The rockefeller university

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (Yxx Phi) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor-and ligand-specific manner. Destruction of YxxF abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxF in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxF in UNC93B1.

Differentiation of CD4(+) T cells into type 1 or type 2 subsets is mediated by the expression of the opposing lineage defining transcription factors T-bet and GATA-3. However, the existence of GATA-3(+) T-bet(+) CD4(+) T cells in mice suggests functional plasticity of these subsets. Little is known about type 1 and type 2 plasticity of human T-cell subsets in vivo. Here, we show that in the xenogeneic environment of humanized mice, which lacks a functional immune-regulatory network, human CD4(+) and, notably, CD8(+) T cells preferentially differentiate into interleukin (IL)-4(+) GATA-3(+) and IL-4(+) interferon-gamma(+) GATA-3(+) T-bet(+) subsets. Treatment with recombinant human IL-12 or expansion of IL-12-producing human dendritic cells in vivo reverted this phenotype and led to the down-regulation of GATA-3 expression. These changes also correlated with improved antiviral immune responses in humanized mice. In conclusion, our study shows the capacity of human CD4(+) and CD8(+) T cells for stable co-expression of GATA-3 and T-bet in humanized mice and reveals a critical role for IL-12 in regulating this phenotype.

1. The coffee berry borer Hypothenemus hampei (Coleoptera, Curculionidae) (Ferrari) is the most important pest of coffee production worldwide. 2. The hypothesis that the tropical fire ant, Solenopsis geminata Westwood, indirectly protects the coffee berry borer by suppressing other ant species that are the coffee berry borer's primary predators was tested. 3. It was found that removing S. geminata from coffee plots significantly increased the disappearance of adult coffee berry borer beetles from coffee berries compared with control plots. An average of 6% of beetles disappeared from plots with S. geminata whereas 23% of beetles disappeared from plots from which S. geminata was removed. This pattern was observed on two shade coffee farms with marked differences in ant species composition, one in the rainforest in central Costa Rica and one in the cloudforest in northwest Costa Rica. 4. If the results of this small-scale study can be replicated on the farm level, then S. geminata suppression may represent a new management technique for the coffee berry borer throughout Central and South America.

Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to alpha interferon (IFN-alpha), telaprevir, daclatasvir, cyclosporine, and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug resistance mutations in the mutant spectrum of the initial or passaged populations but to increased replicative fitness acquired during passage. Fitness increases did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply that viral replicative fitness is a mechanism of multidrug resistance in HCV. IMPORTANCE Viral drug resistance is usually attributed to the presence of amino acid substitutions in the protein targeted by the drug. In the present study with HCV, we show that high viral replicative fitness can confer a general drug resistance phenotype to the virus. The results exclude the possibility that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why the virus is less sensitive to drug treatments in prolonged chronic HCV infections that favor increases in replicative fitness.

Glucose-dependent insulinotropic polypeptide (GIP), an incretin hormone secreted from gastrointestinal K cells in response to food intake, has an important role in the control of whole-body metabolism. GIP signals through activation of the GIP receptor (GIPR), a G-protein-coupled receptor (GPCR). Dysregulation of this pathway has been implicated in the development of metabolic disease. Here we demonstrate that GIPR is constitutively trafficked between the plasma membrane and intracellular compartments of both GIP-stimulated and unstimulated adipocytes. GIP induces a downregulation of plasma membrane GIPR by slowing GIPR recycling without affecting internalization kinetics. This transient reduction in the expression of GIPR in the plasma membrane correlates with desensitization to the effects of GIP. A naturally occurring variant of GIPR (E354Q) associated with an increased incidence of insulin resistance, type 2 diabetes, and cardiovascular disease in humans responds to GIP stimulation with an exaggerated downregulation from the plasma membrane and a delayed recovery of GIP sensitivity following cessation of GIP stimulation. This perturbation in the desensitization-resensitization cycle of the GIPR variant, revealed in studies of cultured adipocytes, may contribute to the link of the E354Q variant to metabolic disease.

