1. Multiancestry association study identifies new asthma risk loci that colocalize with immune-cell enhancer marks.
Florence Demenais at Genetic Variation and Human Diseases Unit (UMR-946) in Paris, France and his colleagues examined common variation in asthma risk by conducting a meta-analysis of worldwide asthma genome-wide association studies (23,948 asthma cases, 118,538 controls) of individuals from ethnically diverse populations. They identified five new asthma loci, found two new associations at two known asthma loci, established asthma associations at two loci previously implicated in the comorbidity of asthma plus hay fever, and confirmed nine known loci. Investigation of pleiotropy showed large overlaps in genetic variants with autoimmune and inflammatory diseases. The enrichment in enhancer marks at asthma risk loci, especially in immune cells, suggested a major role of these loci in the regulation of immunologically related mechanisms.
Read more, please click https://www.nature.com/articles/s41588-017-0014-7
2. Protein-altering variants associated with body mass index implicate pathways that control energy intake and expenditure in obesity.
Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here Valérie Turcot at University of Montreal in Quebec, Canada and his colleagues combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. They identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2 and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4R and KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4R mutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity.
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3. Live-cell measurements of kinase activity in single cells using translocation reporters.
Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. Takamasa Kudo at Stanford University in Stanford, California, USA and his colleagues have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization. The negative charge introduced by phosphorylation of the substrate is used to directly modulate nuclear import and export, thereby regulating the reporter’s distribution between the cytoplasm and nucleus. The relative cytoplasmic versus nuclear fluorescence of the KTR construct (the C/N ratio) is used as a proxy for the kinase activity in living, single cells. Multiple KTRs can be studied in the same cell by fusing them to different fluorescent proteins. Here, they present a protocol to execute and analyze live-cell microscopy experiments using KTRs. They describe strategies for development of new KTRs and procedures for lentiviral expression of KTRs in a cell line of choice. Cells are then plated in a 96-well plate, from which multichannel fluorescent images are acquired with automated time-lapse microscopy. They provide detailed guidance for a computational analysis and parameterization pipeline. The entire procedure, from virus production to data analysis, can be completed in ∼10 d.
Read more, please click https://www.nature.com/articles/nprot.2017.128
4. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.
CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1–10% and therefore not suitable for routine use. Hiromi Miura at School of Medicine, Tokai University in Kanagawa, Japan and her colleagues recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. They call this method efficient additions with ssDNA inserts–CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30–60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.
Read more, please click https://www.nature.com/articles/nprot.2017.153
5. Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics.
Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, Kelly M. Martinovich at The University of Western Australia in Crawley, Western Australia, Australia and her colleagues show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Their findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.
Read more, please click https://www.nature.com/articles/s41598-017-17952-4