Genome-wide Screening Technology Research Articles

Inhibition of Macrophage Pyroptosis─A New Therapeutic Strategy to Alleviate T-2 Toxin-Induced Subacute Liver Injury by Directly Competing with the Key Target.

Multiple compounds are related to the development of liver injury, such as toxins, drugs, and environmental pollutants. Although there are reports that the T-2 toxin can cause liver injury, its toxic mechanism remains unclear, which further impedes the development of effective antidotes. In this study, CRISPR-Cas9 genome-wide screening technology was used to identify transformation-related protein 53 inducible nuclear protein 1 (trp53inp1) as a toxic target of the T-2 toxin. Mechanism studies have shown that the T-2 toxin induced pyroptosis of macrophages (J774A.1 cells) by activating the trp53inp1/NF-κB/NLRP3/GSDMD-N pathway, leading to a subacute liver injury. Also, the new drug berberine (BER) identified through virtual screening significantly alleviated the subacute liver injury by competitively binding trp53inp1 via His224; the effect was better than those of the positive control drugs N-acetylcysteine (NAC) and disulfiram (DSF). In summary, the above results indicate that trp53inp1 is a key target for T-2 toxin to induce subacute liver injury and that inhibiting macrophage pyroptosis is a new method for treating liver injury. In addition, this study provides a new method and strategy for the discovery of key disease targets and the search for effective drugs.

High prevalence of multilocus pathogenic variation in neurodevelopmental disorders in the Turkish population

Neurodevelopmental disorders (NDDs) are clinically and genetically heterogenous; many such disorders are secondary to perturbation in brain development and/or function. The prevalence of NDDs is > 3%, resulting in significant sociocultural and economic challenges to society. With recent advances in family-based genomics, rare-variant analyses, and further exploration of the Clan Genomics hypothesis, there has been a logarithmic explosion in neurogenetic "disease-associated genes" molecular etiology and biology of NDDs; however, the majority of NDDs remain molecularly undiagnosed. We applied genome-wide screening technologies, including exome sequencing (ES) and whole-genome sequencing (WGS), to identify the molecular etiology of 234 newly enrolled subjects and 20 previously unsolved Turkish NDD families. In 176 of the 234 studied families (75.2%), a plausible and genetically parsimonious molecular etiology was identified. Out of 176 solved families, deleterious variants were identified in 218 distinct genes, further documenting the enormous genetic heterogeneity and diverse perturbations in human biology underlying NDDs. We propose 86 candidate disease-trait-associated genes for an NDD phenotype. Importantly, on the basis of objective and internally established variant prioritization criteria, we identified 51 families (51/176= 28.9%) with multilocus pathogenic variation (MPV), mostly driven by runs of homozygosity (ROHs) - reflecting genomic segments/haplotypes that are identical-by-descent. Furthermore, with the use of additional bioinformatic tools and expansion of ES to additional family members, we established a molecular diagnosis in 5 out of 20 families (25%) who remained undiagnosed in our previously studied NDD cohort emanating from Turkey.

Unbiased Screens Show CD8+ T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein

Developing effective strategies to prevent or treat coronavirus disease 2019 (COVID-19) requires understanding the natural immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8+ T cells of COVID-19 patients. In total, we identified 3–8 epitopes for each of the 6 most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8+ T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8+ T cell immunity to SARS-CoV-2.

Applications of Genome-Wide Screening and Systems Biology Approaches in Drug Repositioning.

Simple SummaryDrug repurposing is an accelerated route for drug development and a promising approach for finding medications for orphan and common diseases. Here, we compiled databases that comprise both computationally- or experimentally-derived data, and categorized them based on quiddity and origin of data, further focusing on those that present high throughput omic data or drug screens. These databases were then contextualized with genome-wide screening methods such as CRISPR/Cas9 and RNA interference, as well as state of art systems biology approaches that enable systematic characterizations of multi-omic data to find new indications for approved drugs or those that reached the latest phases of clinical trials.Modern drug discovery through de novo drug discovery entails high financial costs, low success rates, and lengthy trial periods. Drug repositioning presents a suitable approach for overcoming these issues by re-evaluating biological targets and modes of action of approved drugs. Coupling high-throughput technologies with genome-wide essentiality screens, network analysis, genome-scale metabolic modeling, and machine learning techniques enables the proposal of new drug–target signatures and uncovers unanticipated modes of action for available drugs. Here, we discuss the current issues associated with drug repositioning in light of curated high-throughput multi-omic databases, genome-wide screening technologies, and their application in systems biology/medicine approaches.

