The rockefeller university

The c-fos gene responds to extracellular stimuli and undergoes robust but transient transcriptional activation. Here we show that heterogeneous nuclear ribonucleoprotein R (hnRNP R) facilitates transcription reinitiation of the c-fos promoter in vitro in cooperation with Mediator. Consistently, hnRNP R interacts with the Scaffold components (Mediator, TBP, and TFIIH) as well as TFIIB, which recruits RNA polymerase II (Pol II) and TFIIF to Scaffold. The cooperative action of hnRNP R and Mediator is diminished by the cyclin-dependent kinase 8 (CDK8) module, which is comprised of CDK8, Cyclin C, MED12 and MED13 of the Mediator subunits. Interestingly, we find that the length of the G-free cassettes, and thereby their transcripts, influences the hnRNP R-mediated facilitation of reinitiation. Indeed, indicative of a possible role of the transcript in facilitating transcription reinitiation, the RNA transcript produced from the G-free cassette interacts with hnRNP R through its RNA recognition motifs (RRMs) and arginine-glycine-glycine (RGG) domain. Mutational analyses of hnRNP R indicate that facilitation of initiation and reinitiation requires distinct domains of hnRNP R. Knockdown of hnRNP R in mouse cells compromised rapid induction of the c-fos gene but did not affect transcription of constitutive genes. Together, these results suggest an important role for hnRNP R in regulating robust response of the c-fos gene.

We report on a measurement of the top-quark electric charge in t (t) over bar events in which one W boson originating from the top-quark pair decays into leptons and the other into hadrons. The event sample was collected by the CDF II detector in root s = 1.96 TeV proton-antiproton collisions and corresponds to 5.6 fb(-1). We find the data to be consistent with the standard model and exclude the existence of an exotic quark with -4/3 electric charge and mass of the conventional top quark at the 99% confidence level.

Mouse models of prostate cancer (PCa) are critical for understanding the biology of PCa initiation, progression, and treatment modalities. Here, we summarize recent advances in PCa mouse models that led to new insights into specific gene functions in PCa. For example, the study of transgenic mice with TMPRSS2/ERG, an androgen-regulated fusion protein, revealed its role in developing PCa precursor lesions, prostate intraepithelial neoplasia; however, it is not sufficient for PCa development. Double deficiency of Pten and Smad4 leads to a high incidence of metastatic PCa. Targeted deletion of Pten in castration-resistant Nkx3-1-expressing cells results in rapid carcinoma formation after androgen-mediated regeneration, indicating that progenitor cells with luminal characteristics can play a role in initiation of PCa. Transgenic mice with activated oncogenes, growth factors, and steroid hormone receptors or inactivated tumor suppressors continue to provide insights into disease progression from initiation to metastasis. Further development of new PCa models with spatial and temporal regulation of candidate gene expression will probably enhance our understanding of the complex events that lead to PCa initiation and progression, thereby invoking novel strategies to combat this common disease in men.

The development of an effective vaccine preventing HIV-1 infection remains elusive. Thus, the development of novel approaches capable of preventing HIV-1 transmission is of paramount importance. However, this is partly hindered by the lack of an easily accessible small-animal model to rapidly measure viral entry. Here, we report the generation of a human CD4- and human CCR5-expressing transgenic luciferase reporter mouse that facilitates measurement of peritoneal and genitomucosal HIV-1 pseudovirus entry in vivo. We show that antibodies and antiretrovirals mediate preexposure protection in this mouse model and that the serum antibody concentration required for protection from cervicovaginal infection is comparable to that required to protect macaques. Our results suggest that this system represents a model for the preclinical evaluation of prophylactic or vaccine candidates. It further supports the idea that broadly neutralizing antibodies should be evaluated for use as preexposure prophylaxis in clinical trials.

The number of genetically defined Primary Immunodeficiency Diseases (PID) has increased exponentially, especially in the past decade. The biennial classification published by the IUIS PID expert committee is therefore quickly expanding, providing valuable information regarding the disease-causing genotypes, the immunological anomalies, and the associated clinical features of PIDs. These are grouped in eight, somewhat overlapping, categories of immune dysfunction. However, based on this immunological classification, the diagnosis of a specific PID from the clinician's observation of an individual clinical and/or immunological phenotype remains difficult, especially for non-PID specialists. The purpose of this work is to suggest a phenotypic classification that forms the basis for diagnostic trees, leading the physician to particular groups of PIDs, starting from clinical features and combining routine immunological investigations along the way. We present 8 colored diagnostic figures that correspond to the 8 PID groups in the IUIS Classification, including all the PIDs cited in the 2011 update of the IUIS classification and most of those reported since.

