Delayed Differentiation Makes Many Models Compatible with Data for CD8+ T Cell Differentiation

Upon antigen stimulation, naïve CD8+ T cells differentiate into short-lived effectors and longer-lived memory T cells. The kinetics of expansion of antigen-specific CD8+ T cells is highly reproducible at the population level, but the fate of individual naïve cells is stochastic, as individual naïve CD8+ T cells produce different numbers of effector and memory cells. Using mathematical models to analyse experimental data on tracing the fate of individual naïve T cells, it was previously shown that a linear model where naïve CD8+ T cells first differentiate into memory precursors that subsequently differentiate into effector cells describes the data best. However, this ‘memory first’ linear model assumed that the proliferation and differentiation events were distributed exponentially, whereas several studies indicate that differentiation of CD8+ T cell subsets need not follow an exponential distribution. Here we investigate the effect of delayed differentiation by adding intermediate compartments and use similar ordinary differential equations and Gillespie simulations to evaluate alternate models of CD8+ T cell differentiation. Models where a substantial fraction of the naïve CD8+ T cells directly differentiate into effector cells, without going through a memory phase, exhibit population dynamics that are very similar to the original ‘memory first’ linear model. Because alternate models with delayed differentiation perform better than those without a delay, we conclude that non-exponential forms of cellular differentiation need to be considered when comparing models. Hence the exact pathway for the differentiation of naïve CD8+ T cells into effector and memory T cells remains an open question

A Genome-Wide Analysis of FRT-Like Sequences in the Human Genome

Efficient and precise genome manipulations can be achieved by the Flp/FRT system of site-specific DNA recombination. Applications of this system are limited, however, to cases when target sites for Flp recombinase, FRT sites, are pre-introduced into a genome locale of interest. To expand use of the Flp/FRT system in genome engineering, variants of Flp recombinase can be evolved to recognize pre-existing genomic sequences that resemble FRT and thus can serve as recombination sites. To understand the distribution and sequence properties of genomic FRT-like sites, we performed a genome-wide analysis of FRT-like sites in the human genome using the experimentally-derived parameters. Out of 642,151 identified FRT-like sequences, 581,157 sequences were unique and 12,452 sequences had at least one exact duplicate. Duplicated FRT-like sequences are located mostly within LINE1, but also within LTRs of endogenous retroviruses, Alu repeats and other repetitive DNA sequences. The unique FRT-like sequences were classified based on the number of matches to FRT within the first four proximal bases pairs of the Flp binding elements of FRT and the nature of mismatched base pairs in the same region. The data obtained will be useful for the emerging field of genome engineering

Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection

The molecular details of Chlamydia trachomatis binding, entry, and spread are incompletely understood, but heparan sulfate proteoglycans (HSPGs) play a role in the initial binding steps. As cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated the role of HSPG-dependent growth factors in C. trachomatis infection. Here, we report a novel finding that Fibroblast Growth Factor 2 (FGF2) is necessary and sufficient to enhance C. trachomatis binding to host cells in an HSPG-dependent manner. FGF2 binds directly to elementary bodies (EBs) where it may function as a bridging molecule to facilitate interactions of EBs with the FGF receptor (FGFR) on the cell surface. Upon EB binding, FGFR is activated locally and contributes to bacterial uptake into non-phagocytic cells. We further show that C. trachomatis infection stimulates fgf2 transcription and enhances production and release of FGF2 through a pathway that requires bacterial protein synthesis and activation of the Erk1/2 signaling pathway but that is independent of FGFR activation. Intracellular replication of the bacteria results in host proteosome-mediated degradation of the high molecular weight (HMW) isoforms of FGF2 and increased amounts of the low molecular weight (LMW) isoforms, which are released upon host cell death. Finally, we demonstrate the in vivo relevance of these findings by showing that conditioned medium from C. trachomatis infected cells is enriched for LMW FGF2, accounting for its ability to enhance C. trachomatis infectivity in additional rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway to enhance bacterial infection and spread

Induction of Interferon-Stimulated Genes by Chlamydia pneumoniae in Fibroblasts Is Mediated by Intracellular Nucleotide-Sensing Receptors