COVID-19 Patients Form Memory CD8+ T Cells that Recognize a Small Set of Shared Immunodominant Epitopes in SARS-CoV-2

Development of effective strategies to detect, treat, or prevent COVID-19 requires a robust understanding of natural immunity to SARS-CoV-2, including the cellular response mediated by T cells. We used an unbiased, genome-wide screening technology to comprehensively identify the specific epitopes in SARS-CoV-2 that are recognized by the memory CD8+ T cells of 25 COVID-19 convalescent patients. For each of six HLA types examined, patient T cells recognized 3–8 immunodominant epitopes that are broadly shared among patients, and single-cell sequencing revealed common structural features of TCRs recognizing these epitopes. We detected minimal cross-reactivity to the endemic coronaviruses that cause the common cold, arguing that pre-existing immunity to other coronaviruses does not significantly shape CD8+ T cell responses to SARS-CoV-2. Notably, only 3 of the 29 immunodominant epitopes we identified reside in the Spike protein, highlighting the need for second-generation vaccines that recapitulate natural CD8+ T cell immunity to SARS-CoV-2.Funding: This work was supported by TScan Therapeutics, a privately-owned biotechnology company.Ethical Approval: The study was conducted in accordance with the Declaration of Helsinki (1996), approved by the Atlantic Health System Institutional Review Board and the Ochsner Clinic Foundation Institutional Review Board and registered at clinicaltrials.gov (# NCT04397900).

Canthin-6-One Accelerates Alpha-Synuclein Degradation by Enhancing UPS Activity: Drug Target Identification by CRISPR-Cas9 Whole Genome-Wide Screening Technology.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of protein aggregates (namely Lewy bodies) in dopaminergic neurons in the substantia nigra region of the brain. Alpha-synuclein (α-syn) is the major component of Lewy bodies in PD patients, and impairment of the ubiquitin-proteasome system has been linked to its accumulation. In this work, we developed a tetracycline–inducible expression system, with simultaneous induced expression of α-syn-EGFP and a bright red fluorescent protein marker (mCherry) to screen for potential compounds for degrading α-syn. We identified canthin-6-one as an α-syn lowering compound which promoted both wild type and mutants α-syn degradation in an ubiquitin-proteasome-system (UPS) dependent manner. By CRISPR/Cas9 genome-wide screening technology, we identified RPN2/PSMD1, the 26S proteasome non-ATPase regulatory subunit 1, as the targeting gene for pharmacological activity of canthin-6-one. Finally, we showed that canthin-6-one up-regulates PSMD1 and enhances UPS function by activating PKA.

Genetic and Chemical Screenings Identify HDAC3 as a Key Regulator inHepatic Differentiation of Human Pluripotent Stem Cells.

SummaryHepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) offer a promising cell resource for disease modeling and transplantation. However, differentiated HLCs exhibit an immature phenotype and comprise a heterogeneous population. Thus, a better understanding of HLC differentiation will improve the likelihood of future application. Here, by taking advantage of CRISPR-Cas9-based genome-wide screening technology and a high-throughput hPSC screening platform with a reporter readout, we identified several potential genetic regulators of HLC differentiation. By using a chemical screening approach within our platform, we also identified compounds that can further promote HLC differentiation and preserve the characteristics of in vitro cultured primary hepatocytes. Remarkably, both screenings identified histone deacetylase 3 (HDAC3) as a key regulator in hepatic differentiation. Mechanistically, HDAC3 formed a complex with liver transcriptional factors, e.g., HNF4, and co-regulated the transcriptional program during hepatic differentiation. This study highlights a broadly useful approach for studying and optimizing hPSC differentiation.

Abstract 4359: Systematic approach to methylation biomarker development in chronic lymphocytic leukemia, breast and lung cancers

