Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes.

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLINDEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18-StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies

Genome-wide interaction study of early-life smoking exposure on time-to-asthma onset in childhood

BACKGROUND: Asthma, a heterogeneous disease with variable age of onset, results from the interplay between genetic and environmental factors. Early-life tobacco smoke (ELTS) exposure is a major asthma risk factor. Only a few genetic loci have been reported to interact with ELTS exposure in asthma. OBJECTIVE: Our aim was to identify new loci interacting with ELTS exposure on time-to-asthma onset (TAO) in childhood. METHODS: We conducted genome-wide interaction analyses of ELTS exposure on time-to-asthma onset in childhood in five European-ancestry studies (totaling 8,273 subjects) using Cox proportional-hazard model. The results of all five genome-wide analyses were meta-analyzed. RESULTS: The 13q21 locus showed genome-wide significant interaction with ELTS exposure (P=4.3x10-8 for rs7334050 within KLHL1 with consistent results across the five studies). Suggestive interactions (P<5x10-6 ) were found at three other loci: 20p12 (rs13037508 within MACROD2; P=4.9x10-7 ), 14q22 (rs7493885 near NIN; P=2.9x10-6 ) and 2p22 (rs232542 near CYP1B1; P=4.1x10-6 ). Functional annotations and the literature showed that the lead SNPs at these four loci influence DNA methylation in the blood and are located nearby CpG sites reported to be associated with exposure to tobacco smoke components, which strongly support our findings. CONCLUSION AND CLINICAL RELEVANCE: We identified novel candidate genes interacting with ELTS exposure on time-to-asthma onset in childhood. These genes have plausible biological relevance related to tobacco smoke exposure. Further epigenetic and functional studies are needed to confirm these findings and to shed light on the underlying mechanisms. This article is protected by copyright. All rights reserved

The SWI/SNF ATP-Dependent Chromatin Remodeling Complex in Arabidopsis Responds to Environmental Changes in Temperature-Dependent Manner

SWI/SNF ATP-dependent chromatin remodeling complexes (CRCs) play important roles in the regulation of transcription, cell cycle, DNA replication, repair, and hormone signaling in eukaryotes. The core of SWI/SNF CRCs composed of a SWI2/SNF2 type ATPase, a SNF5 and two of SWI3 subunits is sufficient for execution of nucleosome remodeling in vitro. The Arabidopsis genome encodes four SWI2/SNF2 ATPases, four SWI3, a single SNF5 and two SWP73 subunits. Genes of the core SWI/SNF components have critical but not fully overlapping roles during plant growth, embryogenesis, and sporophyte development. Here we show that the Arabidopsis swi3c mutant exhibits a phenotypic reversion when grown at lower temperature resulting in partial restoration of its embryo, root development and fertility defects. Our data indicates that the swi3c mutation alters the expression of several genes engaged in low temperature responses. The location of SWI3C-containing SWI/SNF CRCs on the ICE1, MYB15 and CBF1 target genes depends on the temperature conditions, and the swi3c mutation thus also influences the transcription of several cold-responsive (COR) genes. These findings, together with genetic analysis of swi3c/ice1 double mutant and enhanced freezing tolerance of swi3c plants illustrate that SWI/SNF CRCs contribute to fine-tuning of plant growth responses to different temperature regimes

Genome-wide interaction study of early-life smoking exposure on time-to-asthma onset in childhood

Background: Asthma, a heterogeneous disease with variable age of onset, results from the interplay between genetic and environmental factors. Early-life tobacco smoke (ELTS) exposure is a major asthma risk factor. Only a few genetic loci have been reported to interact with ELTS exposure in asthma. Objective: Our aim was to identify new loci interacting with ELTS exposure on time-to-asthma onset (TAO) in childhood.Methods: We conducted genome-wide interaction analyses of ELTS exposure on time-to-asthma onset in childhood in five European-ancestry studies (totaling 8,273 subjects) using Cox proportional-hazard model. The results of all five genome-wide analyses were meta-analyzed.Results: The 13q21 locus showed genome-wide significant interaction with ELTS exposure (P=4.3x10-8 for rs7334050 within KLHL1 with consistent results across the five studies). Suggestive interactions (P&lt;5x10-6) were found at three other loci: 20p12 (rs13037508 within MACROD2; P=4.9x10-7), 14q22 (rs7493885 near NIN; P=2.9x10-6) and 2p22 (rs232542 near CYP1B1; P=4.1x10-6). Functional annotations and the literature showed that the lead SNPs at these four loci influence DNA methylation in the blood and are located nearby CpG sites reported to be associated with exposure to tobacco smoke components, which strongly support our findings.Conclusion and Clinical Relevance: We identified novel candidate genes interacting with ELTS exposure on time-to-asthma onset in childhood. These genes have plausible biological relevance related to tobacco smoke exposure. Further epigenetic and functional studies are needed to confirm these findings and to shed light on the underlying mechanisms

Identification of Novel and Rare Variants Associated with Handgrip Strength Using Whole Genome Sequence Data from the NHLBI Trans-Omics in Precision Medicine (TOPMed) Program

Handgrip strength is a widely used measure of muscle strength and a predictor of a range of morbidities including cardiovascular diseases and all-cause mortality. Previous genome-wide association studies of handgrip strength have focused on common variants primarily in persons of European descent. We aimed to identify rare and ancestry-specific genetic variants associated with handgrip strength by conducting whole-genome sequence association analyses using 13,552 participants from six studies representing diverse population groups from the Trans-Omics in Precision Medicine (TOPMed) Program. By leveraging multiple handgrip strength measures performed in study participants over time, we increased our effective sample size by 7-12%. Single-variant analyses identified ten handgrip strength loci among African-Americans: four rare variants, five low-frequency variants, and one common variant. One significant and four suggestive genes were identified associated with handgrip strength when aggregating rare and functional variants; all associations were ancestry-specific. We additionally leveraged the different ancestries available in the UK Biobank to further explore the ancestry-specific association signals from the single-variant association analyses. In conclusion, our study identified 11 new loci associated with handgrip strength with rare and/or ancestry-specific genetic variations, highlighting the added value of whole-genome sequencing in diverse samples. Several of the associations identified using single-variant or aggregate analyses lie in genes with a function relevant to the brain or muscle or were reported to be associated with muscle or age-related traits. Further studies in samples with sequence data and diverse ancestries are needed to confirm these findings

Does the implementation of a Quality Improvement Care Bundle reduce the incidence of acute kidney injury in patients undergoing emergency laparotomy?

Previous work has demonstrated a survival improvement following the introduction of an enhanced recovery protocol in patients undergoing emergency laparotomy (the emergency laparotomy pathway quality improvement care (ELPQuiC) bundle). Implementation of this bundle increased the use of intra-operative goal directed fluid therapy and ICU admission, both evidence-based strategies recommended to improve kidney outcomes. The aim of this study was to determine if the observed mortality benefit could be explained by a difference in the incidence of AKI pre- and post-implementation of the protocol.This article is freely available via Open Access. Click on the Publisher URL to access the full-text via the publisher's site

Genome-wide analysis of mitochondrial DNA copy number reveals loci implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation

Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses was used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 × 10(–15)) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 × 10(–8)) and mtDNA replication (p = 1.2 × 10(–7)). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell-related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 × 10(–4)). SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00439-021-02394-w