Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (F) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines. (c) 2009 Elsevier B.V. All rights reserved.

Expression of genes encoding cytosolic and endoplasmic reticulum HSP90 proteins in the aquatic fungus Blastocladiella emersonii

HSP90 proteins are important molecular chaperones involved in multiple cellular processes. This work reports the characterization of cDNAs encoding two distinct HSP90 proteins (named HSP90A and HSP90B) from the chytridiomycete Blastocladiella emersonii. Deduced amino acid sequences of HSP90A and HSP90B exhibit signatures of the cytosolic and endoplasmic reticulum (ER) HSP90 proteins, respectively. A genomic clone encoding HSP90A was also characterized indicating the presence of a single intron of 184 bp interrupting the coding region, located near the amino-terminus of the protein. Expression of both HSP90A and HSP90B genes increases significantly during heat shock at 38 degrees C, with highest induction ratios observed in cells stressed during germination of the fungus. Changes in the amount of HSP90A transcript were also evaluated during B. emersonii life cycle at physiological temperature (27 degrees C), and its levels were found to increase both during germination and sporulation of the fungus. HSP90A protein levels were analyzed during B. emersonii life cycle and significant changes were observed only during sporulation. Furthermore, during heat stress a large increase in the amount of HSP90A protein was observed. Induction of HSP90A and HSP90B genes during heat stress indicates the importance of both genes in the response to high temperature in B. emersonii. (C) 2008 Elsevier B.V. All rights reserved.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.

Directed Differentiation of Neural Progenitors into Neurons Is Accompanied by Altered Expression of P2X Purinergic Receptors

Neural differentiation has been extensively studied in vitro in a model termed neurospheres, which consists of aggregates of neural progenitor cells. Previous studies suggest that they have a great potential for the treatment of neurological disorders. One of the major challenges for scientists is to control cell fate and develop ideal culture conditions for neurosphere expansion in vitro, without altering their features. Similar to human neural progenitors, rat neurospheres cultured in the absence of epidermal and fibroblast growth factors for a short period increased the levels of beta-3 tubulin and decreased the expression of glial fibrillary acidic protein and nestin, compared to neurospheres cultured in the presence of these factors. In this work, we show that rat neurospheres cultured in suspension under mitogen-free condition presented significant higher expression of P2X2 and P2X6 receptor subunits, when compared to cells cultured in the presence of growth factors, suggesting a direct relationship between P2X2/6 receptor expression and induction of neuronal differentiation in mitogen-free cultured rat neurospheres.

Functional Expression of Chemoreceptors with the Help of a Guanine Nucleotide Exchange Factor

Odorant receptors and other chemoreceptors are usually poorly expressed in the plasma membrane of heterologous cells. A key point of regulation in G protein-mediated signaling is the interconversion between the active GTP-bound and inactive GDP-bound states of the G alpha subunit, which regulatory proteins, such as guanine nucleotide exchange factors (GEFs), can control. GEFs stimulate formation of the GTP-bound state of G alpha and therefore are considered to work as positive regulators of G protein-coupled receptor signaling. Ric-8B, a GEF that is specifically expressed in olfactory sensory neurons, promotes functional expression of odorant receptors in HEK293T cells because it amplifies the initially low receptor signaling through G alpha olf. This same strategy could be used to functionally express other types of chemoreceptors.

Tough, sensitive and sincere : how adolescent boys manage masculinities and emotions

This study aimed to explore adolescent boys’ views of masculinity and emotion management and their potential effects on well-being. Interviews with 33 adolescent boys aged 16–17 years in Sweden were analysed using grounded theory. We found two main categories of masculine conceptions in adolescent boys: gender-normative masculinity with emphasis on group-based values, and non-gender-normative masculinity based on personal values. Gender-normative masculinity comprised two seemingly opposite emotional masculinity orientations, one towards toughness and the other towards sensitivity, both of which were highly influenced by contextual and situational group norms and demands, despite their expressions contrasting each other. Non-gender-normative masculinity included an orientation towards sincerity emphasising the personal values of the boys; emotions were expressed more independently of peer group norms. Our findings suggest that different masculinities and the expression of emotions are strongly intertwined and that managing emotions is vital for well-being.

