Browsing by Author "Simpson, Edward"

Now showing 1 - 10 of 11
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    A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle
    (Elsevier, 2023-03-31) Wang, Ruizhong; Kumar, Brijesh; Bhat-Nakshatri, Poornima; Khatpe, Aditi S.; Murphy, Michael P.; Wanczyk, Kristen E.; Simpson, Edward; Chen, Duojiao; Gao, Hongyu; Liu, Yunlong; Doud, Emma H.; Mosley, Amber L.; Nakshatri, Harikrishna; Surgery, School of Medicine
    Skeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized in vivo skeletal muscle model system to study cancer cachexia.
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    Differential Splicing of Skipped Exons Predicts Drug Response in Cancer Cell Lines
    (Elsevier, 2021-12) Simpson, Edward; Chen, Steven; Reiter, Jill L.; Liu, Yunlong; BioHealth Informatics, School of Informatics and Computing
    Alternative splicing of pre-mRNA transcripts is an important regulatory mechanism that increases the diversity of gene products in eukaryotes. Various studies have linked specific transcript isoforms to altered drug response in cancer; however, few algorithms have incorporated splicing information into drug response prediction. In this study, we evaluated whether basal-level splicing information could be used to predict drug sensitivity by constructing doxorubicin-sensitivity classification models with splicing and expression data. We detailed splicing differences between sensitive and resistant cell lines by implementing quasi-binomial generalized linear modeling (QBGLM) and found altered inclusion of 277 skipped exons. We additionally conducted RNA-binding protein (RBP) binding motif enrichment and differential expression analysis to characterize cis- and trans-acting elements that potentially influence doxorubicin response-mediating splicing alterations. Our results showed that a classification model built with skipped exon data exhibited strong predictive power. We discovered an association between differentially spliced events and epithelial-mesenchymal transition (EMT) and observed motif enrichment, as well as differential expression of RBFOX and ELAVL RBP family members. Our work demonstrates the potential of incorporating splicing data into drug response algorithms and the utility of a QBGLM approach for fast, scalable identification of relevant splicing differences between large groups of samples.
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    Impact of Proinflammatory Cytokines on Alternative Splicing Patterns in Human Islets
    (American Diabetes Association, 2021) Wu, Wenting; Syed, Farooq; Simpson, Edward; Lee, Chih-Chun; Liu, Jing; Chang, Garrick; Dong, Chuanpeng; Seitz, Clayton; Eizirik, Decio L.; Mirmira, Raghavendra G.; Liu, Yunlong; Evans-Molina, Carmella; Medical and Molecular Genetics, School of Medicine
    Alternative splicing (AS) within the β-cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We used a computational strategy to prioritize pathogenic splicing events in human islets treated with interleukin-1β plus interferon-γ as an ex vivo model of T1D and coupled this analysis with a k-mer–based approach to predict RNA-binding proteins involved in AS. In total, 969 AS events were identified in cytokine-treated islets, with a majority (44.8%) involving a skipped exon. ExonImpact identified 129 events predicted to affect protein structure. AS occurred with high frequency in MHC class II–related mRNAs, and targeted quantitative PCR validated reduced inclusion of exon 5 in the MHC class II gene HLA-DMB. Single-molecule RNA fluorescence in situ hybridization confirmed increased HLA-DMB splicing in β-cells from human donors with established T1D and autoantibody positivity. Serine/arginine-rich splicing factor 2 was implicated in 37.2% of potentially pathogenic events, including exon 5 exclusion in HLA-DMB. Together, these data suggest that dynamic control of AS plays a role in the β-cell response to inflammatory signals during T1D evolution.
