90 results on '"Chiang DY"'
Search Results
2. Von Hippel-Lindau status influences phenotype of liver cancers arising from PTEN loss
- Author
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Sendor AB, Hacker KE, Chen S, Corona AL, Sen O, Chiang DY, Snavely A, Rogers AB, Montgomery SA, Rathmell WK, and McRee AJ
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lcsh:Diseases of the digestive system. Gastroenterology ,lcsh:RC799-869 ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,lcsh:RC254-282 - Abstract
Adam B Sendor,1 Kathryn E Hacker,1 Shufen Chen,1 Armando L Corona,1 Oishee Sen,1 Derek Y Chiang,1 Anna Snavely,1 Arlin B Rogers,2 Stephanie A Montgomery,1 W Kimryn Rathmell,1 Autumn J McRee11Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA; 2Section of Pathology, Department of Biomedical Sciences, Cummings School of Veterinary Medicine, Tufts University, Boston, MA, USABackground: PTEN loss contributes to the development of liver diseases including hepatic steatosis and both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The factors that influence the penetrance of these conditions are unclear. We explored the influence of sustained hypoxia signaling through co-deletion of Pten and Vhl in a murine model.Methods: We used a CreER-linked Keratin 18 mouse model to conditionally delete Pten, Vhl or both in somatic cells of adult mice, evaluating the resultant tumors by histology and gene expression microarray. Existing sets of gene expression data for human HCC and CC were examined for pathways related to those observed in the murine tumors, and a cohort of human CC samples was evaluated for relationships between HIF-1α expression and clinical outcomes.Results: Both Pten deletion genotypes developed liver tumors, but with differing phenotypes. Pten deletion alone led to large hepatic tumors with widespread hepatosteatosis. Co-deletion of Pten and Vhl with the Keratin 18 promoter resulted in reduced steatosis and a reduced tumor burden that was characterized by a trabecular architecture similar to CC. Genes associated with hepatic steatosis were coordinately expressed in the human HCC dataset, while genes involved in hypoxia response were upregulated in tumors from the human CC dataset. HIF-1α expression and overall survival were examined in an independent cohort of human CC tumors with no statistical differences uncovered.Conclusion: Pten deletion in Keratin 18 expressing cells leads to aggressive tumor formation and widespread steatosis in mouse livers. Co-deletion of Vhl and Pten results in lower tumor burden with gene expression profiling suggesting a switch from a profile of lipid deposition to an expression profile more consistent with upregulation of the hypoxia response pathway. A relationship between tumor hypoxia signaling and altered hepatic steatotic response suggests that competing influences may alter tumor phenotypes.Keywords: Von Hippel-Lindau (VHL), phosphatase and tension homologue deleted on chromosome 10 (PTEN), cholangiocarcinoma (CC), hepatocellular-cholangiocarcinoma (HCC)
- Published
- 2015
3. Single-Cell Analysis Identifies NOTCH3-Mediated Interactions between Stromal Cells That Promote Microenvironment Remodeling and Invasion in Lung Adenocarcinoma.
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Xiang H, Pan Y, Sze MA, Wlodarska M, Li L, van de Mark KA, Qamar H, Moure CJ, Linn DE, Hai J, Huo Y, Clarke J, Tan TG, Ho S, Teng KW, Ramli MN, Nebozhyn M, Zhang C, Barlow J, Gustafson CE, Gornisiewicz S, Albertson TP, Korle SL, Bueno R, Moy LY, Vollmann EH, Chiang DY, Brandish PE, and Loboda A
- Subjects
- Humans, Cell Communication, Signal Transduction, Adenocarcinoma of Lung pathology, Adenocarcinoma of Lung metabolism, Adenocarcinoma of Lung genetics, Cancer-Associated Fibroblasts metabolism, Cancer-Associated Fibroblasts pathology, Lung Neoplasms pathology, Lung Neoplasms metabolism, Lung Neoplasms genetics, Neoplasm Invasiveness, Receptor, Notch3 metabolism, Receptor, Notch3 genetics, Single-Cell Analysis, Stromal Cells metabolism, Stromal Cells pathology, Tumor Microenvironment
- Abstract
Cancer immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD); however, a significant proportion of patients do not respond. Recent transcriptomic studies to understand determinants of immunotherapy response have pinpointed stromal-mediated resistance mechanisms. To gain a better understanding of stromal biology at the cellular and molecular level in LUAD, we performed single-cell RNA sequencing of 256,379 cells, including 13,857 mesenchymal cells, from 9 treatment-naïve patients. Among the mesenchymal cell subsets, FAP+PDPN+ cancer-associated fibroblasts (CAF) and ACTA2+MCAM+ pericytes were enriched in tumors and differentiated from lung-resident fibroblasts. Imaging mass cytometry revealed that both subsets were topographically adjacent to the perivascular niche and had close spatial interactions with endothelial cells (EC). Modeling of ligand and receptor interactomes between mesenchymal and ECs identified that NOTCH signaling drives these cell-to-cell interactions in tumors, with pericytes and CAFs as the signal receivers and arterial and PLVAPhigh immature neovascular ECs as the signal senders. Either pharmacologically blocking NOTCH signaling or genetically depleting NOTCH3 levels in mesenchymal cells significantly reduced collagen production and suppressed cell invasion. Bulk RNA sequencing data demonstrated that NOTCH3 expression correlated with poor survival in stroma-rich patients and that a T cell-inflamed gene signature only predicted survival in patients with low NOTCH3. Collectively, this study provides valuable insights into the role of NOTCH3 in regulating tumor stroma biology, warranting further studies to elucidate the clinical implications of targeting NOTCH3 signaling., Significance: NOTCH3 signaling activates tumor-associated mesenchymal cells, increases collagen production, and augments cell invasion in lung adenocarcinoma, suggesting its critical role in remodeling tumor stroma., (©2024 The Authors; Published by the American Association for Cancer Research.)
- Published
- 2024
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4. The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction.
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Chiang DY, Verkerk AO, Victorio R, Shneyer BI, van der Vaart B, Jouni M, Narendran N, Kc A, Sampognaro JR, Vetrano-Olsen F, Oh JS, Buys E, de Jonge B, Shah DA, Kiviniemi T, Burridge PW, Bezzina CR, Akhmanova A, and MacRae CA
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- Animals, Humans, Action Potentials, Genome-Wide Association Study, Microtubule-Associated Proteins genetics, Microtubules metabolism, Myocytes, Cardiac metabolism, NAV1.5 Voltage-Gated Sodium Channel genetics, NAV1.5 Voltage-Gated Sodium Channel metabolism, Tubulin genetics, Tubulin metabolism, Zebrafish genetics, Zebrafish metabolism, Brugada Syndrome genetics, Brugada Syndrome metabolism, Induced Pluripotent Stem Cells metabolism, Voltage-Gated Sodium Channels metabolism
- Abstract
Background: Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2 , which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome., Methods: A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively., Results: Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential., Conclusions: Genetic ablation of mapre2 led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction., Competing Interests: Disclosures None.
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- 2024
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5. Modeling of large-scale hoxbb cluster deletions in zebrafish uncovers a role for segmentation pathways in atrioventricular boundary specification.
