MARIS: Method for Analyzing RNA following Intracellular Sorting

Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.Howard Hughes Medical InstituteHarvard Stem Cell InstituteNational Institutes of Health (U.S.) (grant 2U01DK07247307)National Institutes of Health (U.S.) (grant RL1DK081184)National Institutes of Health (U.S.) (grant 1U01HL10040804

Rethinking cultural diversity in the UK film sector:practices in community filmmaking

© The Author(s) 2017. Academic, policy and industry debates have tended to focus on the mainstream film sector when discussing cultural diversity. One of the persistent challenges for the sector has been how to diversify cultural representation and participation. This article suggests that participatory modes of community filmmaking make an important contribution to cultural diversity. Drawing on an evidence base derived from qualitative research conducted in three English regions, the article shifts the spotlight away from the mainstream and onto the margins of the film sector in order to explore more ‘bottom-up’ approaches to cultural diversity. It examines how community filmmakers interpret and engage with questions of cultural diversity and how this connects to the participatory and business practices that they adopt. The findings highlight the significance of processes of practice in how mediated cultural diversity manifests itself and the value of community filmmaking in contributing to wider cultural diversity debates and practices.The authors thank the Arts and Humanities Research Council (UK) for funding the ‘Community Filmmaking and Cultural Diversity: Practice, Innovation and Policy project’ (2013–2014)

Single Cell Genome Amplification Accelerates Identification of the Apratoxin Biosynthetic Pathway from a Complex Microbial Assemblage

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites

Relationship Between Early Childhood Oral Health Impact Scale, Child's Dental Status and Parental Psychological Profiles.

BACKGROUND: The aim of this study was to assess the influence of a child's dental status and parental psychological profiles on parental perception of their child's oral health. MATERIALS: This study involved 164 parents of children under five years of age, who filled out the life quality assessment test regarding oral health (ECOHIS test), and Depression, Anxiety and Stress Scale (DASS) questionnaire. The child's oral status was also examined and recorded. RESULTS: Results of the ECOHIS appeared to correlate positively with the number of active caries (R = 0.457; P <0.001). In addition, the ECOHIS showed a significant correlation with the DASS test results (R = 0.356; P <0.001 for depression, R = 0.247; P <0.001 for anxiety and R = 0.235; P <0.001 for the stress result). CONCLUSION: The dental health of a child affects his/her quality of life and the parental quality of life including their psychological status