The rockefeller university

Vertebrate centrioles lose their geometric scaffold, the cartwheel, during mitosis, concurrently with gaining the ability to recruit the pericentriolar material (PCM) and thereby function as the centrosome. Cartwheel removal has recently been implicated in centriole duplication, but whether "cartwheel-less'' centrioles are intrinsically stable or must be maintained through other modifications remains unclear. Here, we identify a newborn centriole-enriched protein, KIAA1731/CEP295, specifically mediating centriole-to-centrosome conversion but dispensable for cartwheel removal. In the absence of CEP295, centrioles form in the S/G2 phase and lose their associated cartwheel in mitosis but cannot be converted to centrosomes, uncoupling the two events. Strikingly, centrioles devoid of both the PCM and the cartwheel progressively lose centriolar components, whereas centrioles associating with either the cartwheel or PCM alone can exist stably. Thus, cartwheel removal can have grave repercussions to centriole stability, and centriole-to-centrosome conversion mediated by CEP295 must occur in parallel to maintain cartwheel-less centrioles for duplication.

Background: Allergic contact dermatitis (ACD) is the most common occupational disease. Although murine contact hypersensitivity provides a framework for understanding ACD, it carries important differences from its human counterpart. Unlike the contact hypersensitivity model, which is induced by potent sensitizers (ie, dinitrofluorobenzene), human ACD is induced by weak-to-moderate sensitizers (ie, nickel), which cannot induce reactions in mice. Distinct hapten-specific immune-polarizing responses to potent inducers were suggested in mice, with unclear relevance to human ACD. Objective: We explored the possibility of distinct T-cell polarization responses in skin to common clinically relevant ACD allergens. Methods: Gene-expression and cellular studies were performed on common allergens (ie, nickel, fragrance, and rubber) compared with petrolatum-occluded skin, using RT-PCR, gene arrays, and immunohistochemistry. Results: Despite similar clinical reactions in all allergen groups, distinct immune polarizations characterized different allergens. Although the common ACD transcriptome consisted of 149 differentially expressed genes across all allergens versus petrolatum, a much larger gene set was uniquely altered by individual allergens. Nickel demonstrated the highest immune activation, with potent inductions of innate immunity, T(H)1/T(H)17 and a T(H)22 component. Fragrance, and to a lesser extent rubber, demonstrated a strong T(H)2 bias, some T(H)22 polarization, and smaller T(H)1/T(H)17 contributions. Conclusions: Our study offers new insights into the pathogenesis of ACD, expanding the understanding of T-cell activation and associated cytokines in allergen-reactive tissues. It is the first study that defines the common transcriptome of clinically relevant sensitizers in human skin and identifies unique pathways preferentially activated by different allergens, suggesting that ACD cannot be considered a single entity.

Asymmetric cell divisions allow stem cells to balance proliferation and differentiation. During embryogenesis, murine epidermis expands rapidly from a single layer of unspecified basal layer progenitors to a stratified, differentiated epithelium. Morphogenesis involves perpendicular (asymmetric) divisions and the spindle orientation protein LGN, but little is known about how the apical localization of LGN is regulated. Here, we combine conventional genetics and lentiviral-mediated in vivo RNAi to explore the functions of the LGN-interacting proteins Par3, mInsc and G alpha(i3). Whereas loss of each gene alone leads to randomized division angles, combined loss of Gnai3 and mInsc causes a phenotype of mostly planar divisions, akin to loss of LGN. These findings lend experimental support for the hitherto untested model that Par3-mInsc and G alpha(i3) act cooperatively to polarize LGN and promote perpendicular divisions. Finally, we uncover a developmental switch between delamination-driven early stratification and spindle-orientation-dependent differentiation that occurs around E15, revealing a two-step mechanism underlying epidermal maturation.

We study the motion of a particle on the two-dimensional honeycomb lattice, whose sites are occupied by either flipping rotators or flipping mirrors, which scatter the particle according to a deterministic rule. For both types of scatterers we find a new type of motion that has not been observed in a Lorentz Lattice gas, where the particle's trajectory is a self-avoiding walk between returns to its initial position. We show that this behavior is a consequence of the deterministic scattering rule and the particular class of initial scatterer configurations we consider. Since self-avoiding walks are one of the main tools used to model the growth of crystals and polymers, the particle's motion in this class of systems is potentially important for the study of these processes.

A search for WV gamma triple vector boson production is presented based on events containing a W boson decaying to a muon or an electron and a neutrino, a second V (W or Z) boson, and a photon. The data correspond to an integrated luminosity of 19.3 fb(-1) collected in 2012 with the CMS detector at the LHC in pp collisions at root s = 8 TeV. An upper limit of 311 fb on the cross section for the WV gamma production process is obtained at 95% confidence level for photons with a transverse energy above 30 GeV and with an absolute value of pseudorapidity of less than 1.44. This limit is approximately a factor of 3.4 larger than the standard model predictions that are based on next-to-leading order QCD calculations. Since no evidence of anomalous WW gamma gamma or WWZ gamma quartic gauge boson couplings is found, this paper presents the first experimental limits on the dimension-eight parameter f(T,0) and the CP-conserving WWZ gamma parameters kappa(W)(0) and kappa(W)(C). Limits are also obtained for the WW gamma gamma parameters a(0)(W) and a(C)(W).

Protein function is largely dependent on coordinated and dynamic interactions of the protein with biomolecules including other proteins, nucleic acids and lipids. Although powerful methods for global profiling of protein-protein and protein-nucleic acid interactions are available, proteome-wide mapping of protein-lipid interactions is still challenging and rarely performed. The emergence of bifunctional lipid probes with photoactivatable and clickable groups offers new chemical tools for globally profiling protein-lipid interactions under cellular contexts. In this review, we summarize recent advances in the development of bifunctional lipid probes for studying protein-lipid interactions. We also highlight how in vivo photocrosslinking reactions contribute to the characterization of lipid-binding proteins and lipidation-mediated protein-protein interactions.