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Hu P, Wang B, Jin D, Gu Y, He H, Meng X, Zhu W, Chiang DY, Li W, MacRae CA, and Zu Y
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- Animals, Humans, Heart, Transcription Factors metabolism, Gene Expression Regulation, Developmental, Zebrafish genetics, Zebrafish metabolism, Zebrafish Proteins genetics, Zebrafish Proteins metabolism
- Abstract
Hox genes orchestrate the segmental specification of the muscular circulatory system in invertebrates but it has not proven straightforward to decipher segmental parallels in the vertebrate heart. Recently, patients with HOXB gene cluster deletion were found to exhibit abnormalities including atrioventricular canal defects. Using CRISPR, we established a mutant with the orthologous hoxbb cluster deletion in zebrafish. The mutant exhibited heart failure and atrioventricular regurgitation at 5 days. Analyzing the four genes in the hoxbb cluster, isolated deletion of hoxb1b
-/- recapitulated the cardiac abnormalities, supporting hoxb1b as the causal gene. Both in situ and in vitro data indicated that hoxb1b regulates gata5 to inhibit hand2 expression and ultimately is required to pattern the vertebrate atrioventricular boundary. Together, these data reveal a role for segmental specification in vertebrate cardiac development and highlight the utility of CRISPR techniques for efficiently exploring the function of large structural genomic lesions., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)- Published
- 2023
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6. Outlining cardiac ion channel protein interactors and their signature in the human electrocardiogram.
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Maurya S, Mills RW, Kahnert K, Chiang DY, Bertoli G, Lundegaard PR, Duran MP, Zhang M, Rothenberg E, George AL Jr, MacRae CA, Delmar M, and Lundby A
- Abstract
Protein-protein interactions are essential for normal cellular processes and signaling events. Defining these interaction networks is therefore crucial for understanding complex cellular functions and interpretation of disease-associated gene variants. We need to build a comprehensive picture of the interactions, their affinities and interdependencies in the specific organ to decipher hitherto poorly understood signaling mechanisms through ion channels. Here we report the experimental identification of the ensemble of protein interactors for 13 types of ion channels in murine cardiac tissue. Of these, we validated the functional importance of ten interactors on cardiac electrophysiology through genetic knockouts in zebrafish, gene silencing in mice, super-resolution microscopy and patch clamp experiments. Furthermore, we establish a computational framework to reconstruct human cardiomyocyte ion channel networks from deep proteome mapping of human heart tissue and human heart single-cell gene expression data. Finally, we integrate the ion channel interactome with human population genetics data to identify proteins that influence the electrocardiogram (ECG). We demonstrate that the combined channel network is enriched for proteins influencing the ECG, with 44% of the network proteins significantly associated with an ECG phenotype. Altogether, we define interactomes of 13 major cardiac ion channels, contextualize their relevance to human electrophysiology and validate functional roles of ten interactors, including two regulators of the sodium current (epsin-2 and gelsolin). Overall, our data provide a roadmap for our understanding of the molecular machinery that regulates cardiac electrophysiology., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2023.)
- Published
- 2023
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7. Atrial Standstill in the Pediatric Population: A Multi-Institution Collaboration.
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Howard TS, Chiang DY, Ceresnak SR, Ladouceur VB, Whitehill RD, Czosek RJ, Knilans TK, Ahnfeldt AM, Borresen ML, Jaeggi E, Udupa S, Gow R, Moore JP, Galloti RG, Mah DY, Kim JJ, Valdes SO, Milewicz DM, and Miyake CY
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- Humans, Child, Child, Preschool, Heart Atria diagnostic imaging, Heart Block, Anticoagulants, Atrial Fibrillation, Heart Arrest
- Abstract
Background: Atrial standstill (AS) is a rare condition characterized by absence of electrical activity within the atria. Studies to date have been limited., Objectives: The authors sought to describe the clinical characteristics, genetics, and outcomes of patients with AS., Methods: This was a retrospective multicenter study of patients <18 years at AS diagnosis, defined as absence of atrial activity documented during an electrophysiology study, device placement, or noninvasive rhythm tracings and confirmed by echocardiogram. Patients with acquired disorders were excluded. Clinical details and genetic variants were recorded and analyzed., Results: Twenty patients were diagnosed at a median age of 6.6 years (IQR: 2.9-10.8 years). Arrhythmias included 16 (80%) with atrial/supraventricular arrhythmias and 8 (40%) with ventricular tachycardia, including 4 with cardiac arrests. A type 1 Brugada pattern was documented in 4. Pacemakers were implanted in 18 (90%). Although atrial leads were attempted in 15, only 4 achieved pacing at implantation. During a median follow-up of 6.9 years (IQR: 1.2-13.3 years), 7 (35%) had thromboembolic events. Of these, none had atrial pacing, 6 were not on anticoagulation, and 1 was on aspirin. Genetic testing identified SCN5A variants in 13 patients (65%). Analyses suggest SCN5A loss-of-function may be one mechanism driving AS. Ventricular arrhythmias and cardiac arrest were more commonly seen in patients with biallelic SCN5A variants., Conclusions: AS may be associated with loss-of-function SCN5A variants. Patients demonstrate atrial and ventricular arrhythmias, and may present challenges during device placement. Patients without the capacity for atrial pacing are at risk for thromboembolic events and warrant anticoagulation., Competing Interests: Funding Support and Author Disclosures Dr Miyake is supported by National Heart, Lung, and Blood Institute grant K23HL136932. Dr Chiang is supported by the Leducq Foundation and a National Institutes of Health T32 grant from the National Human Genome Research Institute (#1T32HG010464). The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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8. Parallel single-cell and bulk transcriptome analyses reveal key features of the gastric tumor microenvironment.
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Kang B, Camps J, Fan B, Jiang H, Ibrahim MM, Hu X, Qin S, Kirchhoff D, Chiang DY, Wang S, Ye Y, Shen Z, Bu Z, Zhang Z, and Roider HG
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- Humans, Endothelial Cells, Tumor Microenvironment, Gene Expression Profiling, Transcriptome, Single-Cell Analysis, Stomach Neoplasms genetics
- Abstract
Background: The tumor microenvironment (TME) has been shown to strongly influence treatment outcome for cancer patients in various indications and to influence the overall survival. However, the cells forming the TME in gastric cancer have not been extensively characterized., Results: We combine bulk and single-cell RNA sequencing from tumors and matched normal tissue of 24 treatment-naïve GC patients to better understand which cell types and transcriptional programs are associated with malignant transformation of the stomach. Clustering 96,623 cells of non-epithelial origin reveals 81 well-defined TME cell types. We find that activated fibroblasts and endothelial cells are most prominently overrepresented in tumors. Intercellular network reconstruction and survival analysis of an independent cohort imply the importance of these cell types together with immunosuppressive myeloid cell subsets and regulatory T cells in establishing an immunosuppressive microenvironment that correlates with worsened prognosis and lack of response in anti-PD1-treated patients. In contrast, we find a subset of IFNγ activated T cells and HLA-II expressing macrophages that are linked to treatment response and increased overall survival., Conclusions: Our gastric cancer single-cell TME compendium together with the matched bulk transcriptome data provides a unique resource for the identification of new potential biomarkers for patient stratification. This study helps further to elucidate the mechanism of gastric cancer and provides insights for therapy., (© 2022. The Author(s).)
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- 2022
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9. Author Correction: Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.