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    Innate immune activation restricts priming and protective efficacy of the radiation-attenuated PfSPZ malaria vaccine
    (American Society for Clinical Investigation, 2024-04-30) Senkpeil, Leetah; Bhardwaj, Jyoti; Little, Morgan R.; Holla, Prasida; Upadhye, Aditi; Fusco, Elizabeth M.; Swanson, Phillip A., II; Wiegand, Ryan E.; Macklin, Michael D.; Bi, Kevin; Flynn, Barbara J.; Yamamoto, Ayako; Gaskin, Erik L.; Sather, D. Noah; Oblak, Adrian L.; Simpson, Edward; Gao, Hongyu; Haining, W. Nicholas; Yates, Kathleen B.; Liu, Xiaowen; Murshedkar, Tooba; Richie, Thomas L.; Sim, B. Kim Lee; Otieno, Kephas; Kariuki, Simon; Xuei, Xiaoling; Liu, Yunlong; Polidoro, Rafael B.; Hoffman, Stephen L.; Oneko, Martina; Steinhardt, Laura C.; Schmidt, Nathan W.; Seder, Robert A.; Tran, Tuan M.; Medicine, School of Medicine
    A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole-sporozoite PfSPZ vaccine in African infants. Innate immune activation and myeloid signatures at prevaccination baseline correlated with protection from P. falciparum parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ vaccine dose. Machine learning identified spliceosome, proteosome, and resting DC signatures as prevaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline circumsporozoite protein-specific (CSP-specific) IgG predicted nonprotection. Prevaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T cell responses after vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naive mice while diminishing the CD8+ T cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity by whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggests that PfSPZ vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.
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    Intragenic CFTR Duplication and 5T/12TG Variant in a Patient with Non-Classic Cystic Fibrosis
    (SpringerNature, 2016-12-20) Celestino-Soper, Patricia B. S.; Simpson, Edward; Brink, Danika Tumbleson; Lynnes, Ty C.; Dlouhy, Stephen; Vatta, Matteo; Yeley, Jana; Brown, Cynthia; Bai, Shaochun; Department of Medical and Molecular Genetics, IU School of Medicine
    Cystic fibrosis (CF) is an autosomal recessive disorder characterized by the accumulation of sticky and heavy mucus that can damage several organs. CF shows variable expressivity in affected individuals, but it typically causes respiratory and digestive complications as well as congenital bilateral absence of the vas deferens in males. Individuals with classic CF usually have variants that produce a defective protein from both alleles of the CFTR gene. Individuals with other variants may present with classic, non-classic, or milder forms of CF due to lower levels of functional CFTR protein. This article reports the genetic analysis of a female with features of asthma and mild or non-classic CF. CFTR sequencing demonstrated that she is a carrier for a maternally derived 5T/12TG variant. Deletion/duplication analysis by multiplex ligation-dependent probe amplification (MLPA) showed the presence of an intragenic paternally derived duplication involving exons 7-11 of the CFTR gene. This duplication is predicted to result in the production of a truncated CFTR protein lacking the terminal part of the nucleotide-binding domain 1 (NBD1) and thus is likely to be a non-functioning allele. The combination of this large intragenic duplication and 5T/12TG is the probable cause of the mild or non-classic CF features in this individual.
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    MicroRNA sequencing in patients with coronary artery disease - considerations for use as biomarker for thrombotic risk.
    (Wiley, 2022-08) Onuoha, Chimnonso P.; Ipe, Joseph; Simpson, Edward; Liu, Yunlong; Skaar, Todd C.; Kreutz, Rolf P.
    MicroRNAs (miRNAs) are small RNAs integral in the regulation of gene expression. Analysis of circulating miRNA levels may identify patients with coronary artery disease (CAD) at risk for recurrent myocardial infarction (MI) after percutaneous coronary interventions (PCIs). Subjects with CAD were selected from the GENCATH cardiac catheterization biobank. Subjects with recurrent MI after PCI were compared with those without recurrent MI during follow-up in the initial (n = 48) and replication cohort (n = 67). Next generation MiRNA sequencing was performed on plasma samples and whole blood samples fixed with PAXGENE tubes upon collection. Overall, 164 miRNAs derived from whole blood were differentially expressed in the replication cohort between subjects with and without recurrent MI events (p < 0.05), with 69 remaining significant after false-discovery rate (FDR) correction. None of the miRNAs in plasma was significantly different by FDR among subjects with and without MI. Overall, correlation between direction of effects between plasma and whole blood assays was variable, and only two miRNAs were concordant and significant in both. Associations of miRNA with vascular disease, MI, and thrombosis were further explored. MiRNA profiling has potential as the future biomarker for disease prognosis and treatment response marker in secondary treatment of patients with CAD after PCI. Whole blood may be the preferred sample source as compared to plasma.