OMG!: characterizing the role of emotions in the sharing of online commercial videos

"Se tornar viral" é visto pelos comerciantes como o novo Graal para alcançar grandes comunidades online. Neste contexto viral, vídeos têm um papel especial dada a forte capacidade deles de se espalhar exponencialmente pela internet através do compartilhamento social. Cada ano se vê a quebra de novos recordes através deste tipo de viralidade. Em março de 2012, o vídeo "Kony 2012" envolvendo a ação unida contra o líder de milícia Africano epônimo, atingiu mais de 34 milhões de visualizações em seu primeiro dia de lançamento. Em dezembro de 2012, o vídeo-clipe da música "Gangnam Style" tornou-se o primeiro vídeo do YouTube a alcançar mais de um bilhão de visualizações, totalizando mais de 1,4 bilhões de visualizações em março de 2013. Tais ilustrações mostram claramente a nova escala que a internet deu ao fenômeno do boca-a-boca. Os comerciantes entenderam o potencial fantástico dos vídeos virais e tentaram aproveitar o fenômeno de modo a reproduzi-lo para fins comerciais. Esta pesquisa oferece para os acadêmicos e os profissionais de marketing uma análise dos determinantes do compartilhamento de vídeos comerciais online. Mais especificamente, o foco da dissertação foi definido sobre o papel das emoções no compartilhamento, para identificar quais delas levam e como levam à partilha de vídeos comerciais online. A pesquisa foi realizada a partir de dois métodos científicos: uma pesquisa e uma análise de texto sobre a atribuição de emoções para comentários dos vídeos mais compartilhados do YouTube. A pesquisa confirma, com novos métodos, hipóteses previamente testadas e validadas por acadêmicos. Ela mostra que a positividade e a força das emoções são determinantes de compartilhamento maiores do que a negatividade e a fraqueza (Lindgreen and Vanhamme, 2005; Dobele et al., 2007). A dissertação também argumenta que o conteúdo do vídeo, bem como o contexto são determinantes significativos de compartilhamento de vídeo (Laskey et al., 1989; Taylor, 1999). Além de validar teorias existentes, a pesquisa trouxe novos conceitos para a discussão, especialmente o papel da dimensão força / fraqueza de emoções para analisar o fenômeno viral, e a importância de uma clara "chamada à ação" incluída no vídeo para aumentar a sua partilha. Estes novos conceitos enriquecem a literatura do tema – que evolui muito rapidamente – e preparam o caminho para futuras pesquisas.

Expression of the sigma35 and cry2AB genes involved in Bacillus thuringiensis virulence

Muitos genes estão envolvidos nos mecanismos de esporulação da bactéria Bacillus thuringiensis. A regulação e expressão desses genes resultam em uma produção massiva da proteína Cry, responsável pela morte das larvas de muitos insetos. Neste trabalho monitorou-se a expressão de genes de Bacillus thuringiensis, ao longo de três fases de seu desenvolvimento. Foram construídos macroarrays de DNA dos genes selecionados, cujas seqüências estão disponibilizadas no GenBank. Estes genes foram hibridizados com cDNAs obtidos de B. thuringiensis kurstaki HD-1. As sondas de cDNA foram sintetizadas a partir da transcrição reversa do RNA da bactéria, extraído durante as fases de crescimento logarítmico, estacionária e esporulativa, marcadas com 33PadCTP. A expressão diferencial encontrada foi significativa para dois genes de B.thuringiensis, um relacionado aos fatores sigma (sigma35) e outro ao gene cry (cry2Ab). Detectaram-se diferenças entre as médias de expressão do fator sigma e do gene cry2Ab. Os valores máximos de expressão diferencial foram obtidos para o gene codificador do fator sigma35 na fase log e na fase esporulativa. Na análise de médias observou-se expressão do gene cry2Ab apenas na fase log; no entanto, de forma bem mais baixa quando comparado com a expressão de sigma35, nas três fases.

Expression of matrix metalloproteinases, type IV collagen, and interleukin-10 in rabbits treated with morphine after lamellar keratectomy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aberrant expression of E-cadherin and beta-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer

Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and beta-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n - 6) and control pregnancies (n - 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or beta-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total beta-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-beta-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/beta-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and beta-catenin proteins, along with defective beta-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

Expression of MyoD, myogenin, myostatin and Hsp70 transcripts in chicken embryos submitted to mild cold or heat

The expression of the MyoD, myogenin, myostatin and Hsp70 genes was estimated in chicken embryos submitted to mild cold (36 +/- 0.5degreesC) or heat (44 +/- 0.5degreesC) for 1 h. 2. Marked decreases in MyoD, myogenin and myostatin transcript levels were observed in embryos exposed to high temperature, contrasting to the higher expression of the Hsp70 mRNA detected in heat-stressed embryos. 3. The exposure of chicken embryos to low temperature significantly affected only the abundance of myogenin mRNA. 4. These findings suggest that myogenic proliferation and differentiation events are compromised by variations in environmental temperature during avian embryogenesis. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor), Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.