- Author
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Barc J, Tadros R, Glinge C, Chiang DY, Jouni M, Simonet F, Jurgens SJ, Baudic M, Nicastro M, Potet F, Offerhaus JA, Walsh R, Choi SH, Verkerk AO, Mizusawa Y, Anys S, Minois D, Arnaud M, Duchateau J, Wijeyeratne YD, Muir A, Papadakis M, Castelletti S, Torchio M, Ortuño CG, Lacunza J, Giachino DF, Cerrato N, Martins RP, Campuzano O, Van Dooren S, Thollet A, Kyndt F, Mazzanti A, Clémenty N, Bisson A, Corveleyn A, Stallmeyer B, Dittmann S, Saenen J, Noël A, Honarbakhsh S, Rudic B, Marzak H, Rowe MK, Federspiel C, Le Page S, Placide L, Milhem A, Barajas-Martinez H, Beckmann BM, Krapels IP, Steinfurt J, Winkel BG, Jabbari R, Shoemaker MB, Boukens BJ, Škorić-Milosavljević D, Bikker H, Manevy F, Lichtner P, Ribasés M, Meitinger T, Müller-Nurasyid M, Veldink JH, van den Berg LH, Van Damme P, Cusi D, Lanzani C, Rigade S, Charpentier E, Baron E, Bonnaud S, Lecointe S, Donnart A, Le Marec H, Chatel S, Karakachoff M, Bézieau S, London B, Tfelt-Hansen J, Roden D, Odening KE, Cerrone M, Chinitz LA, Volders PG, van de Berg MP, Laurent G, Faivre L, Antzelevitch C, Kääb S, Arnaout AA, Dupuis JM, Pasquie JL, Billon O, Roberts JD, Jesel L, Borggrefe M, Lambiase PD, Mansourati J, Loeys B, Leenhardt A, Guicheney P, Maury P, Schulze-Bahr E, Robyns T, Breckpot J, Babuty D, Priori SG, Napolitano C, de Asmundis C, Brugada P, Brugada R, Arbelo E, Brugada J, Mabo P, Behar N, Giustetto C, Molina MS, Gimeno JR, Hasdemir C, Schwartz PJ, Crotti L, McKeown PP, Sharma S, Behr ER, Haissaguerre M, Sacher F, Rooryck C, Tan HL, Remme CA, Postema PG, Delmar M, Ellinor PT, Lubitz SA, Gourraud JB, Tanck MW, George AL Jr, MacRae CA, Burridge PW, Dina C, Probst V, Wilde AA, Schott JJ, Redon R, and Bezzina CR
- Published
- 2022
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10. Reverse Translating Molecular Determinants of Anti-Programmed Death 1 Immunotherapy Response in Mouse Syngeneic Tumor Models.
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Georgiev P, Muise ES, Linn DE, Hinton MC, Wang Y, Cai M, Cadzow L, Wilson DC, Sukumar S, Caniga M, Chen L, Xiao H, Yearley JH, Sriram V, Nebozhyn M, Sathe M, Blumenschein WM, Kerr KS, Hirsch HA, Javaid S, Olow AK, Moy LY, Chiang DY, Loboda A, Cristescu R, Sadekova S, Long BJ, McClanahan TK, and Pinheiro EM
- Subjects
- Animals, Biomarkers, Tumor genetics, Cell Line, Tumor, Disease Models, Animal, Humans, Immune Checkpoint Inhibitors, Mice, Tumor Microenvironment, B7-H1 Antigen antagonists & inhibitors, Immunotherapy, Neoplasms drug therapy, Neoplasms genetics, Programmed Cell Death 1 Receptor antagonists & inhibitors
- Abstract
Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell-inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell-inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFβ biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies., (©2021 The Authors; Published by the American Association for Cancer Research.)
- Published
- 2022
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11. Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.
- Author
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Barc J, Tadros R, Glinge C, Chiang DY, Jouni M, Simonet F, Jurgens SJ, Baudic M, Nicastro M, Potet F, Offerhaus JA, Walsh R, Choi SH, Verkerk AO, Mizusawa Y, Anys S, Minois D, Arnaud M, Duchateau J, Wijeyeratne YD, Muir A, Papadakis M, Castelletti S, Torchio M, Ortuño CG, Lacunza J, Giachino DF, Cerrato N, Martins RP, Campuzano O, Van Dooren S, Thollet A, Kyndt F, Mazzanti A, Clémenty N, Bisson A, Corveleyn A, Stallmeyer B, Dittmann S, Saenen J, Noël A, Honarbakhsh S, Rudic B, Marzak H, Rowe MK, Federspiel C, Le Page S, Placide L, Milhem A, Barajas-Martinez H, Beckmann BM, Krapels IP, Steinfurt J, Winkel BG, Jabbari R, Shoemaker MB, Boukens BJ, Škorić-Milosavljević D, Bikker H, Manevy F, Lichtner P, Ribasés M, Meitinger T, Müller-Nurasyid M, Veldink JH, van den Berg LH, Van Damme P, Cusi D, Lanzani C, Rigade S, Charpentier E, Baron E, Bonnaud S, Lecointe S, Donnart A, Le Marec H, Chatel S, Karakachoff M, Bézieau S, London B, Tfelt-Hansen J, Roden D, Odening KE, Cerrone M, Chinitz LA, Volders PG, van de Berg MP, Laurent G, Faivre L, Antzelevitch C, Kääb S, Arnaout AA, Dupuis JM, Pasquie JL, Billon O, Roberts JD, Jesel L, Borggrefe M, Lambiase PD, Mansourati J, Loeys B, Leenhardt A, Guicheney P, Maury P, Schulze-Bahr E, Robyns T, Breckpot J, Babuty D, Priori SG, Napolitano C, de Asmundis C, Brugada P, Brugada R, Arbelo E, Brugada J, Mabo P, Behar N, Giustetto C, Molina MS, Gimeno JR, Hasdemir C, Schwartz PJ, Crotti L, McKeown PP, Sharma S, Behr ER, Haissaguerre M, Sacher F, Rooryck C, Tan HL, Remme CA, Postema PG, Delmar M, Ellinor PT, Lubitz SA, Gourraud JB, Tanck MW, George AL Jr, MacRae CA, Burridge PW, Dina C, Probst V, Wilde AA, Schott JJ, Redon R, and Bezzina CR
- Subjects
- Alleles, Disease Susceptibility complications, Genetic Predisposition to Disease, Genome-Wide Association Study, Humans, Microtubule-Associated Proteins genetics, Mutation, NAV1.5 Voltage-Gated Sodium Channel genetics, NAV1.5 Voltage-Gated Sodium Channel metabolism, Young Adult, Brugada Syndrome complications, Brugada Syndrome genetics, Brugada Syndrome metabolism
- Abstract
Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel Na
V 1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV 1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)- Published
- 2022
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12. Targeting the Microtubule EB1-CLASP2 Complex Modulates Na V 1.5 at Intercalated Discs.
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Marchal GA, Jouni M, Chiang DY, Pérez-Hernández M, Podliesna S, Yu N, Casini S, Potet F, Veerman CC, Klerk M, Lodder EM, Mengarelli I, Guan K, Vanoye CG, Rothenberg E, Charpentier F, Redon R, George AL Jr, Verkerk AO, Bezzina CR, MacRae CA, Burridge PW, Delmar M, Galjart N, Portero V, and Remme CA
- Subjects
- Action Potentials, Animals, Arrhythmias, Cardiac metabolism, Cells, Cultured, Glycogen Synthase Kinase 3 beta metabolism, HEK293 Cells, Humans, Loss of Function Mutation, Male, Mice, Mice, Inbred C57BL, Microtubule-Associated Proteins genetics, Myocytes, Cardiac physiology, NAV1.5 Voltage-Gated Sodium Channel genetics, Protein Transport, Zebrafish, Arrhythmias, Cardiac genetics, Microtubule-Associated Proteins metabolism, Myocytes, Cardiac metabolism, NAV1.5 Voltage-Gated Sodium Channel metabolism
- Abstract
[Figure: see text].