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however, differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer Layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development.

IL-17 is a proinflammatory cytokine implicated in the pathogenesis of autoimmune diseases including psoriasis. ACT1 is an essential adaptor molecule in the IL-17 signaling pathway. A missense single nucleotide polymorphism (rs33980500; SNP-D10N) that resulted in the substitution of an asparagine for an aspartic acid at position 10 of ACT1 (ACT1-D10N) is associated with psoriasis susceptibility. Due to alternative splicing in humans, SNP-D10N encodes two mutated ACT1 proteins, ACT1-D10N and ACT1-D19N. Although both ACT1 isoforms are Hsp90 client proteins, the nine additional amino acids in ACT1-D19N provide an additional Hsp90 binding site that is absent in ACT1-D10N. Therefore, whereas ACT1-D10N is a dead protein that is unable to transduce IL-17 signals for gene expression, ACT1-D19N is fully responsive to IL-17. Intriguingly, the two ACT1 isoforms are differentially expressed in ACT1 (D10N/D10N) fibroblasts and T cells. Fibroblasts express both isoforms equally, enabling ACT1-D19N to compensate for the loss of ACT1-D10N function. ACT1(D10N/D10N) T cells, however, express predominantly ACT1-D10N. Lacking this compensatory mechanism, ACT1(D10N/D10N) T cells behave like ACT1-deficient T cells, exhibiting a dysregulated and hyperactive Th17 phenotype with overproduction of IL-22 and IL-17. The hyperactive Th17 response combined with fully responsive fibroblasts likely synergized to contribute to psoriasis susceptibility in SNP-D10N patients.

Hepatitis B virus (HBV) chronically infects 400 million people worldwide and is a leading driver of end-stage liver disease and liver cancer. Research into the biology and treatment of HBV requires an in vitro cell-culture system that supports the infection of human hepatocytes, and accurately recapitulates virus-host interactions. Here, we report that micropatterned cocultures of primary human hepatocytes with stromal cells (MPCCs) reliably support productive HBV infection, and infection can be enhanced by blocking elements of the hepatocyte innate immune response associated with the induction of IFN-stimulated genes. MPCCs maintain prolonged, productive infection and represent a facile platform for studying virus-host interactions and for developing antiviral interventions. Hepatocytes obtained from different human donors vary dramatically in their permissiveness to HBV infection, suggesting that factors-such as divergence in genetic susceptibility to infection-may influence infection in vitro. To establish a complementary, renewable system on an isogenic background in which candidate genetics can be interrogated, we show that inducible pluripotent stem cells differentiated into hepatocyte-like cells (iHeps) support HBV infection that can also be enhanced by blocking interferon-stimulated gene induction. Notably, the emergence of the capacity to support HBV transcriptional activity and initial permissiveness for infection are marked by distinct stages of iHep differentiation, suggesting that infection of iHeps can be used both to study HBV, and conversely to assess the degree of iHep differentiation. Our work demonstrates the utility of these infectious systems for studying HBV biology and the virus' interactions with host hepatocyte genetics and physiology.

Background: The reproducible nature of HIV-1 escape from HLA-restricted CD8+ T-cell responses allows the identification of HLA-associated viral polymorphisms "at the population level" - that is, via analysis of cross-sectional, linked HLA/HIV-1 genotypes by statistical association. However, elucidating their timing of selection traditionally requires detailed longitudinal studies, which are challenging to undertake on a large scale. We investigate whether the extent and relative timecourse of immune-driven HIV adaptation can be inferred via comparative cross-sectional analysis of independent early and chronic infection cohorts. Results: Similarly-powered datasets of linked HLA/HIV-1 genotypes from individuals with early (median < 3 months) and chronic untreated HIV-1 subtype B infection, matched for size (N > 200/dataset), HLA class I and HIV-1 Gag/Pol/Nef diversity, were established. These datasets were first used to define a list of 162 known HLA-associated polymorphisms detectable at the population level in cohorts of the present size and host/viral genetic composition. Of these 162 known HLA-associated polymorphisms, 15% (occurring at 14 Gag, Pol and Nef codons) were already detectable via statistical association in the early infection dataset at p <= 0.01 (q < 0.2) - identifying them as the most consistently rapidly escaping sites in HIV-1. Among these were known rapidly-escaping sites (e. g. B*57-Gag-T242N) and others not previously appreciated to be reproducibly rapidly selected (e. g. A*31:01-associated adaptations at Gag codons 397, 401 and 403). Escape prevalence in early infection correlated strongly with first-year escape rates (Pearson's R = 0.68, p = 0.0001), supporting cross-sectional parameters as reliable indicators of longitudinally-derived measures. Comparative analysis of early and chronic datasets revealed that, on average, the prevalence of HLA-associated polymorphisms more than doubles between these two infection stages in persons harboring the relevant HLA (p < 0.0001, consistent with frequent and reproducible escape), but remains relatively stable in persons lacking the HLA (p = 0.15, consistent with slow reversion). Published HLA-specific Hazard Ratios for progression to AIDS correlated positively with average escape prevalence in early infection (Pearson's R = 0.53, p = 0.028), consistent with high early within-host HIV-1 adaptation (via rapid escape and/or frequent polymorphism transmission) as a correlate of progression. Conclusion: Cross-sectional host/viral genotype datasets represent an underutilized resource to identify reproducible early pathways of HIV-1 adaptation and identify correlates of protective immunity.