Abstract There is no doubt that testing for disease specific methylation changes can guide disease risk assessment, facilitate detection of the disease, support prognostication and personalization of treatment as well as guide post treatment patient care (1). Nevertheless, the use of methylation biomarkers in standard patient care is still marginal. Difficulties in implementation of methylation biomarker testing in standard in-vitro diagnostics are mainly attributed to challenges in establishment of the systematic approach allowing for efficient discovery and subsequent validation of clinical utility of potential disease specific methylation biomarkers. We have combined state-of-the-art genome wide methylation screening technologies (including: newest Illumina MethylationEPIC 850k BeadChip) enabling methylation biomarker discovery with the cost and time efficient locus specific technologies to streamline the development, validation and implementation of methylation biomarkers for clinical disease management. With the use of technologies that in an affordable fashion enable screening for genome wide methylation changes in a substantial number of clinical samples, we were able to discover clinically relevant disease specific methylation signatures e.g. in CLL. The high technical complexity of the genome wide screen technologies prevents them for being straightforward applicable in diagnostic settings. Thus we used techniques such as Methylation-Sensitive High Resolution Melting (MS-HRM) (2) to develop assays fulfilling the requirements for diagnostic applications. Subsequently, we evaluated the clinical relevance of the most promising biomarker candidates in chronic lymphocytic leukaemia, breast and lung cancers, using these assays (3, 4). Overall, our workflow allows fast and efficient validation of not only already existing biomarker candidates but also discovery of new disease specific methylation changes that show promise for clinical implementation.

Abstract 2308: Heterogeneous and low-level methylation of novel biomarker candidates for breast cancer clinical management

Abstract Breast cancer is a highly heterogeneous malignancy, which is best exemplified by the fact that despite similar clinical and pathological features large number breast cancer patients experience different outcome of the disease. Therefore there is an urgent need for identification of new biomarkers to stratify patients for personalized treatment. Methylation of gene promoters regulates genes transcription and provides normal cell function. At the same time, changes of the normal pattern of methylation are directly involved in malignant transformation, and have been shown to provide a good source of clinical biomarkers for cancer management. Nevertheless it is still challenging to firstly identify methylation changes suitable for clinical biomarkers and secondly evaluate the clinical specificity and sensitivity of those biomarkers. We used ultra high-resolution microarrays to compare genome wide methylation patterns of breast carcinomas (N=20) with non-malignant breast tissue (N=5) and identified 1000+ differentially methylated regions (DMR) in the genome, which undergo methylation changes during breast cancer development. The results of genome wide screening technologies (due to the high complexity of those methods) require validation with PCR based technology. Therefore, we validated 19 of the regions identified in the genome wide screening with the Methylation Sensitive High Resolution Melting (MS-HRM) technique in a panel of breast carcinomas (N=275) and non-malignant controls (N=74). The validation experiments corroborated the results, confirming that methylation changes within the identified regions can potentially be used as biomarkers. Interestingly, we have observed two types of methylation gain at the validated loci: 1) loci undergoing predominantly complete methylation, and 2) loci acquiring mainly heterogeneous methylation during carcinogenesis. It can be speculated that the different types of methylation gain have different physiological consequences for transcriptional activity of the affected genes. Consequently the involvement of the gene in carcinogenesis may depend on the type of methylation acquired. However more research is needed to elaborate these findings. Our validation experiments also showed that almost all tested DMRs (17 out of 19) displayed low-level methylation in non-malignant breast tissue. The physiological consequences of the low level methylation of non-malignant tissue are largely unknown. These changes does not seem to have immediate pathological consequences but may predispose to breast cancer and hence have significant impact on evaluation of the clinical sensitivity and specificity of methylation biomarkers. Summarizing in our study, we have identified two phenomena that have significant consequences for the locus specific methylation biomarker development and need to be further researched. Citation Format: Tomasz K. Wojdacz, Johanne A. Windeløv, Britta B. Thestrup, Tine E. Damsgaard, Jens Overgaard, Lise Lotte Hansen. Heterogeneous and low-level methylation of novel biomarker candidates for breast cancer clinical management. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2308. doi:10.1158/1538-7445.AM2014-2308

Genetic requirements for Moraxella catarrhalis growth under iron‐limiting conditions

Iron sequestration by the human host is a first line defence against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron starvation during colonization. We determined the genetic requirements for M. catarrhalis BBH18 growth during iron starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). By subjecting a large random transposon mutant library to growth under iron-limiting conditions, mutants of the MCR_0996-rhlB-yggW operon, rnd, and MCR_0457 were negatively selected. Growth experiments using directed mutants confirmed the GAF phenotypes with ΔyggW (putative haem-shuttling protein) and ΔMCR_0457 (hypothetical protein) most severely attenuated during iron starvation, phenotypes which were restored upon genetic complementation of the deleted genes. Deletion of yggW resulted in similar attenuated phenotypes in three additional strains. Transcriptional profiles of ΔyggW and ΔMCR_0457 were highly altered with 393 and 192 differentially expressed genes respectively. In all five mutants, expression of nitrate reductase genes was increased and of nitrite reductase decreased, suggesting an impaired aerobic respiration. Alteration of iron metabolism may affect nasopharyngeal colonization as adherence of all mutants to respiratory tract epithelial cells was attenuated. In conclusion, we elucidated the genetic requirements for M. catarrhalis growth during iron starvation and characterized the roles of the identified genes in bacterial growth and host interaction.