- Published
- 2021
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13. Phosphorylation-Dependent Interactome of Ryanodine Receptor Type 2 in the Heart.
- Author
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Chiang DY, Lahiri S, Wang G, Karch J, Wang MC, Jung SY, Heck AJR, Scholten A, and Wehrens XHT
- Abstract
Hyperphosphorylation of the calcium release channel/ryanodine receptor type 2 (RyR2) at serine 2814 (S2814) is associated with multiple cardiac diseases including atrial fibrillation and heart failure. Despite recent advances, the molecular mechanisms driving pathological changes associated with RyR2 S2814 phosphorylation are still not well understood. Methods: Using affinity-purification coupled to mass spectrometry (AP-MS), we investigated the RyR2 interactome in ventricles from wild-type (WT) mice and two S2814 knock-in mutants: the unphosphorylated alanine mutant (S2814A) and hyperphosphorylated mimic aspartic acid mutant (S2814D). Western blots were used for validation. Results: In WT mouse ventricular lysates, we identified 22 proteins which were enriched with RyR2 pull-down relative to both IgG control and no antibody (beads-only) pull-downs. Parallel AP-MS using WT, S2814A, and S2814D mouse ventricles identified 72 proteins, with 20 being high confidence RyR2 interactors. Of these, 14 had an increase in their binding to RyR2 S2814A but a decrease in their binding to RyR2 S2814D. We independently validated three protein hits, Idh3b, Aifm1, and Cpt1b, as RyR2 interactors by western blots and showed that Aifm1 and Idh3b had significantly decreased binding to RyR2 S2814D compared to WT and S2814A, consistent with MS findings. Conclusion: By applying state-of-the-art proteomic approaches, we discovered a number of novel RyR2 interactors in the mouse heart. In addition, we found and defined specific alterations in the RyR2 interactome that were dependent on the phosphorylation status of RyR2 at S2814. These findings yield mechanistic insights into RyR2 regulation which may guide future drug designs.
- Published
- 2021
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14. ILT3 (LILRB4) Promotes the Immunosuppressive Function of Tumor-Educated Human Monocytic Myeloid-Derived Suppressor Cells.
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Singh L, Muise ES, Bhattacharya A, Grein J, Javaid S, Stivers P, Zhang J, Qu Y, Joyce-Shaikh B, Loboda A, Zhang C, Meehl M, Chiang DY, Ranganath SH, Rosenzweig M, and Brandish PE
- Subjects
- Animals, Female, Heterografts, Humans, Melanoma metabolism, Membrane Glycoproteins metabolism, Mice, Monocytes metabolism, Myeloid-Derived Suppressor Cells metabolism, Receptors, Immunologic metabolism, Melanoma immunology, Membrane Glycoproteins immunology, Monocytes immunology, Myeloid-Derived Suppressor Cells immunology, Receptors, Immunologic immunology
- Abstract
Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro . We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8
+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion. IMPLICATIONS: These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors., (©2020 American Association for Cancer Research.)- Published
- 2021
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15. The endosomal trafficking regulator LITAF controls the cardiac Nav1.5 channel via the ubiquitin ligase NEDD4-2.
- Author
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Turan NN, Moshal KS, Roder K, Baggett BC, Kabakov AY, Dhakal S, Teramoto R, Chiang DY, Zhong M, Xie A, Lu Y, Dudley SC Jr, MacRae CA, Karma A, and Koren G
- Subjects
- Action Potentials, Animals, Genome-Wide Association Study, Humans, Myocytes, Cardiac cytology, NAV1.5 Voltage-Gated Sodium Channel genetics, Nedd4 Ubiquitin Protein Ligases genetics, Nuclear Proteins genetics, Protein Binding, Protein Transport, Rabbits, Transcription Factors genetics, Ubiquitination, Zebrafish, Endosomes metabolism, Myocytes, Cardiac metabolism, NAV1.5 Voltage-Gated Sodium Channel metabolism, Nedd4 Ubiquitin Protein Ligases metabolism, Nuclear Proteins metabolism, Transcription Factors metabolism, Ubiquitin metabolism
- Abstract
The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current I
Na and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on INa We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Turan et al.)- Published
- 2020
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16. Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases.
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Hayashi K, Teramoto R, Nomura A, Asano Y, Beerens M, Kurata Y, Kobayashi I, Fujino N, Furusho H, Sakata K, Onoue K, Chiang DY, Kiviniemi TO, Buys E, Sips P, Burch ML, Zhao Y, Kelly AE, Namura M, Kita Y, Tsuchiya T, Kaku B, Oe K, Takeda Y, Konno T, Inoue M, Fujita T, Kato T, Funada A, Tada H, Hodatsu A, Nakanishi C, Sakamoto Y, Tsuda T, Nagata Y, Tanaka Y, Okada H, Usuda K, Cui S, Saito Y, MacRae CA, Takashima S, Yamagishi M, Kawashiri MA, and Takamura M
- Subjects
- Action Potentials genetics, Adult, Age of Onset, Aged, Animals, Cardiac Conduction System Disease epidemiology, Cardiac Conduction System Disease metabolism, Cardiac Conduction System Disease physiopathology, Case-Control Studies, Computer Simulation, ERG1 Potassium Channel genetics, Female, Gene Frequency, Genetic Association Studies, Genetic Predisposition to Disease, Humans, Japan epidemiology, Lamin Type A genetics, Male, Membrane Proteins genetics, Middle Aged, Models, Cardiovascular, Myocytes, Cardiac metabolism, NAV1.5 Voltage-Gated Sodium Channel genetics, NAV1.8 Voltage-Gated Sodium Channel genetics, Nuclear Proteins genetics, Phenotype, Predictive Value of Tests, Risk Assessment, Risk Factors, Young Adult, Zebrafish genetics, Zebrafish metabolism, Zebrafish Proteins genetics, Zebrafish Proteins metabolism, Cardiac Conduction System Disease genetics, Genetic Variation, Heart Rate genetics, Exome Sequencing
- Abstract
Aims: The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them., Methods and Results: We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analysed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as 'pathogenic' by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that two variants in KCNH2 and SCN5A, four variants in SCN10A, and one variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from 'Uncertain significance' to 'Likely pathogenic' in six probands., Conclusion: Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2020
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17. Loss of SPEG Inhibitory Phosphorylation of Ryanodine Receptor Type-2 Promotes Atrial Fibrillation.