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    A Multi-Omic Analysis of the Dorsal Striatum in an Animal Model of Divergent Genetic Risk for Alcohol Use Disorder
    (Wiley, 2021) Grecco, Gregory G.; Haggerty, David L.; Doud, Emma H.; Fritz, Brandon M.; Yin, Fuqin; Hoffman, Hunter; Mosley, Amber L.; Simpson, Edward; Liu, Yunlong; Baucum, Anthony J., II.; Atwood, Brady K.; Pharmacology and Toxicology, School of Medicine
    The development of selectively bred high and low alcohol-preferring mice (HAP and LAP, respectively) has allowed for an assessment of the polygenetic risk for pathological alcohol consumption and phenotypes associated with alcohol use disorder (AUD). Accumulating evidence indicates that the dorsal striatum (DS) is a central node in the neurocircuitry underlying addictive processes. Therefore, knowledge of differential gene, protein, and phosphorylated protein expression in the DS of HAP and LAP mice may foster new insights into how aberrant DS functioning may contribute to AUD-related phenotypes. To begin to elucidate these basal differences, a complementary and integrated analysis of DS tissue from alcohol-naïve male and female HAP and LAP mice was performed using RNA sequencing, quantitative proteomics, and phosphoproteomics. These datasets were subjected to a thorough analysis of gene ontology, pathway enrichment, and hub gene assessment. Analyses identified 2,108, 390, and 521 significant differentially expressed genes, proteins, and phosphopeptides, respectively between the two lines. Network analyses revealed an enrichment in the differential expression of genes, proteins, and phosphorylated proteins connected to cellular organization, cytoskeletal protein binding, and pathways involved in synaptic transmission and functioning. These findings suggest that the selective breeding to generate HAP and LAP mice may lead to a rearrangement of synaptic architecture which could alter DS neurotransmission and plasticity differentially between mouse lines. These rich datasets will serve as an excellent resource to inform future studies on how inherited differences in gene, protein, and phosphorylated protein expression contribute to AUD-related phenotypes.
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    Prostaglandin E2 Enhances Aged Hematopoietic Stem Cell Function
    (Springer, 2021-10) Patterson, Andrea M.; Plett, P. Artur; Sampson, Carol H.; Simpson, Edward; Liu, Yunlong; Pelus, Louis M.; Orschell, Christie M.; Medicine, School of Medicine
    Aging of hematopoiesis is associated with increased frequency and clonality of hematopoietic stem cells (HSCs), along with functional compromise and myeloid bias, with donor age being a significant variable in survival after HSC transplantation. No clinical methods currently exist to enhance aged HSC function, and little is known regarding how aging affects molecular responses of HSCs to biological stimuli. Exposure of HSCs from young fish, mice, nonhuman primates, and humans to 16,16-dimethyl prostaglandin E2 (dmPGE2) enhances transplantation, but the effect of dmPGE2 on aged HSCs is unknown. Here we show that ex vivo pulse of bone marrow cells from young adult (3 mo) and aged (25 mo) mice with dmPGE2 prior to serial competitive transplantation significantly enhanced long-term repopulation from aged grafts in primary and secondary transplantation (27 % increase in chimerism) to a similar degree as young grafts (21 % increase in chimerism; both p < 0.05). RNA sequencing of phenotypically-isolated HSCs indicated that the molecular responses to dmPGE2 are similar in young and old, including CREB1 activation and increased cell survival and homeostasis. Common genes within these pathways identified likely key mediators of HSC enhancement by dmPGE2 and age-related signaling differences. HSC expression of the PGE2 receptor EP4, implicated in HSC function, increased with age in both mRNA and surface protein. This work suggests that aging does not alter the major dmPGE2 response pathways in HSCs which mediate enhancement of both young and old HSC function, with significant implications for expanding the therapeutic potential of elderly HSC transplantation.
