Bioorthogonal mimetics of palmitoyl-CoA and myristoyl-CoA and their subsequent isolation by click chemistry and characterization by mass spectrometry reveal novel acylated host-proteins modified by HIV-1 infection.

Protein acylation plays a critical role in protein localization and function. Acylation is essential for human immunodeficiency virus 1 (HIV-1) assembly and budding of HIV-1 from the plasma membrane in lipid raft microdomains and is mediated by myristoylation of the Gag polyprotein and the copackaging of the envelope protein is facilitated by colocalization mediated by palmitoylation. Since the viral accessory protein NEF has been shown to alter the substrate specificity of myristoyl transferases, and alter cargo trafficking lipid rafts, we hypothesized that HIV-1 infection may alter protein acylation globally. To test this hypothesis, we labeled HIV-1 infected cells with biomimetics of acyl azides, which are incorporated in a manner analogous to natural acyl-Co-A. A terminal azide group allowed us to use a copper catalyzed click chemistry to conjugate the incorporated modifications to a number of substrates to carry out SDS-PAGE, fluorescence microscopy, and enrichment for LC-MS/MS. Using LC-MS/MS, we identified 103 and 174 proteins from the myristic and palmitic azide enrichments, with 27 and 45 proteins respectively that differentiated HIV-1 infected from uninfected cells. This approach has provided us with important insights into HIV-1 biology and is widely applicable to many virological systems.

Ig-like transcript 7, but not bone marrow stromal cell antigen 2 (also known as HM1.24, tetherin, or CD317), modulates plasmacytoid dendritic cell function in primary human blood leukocytes.

The Ig-like transcript (ILT) 7 is a surface molecule selectively expressed by human plasmacytoid dendritic cells (pDCs). ILT7 cross-linking suppresses pDC activation and type I IFN (IFN-I) secretion following TLR7/9 engagement. The bone marrow stromal cell Ag 2 (BST2, aka HM1.24, tetherin, or CD317) is expressed by different cell types upon exposure to IFN-I and is a natural ligand for ILT7. In this study, we show that ILT7 expression decreased spontaneously in pDCs upon in vitro culture, which correlates with pDC differentiation measured as increased side scatter properties and CCR7 expression. TLR7/9 ligands, as well as HIV, induced BST2 upregulation on all tested cell types except T cells, which required TCR stimulation to respond to TLR9L-induced IFN-I. IFN-γ, IL-4, IL-10, and TNF-α had only marginal effects on BST2 expression in blood leukocytes compared with TLR9L. Preincubation with ILT7 cross-linking Ab inhibited IFN-I production in PBMCs treated with TLR7/9L or HIV, whereas BST2 blockade did not affect IFN-I responses even when BST2 upregulation was further boosted with TCR agonists or immunoregulatory cytokines. Our data indicate that BST2-mediated ILT7 cross-linking may act as a homeostatic regulatory mechanism on immature circulating pDC, rather than a negative feedback for activated mature pDCs that have downregulated ILT7.

The conserved set of host proteins incorporated into HIV-1 virions suggests a common egress pathway in multiple cell types.

HIV-1 incorporates a large array of host proteins into virions. Determining the host protein composition in HIV virions has technical difficulties, including copurification of microvesicles. We developed an alternative purification technique using cholesterol that differentially modulates the density of virions and microvesicles (density modification, DM) allowing for high-yield virion purification that is essential for tandem mass spectrometric and quantitative proteomic (iTRAQ) analysis. DM purified virions were analyzed using iTRAQ and validated against Optiprep (60% iodixanol) purified virions. We were able to characterize host protein incorporation in DM-purified HIV particles derived from CD4+ T-cell lines; we compared this data set to a reprocessed data set of monocyte-derived macrophages (MDM) derived HIV-1 using the same bioinformatics pipeline. Seventy-nine clustered proteins were shared between the MDM derived and T-cell derived data set. These clusters included an extensive collection of actin isoforms, HLA proteins, chaperones, and a handful of other proteins, many of which have previously been documented to interact with viral proteins. Other proteins of note were ERM proteins, the dynamin domain containing protein EH4, a phosphodiesterase, and cyclophilin A. As these proteins are incorporated in virions produced in both cell types, we hypothesize that these proteins may have direct interactions with viral proteins or may be important in the viral life cycle. Additionally, identified common set proteins are predicted to interact with >1000 related human proteins. Many of these secondary interacting proteins are reported to be incorporated into virions, including ERM proteins and adhesion molecules. Thus, only a few direct interactions between host and viral proteins may dictate the host protein composition in virions. Ultimately, interaction and expression differences in host proteins between cell types may drive virion phenotypic diversity, despite conserved viral protein-host protein interactions between cell types.

Overactivation of plasmacytoid dendritic cells inhibits antiviral T-cell responses: a model for HIV immunopathogenesis.