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Campbell HM, Quick AP, Abu-Taha I, Chiang DY, Kramm CF, Word TA, Brandenburg S, Hulsurkar M, Alsina KM, Liu HB, Martin B, Uhlenkamp D, Moore OM, Lahiri SK, Corradini E, Kamler M, Heck AJR, Lehnart SE, Dobrev D, and Wehrens XHT
- Subjects
- Animals, Atrial Fibrillation genetics, Female, Humans, Male, Mice, Mice, Knockout, Muscle Proteins genetics, Myosin-Light-Chain Kinase genetics, Phosphorylation, Protein Serine-Threonine Kinases genetics, Ryanodine Receptor Calcium Release Channel genetics, Sarcoplasmic Reticulum genetics, Sarcoplasmic Reticulum metabolism, Atrial Fibrillation metabolism, Calcium Signaling, Muscle Proteins metabolism, Myocardium metabolism, Myosin-Light-Chain Kinase metabolism, Protein Serine-Threonine Kinases metabolism, Ryanodine Receptor Calcium Release Channel metabolism
- Abstract
Background: Enhanced diastolic calcium (Ca
2+ ) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca2+ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca2+ /calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis., Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca2+ leak in atrial cardiomyocytes was assessed using confocal Ca2+ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9., Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca2+ spark frequency in atrial cardiomyocytes with Ca2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca2+ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF., Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca2+ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.- Published
- 2020
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18. A community-based lung cancer rapid tissue donation protocol provides high-quality drug-resistant specimens for proteogenomic analyses.
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Boyle TA, Quinn GP, Schabath MB, Muñoz-Antonia T, Saller JJ, Duarte LF, Hair LS, Teer JK, Chiang DY, Leary R, Wong CC, Savchenko A, Singh AP, Charette L, Mendell K, Gorgun G, Antonia SJ, Chiappori AA, Creelan BC, Gray JE, and Haura EB
- Subjects
- Adenocarcinoma of Lung genetics, Adenocarcinoma of Lung mortality, Adenocarcinoma of Lung pathology, Aged, B7-H1 Antigen genetics, Biomarkers, Tumor analysis, Evolution, Molecular, Female, Florida, Genetic Heterogeneity, High-Throughput Nucleotide Sequencing, Humans, Immunohistochemistry, Lung pathology, Lung Neoplasms genetics, Lung Neoplasms mortality, Lung Neoplasms pathology, Male, Middle Aged, Oncogene Proteins, Fusion genetics, Phosphotransferases (Alcohol Group Acceptor) genetics, Proto-Oncogene Proteins B-raf genetics, Adenocarcinoma of Lung drug therapy, Biomarkers, Tumor genetics, Community-Based Participatory Research methods, Drug Resistance, Neoplasm genetics, Lung Neoplasms drug therapy, Tissue and Organ Procurement methods
- Abstract
Background: For the advancement of cancer research, the collection of tissue specimens from drug-resistant tumors after targeted therapy is crucial. Although patients with lung cancer are often provided targeted therapy, post-therapy specimens are not routinely collected due to the risks of collection, limiting the study of targeted therapy resistance mechanisms. Posthumous rapid tissue donation (RTD) is an expedient collection process that provides an opportunity to understand treatment-resistant lung cancers., Methods: Consent to participate in the thoracic RTD protocol was obtained during patient care. When death occurred, tumor and paired non-tumor, cytology, and blood specimens were collected within 48 hours and preserved as formalin-fixed and frozen specimens. Tissue sections were evaluated with hematoxylin and eosin staining and immunohistochemistry (IHC) against multiple biomarkers, including various programmed death ligand 1 (PD-L1) clones. Next-generation sequencing was performed on 13 specimens from 5 patients., Results: Postmortem specimens (N = 180) were well preserved from 9 patients with lung cancer. PD-L1 IHC revealed heterogeneity within and between tumors. An AGK-BRAF fusion was newly identified in tumor from a donor with a known echinoderm microtubule-associated protein-like 4 to anaplastic lymphoma kinase (EML4-ALK) fusion and history of anaplastic lymphoma kinase (ALK) inhibitor therapy. RNA expression analysis revealed a clonal genetic origin of metastatic cancer cells., Conclusions: Post-therapy specimens demonstrated PD-L1 heterogeneity and an acyl glycerol kinase to B-rapidly accelerated fibrosarcoma (AGK-BRAF) fusion in a patient with an EML4-ALK-positive lung adenocarcinoma as a potential resistance mechanism to ALK inhibitor therapy. Rapid tissue donation collection of postmortem tissue from lung cancer patients is a novel approach to cancer research that enables studies of molecular evolution and drug resistance., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)
- Published
- 2020
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19. LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase.
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Moshal KS, Roder K, Kabakov AY, Werdich AA, Chiang DY, Turan NN, Xie A, Kim TY, Cooper LL, Lu Y, Zhong M, Li W, Terentyev D, Choi BR, Karma A, MacRae CA, and Koren G
- Subjects
- Animals, Calcium metabolism, Calcium Channels, L-Type genetics, DNA-Binding Proteins genetics, Heart embryology, Humans, Membrane Proteins genetics, Myocytes, Cardiac enzymology, Nedd4 Ubiquitin Protein Ligases genetics, Nuclear Proteins genetics, Nuclear Proteins metabolism, Rabbits, Transcription Factors genetics, Transcription Factors metabolism, Ubiquitination, Zebrafish genetics, Zebrafish growth & development, Zebrafish Proteins genetics, Calcium Channels, L-Type metabolism, DNA-Binding Proteins metabolism, Membrane Proteins metabolism, Nedd4 Ubiquitin Protein Ligases metabolism, Zebrafish metabolism, Zebrafish Proteins metabolism
- Abstract
Background: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation., Methods: Optical mapping was performed in zebrafish hearts to determine Ca
2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+ handling. L-type calcium channel (LTCC) current ( ICa,L ) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay., Results: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD ( neural precursor cell expressed developmentally downregulated protein ) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes., Conclusions: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.- Published
- 2019
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20. Loss of Protein Phosphatase 1 Regulatory Subunit PPP1R3A Promotes Atrial Fibrillation.
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Alsina KM, Hulsurkar M, Brandenburg S, Kownatzki-Danger D, Lenz C, Urlaub H, Abu-Taha I, Kamler M, Chiang DY, Lahiri SK, Reynolds JO, Quick AP, Scott L Jr, Word TA, Gelves MD, Heck AJR, Li N, Dobrev D, Lehnart SE, and Wehrens XHT
- Subjects
- Animals, Atrial Fibrillation genetics, Calcium metabolism, Calcium-Binding Proteins metabolism, Cells, Cultured, Disease Models, Animal, Disease Susceptibility, Humans, Mice, Mice, Knockout, Phosphoprotein Phosphatases genetics, Protein Phosphatase 1 metabolism, Proteomics, Ryanodine Receptor Calcium Release Channel metabolism, Sarcoplasmic Reticulum metabolism, Signal Transduction, Atrial Fibrillation metabolism, Myocytes, Cardiac physiology, Phosphoprotein Phosphatases metabolism
- Abstract
Background: Abnormal calcium (Ca
2+ ) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN., Methods: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln-knockout mice. Ppp1r3a-knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a-knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca2+ imaging., Results: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF., Conclusions: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.- Published
- 2019
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21. Rearrangement of the Protein Phosphatase 1 Interactome During Heart Failure Progression.
