The rockefeller university

The conserved oligomeric Golgi (COG) complex orchestrates vesicular trafficking to and within the Golgi apparatus. Here, we use negative-stain electron microscopy to elucidate the architecture of the hetero-octameric COG complex from Saccharomyces cerevisiae. Intact COG has an intricate shape, with four (or possibly five) flexible legs, that differs strikingly from that of the exocyst complex and appears to be well suited for vesicle capture and fusion.

The persistence of tick-borne encephalitis virus (TBEV) in nature is maintained by numerous species of reservoir hosts, multiple transmissions between vertebrates and invertebrates, and the virus adaptation to its hosts. Our Aim: was to compare TBEV isolates from ticks and small wild mammals to estimate their roles in the circulation of the viral subtypes. Methods: TBEV isolates from two species of ixodid ticks, four species of rodents, and one species of shrews in the Novosibirsk region, South-Western Siberia, Russia, were analyzed using bioassay, hemagglutination, hemagglutination inhibition, neutralization tests, ELISA, reverse transcription with real-time PCR, and phylogenetic analysis. Results: TBEV RNA and/or protein E were found in 70.9% +/- 3.0% of mammals and in 3.8% +/- 0.4% of ticks. The TBEV infection rate, main subtypes, and neurovirulence were similar between ixodid tick species. However, the proportions of the virus that were pathogenic for laboratory mice and of the Far-Eastern (FE) subtype, as well as the viral loads with the Siberian and the European subtypes for the TBEV in Ixodes pavlovskyi Pomerantsev, 1946 were higher than in Ixodes persulcatus (P. Schulze, 1930). Percentages of infected Myodes rutilus, Sicista betulina, and Sorex araneus exceeded those of Apodemus agrarius and Myodes rufocanus. Larvae and nymphs of ticks were found mainly on rodents, especially on Myodes rufocanus and S. betulina. The proportion of TBEV-mixed infections with different subtypes in the infected ticks (55.9% +/- 6.5%) was higher than in small mammals (36.1% +/- 4.0%) (p < 0.01). Conclusions: Molecular typing revealed mono-or mixed infection with three main subtypes of TBEV in ticks and small mammals. The Siberian subtype was more common in ixodid ticks, and the FE subtype was more common in small mammals (p < 0.001). TBEV isolates of the European subtype were rare. TBEV infection among different species of small mammals did not correlate with their infestation rate with ticks in the Novosibirsk region, Russia.

Chikungunya virus (CHIKV) and related alphaviruses cause epidemics of acute and chronic musculoskeletal disease. To investigate the mechanisms underlying the failure of immune clearance of CHIKV, we studied mice infected with an attenuated CHIKV strain (181/25) and the pathogenic parental strain (AF15561), which differ by five amino acids. Whereas AF15561 infection of wild-type mice results in viral persistence in joint tissues, 181/25 is cleared. In contrast, 181/25 infection of mu MT mice lacking mature B cells results in viral persistence in joint tissues, suggesting that virus-specific antibody is required for clearance of infection. Mapping studies demonstrated that a highly conserved glycine at position 82 in the A domain of the E2 glycoprotein impedes clearance and neutralization of multiple CHIKV strains. Remarkably, murine and human antibodies targeting E2 domain B failed to neutralize pathogenic CHIKV strains efficiently. Our data suggest that pathogenic CHIKV strains evade E2 domain-B-neutralizing antibodies to establish persistence.

Chronic granulomatous disease (CGD) is an inherited mutational defect in any of the NADPH oxidase complex, CYBB (gp91-phox), NCF1 (p47-phox), CYBA (p22-phox), NCF2 (p67-phox), or NCF4 (p40-phox) leading to inability of phagocytes to perform effective respiratory burst and thus diminished killing of bacteria and fungi. The identification of defective proteins aids in establishing a diagnosis prior to genetic analysis, which is rather labor-intensive, expensive, and time-consuming. The present study aims at assessing the NADPH proteins by performing the intracellular staining with specific monoclonal antibodies and their assessment on flow cytometry. The use of flow cytometry is less laborious and faster to perform than western blot. It also confirms the diagnosis of CGD and detects the affected components allowing proper management of patients. Twenty-eight patients from 25 different kindred, clinically suspected as CGD were recruited in Egypt. Dihydrorhodamine test was performed to confirm the diagnosis of the patients. Intracellular staining of NADPH components using specific monoclonal antibodies was performed followed by flow cytometric analysis. The present study revealed that the most common defective protein in our cohort is p22-phox, found in 13 patients (46.4 % of cases) followed by p47-phox in 8 patients (28.6 %), gp91-phox in 5 patients (17.9 %), and finally p67-phox in 2 patients (7.1 %). In countries with limited resources and yet large number of CGD patients, the analysis of the defective proteins by flow cytometry is an optimum solution for confirming the diagnosis and is a step for targeted sequencing in families seeking prenatal diagnosis.