Evolutionary dynamics in nature constitute an immensely complex non-equilibrium process. We review the application of physical models of evolution, by focusing on adaptation, extinction, and ecology. In each case, we examine key concepts by working through examples. Adaptation is discussed in the context of bacterial evolution, with a view toward the relationship between growth rates, mutation rates, selection strength, and environmental changes. Extinction dynamics for an isolated population are reviewed, with emphasis on the relation between timescales of extinction, population size, and temporally correlated noise. Ecological models are discussed by focusing on the effect of spatial interspecies interactions on diversity. Connections between physical processes-such as diffusion, turbulence, and localization-and evolutionary phenomena are highlighted.

The hair bundle, an apical specialization of the hair cell composed of several rows of regularly organized stereocilia and a kinocilium, is essential for mechanotransduction in the ear. Its precise organization allows the hair bundle to convert mechanical stimuli to electrical signals; mutations that alter the bundle's morphology often cause deafness. However, little is known about the proteins involved in the process of morphogenesis and how the structure of the bundle arises through interactions between these molecules. We present a mathematical model based on simple reaction-diffusion mechanisms that can reproduce the shape and organization of the hair bundle. This model suggests that the boundary of the cell and the kinocilium act as signaling centers that establish the bundle's shape. The interaction of two proteins forms a hexagonal Turing patterna periodic modulation of the concentrations of the morphogens, sustained by local activation and long-range inhibition of the reactants that sets a blueprint for the location of the stereocilia. Finally we use this model to predict how different alterations to the system might impact the shape and organization of the hair bundle.

Hepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to the case for other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with small interfering RNA (siRNA) knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a cocrystal structure of a core MOBKL1B-NS5A peptide complex at 1.95 angstrom, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18-amino-acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, which indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches, we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data. IMPORTANCE Our study illustrates an underappreciated shortcoming of siRNA gene knockdown technology. We initially identified a cellular protein, MOBKL1B, as a binding partner with the NS5A protein of hepatitis C virus (HCV). MOBKL1B siRNA, but not irrelevant RNA, treatment was associated with both reduced virus replication and the absence of MOBKL1B. Believing that HCV replication depended on the MOBKL1B-NS5A interaction, we carried out structural and biochemical analyses. Unexpectedly, an HCV variant lacking the MOBKL1B-NS5A interaction could not replicate after cells were treated with MOBKL1B siRNA. By repeating the MOBKL1B siRNA knockdowns and including seed sequence-matched siRNA instead of irrelevant siRNA as a control, we found that the MOBKL1B siRNAs utilized had off-target inhibitory effects on virus replication. Collectively, our results suggest that stricter controls must be utilized in all RNA interference (RNAi)-mediated gene knockdown experiments to ensure sound conclusions and a reliable scientific knowledge database.

Observation of the diphoton decay mode of the recently discovered Higgs boson and measurement of some of its properties are reported. The analysis uses the entire dataset collected by the CMS experiment in proton-proton collisions during the 2011 and 2012 LHC running periods. The data samples correspond to integrated luminosities of 5.1 fb(-1) at root s = 7 TeV and 19.7 fb(-1) at 8 TeV. A clear signal is observed in the diphoton channel at a mass close to 125 GeV with a local significance of 5.7 sigma, where a significance of 5.2 sigma is expected for the standard model Higgs boson. The mass is measured to be 124.70 +/- 0.34 GeV = 124.70 +/- 0.31 (stat) +/- 0.15 (syst) GeV, and the best-fit signal strength relative to the standard model prediction is 1.14(-0.23)(+0.26) = 1.14 +/- 0.21 (stat) (-0.05)(+0.09) (syst) (+0.13)(-0.09) (theo). Additional measurements include the signal strength modifiers associated with different production mechanisms, and hypothesis tests between spin-0 and spin-2 models.