- Author
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Chiang DY, Alsina KM, Corradini E, Fitzpatrick M, Ni L, Lahiri SK, Reynolds JO, Pan X, Scott L Jr, Heck AJR, and Wehrens XHT
- Subjects
- Animals, Calcium Signaling, Dependovirus genetics, Disease Models, Animal, Disease Progression, Female, Genetic Vectors, HeLa Cells, Heart Failure genetics, Heart Failure pathology, Heart Failure physiopathology, Humans, Male, Mice, Inbred C57BL, Myocytes, Cardiac pathology, Protein Binding, Protein Phosphatase 1 deficiency, Protein Phosphatase 1 genetics, RNA Interference, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Time Factors, Heart Failure enzymology, Myocytes, Cardiac enzymology, Protein Interaction Maps, Protein Phosphatase 1 metabolism
- Abstract
Background: Heart failure (HF) is a complex disease with a rising prevalence despite advances in treatment. Protein phosphatase 1 (PP1) has long been implicated in HF pathogenesis, but its exact role is both unclear and controversial. Most previous studies measured only the PP1 catalytic subunit (PP1c) without investigating its diverse set of interactors, which confer localization and substrate specificity to the holoenzyme. In this study, we define the PP1 interactome in cardiac tissue and test the hypothesis that this interactome becomes rearranged during HF progression at the level of specific PP1c interactors., Methods: Mice were subjected to transverse aortic constriction and grouped on the basis of ejection fraction into sham, hypertrophy, moderate HF (ejection fraction, 30%-40%), and severe HF (ejection fraction <30%). Cardiac lysates were subjected to affinity purification with anti-PP1c antibodies followed by high-resolution mass spectrometry. PP1 regulatory subunit 7 (Ppp1r7) was knocked down in mouse cardiomyocytes and HeLa cells with adeno-associated virus serotype 9 and siRNA, respectively. Calcium imaging was performed on isolated ventricular myocytes., Results: Seventy-one and 98 PP1c interactors were quantified from mouse cardiac and HeLa lysates, respectively, including many novel interactors and protein complexes. This represents the largest reproducible PP1 interactome data set ever captured from any tissue, including both primary and secondary/tertiary interactors. Nine PP1c interactors with changes in their binding to PP1c were strongly associated with HF progression, including 2 known (Ppp1r7 and Ppp1r18) and 7 novel interactors. Within the entire cardiac PP1 interactome, Ppp1r7 had the highest binding to PP1c. Cardiac-specific knockdown in mice led to cardiac dysfunction and disruption of calcium release from the sarcoplasmic reticulum., Conclusions: PP1 is best studied at the level of its interactome, which undergoes significant rearrangement during HF progression. The 9 key interactors that are associated with HF progression may represent potential targets in HF therapy. In particular, Ppp1r7 may play a central role in regulating the PP1 interactome by acting as a competitive molecular "sponge" of PP1c.
- Published
- 2018
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22. Elucidating therapeutic molecular targets in premenopausal Asian women with recurrent breast cancers.
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Yap YS, Singh AP, Lim JHC, Ahn JH, Jung KH, Kim J, Dent RA, Ng RCH, Kim SB, and Chiang DY
- Abstract
Breast cancer is an increasing problem in Asia, with a higher proportion of premenopausal patients who are at higher risk of recurrence. Targeted sequencing was performed on DNA extracted from primary tumor specimens of 63 premenopausal Asian patients who relapsed after initial diagnosis of non-metastatic breast cancer. The most prevalent alterations included: TP53 (65%); PIK3CA (32%); GATA3 (29%); ERBB2 (27%); MYC (25%); KMT2C (21%); MCL1 (17%); PRKDC, TPR, BRIP1 (14%); MDM4, PCDH15, PRKAR1A, CDKN1B (13%); CCND1, KMT2D, STK11 , and MLH1 (11%). Sixty of the 63 patients (95%) had at least one genetic alteration in a signaling pathway related to cell cycle or p53 signaling. The presence of MCL1 amplification, HIF-1-alpha transcription factor network pathway alterations, and direct p53 effectors pathway alterations were independent predictors of inferior overall survival from initial diagnosis. Comparison with non-Asian premenopausal tumors in The Cancer Genome Atlas (TCGA) revealed a higher prevalence of TP53 mutations among HER2-positive cancers, and more frequent TP53, TET2 , and CDK12 mutations among hormone receptor-positive HER2-negative cancers in our cohort. Given the limited number of non-Asian premenopausal breast cancers that had relapsed in TCGA, we compared the frequency of mutations in our cohort with 43 premenopausal specimens from both TCGA and International Cancer Genome Consortium that had relapsed. There was a trend toward higher prevalence of TP53 mutations in our cohort. Certain genomic aberrations may be enriched in tumors of poor-prognosis premenopausal Asian breast cancers. The development of novel therapies targeting these aberrations merit further research., Competing Interests: Y.S.Y. provided consultancy and received honoraria from Novartis; A.P.S. and D.Y.C. are employees of Novartis; R.A.D. has received honoraria from Novartis; and S.B.K. has received research support from Novartis. All remaining authors have declared no competing interests.
- Published
- 2018
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23. Wounds That Would Not Heal: Pyoderma Gangrenosum.
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Pinard J, Chiang DY, Mostaghimi A, Granter SR, Merola JF, and Barkoudah E
- Subjects
- Antibodies, Monoclonal adverse effects, Antibodies, Monoclonal, Humanized, Combined Modality Therapy, Diagnosis, Differential, Female, Humans, Immunocompromised Host, Middle Aged, Genital Diseases, Female diagnosis, Genital Diseases, Female therapy, Pyoderma Gangrenosum diagnosis, Pyoderma Gangrenosum therapy
- Published
- 2018
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24. Oxidized CaMKII (Ca 2+ /Calmodulin-Dependent Protein Kinase II) Is Essential for Ventricular Arrhythmia in a Mouse Model of Duchenne Muscular Dystrophy.
- Author
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Wang Q, Quick AP, Cao S, Reynolds J, Chiang DY, Beavers D, Li N, Wang G, Rodney GG, Anderson ME, and Wehrens XHT
- Subjects
- Action Potentials, Animals, Arrhythmias, Cardiac enzymology, Arrhythmias, Cardiac physiopathology, Arrhythmias, Cardiac prevention & control, Calcium metabolism, Calcium Signaling, Calcium-Calmodulin-Dependent Protein Kinase Type 2 genetics, Disease Models, Animal, Heart Rate, Heart Ventricles physiopathology, Mice, Inbred mdx, Mice, Transgenic, Muscular Dystrophy, Duchenne enzymology, Muscular Dystrophy, Duchenne physiopathology, NADPH Oxidase 2 metabolism, Oxidation-Reduction, Oxidative Stress, Reactive Oxygen Species metabolism, Arrhythmias, Cardiac etiology, Calcium-Calmodulin-Dependent Protein Kinase Type 2 metabolism, Heart Ventricles enzymology, Muscular Dystrophy, Duchenne complications
- Abstract
Background: Duchenne muscular dystrophy patients are prone to ventricular arrhythmias, which may be caused by abnormal calcium (Ca
2+ ) homeostasis and elevated reactive oxygen species. CaMKII (Ca2+ /calmodulin-dependent protein kinase II) is vital for normal Ca2+ homeostasis, but excessive CaMKII activity contributes to abnormal Ca2+ homeostasis and arrhythmias in cardiomyocytes. Reactive oxygen species induce CaMKII to become autonomously active. We hypothesized that genetic inhibition of CaMKII oxidation (ox-CaMKII) in a mouse model of Duchenne muscular dystrophy can alleviate abnormal Ca2+ homeostasis, thus, preventing ventricular arrhythmia. The objective of this study was to test if selective loss of ox-CaMKII affects ventricular arrhythmias in the mdx mouse model of Duchenne muscular dystrophy., Methods and Results: 5-(6)-Chloromethyl-2,7-dichlorodihydrofluorescein diacetate staining revealed increased reactive oxygen species production in ventricular myocytes isolated from mdx mice, which coincides with elevated ventricular ox-CaMKII demonstrated by Western blotting. Genetic inhibition of ox-CaMKII by knockin replacement of the regulatory domain methionines with valines (MM-VV [CaMKII M281/282V]) prevented ventricular tachycardia in mdx mice. Confocal calcium imaging of ventricular myocytes isolated from mdx :MM-VV mice revealed normalization of intracellular Ca2+ release events compared with cardiomyocytes from mdx mice. Abnormal action potentials assessed by optical mapping in mdx mice were also alleviated by genetic inhibition of ox-CaMKII. Knockout of the NADPH oxidase regulatory subunit p47phox normalized elevated ox-CaMKII, repaired intracellular Ca2+ homeostasis, and rescued inducible ventricular arrhythmias in mdx mice., Conclusions: Inhibition of reactive oxygen species or ox-CaMKII protects against proarrhythmic intracellular Ca2+ handling and prevents ventricular arrhythmia in a mouse model of Duchenne muscular dystrophy., (© 2018 American Heart Association, Inc.)- Published
- 2018
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25. Genomic Analysis of Nasopharyngeal Carcinoma Reveals TME-Based Subtypes.