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-gamma receptor (IFN-gamma R) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-gamma R2 subunit, this mutation blocks IFN-gamma activity by unknown mechanisms. We show that the lateral diffusion of IFN-gamma R2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-gamma R2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-gamma could not occur. Removing IFN-gamma R2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo.

A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A*0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A*0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naive) mice were protected against subsequent malaria challenge. The level of the HLA-A*0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti -malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo. (C) 2016 The Author(s). Published by Elsevier Ltd.

The APOBEC3 (A3) cytidine deaminases are antiretroviral proteins, whose targets include human immunodeficiency virus type-1 (HIV-1). Their incorporation into viral particles is critical for antiviral activity and is driven by interactions with the RNA molecules that are packaged into virions. However, it is unclear whether A3 proteins preferentially target RNA molecules that are destined to be packaged and if so, how. Using cross-linking immunoprecipitation sequencing (CLIP-seq), we determined the RNA binding preferences of the A3F, A3G and A3H proteins. We found that A3 proteins bind preferentially to RNA segments with particular properties, both in cells and in virions. Specifically, A3 proteins target RNA sequences that are G-rich and/or A-rich and are not scanned by ribosomes during translation. Comparative analyses of HIV-1 Gag, nucleocapsid (NC) and A3 RNA binding to HIV-1 RNA in cells and virions revealed the striking finding that A3 proteins partially mimic the RNA binding specificity of the HIV-1 NC protein. These findings suggest a model for A3 incorporation into HIV-1 virions in which an NC-like RNA binding specificity is determined by nucleotide composition rather than sequence. This model reconciles the promiscuity of A3 RNA binding that has been observed in previous studies with a presumed advantage that would accompany selective binding to RNAs that are destined to be packaged into virions.

The discovery of steroid hormone receptors in brain regions that mediate virtually every aspect of brain function has broadened the definition of 'neuroendocrinology' to include the reciprocal communication between the brain and the body via hormonal and neural pathways. The brain is the central organ of stress and adaptation to stress because it perceives and determines what is threatening, as well as determining the behavioral and physiological responses to the stressor. The adult and developing brain possess remarkable structural and functional plasticity in response to stress, including neurogenesis leading to neuronal replacement, dendritic remodeling, and synapse turnover. Stress causes an imbalance of neural circuitry subserving cognition, decision-making, anxiety and mood that can alter expression of those behaviors and behavioral states. The two Brain Research papers noted in this review played an important role in triggering these advances. This article is part of a Special Issue entitled SI:50th Anniversary Issue. (C) 2016 Published by Elsevier B.V.

N-Myristoyltransferase (NMT) covalently attaches a C14 fatty acid to the N-terminal glycine of proteins and has been proposed as a therapeutic target in cancer. We have recently shown that selective NMT inhibition leads to dose-responsive loss of N-myristoylation on more than 100 protein targets in cells, and cytotoxicity in cancer cells. N-myristoylation lies upstream of multiple pro-proliferative and oncogenic pathways, but to date the complex substrate specificity of NMT has limited determination of which diseases are most likely to respond to a selective NMT inhibitor. We describe here the phenotype of NMT inhibition in HeLa cells and show that cells die through apoptosis following or concurrent with accumulation in the G1 phase. We used quantitative proteomics to map protein expression changes for more than 2700 proteins in response to treatment with an NMT inhibitor in HeLa cells and observed down-regulation of proteins involved in cell cycle regulation and up-regulation of proteins involved in the endoplasmic reticulum stress and unfolded protein response, with similar results in breast (MCF-7, MDA-MB-231) and colon (HCT116) cancer cell lines. This study describes the cellular response to NMT inhibition at the proteome level and provides a starting point for selective targeting of specific diseases with NMT inhibitors, potentially in combination with other targeted agents.

Cell division terminates with cytokinesis and cellular separation. Autosomal-recessive primary microcephaly (PMCPH) is a neurodevelopmental disorder characterized by a reduction in brain and head size at birth in addition to non-progressive intellectual disability. MCPH is genetically heterogeneous, and 16 loci are known to be associated with loss-of-function mutations predominantly affecting centrosomal-associated proteins, but the multiple roles of centrosomes in cellular function has left questions about etiology. Here, we identified three families affected by homozygous missense mutations in CIT, encoding citron rho-interacting kinase (CIT), which has established roles in cytokinesis. All mutations caused substitution of conserved amino acid residues in the kinase domain and impaired kinase activity. Neural progenitors that were differentiated from induced pluripotent stem cells (iPSCs) derived from individuals with these mutations exhibited abnormal cytokinesis with delayed mitosis, multipolar spindles, and increased apoptosis, rescued by CRISPR/Cas9 genome editing. Our results highlight the importance of cytokinesis in the pathology of primary microcephaly.