A delicate balance between immunostimulatory and immunosuppressive signals mediated by dendritic cells (DCs) and other antigen-presenting cells (APCs) regulates the strength and efficacy of antiviral T-cell responses. HIV is a potent activator of plasmacytoid DCs (pDCs), and chronic pDC activation by HIV promotes the pathogenesis of AIDS. Cholesterol is pivotal in maintaining HIV envelope integrity and allowing HIV-cell interaction. By depleting envelope-associated cholesterol to different degrees, we generated virions with reduced ability to activate pDCs. We found that APC activation was dissociated from the induction of type I IFN-α/ß and indoleamine-2,3-dioxygenase (IDO)-mediated immunosuppression in vitro. Extensive cholesterol withdrawal, resulting in partial protein and RNA loss from the virions, rendered HIV a more powerful recall immunogen for stimulating memory CD8 T-cell responses in HIV-exposed, uninfected individuals. These enhanced responses were dependent on the inability of cholesterol-depleted HIV to induce IFN-α/ß.

The meaning of religious beliefs for a group of cancer patients during rehabilitation.

The objective of this exploratory study was to identify how religion influences the survival of a group of cancer patients. The study consisted of an ethnographic case with the participation of six laryngectomized male and female patients between 51 and 72 years old, who had been operated on two to five years earlier. Data were collected by semistructured interviews and analyzed on the basis of the concepts of culture and religion. The results were synthesized into three descriptive categories: the moral representation of cancer, religious beliefs about the cancer trajectory, and negotiation with religion for survival. These categories give rise to the meaning "the hope for a second chance", which emphasizes the importance of religion as part of the support networks that articulate with the patient's coping with the stigma of cancer, with the hope for cure, and with the ways of organizing everyday life, during survival.

Modulation of HIV-1-induced activation of plasmacytoid dendritic cells by 6-desfluoroquinolones.

Chronic activation of plasmacytoid dendritic cells (pDCs) is an important contributor to the immunopathogenesis of HIV infection. The quinolone derivative chloroquine (CQ) prevents endosomal acidification, required for toll-like receptor sensing of HIV by pDCs, and is currently under clinical trial as an immunotherapeutic approach. We tested three different 6-desfluoroquinolones (6-DFQs), structurally related to CQ and endowed with antiretroviral activity, for their ability to inhibit HIV-induced pDC activation and interferon (IFN)-α production in peripheral blood mononuclear cells (PBMCs) in vitro. PBMCs from six healthy donors were cultured overnight with aldrithiol-2 (AT-2)-inactivated HIV-1MN in the presence or absence of 6-DFQs or CQ. IFN-α production was measured by ELISA; pDC and monocyte activation was analyzed by flow cytometry. Incubation with HIV labeled with the fluorescent dye DyLight-488 (DL488) was used to test virus uptake by flow cytometry. We found that the 6-DFQs effectively inhibited HIV-induced IFN-α similar to CQ, but only 6-DFQs also inhibited the upregulation of the pDC activation marker CD83. Interestingly, HIV-induced expression of the costimulatory molecule CD80 and, to a lesser extent CD86, was further enhanced on pDCs by 6-DFQs, but not CQ. Conversely, 6-DFQs and CQ had similar inhibitory effects on HIV-induced monocyte activation, consistent with the primary mechanism being associated with IFN-α signaling. Finally, 6-DFQs interfered with HIV interaction with pDCs and monocytes, but not myeloid DCs. Our data indicate that 6-DFQs may interfere with pDC-mediated and IFN-α-dependent immunopathogenesis while supporting pDC differentiation into mature antigen-presenting cells by favoring expression of costimulatory molecules.

Trisomy for the Down syndrome 'critical region' is necessary but not sufficient for brain phenotypes of trisomic mice.

Trisomic Ts65Dn mice show direct parallels with many phenotypes of Down syndrome (DS), including effects on the structure of cerebellum and hippocampus. A small segment of Hsa21 known as the 'DS critical region' (DSCR) has been held to contain a gene or genes sufficient to cause impairment in learning and memory tasks involving the hippocampus. To test this hypothesis, we developed Ts1Rhr and Ms1Rhr mouse models that are, respectively, trisomic and monosomic for this region. Here, we show that trisomy for the DSCR alone is not sufficient to produce the structural and functional features of hippocampal impairment that are seen in the Ts65Dn mouse and DS. However, when the critical region is returned to normal dosage in trisomic Ms1Rhr/Ts65Dn mice, performance in the Morris water maze is identical to euploid, demonstrating that this region is necessary for the phenotype. Thus, although the prediction of the critical region hypothesis was disproved, novel gene dosage effects were identified, which help to define how trisomy for this segment of the chromosome contributes to phenotypes of DS.