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Zhang L, MacIsaac KD, Zhou T, Huang PY, Xin C, Dobson JR, Yu K, Chiang DY, Fan Y, Pelletier M, Wang Y, Jaeger S, Krishnamurthy Radhakrishnan V, JeBailey L, Skewes-Cox P, Zhang J, Fang W, Huang Y, Zhao H, Zhao Y, Li E, Peng B, Huang A, Dranoff G, Hammerman PS, Engelman J, Bitter H, Zeng YX, and Yao Y
- Subjects
- Adult, Aged, Carcinoma pathology, Carcinoma virology, Cell Proliferation genetics, Cyclin-Dependent Kinase Inhibitor p16, Cyclin-Dependent Kinase Inhibitor p18 genetics, Disease-Free Survival, Female, Gene Expression Regulation, Neoplastic, Genomics, Herpesvirus 4, Human genetics, Herpesvirus 4, Human pathogenicity, Humans, Lymphocytes, Tumor-Infiltrating metabolism, Lymphocytes, Tumor-Infiltrating pathology, Male, Middle Aged, Mutation, NF-kappa B genetics, Nasopharyngeal Carcinoma, Nasopharyngeal Neoplasms pathology, Nasopharyngeal Neoplasms virology, Transforming Growth Factor beta genetics, Wnt Signaling Pathway genetics, Carcinoma genetics, Genome, Human genetics, Nasopharyngeal Neoplasms genetics, Prognosis, Tumor Microenvironment genetics
- Abstract
Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated cancer characterized by a poor prognosis and a high level of lymphocyte infiltrate. Genetic hallmarks of NPC are not completely known but include deletion of the p16 ( CDKN2A ) locus and mutations in NF-κB pathway components, with a relatively low total mutational load. To better understand the genetic landscape, an integrated genomic analysis was performed using a large clinical cohort of treatment-naïve NPC tumor specimens. This genomic analysis was generally concordant with previous studies; however, three subtypes of NPC were identified by differences in immune cell gene expression, prognosis, tumor cell morphology, and genetic characteristics. A gene expression signature of proliferation was poorly prognostic and associated with either higher mutation load or specific EBV gene expression patterns in a subtype-specific manner. Finally, higher levels of stromal tumor-infiltrating lymphocytes associated with good prognosis and lower expression of a WNT and TGFβ pathway activation signature. Implications: This study represents the first integrated analysis of mutation, copy number, and gene expression data in NPC and suggests how tumor genetics and EBV infection influence the tumor microenvironment in this disease. These insights should be considered for guiding immunotherapy treatment strategies in this disease. Mol Cancer Res; 15(12); 1722-32. ©2017 AACR ., (©2017 American Association for Cancer Research.)
- Published
- 2017
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26. Progress towards molecular patient stratification of hepatocellular carcinoma: Lost in translation?
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Chiang DY and Villanueva A
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- Carcinoma, Hepatocellular drug therapy, Carcinoma, Hepatocellular immunology, Humans, Liver Neoplasms drug therapy, Liver Neoplasms immunology, Mutation, Carcinoma, Hepatocellular genetics, Liver Neoplasms genetics
- Published
- 2017
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27. SPEG (Striated Muscle Preferentially Expressed Protein Kinase) Is Essential for Cardiac Function by Regulating Junctional Membrane Complex Activity.
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Quick AP, Wang Q, Philippen LE, Barreto-Torres G, Chiang DY, Beavers D, Wang G, Khalid M, Reynolds JO, Campbell HM, Showell J, McCauley MD, Scholten A, and Wehrens XH
- Subjects
- Adult, Aged, Animals, Female, HEK293 Cells, Heart Failure genetics, Heart Failure pathology, Humans, Male, Membrane Proteins genetics, Mice, Mice, Inbred C57BL, Mice, Knockout, Middle Aged, Muscle Proteins genetics, Myosin-Light-Chain Kinase genetics, Heart Failure metabolism, Membrane Proteins metabolism, Muscle Proteins biosynthesis, Muscle Proteins metabolism, Myocytes, Cardiac metabolism, Myosin-Light-Chain Kinase biosynthesis, Proteomics methods
- Abstract
Rationale: Junctional membrane complexes (JMCs) in myocytes are critical microdomains, in which excitation-contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. However, the role of newly identified JMC protein SPEG (striated muscle preferentially expressed protein kinase) remains unclear., Objective: To determine the role of SPEG in healthy and failing adult hearts., Methods and Results: Proteomic analysis of immunoprecipitated JMC proteins ryanodine receptor type 2 and junctophilin-2 (JPH2) followed by mass spectrometry identified the serine-threonine kinase SPEG as the only novel binding partner for both proteins. Real-time polymerase chain reaction revealed the downregulation of SPEG mRNA levels in failing human hearts. A novel cardiac myocyte-specific Speg conditional knockout (MCM-Speg
fl/fl ) model revealed that adult-onset SPEG deficiency results in heart failure (HF). Calcium (Ca2+ ) and transverse-tubule imaging of ventricular myocytes from MCM-Spegfl/fl mice post HF revealed both increased sarcoplasmic reticulum Ca2+ spark frequency and disrupted JMC integrity. Additional studies revealed that transverse-tubule disruption precedes the development of HF development in MCM-Spegfl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM-Spegfl/fl mice, suggesting that loss of SPEG phosphorylation of JPH2 led to transverse-tubule disruption, a precursor of HF development in SPEG-deficient mice., Conclusions: The novel JMC protein SPEG is downregulated in human failing hearts. Acute loss of SPEG in mouse hearts causes JPH2 dephosphorylation and transverse-tubule loss associated with downstream Ca2+ mishandling leading to HF. Our study suggests that SPEG could be a novel target for the treatment of HF., (© 2016 American Heart Association, Inc.)- Published
- 2017
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28. PureCN: copy number calling and SNV classification using targeted short read sequencing.
