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Genetically encoded p-azido-phenylalanine (azF) residues in G protein-coupled receptors (GPCRs) can be targeted with dibenzocyclooctyne-modified (DIBO-modified) fluorescent probes by means of strain-promoted [3+2] azide-alkyne cycloaddition (SpAAC). Here we show that azF residues situated on the transmembrane surfaces of detergent-solubilized receptors exhibit up to 1000-fold rate enhancement relative to azF residues on water-exposed surfaces. We show that the amphipathic moment of the labeling reagent, consisting of hydrophobic DIBO coupled to hydrophilic Alexa dye, results in strong partitioning of the DIBO group into the hydrocarbon core of the detergent micelle and consequently high local reactant concentrations. The observed rate constant for the micelleenhanced SpAAC is comparable with those of the fastest bioorthogonal labeling reactions known. Targeting hydrophobic regions of membrane proteins by use of micelle-enhanced SpAAC should expand the utility of bioorthogonal labeling strategies.

A search is presented for particle dark matter produced in association with a pair of top quarks in pp collisions at a centre-of-mass energy of root s = 8TeV. The data were collected with the CMS detector at the LHC and correspond to an integrated luminosity of 19.7 fb(-1). This search requires the presence of one lepton, multiple jets, and large missing transverse energy. No excess of events is found above the SM expectation, and upper limits are derived on the production cross section. Interpreting the findings in the context of a scalar contact interaction between fermionic dark matter particles and top quarks, lower limits on the interaction scale are set. These limits are also interpreted in terms of the dark matter-nucleon scattering cross sections for the spin-independent scalar operator and they complement direct searches for dark matter particles in the low mass region.

The second-order azimuthal anisotropy Fourier harmonics, nu(2), are obtained in p-Pb and PbPb collisions over a wide pseudorapidity (.) range based on correlations among six or more charged particles. The p-Pb data, corresponding to an integrated luminosity of 35 nb(-1), were collected during the 2013 LHC p-Pb run at a nucleon-nucleon center-of-mass energy of 5.02 TeV by the CMS experiment. A sample of semiperipheral PbPb collision data at root S-NN = 2.76 TeV, corresponding to an integrated luminosity of 2.5 mu b(-1) and covering a similar range of particle multiplicities as the p-Pb data, is also analyzed for comparison. The six-and eight-particle cumulant and the Lee-Yang zeros methods are used to extract the nu(2) coefficients, extending previous studies of two-and four-particle correlations. For both the p-Pb and PbPb systems, the nu(2) values obtained with correlations among more than four particles are consistent with previously published four-particle results. These data support the interpretation of a collective origin for the previously observed long-range (large..) correlations in both systems. The ratios of nu(2) values corresponding to correlations including different numbers of particles are compared to theoretical predictions that assume a hydrodynamic behavior of a p-Pb system dominated by fluctuations in the positions of participant nucleons. These results provide new insights into the multiparticle dynamics of collision systems with a very small overlapping region.

Central memory T (T-CM) cells in lymph nodes (LNs) and resident memory T (T-RM) cells in peripheral tissues have distinct roles in protective immunity(1-5). Both are generated after primary infections, but their clonal origins have been unclear. To address this question, we immunized mice through the skin with a protein antigen, a chemical hapten, or a non-replicating poxvirus. We then analyzed antigen-activated T cells from different tissues using high-throughput sequencing (HIS) of the gene encoding the T cell receptor (TCR) beta-chain (Trb, also known as Tcrb) using CDR3 sequences to simultaneously track thousands of unique T cells(6). For every abundant T-RM cell clone generated in the skin, an abundant T-CM cell clone bearing the identical TCR was present in the LNs. Thus, antigen-reactive skin T-RM and LN T-CM cell clones were derived from a common naive T cell precursor after skin immunization, generating overlapping TCR repertoires. Although they bore the same TCR, T-RM cells mediated rapid contact hypersensitivity(7) responses, whereas T-CM cells mediated delayed and attenuated responses. Studies in human subjects confirmed the generation of skin T-RM cells in allergic contact dermatitis. Thus, immunization through skin simultaneously generates skin T-RM and LN T-CM cells in similar numbers from the same naive T cells.

During 1 year, experimentally naive C57BL/6NCrl weanlings born to timed-pregnant dams from a single vendor demonstrated markedly increased mortality associated with runting, abnormal gait, and decreased activity. Gram-positive, aerobic, a-hemolytic, coccoid bacteria were isolated from the meninges (n = 16), blood (n = 1), and kidneys (n = 1) of clinically affected weanlings (n = 15); from the uterus (n = 1), meninges (n = 1), and oral cavity (n = 2) of 3 dams; and from the meninges and oral cavity of a clinically affected 86-d-old mouse in the same colony. Multifocal, necrosuppurative meningoencephalitis and ventriculitis with intralesional gram-positive coccoid bacteria were present in all but 2 affected animals. The bacterium also was isolated from the oral cavity of an asymptomatic timed-pregnant dam (1 of 23) from the same vendor and from 8 mice at the vendor's facility. All isolates (n = 25) were identified by using 2 semiautomated rapid-identification systems, one of which consistently identified the causative bacterium as Aerococcus viridans 2 (n = 12) or 3 (n = 13), with probabilities of 55.7% to 98.3%. The bacterium did not grow in 6.5% NaCl at 10 degrees C, thus suggesting a Streptococcus species. Partial 16S rRNA sequencing of 4 isolates suggested S. hyointestinalis (probability, 93.4%) and S. gallinaceus (99.5%). Full 16S rRNA sequences for 3 isolates identified the bacterium as a novel Streptococcus species most closely related to S. acidominimus strain LGM (96.5%) and Streptococcus species strain Smarlab 3301444 (96.3%) and for which we propose the name S. azizii.