Avaliação das condições socioeconômicas, dos parâmetros clínicos e da autopercepção em saúde bucal dos pacientes atendidos na clínica de Graduação em Odontologia / Evaluation of socio-economic conditions, clinical parameters, and oral health self perception of patients treated at the clinic of the Dentistry

Entendendo que a condição de saúde bucal pode afetar a qualidade de vida (QdV) das pessoas, este estudo busca avaliar a autopercepção dos pacientes atendidos na Clínica Odontológica das Faculdades Integradas São Pedro (FAESA, Vitória-ES), no que se refere à saúde bucal, e como esta interfere no seu dia a dia e na sua QdV, a partir do preenchimento do questionário deOral Heath Impact Profile, versão simplificada (OHIP-14), correlacionando com as condições socioeconômicas e os parâmetros clínicos (CPO-D - Dentes Cariados Perdidos e Obturados e oCommunity Periodontal Index and Treatment Needs - CPITN ou IP - Índice Periodontal). O índice OHIP-14 foi obtido pelo método aditivo, sendo os pacientes distribuídos segundo gênero, idade, grau de instrução e renda, com nível de significância estatística de 5%. O teste de comparação utilizado foi o teste paramétrico ôtõ para médias e ANOVA (análise de variância). A amostra foi de 150 pacientes coletados. Concluímos que a maior percepção do impacto foi encontrada no grupo feminino e que quanto maior a idade, maior o impacto na saúde bucal (CPO-D e IP) e maior a autopercepção (OHIP-14). Esta pesquisa foi aprovada pelo Comitê de Ética em Pesquisa com Seres Humanos (CEP) û FAESA û protocolo nº 025/2011.

Understanding that oral health can affect the quality of life (QoL) of people, the aim of this study was to evaluate the perception of patients treated at the clinic of the School of Dentistry ôFaculdades Integradas São Pedroõ ( Vitória - ES - Brazil), regarding oral health and how this interferes in their daily lives and QoL. It was applied a simplified version of the Oral Heath Impact Profile (OHIP-14), correlating obtained data with socioeconomic status and clinical parameters (DMFT - decayed missing and filled teeth and Community Periodontal Index and Treatment Needs - CPITN or IP - Periodontal Index). The OHIP-14 index was obtained by the additive method, and patients were divided according to gender, age, education level and income, with statistical significance level of 5%. T-test (means comparison) and ANOVA (analysis of variance) were applied. The sample composed by 150 patients. It was verified that women perceive a greater impact was found in females and that the higher the age, the greater the impact on dental health (DMFT and IP) and greater self-awareness (OHIP-14). This study was approved by the Ethics in Human Research (CEP) - FAESA - Protocol No. 025/2011.

The meaning of religious beliefs for a group of cancer patients during rehabilitation

The objective of this exploratory study was to identify how religion influences the survival of a group of cancer patients. The study consisted of an ethnographic case with the participation of six laryngectomized male and female patients between 51 and 72 years old, who had been operated on two to five years earlier. Data were collected by semistructured interviews and analyzed on the basis of the concepts of culture and religion. The results were synthesized into three descriptive categories: the moral representation of cancer, religious beliefs about the cancer trajectory, and negotiation with religion for survival. These categories give rise to the meaning "the hope for a second chance", which emphasizes the importance of religion as part of the support networks that articulate with the patient's coping with the stigma of cancer, with the hope for cure, and with the ways of organizing everyday life, during survival.

La finalidad de este estudio exploratorio fue identificar cómo la religión influencia la supervivencia de un grupo de pacientes oncológicos. Consistió en un estudio de caso etnográfico con la participación de seis laringectomizados, de ambos sexos, con edad de 51 a 72 años, que habían sido operados de dos a cinco años antes. Los datos fueron recogidos por entrevistas semi-estructuradas y analizados según los conceptos de cultura y religión. Sintetizamos los resultados en tres categorias descriptivas: la representación moral del cáncer, las creencias religiosas en el trayecto del cáncer y la negociación con la religión por la supervivencia. El significado que resulta - "la expectativa por una segunda oportunidad" - enfatiza la importancia de la religión como parte de las redes de apoyo que se encadenan con la conciliación con el estigma del cáncer, con la expectativa de cura y con las formas de arreglar la vida cotidiana, en la supervivenvia.

Este estudo exploratório teve o objetivo de identificar como a religião influencia a sobrevivência de um grupo de pacientes oncológicos. Consistiu em estudo de caso etnográfico, com a participação de seis laringectomizados, de ambos os sexos, na faixa etária de 51 a 72 anos, operados de dois a cinco anos. Os dados foram coletados por entrevistas semi-estruturadas e analisados segundo os conceitos de cultura e religião. Sintetizou-se os resultados em três categorias descritivas: a representação moral do câncer, as crenças religiosas na trajetória do câncer e a negociação com a religião para a sobrevivência. O significado que emerge - "a expectativa por uma segunda chance" - enfatiza a importância da religião como parte das redes de apoio que se articulam com o enfrentamento do estigma do câncer, com a expectativa da cura e com as formas de organizar a vida cotidiana, na sobrevivência.