BACKGROUND: Recognition of microorganisms by the innate immune system is mediated by pattern recognition receptors, including Toll-like receptors and cytoplasmic RIG-I-like receptors. Chlamydia, which include several human pathogenic species, are obligate intracellular gram-negative bacteria that replicate in cytoplasmic vacuoles. The infection triggers a host response contributing to both bacterial clearance and tissue damage. For instance, type I interferons (IFN)s have been demonstrated to exacerbate the course of Chlamydial lung infections in mice. METHODS/PRINCIPAL FINDINGS: Here we show that Chlamydia pneumoniae induces expression of IFN-stimulated genes (ISG)s dependent on recognition by nucleotide-sensing Toll-like receptors and RIG-I-like receptors, localized in endosomes and the cytoplasm, respectively. The ISG response was induced with a delayed kinetics, compared to virus infections, and was dependent on bacterial replication and the bacterial type III secretion system (T3SS). CONCLUSIONS/SIGNIFICANCE: Activation of the IFN response during C. pneumoniae infection is mediated by intracellular nucleotide-sensing PRRs, which operate through a mechanism dependent on the bacterial T3SS. Strategies to inhibit the chlamydial T3SS may be used to limit the detrimental effects of the type I IFN system in the host response to Chlamydia infection

Extensions of MADM (Mosaic Analysis with Double Markers) in Mice

Mosaic Analysis with Double Markers (MADM) is a method for generating genetically mosaic mice, in which sibling mutant and wild-type cells are labeled with different fluorescent markers. It is a powerful tool that enables analysis of gene function at the single cell level in vivo. It requires transgenic cassettes to be located between the centromere and the mutation in the gene of interest on the same chromosome. Here we compare procedures for introduction of MADM cassettes into new loci in the mouse genome, and describe new approaches for expanding the utility of MADM. We show that: 1) Targeted homologous recombination outperforms random transgenesis in generation of reliably expressed MADM cassettes, 2) MADM cassettes in new genomic loci need to be validated for biallelic and ubiquitous expression, 3) Recombination between MADM cassettes on different chromosomes can be used to study reciprocal chromosomal deletions/duplications, and 4) MADM can be modified to permit transgene expression by combining it with a binary expression system. The advances described in this study expand current, and enable new and more versatile applications of MADM

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Search for direct stau production in events with two hadronic tau-leptons in root s=13 TeV pp collisions with the ATLAS detector

A search for the direct production of the supersymmetric partners ofτ-leptons (staus) in final stateswith two hadronically decayingτ-leptons is presented. The analysis uses a dataset of pp collisions corresponding to an integrated luminosity of139fb−1, recorded with the ATLAS detector at the LargeHadron Collider at a center-of-mass energy of 13 TeV. No significant deviation from the expected StandardModel background is observed. Limits are derived in scenarios of direct production of stau pairs with eachstau decaying into the stable lightest neutralino and oneτ-lepton in simplified models where the two staumass eigenstates are degenerate. Stau masses from 120 GeV to 390 GeV are excluded at 95% confidencelevel for a massless lightest neutralino

Expected Performance of the ATLAS Experiment - Detector, Trigger and Physics

A detailed study is presented of the expected performance of the ATLAS detector. The reconstruction of tracks, leptons, photons, missing energy and jets is investigated, together with the performance of b-tagging and the trigger. The physics potential for a variety of interesting physics processes, within the Standard Model and beyond, is examined. The study comprises a series of notes based on simulations of the detector and physics processes, with particular emphasis given to the data expected from the first years of operation of the LHC at CERN

Ptc6 is required for proper rapamycin-induced down-regulation of the genes coding for ribosomal and rRNA processing proteins in S. cerevisiae

Ptc6 is one of the seven components (Ptc1-Ptc7) of the protein phosphatase 2C family in the yeast Saccharomyces cerevisiae. In contrast to other type 2C phosphatases, the cellular role of this isoform is poorly understood. We present here a comprehensive characterization of this gene product. Cells lacking Ptc6 are sensitive to zinc ions, and somewhat tolerant to cell-wall damaging agents and to Li+. Ptc6 mutants are sensitive to rapamycin, albeit to lesser extent than ptc1 cells. This phenotype is not rescued by overexpression of PTC1 and mutation of ptc6 does not reproduce the characteristic geneti