Passively administered anti-tumor monoclonal antibodies (mAbs) rapidly kill tumor targets via Fc gamma R-mediated cytotoxicity (ADCC), a short-term process. However, anti-tumor mAb treatment can also induce a vaccinal effect, in which mAb-mediated tumor death induces a long-term anti-tumor cellular immune response. To determine how such responses are generated, we utilized a murine model of an anti-tumor vaccinal effect against a model neoantigen. We demonstrate that Fc gamma R expression by CD11c(+) antigen-presenting cells is required to generate antitumor T cell responses upon ADCC-mediated tumor clearance. Using Fc gamma R-humanized mice, we demonstrate that anti-tumor human (h)IgG1 must engage hFc gamma RIIIA on macrophages to mediate ADCC, but also engage hFc gamma RIIA, the sole hFc gamma R expressed by human dendritic cells (DCs), to generate a potent vaccinal effect. Thus, while next-generation antitumor antibodies with enhanced binding to only hFc gamma RIIIA are now in clinical use, ideal anti-tumor antibodies must be optimized for both cytotoxic effects as well as hFc gamma RIIA engagement on DCs to stimulate long-term anti-tumor cellular immunity.

The differential cross sections for the production of photons in Z -> mu(+)mu(-)gamma decays are presented as a function of the transverse energy of the photon and its separation from the nearest muon. The data for these measurements are collected with the CMS detector and correspond to an integrated luminosity of 4.7 fb(-1) of pp collisions at root s = 7 TeV delivered by the CERN LHC. The cross sections are compared to simulations with POWHEG and PYTHIA, where PYTHIA is used to simulate parton showers and final- state photons. These simulations match the data to better than 5%. ffiffiffi

Background: Genomic profiling of lesional and nonlesional skin of patients with atopic dermatitis (AD) using microarrays has led to increased understanding of AD and identification of novel therapeutic targets. However, the limitations of microarrays might decrease detection of AD genes. These limitations might be lessened with next-generation RNA sequencing (RNA-seq). Objective: We sought to define the lesional AD transcriptome using RNA-seq and compare it using microarrays performed on the same cohort. Methods: RNA-seq and microarrays were performed to identify differentially expressed genes (criteria: fold change, >= 2.0; false discovery rate <= 0.05) in lesional versus nonlesional skin from 18 patients with moderate-to-severe AD, with real-time PCR (RT-PCR) and immunohistochemistry used for validation. Results: Both platforms showed robust disease transcriptomes and correlated well with RT-PCR. The common AD transcriptome identified by using both techniques contained 217 genes, including inflammatory (S100A8/A9/A12, CXCL1, and 2'-5'-oligoadenylate synthetase-like [OASL]) and barrier (MKi67, keratin 16 [K16], and claudin 8 [CLDN8]) AD-related genes. Although fold change estimates determined by using RNA-seq showed somewhat better agreement with RT-PCR (intraclass correlation coefficient, 0.57 and 0.70 for microarrays and RNA-seq vs RT-PCR, respectively), bias was not eliminated. Among genes uniquely identified by using RNA-seq were triggering receptor expressed on myeloid cells 1 (TREM-1) signaling (eg, CCL2, CCL3, and single immunoglobulin domain IL1R1 related [SIGIRR]) and IL-36 isoform genes. TREM-1 is a surface receptor implicated in innate and adaptive immunity that amplifies infection-related inflammation. Conclusions: This is the first report of a lesional AD phenotype using RNA-seq and the first direct comparison between platforms in this disease. Both platforms robustly characterize the AD transcriptome. Through RNA-seq, we unraveled novel disease pathology, including increased expression of the novel TREM-1 pathway and the IL-36 cytokine in patients with AD.

Enzyme-mediated modifications at the wobble position of tRNAs are essential for the translation of the genetic code. We report the genetic, biochemical and structural characterization of CmoB, the enzyme that recognizes the unique metabolite carboxy-S-adenosine-L-methionine (Cx-SAM) and catalyzes a carboxymethyl transfer reaction resulting in formation of 5-oxyacetyluridine at the wobble position of tRNAs. CmoB is distinctive in that it is the only known member of the SAM-dependent methyltransferase (SDMT) superfamily that utilizes a naturally occurring SAM analog as the alkyl donor to fulfill a biologically meaningful function. Biochemical and genetic studies define the in vitro and in vivo selectivity for Cx-SAM as alkyl donor over the vastly more abundant SAM. Complementary high-resolution structures of the apo- and Cx-SAM bound CmoB reveal the determinants responsible for this remarkable discrimination. Together, these studies provide mechanistic insight into the enzymatic and non-enzymatic feature of this alkyl transfer reaction which affords the broadened specificity required for tRNAs to recognize multiple synonymous codons.

The double-differential cross sections of promptly produced J/psi and psi(2S) mesons are measured in pp collisions at root s = 7 TeV, as a function of transverse momentum p(T) and absolute rapidity y. The analysis uses J/psi and psi(2S) dimuon samples collected by the CMS experiment, corresponding to integrated luminosities of 4.55 and 4.90 fb(-1), respectively. The results are based on a two-dimensional analysis of the dimuon invariant mass and decay length, and extend to p(T) = 120 and 100 GeV for the J/psi and psi(2S), respectively, when integrated over the interval y < 1.2. The ratio of the psi(2S) to J/psi cross sections is also reported for y < 1.2, over the range 10 < p(T) < 100 GeV. These are the highest p(T) values for which the cross sections and ratio have been measured.