Negative Regulators of Insulin Signaling Revealed in a Genome-Wide Functional Screen

BackgroundType 2 diabetes develops due to a combination of insulin resistance and β-cell failure and current therapeutics aim at both of these underlying causes. Several negative regulators of insulin signaling are known and are the subject of drug discovery efforts. We sought to identify novel contributors to insulin resistance and hence potentially novel targets for therapeutic intervention.MethodologyAn arrayed cDNA library encoding 18,441 human transcripts was screened for inhibitors of insulin signaling and revealed known inhibitors and numerous potential novel regulators. The novel hits included proteins of various functional classes such as kinases, phosphatases, transcription factors, and GTPase associated proteins. A series of secondary assays confirmed the relevance of the primary screen hits to insulin signaling and provided further insight into their modes of action.Conclusion/SignificanceAmong the novel hits was PALD (KIAA1274, paladin), a previously uncharacterized protein that when overexpressed led to inhibition of insulin's ability to down regulate a FOXO1A-driven reporter gene, reduced upstream insulin-stimulated AKT phosphorylation, and decreased insulin receptor (IR) abundance. Conversely, knockdown of PALD gene expression resulted in increased IR abundance, enhanced insulin-stimulated AKT phosphorylation, and an improvement in insulin's ability to suppress FOXO1A-driven reporter gene activity. The present data demonstrate that the application of arrayed genome-wide screening technologies to insulin signaling is fruitful and is likely to reveal novel drug targets for insulin resistance and the metabolic syndrome.

Genome variability in European and American bison detected using the BovineSNP50 BeadChip

The remaining wild populations of bison have all been through severe bottlenecks. The genomic consequences of these bottlenecks present an interesting area to study. Using a very large panel of SNPs developed in Bos taurus we have carried out a genome-wide screening on the European bison (Bison bonasus; EB) and on two subspecies of American bison: the plains bison (B. bison bison; PB) and the wood bison (B. bison athabascae; WB). One hundred bison samples were genotyped for 52,978 SNPs along with seven breeds of domestic bovine Bos taurus. Only 2,209 of the SNPs were polymorphic in the bison when EB, PB and WB were pooled and only 929 SNPs were polymorphic in EB. Larger numbers of polymorphic SNPs were found in PB (1,403 SNPs) and WB (1,524 SNPs). Also the expected heterozygosity was lower in EB (HE = 0.135) than in WB (HE = 0.197) and PB (HE = 0.199). The polymorphic SNPs were not randomly distributed in the bison, but were aggregated and separated from each other by regions with low haplotype diversity (haplotype blocks). Based on our results we suggest that the utilization of genome-wide screening technologies holds large potential to radically change the breeding practices in captive or managed populations of threatened populations and advocate for developing marker assisted selected strategies in such populations.

Depauperate genetic variability detected in the American and European bison using genomic techniques

A total of 929 polymorphic SNPs in EB (out of 54, 000 SNPs screened using a BovineSNP50 Illumina Genotyping BeadChip), and 1, 524 and 1, 403 polymorphic SNPs in WB and PB, respectively, were analysed. EB, WB and PB have all undergone recent drastic reductions in population size. Accordingly, they exhibited extremely depauperate genomes, deviations from genetic equilibrium and a genome organization consisting of a mosaic of haplotype blocks: regions with low haplotype diversity and high levels of linkage disequilibrium. No evidence for positive or stabilizing selection was found in EB, WB and PB, likely reflecting drift overwhelming selection. We suggest that utilization of genome-wide screening technologies, followed by utilization of less expensive techniques (e.g. VeraCode and Fluidigm EP1), holds large potential for genetic monitoring of populations. Additionally, these techniques will allow radical improvements of breeding practices in captive or managed populations, otherwise hampered by the limited availability of polymorphic markers. This result in improved possibilities for 1) estimating genetic relationships among individuals and 2) designing breeding strategies which attempt to preserve or reduce polymorphism in ecologically relevant genes and/or entire blocks.ReviewersThis article was reviewed by: Fyodor Kondrashov and Shamil Sunyaev