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    Structural and genome-wide analyses suggest that transposon-derived protein SETMAR alters transcription and splicing
    (Elsevier, 2022) Chen, Qiujia; Bates, Alison M.; Hanquier, Jocelyne N.; Simpson, Edward; Rusch, Douglas B.; Podicheti, Ram; Liu, Yunlong; Wek, Ronald C.; Cornett, Evan M.; Georgiadis, Millie M.; Biochemistry and Molecular Biology, School of Medicine
    Extensive portions of the human genome have unknown function, including those derived from transposable elements. One such element, the DNA transposon Hsmar1, entered the primate lineage approximately 50 million years ago leaving behind terminal inverted repeat (TIR) sequences and a single intact copy of the Hsmar1 transposase, which retains its ancestral TIR-DNA-binding activity, and is fused with a lysine methyltransferase SET domain to constitute the chimeric SETMAR gene. Here, we provide a structural basis for recognition of TIRs by SETMAR and investigate the function of SETMAR through genome-wide approaches. As elucidated in our 2.37 Å crystal structure, SETMAR forms a dimeric complex with each DNA-binding domain bound specifically to TIR-DNA through the formation of 32 hydrogen bonds. We found that SETMAR recognizes primarily TIR sequences (∼5000 sites) within the human genome as assessed by chromatin immunoprecipitation sequencing analysis. In two SETMAR KO cell lines, we identified 163 shared differentially expressed genes and 233 shared alternative splicing events. Among these genes are several pre-mRNA-splicing factors, transcription factors, and genes associated with neuronal function, and one alternatively spliced primate-specific gene, TMEM14B, which has been identified as a marker for neocortex expansion associated with brain evolution. Taken together, our results suggest a model in which SETMAR impacts differential expression and alternative splicing of genes associated with transcription and neuronal function, potentially through both its TIR-specific DNA-binding and lysine methyltransferase activities, consistent with a role for SETMAR in simian primate development.
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    A Treatment to Eliminate SARS-CoV-2 Replication in Human Airway Epithelial Cells Is Safe for Inhalation as an Aerosol in Healthy Human Subjects
    (American Association for Respiratory Care (AARC), 2020-09-21) Davis, Michael D.; Clemente, Tatiana M.; Giddings, Olivia K.; Ross, Kristie; Cunningham, Rebekah S.; Smith, Laura; Simpson, Edward; Liu, Yunlong; Kloepfer, Kirsten; Ramsey, I. Scott; Zhao, Yi; Robinson, Christopher M.; Gilk, Stacey D.; Gaston, Benjamin; Pediatrics, School of Medicine
    Background: Low airway surface pH is associated with many airway diseases, impairs antimicrobial host defense and worsens airway inflammation. Inhaled Optate is designed to safely to raise airway surface pH and is well-tolerated in humans. Raising intracellular pH partially prevents activation of SARS-CoV-2 in primary normal human airway epithelial (NHAE) cells, decreasing viral replication by several mechanisms. Methods: Here, we grew primary normal human airway epithelial (NHAE) cells from healthy subjects, infected them with SARS-CoV-2 (isolate USA-WA1/2020), and used clinical Optate at concentrations used in humans in vivo to determine whether it would prevent viral infection and replication. Cells were pre-treated with Optate or placebo prior to infection (MOI of 0.1) and viral replication was determined by plaque assay and nucleocapsid (N) protein levels. Healthy human subjects also inhaled Optate as part of a Phase 2a safety trial. Results: Optate almost completely prevented viral replication at each time point between 24 and 120 hours, relative to placebo, both by plaque assay and by N protein expression (p < 0.001). Mechanistically, Optate inhibited expression of major endosomal trafficking genes and raised NHAE intracellular pH. Optate had no effect on NHAE cell viability at any time point. Inhaled Optate was well tolerated in 10 normal subjects, with no change in lung function, vital signs or oxygenation. Conclusions: Inhaled Optate may be well-suited for a clinical trial in patients with a pulmonary SARS-CoV-2 infection. However, it is vitally important for patient safety that formulations designed for inhalation with regards to pH, isotonicity and osmolality be used. An inhalational treatment that safely prevents SARS-CoV-2 viral replication could be helpful for treating patients with pulmonary SARS-CoV-2 infection.