Identification of Novel Regulatory Genes in Acetaminophen Induced Hepatocyte Toxicity by a Genome-Wide CRISPR/Cas9 Screen

Title from PDF of title page viewed May 10, 2019Dissertation advisor: Shui Qing YeVitaIncludes bibliographical references (pages 154-165)Thesis (Ph.D.)--School of Biological Sciences and School of Medicine. University of Missouri--Kansas City, 2018Acetaminophen (APAP) is a commonly used analgesic responsible for over 56,000 overdose-related emergency room visits annually. A long asymptomatic period and limited treatment options result in a high rate of liver failure, generally resulting in either organ transplant or mortality. The underlying molecular mechanisms of injury are not well understood and effective therapy is limited. Identification of previously unknown genetic risk factors would provide new mechanistic insights and new therapeutic targets for APAP induced hepatocyte toxicity or liver injury. This study used a genome-wide CRISPR/Cas9 screen to evaluate genes that are protective against or cause susceptibility to APAP-induced liver injury. HuH7 human hepatocellular carcinoma cells containing CRISPR/Cas9 gene knockouts were treated with 15mM APAP for 30 minutes to 4 days. A gene expression profile was developed based on the 1) top screening hits, 2) overlap with gene expression data of APAP overdosed human patients, and 3) biological interpretation including assessment of known and suspected APAP-associated genes and their therapeutic potential, predicted affected biological pathways, and functionally validated candidate genes. This screen is the first genome-wide CRISPR/Cas9 knockout screen of APAP induced hepatocyte toxicity. The top hits from this screen included numerous genes previously not linked to liver injury. We further demonstrated the implementation of intermediate time points for the identification of early and late response genes. A negative selection screen identified genes involved in fundamental processes, including NAAA, ATG2B, and MYOZ3. A positive selection screen identified numerous genes potentially involved in pathogenic processes, including LZTR1, PGM5, and EEF1D. A top essential pathway at 24 hours of APAP treatment was Regulation of Skeletal Muscle Contraction. We additionally identified 6 genes, 3 novel and 3 known, that have drug-gene interactions favorable for re-purposing existing therapies to treat APAP-induced hepatotoxicity. Collectively, this line of research has illustrated the power of a genome-wide CRISPR/Cas9 screen to systematically identify novel genes involved in APAP induced hepatocyte toxicity and to provide potential new targets to develop novel therapeutic modalities.Introduction -- Review of the literature -- Research question -- Methods -- Results and discussion part 1: CRISPR/Cas9 screen -- Results and discussion part 2: our screen in the context of other acetaminophen data sets -- Results and discussion part 3: acetaminophen-associated single nucleotide polymorphisms in the literature -- Results and discussion Part 4: validation of top candidate genes -- Conclusions and future directions -- Appendix A. Supplementary figures -- Appendix B. Supplementary table

Identification of novel coding single nucleotide polymorphisms associated with acute respiratory distress syndrome

Title from PDF of title page, viewed on July 28, 2014Thesis advisor: Shui Qing YeVitaIncludes bibliographical references (pages 59-61)Thesis (M. S.)--School of Medicine. University of Missouri--Kansas City, 2014ARDS is a lung condition characterized by impaired gas exchange with systemic release of inflammatory mediators, causing inflammation, hypoxemia and multiple organ failure. Disease susceptibility and progression are poorly understood and there are few effective therapeutic options. Existing biomarkers have limited effectiveness as diagnostic and therapeutic targets. Whole-exome sequencing is an effective tool in detection of disease-causing genetic variants in complex genetic conditions such as acute respiratory distress syndrome (ARDS). To identify disease-causing variants in ARDS patients, whole-exome sequencing was performed on 96 patient DNA samples from the National Heart, Lung and Blood Institute's ARDS Network (ARDSnet). By comparing these exome data with 625 participants of the 1000 Genomes Project, we have tentatively identified a number of single nucleotide polymorphisms (SNP) which are potentially associated with ARDS. In this study, we validated three SNPs (rs78142040, rs9605146, and rs3848719) in an additional 117 ARDS patients using TaqMan SNP genotyping assays (Life Technologies) to substantiate their associations with the susceptibility, severity and outcome of ARDS. rs78142040 (C>T) occurs within a histone mark in intron 6 of the Arylsulfatase D gene. rs9605146 (G>A)(also known as rs114989947) causes a coding change (proline to leucine) with a deleterious effect in the XK, Kell blood group complex subunit-related family, member 3 gene. rs3848719 (G>A) is a synonymous SNP in exon 5 of gene Zinc-Finger/Leucine-Zipper Co-Transducer NIF1. rs78142040 and rs9605146 are significantly associated with susceptibility to ARDS[minor allele frequency (MAF): 0.219 versus 0.003 (control), p1×10-4) in our cases. These 3 SNPs have not been previously associated with ARDS and represent potential new genetic biomarkers for ARDS. More validations in larger patient populations and further exploration of underlying molecular mechanisms are warrantedAbstract -- List of illustrations -- List of tables -- Glossary -- Acknowledgments -- Introduction - Review of the literature -- Research question -- Methods -- Results -- Discussion -- Conclusions -- Supplementary figures and tables -- Reference lis