Purpose This study reports the identification of a novel heterozygous IKBA missense mutation ( p.M37K) in a boy presenting with ectodermal dysplasia with immunodeficiency (EDA-ID) who had wild type IKBKG gene encoding NEMO. Our aim was to characterize the clinical course of this I kappa B-alpha gain-of-function mutant and to investigate if the p.M37K substitution affects NF-kappa B activation by interfering with I kappa B-alpha degradation, thus impairing NF-kappa B signaling and causing the EDA-ID phenotype. Methods NF-kappa B signaling was evaluated by measuring I kappa B-alpha degradation in patient fibroblasts. In addition, transientlytransfected HeLa cells expressing either the M37K-mutant I kappa B-alpha allele, the previously characterized S36A-mutant I kappa B-alpha allele, or wild type I kappa B-alpha were evaluated for I kappa B-alpha degradation and NF-kappa B nuclear translocation following stimulation with TNF-alpha. Results Clinical findings revealed a classical ectodermal dysplasia phenotype complicated by recurrent mucocutaneous candidiasis, hypothyroidism, hypopituitarism, and profound combined immunodeficiency with decreased numbers of IL-17 T cells. I kappa B-alpha degradation after TNF-alpha and TLR agonist stimulation was abolished in patient fibroblasts as well as in HeLa cells expressing M37K-I kappa B-alpha similar to cells expressing S36A-I kappa B-alpha resulting in impaired nuclear translocation of NF-kappa B and reduced NF-kappa B dependent luciferase activity compared to cells expressing wild type I kappa B-alpha. Patient whole blood cells failed to secrete IL-6 in response to IL-1 beta, Pam(2)CSK(4), showed reduced responses to LPS and PMA/Ionomycin, and lacked IL-10 production in response to TNF-alpha. Conclusion The novel heterozygous mutation p.M37K in I kappa B-alpha impairs NF-kappa B activation causing autosomal dominant EDA-ID with an expanded clinical phenotype.

Tryptic digestion is an important component of most proteomics experiments, and trypsin is available from many sources with a cost that varies by more than 1000-fold. This high-mass-accuracy LC-MS study benchmarks six commercially available trypsins with respect to autolytic species and sequence specificity. The analysis of autolysis products led to the identification of a number of contaminating proteins and the generation of a list of peptide species that will be present in tryptic digests. Intriguingly, many of the autolysis products were nontryptic peptides, specifically peptides generated by C-terminal cleavage at asparagine residues. Both porcine and bovine trypsins were demonstrated to be tyrosine O-sulfated. Using both a label-free and a tandem mass tag (TMT) labeling approach, a comparison of the digestion of a standard protein mixture using the six trypsins demonstrated that, apart from the least expensive bovine trypsin, the trypsins were equally specific. The semitryptic activity led to a better sequence coverage for abundant substrates at the expense of low-abundance species. The label-free analysis was shown to be more sensitive to unique features from the individual digests that were lost in the TMT-multiplexing study.

The International Psoriasis Council, a global nonprofit organization dedicated to advancing psoriasis research and treatment, led an initiative to better define the pathogenic mechanisms that constitute psoriasis. In September 2012, a workshop was held at the 42nd Annual European Society for Dermatological Research in Venice, Italy. By assembling a panel of global dermatology and immunology experts, the objective was to evaluate the current status of the science explaining the mechanism of disease in psoriasis, e.g. dysregulation of the skin immune system and perturbations of epidermal homeostasis. The workshop consisted of four oral presentations, which addressed key topics in psoriasis, delivered by Herve Bachelez (Paris, France), Antonio Costanzo (Rome, Italy), Michelle Lowes (New York, NY, U.S.A.) and Frank Nestle (London, U.K.). A global expert panel was assembled to stimulate dialogue and debate: Kevin Cooper (Cleveland, OH, U.S.A.), Michel Gilliet (Lausanne, Switzerland), Joerg Prinz (Munich, Germany), Martin Rocken (Tubingen, Germany), Jens Schroeder (Kiel, Germany), Manuelle Viguier (Paris, France), Mayte Suarez-Farinas (New York, NY, U.S.A.) and Cristina Zielinski (Berlin, Germany). Collectively, the presentations demonstrated the significant advances in understanding immune regulation that have occurred over the past decade by virtue of the study of psoriasis subtypes, phenotypic manifestations and genetic associations. Elucidating the pathogenic and genetic basis of psoriasis holds the promise of a complete understanding of disease mechanisms, predictors of treatment response, novel drug development strategies and customized therapeutic regimens for the individual patient.

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.