A Powerful Method for Transcriptional Profiling of Specific Cell Types in Eukaryotes: Laser-Assisted Microdissection and RNA Sequencing

The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms

New Insights into the Organization, Recombination, Expression and Functional Mechanism of Low Molecular Weight Glutenin Subunit Genes in Bread Wheat

The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding

Safety and Reactogenicity of Canarypox ALVAC-HIV (vCP1521) and HIV-1 gp120 AIDSVAX B/E Vaccination in an Efficacy Trial in Thailand

A prime-boost vaccination regimen with ALVAC-HIV (vCP1521) administered intramuscularly at 0, 4, 12, and 24 weeks and gp120 AIDSVAX B/E at 12 and 24 weeks demonstrated modest efficacy of 31.2% for prevention of HIV acquisition in HIV-uninfected adults participating in a community-based efficacy trial in Thailand.Reactogenicity was recorded for 3 days following vaccination. Adverse events were monitored every 6 months for 3.5 years, during which pregnancy outcomes were recorded. Of the 16,402 volunteers, 69% of the participants reported an adverse event any time after the first dose. Only 32.9% experienced an AE within 30 days following any vaccination. Overall adverse event rates and attribution of relatedness did not differ between groups. The frequency of serious adverse events was similar in vaccine (14.3%) and placebo (14.9%) recipients (p = 0.33). None of the 160 deaths (85 in vaccine and 75 in placebo recipients, p = 0.43) was assessed as related to vaccine. The most common cause of death was trauma or traffic accident. Approximately 30% of female participants reported a pregnancy during the study. Abnormal pregnancy outcomes were experienced in 17.1% of vaccine and 14.6% (p = 0.13) of placebo recipients. When the conception occurred within 3 months (estimated) of a vaccination, the majority of these abnormal outcomes were spontaneous or elective abortions among 22.2% and 15.3% of vaccine and placebo pregnant recipients, respectively (p = 0.08). Local reactions occurred in 88.0% of vaccine and 61.0% of placebo recipients (p<0.001) and were more frequent after ALVAC-HIV than AIDSVAX B/E vaccination. Systemic reactions were more frequent in vaccine than placebo recipients (77.2% vs. 59.8%, p<0.001). Local and systemic reactions were mostly mild to moderate, resolving within 3 days.The ALVAC-HIV and AIDSVAX B/E vaccine regimen was found to be safe, well tolerated and suitable for potential large-scale use in Thailand.ClinicalTrials.govNCT00223080

Insights into pathogenic events of HIV-associated Kaposi sarcoma and immune reconstitution syndrome related Kaposi sarcoma

A decrease in the incidence of human immune deficiency virus-associated Kaposi sarcoma (HIV-KS) and regression of some established HIV-KS lesions is evident after the introduction of highly active anti-retroviral treatment (HAART), and is attributed to generalized immune restoration, to the reconstitution of human herpesvirus (HHV)-8 specific cellular immune responses, and to the decrease in HIV Tat protein and HHV-8 loads following HAART. However, a small subset of HIV-seropositive subjects with a low CD4+ T cell count at the time of introduction of HAART, may develop HIV-KS as immune reconstitution inflammatory syndrome (IRIS) within 8 weeks thereafter

Prática esportiva e participação nas aulas de educação física: fatores associados em estudantes de Londrina, Paraná, Brasil

O objetivo deste estudo foi analisar os fatores associados à prática esportiva e participação nas aulas de educação física. A amostra foi composta por 827 jovens, selecionados em dois estágios e estratificados por região da cidade de Londrina, Paraná, Brasil. Foram analisadas a prática esportiva, participação nas aulas de educação física e variáveis sociodemográficas, comportamentais e ambientais. Os fatores associados à participação nas aulas de educação física foram: sexo masculino, idade (10-13 anos), percepção de atividade física igual ou elevada, prática esportiva, movimentação durante as aulas e realizar aulas na quadra. Para a prática esportiva, os fatores associados foram o sexo masculino, idade (10-13 anos), percepção de atividade física igual/elevada, atividade física (≥ 7horas/semana), participação nas aulas de educação física, possuir ≥ 10 amigos, prática de esportes com amigos e frequência de utilização de lugares para a prática. Esses fatores devem ser considerados nos programas de incentivo à prática esportiva e à participação nas aulas de educação física.El objetivo de este estudio fue analizar los factores asociados a la práctica deportiva y participación en las clases de educación física. La muestra consistió en 827 jóvenes; se seleccionó en dos etapas y estratificó por región en la ciudad de Londrina, Paraná, Brasil. Se analizó la práctica deportiva, participación en las clases de educación física y variables sociodemográficas, de comportamiento y ambientales. Los factores asociados a la participación en las clases de educación física fueron: sexo masculino, edad (10-13 años), percepción de actividad física igual o superior, práctica deportiva, ejercicio en las clases y clases en pista deportiva. Para la práctica deportiva, los factores asociados fueron: sexo masculino, edad (10-13 años), percepción de actividad física igual o superior, actividad física (≥ 7 horas/semana), participación en las clases de educación física, tener ≥ 10 amigos, práctica deportiva con amigos y frecuencia de uso de las instalaciones. Estos factores deben ser considerados en programas de incentivos a la práctica deportiva y participación en las clases de educación física.This study aimed to analyze factors associated with participation in sports and physical education. The sample consisted of 827 young people selected in two stages and stratified by neighborhood of Londrina, Paraná State, Brazil. The study analyzed participation in sports and physical education in relation to socio-demographic, behavioral, and environmental variables. Factors associated with participation in physical education included: male gender, age (10-13 years), equal to high self-rated physical activity, participation in sports, moving around during classes, and having classes on the court. Factors associated with participation in sports were male gender, age (10-13years), equal to high self-rated physical activity, physical activity (≥ 7hours/week), participation in physical education, having ≥ 10 friends, participation in sports with friends, and frequency using facilities for sports practice. These factors should be considered in programs to encourage sports and participation in physical education.Universidade Estadual de MaringáUniversidade Estadual de LondrinaLondrinaUniversidade Estadual Paulista Faculdade de CiênciasUniversidade Estadual Paulista Faculdade de Ciência