Taking health geography out of the academy:Measuring academic impact

In recent years the academic landscape has been shifting and significantly affected by the introduction of an ‘impact agenda’. Academics are increasingly expected to demonstrate their broader engagement with the world and evidence related outcomes. Whilst different countries are at various stages along this impact journey, the UK is the first country to link impact to funding outcomes; here impact now accounts for 20% of an academic unit of assessment’s Research Excellence Framework (REF) result. This concept of ‘research impact’ implies that our work can effect change through one or more identifiable events in a direct, preferably linear and certainly measurable manner. In this paper, focusing on impact in social science, and policy-related impact in particular, we argue that such a cause and effect model is inappropriate. Furthermore that impact is not immediate or indeed linear within social science research. Drawing on recent work on alcohol and tobacco environments in Scotland we present a case study of impact, reflect on the process and respond to the challenges of moving beyond ‘business as usual’ public participation towards the measurement of outcomes. In doing so we critique the way in which ‘impact’ is currently measured and suggest a move towards an enlightenment model with greater recognition of process

The mTORC1 inhibitor everolimus prevents and treats Eμ-Myc lymphoma by restoring oncogene-induced senescence

MYC deregulation is common in human cancer. IG-MYC translocations that are modeled in EμMyc mice occur in almost all cases of Burkitt lymphoma as well as in other B-cell lymphoproliferative disorders. Deregulated expression of MYC results in increased mTOR complex 1 (mTORC1) signaling. As tumors with mTORC1 activation are sensitive to mTORC1 inhibition, we used everolimus, a potent and specific mTORC1 inhibitor, to test the requirement for mTORC1 in the initiation and maintenance of EμMyc lymphoma. Everolimus selectively cleared premalignant B cells from the bone marrow and spleen, restored a normal pattern of B-cell differentiation, and strongly protected against lymphoma development. Established EμMyc lymphoma also regressed after everolimus therapy. Therapeutic response correlated with a cellular senescence phenotype and induction of p53 activity. Therefore, mTORC1-dependent evasion of senescence is critical for cellular transformation and tumor maintenance by MYC in B lymphocytes

Conservation, Extensive Heterozygosity, and Convergence of Signaling Potential All Indicate a Critical Role for KIR3DL3 in Higher Primates

Natural killer (NK) cell functions are modulated by polymorphic killer cell immunoglobulin-like receptors (KIR). Among 13 human KIR genes, which vary by presence and copy number, KIR3DL3 is ubiquitously present in every individual across diverse populations. No ligand or function is known for KIR3DL3, but limited knowledge of expression suggests involvement in reproduction, likely during placentation. With 157 human alleles, KIR3DL3 is also highly polymorphic and we show heterozygosity exceeds that of HLA-B in many populations. The external domains of catarrhine primate KIR3DL3 evolved as a conserved lineage distinct from other KIR. Accordingly, and in contrast to other KIR, we show the focus of natural selection does not correspond exclusively to known ligand binding sites. Instead, a strong signal for diversifying selection occurs in the D1 Ig domain at a site involved in receptor aggregation, which we show is polymorphic in humans worldwide, suggesting differential ability for receptor aggregation. Meanwhile in the cytoplasmic tail, the first of two inhibitory tyrosine motifs (ITIM) is conserved, whereas independent genomic events have mutated the second ITIM of KIR3DL3 alleles in all great apes. Together, these findings suggest that KIR3DL3 binds a conserved ligand, and a function requiring both receptor aggregation and inhibitory signal attenuation. In this model KIR3DL3 resembles other NK cell inhibitory receptors having only one ITIM, which interact with bivalent downstream signaling proteins through dimerization. Due to the extensive conservation across species, selection, and other unusual properties, we consider elucidating the ligand and function of KIR3DL3 to be a pressing question

The SPECTRUM Consortium : A new UK Prevention Research Partnership consortium focussed on the commercial determinants of health, the prevention of non-communicable diseases, and the reduction of health inequalities

The main causes of non-communicable diseases (NCDs), health inequalities and health inequity include consumption of unhealthy commodities such as tobacco, alcohol and/or foods high in fat, salt and/or sugar. These exposures are preventable, but the commodities involved are highly profitable. The economic interests of 'Unhealthy Commodity Producers' (UCPs) often conflict with health goals but their role in determining health has received insufficient attention. In order to address this gap, a new research consortium has been established. This open letter introduces the SPECTRUM ( S haping Public h Ealth poli Cies To Reduce ineq Ualities and har M) Consortium: a multi-disciplinary group comprising researchers from 10 United Kingdom (UK) universities and overseas, and partner organisations including three national public health agencies in Great Britain (GB), five multi-agency alliances and two companies providing data and analytic support. Through eight integrated work packages, the Consortium seeks to provide an understanding of the nature of the complex systems underlying the consumption of unhealthy commodities, the role of UCPs in shaping these systems and influencing health and policy, the role of systems-level interventions, and the effectiveness of existing and emerging policies. Co-production is central to the Consortium's approach to advance research and achieve meaningful impact and we will involve the public in the design and delivery of our research. We will also establish and sustain mutually beneficial relationships with policy makers, alongside our partners, to increase the visibility, credibility and impact of our evidence. The Consortium's ultimate aim is to achieve meaningful health benefits for the UK population by reducing harm and inequalities from the consumption of unhealthy commodities over the next five years and beyond

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

A first update on mapping the human genetic architecture of COVID-19

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