are required to have one isolated, high transverse momentum electron or muon, large missing transverse energy, and hadronic jets, at least one of which must be consistent with having originated from a b quark. The measured cross section is 158.1 +/- 2.1 (stat.) +/- 10.2 (syst.) +/- 3.5 (lum.) pb, in agreement with standard model predictions. (C) 2013 CERN. Published by Elsevier B.V. All rights reserved.

Past studies have documented a crosstalk between H2B ubiquitylation (H2Bub) and H3K4 methylation, but little (if any) direct evidence exists explaining the mechanism underlying H2Bub-dependent H3K4 methylation on chromatin templates. Here, we took advantage of an in vitro histone methyltransferase assay employing a reconstituted yeast Set1 complex (ySet1C) and a recombinant chromatin template containing fully ubiquitylated H2B to gain valuable insights. Combined with genetic analyses, we demonstrate that the n-SET domain within Set1, but not Swd2, is essential for H2Bub-dependent H3K4 methylation. Spp1, a homolog of human CFP1, is conditionally involved in this crosstalk. Our findings extend to the human Set1 complex, underscoring the conserved nature of this disease-relevant crosstalk pathway. As not all members of the H3K4 methyltransferase family contain n-SET domains, our studies draw attention to the n-SET domain as a predictor of an H2B ubiquitylation-sensing mechan!

The acute response of human skin to UVB radiation has not been fully characterized. We sought to define the cutaneous response at 24 hours following narrowband UVB (NB-UVB, 312-nm peak), a therapeutically relevant source of UVB, using transcriptional profiling, immunohistochemistry, and immunofluorescence. There were 1,522 unique differentially regulated genes, including upregulated genes encoding antimicrobial peptides (AMPs) (S100A7, S100A12, human beta-defensin 2, and elafin), as well as neutrophil and monocyte/dendritic cell (DC) chemoattractants (IL-8, CXCL1, CCL20, CCL2). Ingenuity pathway analysis demonstrated activation of innate defense and early adaptive immune pathways. Immunohistochemistry confirmed increased epidermal staining for AMPs (S100A7, S100A12, human beta-defensin 2, and elafin). Inflammatory myeloid CD11c(+)BDCA1(-) DCs were increased in irradiated skin, which were immature as shown by minimal colocalization with DC-LAMP, and coexpressed inflammator!

Polyglutamine-binding protein 1 (PQBP1) is a highly conserved protein associated with neurodegenerative disorders. Here, we identify PQBP1 as an alternative messenger RNA (mRNA) splicing (AS) effector capable of influencing splicing of multiple mRNA targets. PQBP1 is associated with many splicing factors, including the key U2 small nuclear ribonucleoprotein (snRNP) component SF3B1 (subunit 1 of the splicing factor 3B [SF3B] protein complex). Loss of functional PQBP1 reduced SF3B1 substrate mRNA association and led to significant changes in AS patterns. Depletion of PQBP1 in primary mouse neurons reduced dendritic outgrowth and altered AS of mRNAs enriched for functions in neuron projection development. Disease-linked PQBP1 mutants were deficient in splicing factor associations and could not complement neurite outgrowth defects. Our results indicate that PQBP1 can affect the AS of multiple mRNAs and indicate specific affected targets whose splice site determination may con!

Depression induces structural and functional synaptic plasticity in brain reward circuits, although the mechanisms promoting these changes and their relevance to behavioral outcomes are unknown. Transcriptional profiling of the nucleus accumbens (NAc) for Rho GTPase related genes, which are known regulators of synaptic structure, revealed a sustained reduction in RAS-related C3 botulinum toxin substrate 1 (Rac1) expression after chronic social defeat stress. This was associated with a repressive chromatin state surrounding the proximal promoter of Rac1. Inhibition of class 1 histone deacetylases (HDACs) with MS-275 rescued both the decrease in Rac1 transcription after social defeat stress and depression-related behavior, such as social avoidance. We found a similar repressive chromatin state surrounding the RAC1 promoter in the NAc of subjects with depression, which corresponded with reduced RAC1 transcription. Viral-mediated reduction of Rac1 expression or inhibition of !

Carbon fixation and production of reductant by cyanobacteria can exceed new biomass synthesis rates when the supply of essential nutrients is limiting. Under these circumstances metabolic balance is achieved by diversion of excess carbon and reductant to synthesis of glycogen, which can accumulate to more than 50% of cellular dry biomass. We discovered that when glycogen synthesis was abolished, by deletion of the gene for glucose-1-phosphate adenylyl transferase (glgC), Synechococcus elongatus PCC 7942 was unable to degrade its phycobilisomes in response to nitrogen stress. Furthermore, nitrogen deprived glgC null cells recalibrated the levels of glycolytic and TCA cycle intermediates. Specifically, succinate, fumarate, and 2-oxoglutarate, a metabolic indicator of cellular nitrogen status andmetabolic effector of the global nitrogen regulator NtcA, accumulated within and were excreted by glgC null cells under nitrogen stress. Moreover, intracellular accumulation and excretion of 2-oxoglutarate from nitrogen stressed glgC null cells coincided temporally with suppression of nblA transcription, while internalization of 2-oxoglutarate by nitrogen deprived wild type cells delayed both nblA expression and phycobilisome degradation. Furthermore, glgC null cells exhibited a non-bleaching phenotype in response to sulfur and phosphate stress. These data indicate that glycogen synthesis is a required component of the global response to nutrient stress. (C) 2013 Elsevier B.V. All rights reserved.