- Author
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Riester M, Singh AP, Brannon AR, Yu K, Campbell CD, Chiang DY, and Morrissey MP
- Abstract
Background: Matched sequencing of both tumor and normal tissue is routinely used to classify variants of uncertain significance (VUS) into somatic vs. germline. However, assays used in molecular diagnostics focus on known somatic alterations in cancer genes and often only sequence tumors. Therefore, an algorithm that reliably classifies variants would be helpful for retrospective exploratory analyses. Contamination of tumor samples with normal cells results in differences in expected allelic fractions of germline and somatic variants, which can be exploited to accurately infer genotypes after adjusting for local copy number. However, existing algorithms for determining tumor purity, ploidy and copy number are not designed for unmatched short read sequencing data., Results: We describe a methodology and corresponding open source software for estimating tumor purity, copy number, loss of heterozygosity (LOH), and contamination, and for classification of single nucleotide variants (SNVs) by somatic status and clonality. This R package, PureCN, is optimized for targeted short read sequencing data, integrates well with standard somatic variant detection pipelines, and has support for matched and unmatched tumor samples. Accuracy is demonstrated on simulated data and on real whole exome sequencing data., Conclusions: Our algorithm provides accurate estimates of tumor purity and ploidy, even if matched normal samples are not available. This in turn allows accurate classification of SNVs. The software is provided as open source (Artistic License 2.0) R/Bioconductor package PureCN (http://bioconductor.org/packages/PureCN/).
- Published
- 2016
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29. Regulating the regulator: Insights into the cardiac protein phosphatase 1 interactome.
- Author
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Chiang DY, Heck AJ, Dobrev D, and Wehrens XH
- Subjects
- Animals, Atrial Fibrillation etiology, Atrial Fibrillation metabolism, Computational Biology methods, Heart Failure etiology, Heart Failure metabolism, Humans, Protein Binding, Protein Interaction Mapping methods, Carrier Proteins metabolism, Heart physiology, Myocardium metabolism, Protein Phosphatase 1 metabolism
- Abstract
Reversible phosphorylation of proteins is a delicate yet dynamic balancing act between kinases and phosphatases, the disturbance of which underlies numerous disease processes. While our understanding of protein kinases has grown tremendously over the past decades, relatively little is known regarding protein phosphatases. This may be because protein kinases are great in number and relatively specific in function, and thereby amenable to be studied in isolation, whereas protein phosphatases are much less abundant and more nonspecific in their function. To achieve subcellular localization and substrate specificity, phosphatases depend on partnering with a large number of regulatory subunits, protein scaffolds and/or other interactors. This added layer of complexity presents a significant barrier to their study, but holds the key to unexplored opportunities for novel pharmacologic intervention. In this review we focus on serine/threonine protein phosphatase type-1 (PP1), which plays an important role in cardiac physiology and pathophysiology. Although much work has been done to investigate the role of PP1 in cardiac diseases including atrial fibrillation and heart failure, most of these studies were limited to examining and manipulating the catalytic subunit(s) of PP1 without adequately considering the PP1 interactors, which give specificity to PP1's functions. To complement these studies, three unbiased methods have been developed and applied to the mapping of the PP1 interactome: bioinformatics approaches, yeast two-hybrid screens, and affinity-purification mass spectrometry. The application of these complementary methods has the potential to generate a detailed cardiac PP1 interactome, which is an important step in identifying novel and targeted pharmacological interventions., Competing Interests: There are no relevant relationships with industry., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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30. Integrative biomarker analyses indicate etiological variations in hepatocellular carcinoma.
- Author
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Zhu AX, Chen D, He W, Kanai M, Voi M, Chen LT, Daniele B, Furuse J, Kang YK, Poon RT, Vogel A, and Chiang DY
- Subjects
- Biomarkers, Tumor, Humans, Proto-Oncogene Proteins c-met, Vascular Endothelial Growth Factor D, Carcinoma, Hepatocellular, Liver Neoplasms
- Abstract
Background & Aims: The purpose of this study was to determine whether biomarkers from baseline plasma and archival tissue specimens collected from patients enrolled in the EVOLVE-1 trial - a randomized phase 3 study of everolimus in hepatocellular carcinoma (HCC) - were associated with prognosis, etiology or ethnicity., Methods: Circulating plasma levels of bFGF, PLGF, VEGF, VEGF-D, c-Kit, collagen IV, sVEGFR1 and VEGFR2 were measured by ELISA (N=503). Protein levels of IGF-1R, c-Met, mTOR, Tsc2 were assayed by immunohistochemistry (N=125). Genomic DNA sequencing was conducted on a panel of 287 cancer-related genes (N=69)., Results: Patients with baseline plasma concentrations of VEGF or sVEGFR1 above the cohort median had significantly shorter overall survival. These plasma biomarkers retained prognostic significance in a multivariate Cox regression model with geographic region, macroscopic vascular invasion and alpha fetoprotein AFP levels. Membranous c-Met protein levels were significantly lower for Asian patients, as well as for hepatitis B viral etiology. The prevalence of genetic changes were similar to previous reports, along with a trend towards higher PTEN and TSC2 mutations among Asians., Conclusions: The angiogenesis biomarkers VEGF and sVEGFR1 were independent prognostic predictors of survival in patients with advanced HCC. Potential differences in c-Met and mTOR pathway activation between Asian and non-Asian patients should be considered in future clinical trials., Lay Summary: Our study demonstrates that circulating angiogenesis biomarkers can predict the survival outcome in patients with advanced hepatocellular carcinoma independent of the clinical variables. There is etiology and ethnicity variation in molecular pathway activation in hepatocellular carcinoma, which should be considered for future clinical trial design of targeted therapy., Clinical Trial Registration Number: NCT01035229., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
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31. Relation of Cardiac Dysfunction to Rhythm Abnormalities in Patients With Duchenne or Becker Muscular Dystrophies.
- Author
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Chiang DY, Allen HD, Kim JJ, Valdes SO, Wang Y, Pignatelli RH, Lotze TE, and Miyake CY
- Subjects
- Adolescent, Adult, Arrhythmias, Cardiac diagnostic imaging, Arrhythmias, Cardiac physiopathology, Cardiomyopathy, Dilated diagnostic imaging, Cardiomyopathy, Dilated physiopathology, Child, Child, Preschool, Female, Follow-Up Studies, Humans, Male, Muscular Dystrophy, Duchenne diagnostic imaging, Muscular Dystrophy, Duchenne physiopathology, Retrospective Studies, Young Adult, Arrhythmias, Cardiac complications, Cardiomyopathy, Dilated complications, Echocardiography methods, Heart Conduction System physiopathology, Heart Rate physiology, Muscular Dystrophy, Duchenne complications
- Abstract
The association between systolic cardiac dysfunction and arrhythmia development in patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) is generally assumed but has not been extensively studied. The purpose of this study was to describe arrhythmias in patients with DMD and BMD in the present era and determine whether arrhythmia development is associated with cardiac dysfunction. This is a single-center retrospective review of 237 Holters from 91 patients with DMD (mean = 17 ± 4 years, range 3 to 27 years) and 64 Holters from 21 patients with BMD (mean = 18 ± 7 years, range 4 to 31 years) with corresponding echocardiography. Holters were stratified by age of patient at the time of study and ejection fraction: normal (≥55%), mild (<55% and ≥45%), moderate (<45% and ≥30%), and severe (<30%). Arrhythmias included frequent atrial and ventricular premature complexes (>10/hr), couplets, and runs of supraventricular and ventricular tachycardias. Arrhythmias occurred in 44% of DMD and 57% of BMD patients and were significantly associated with decrease in cardiac function. Clinically significant arrhythmias (supraventricular tachycardia and ventricular tachycardia) occurred in 10% of all Holters obtained in patients with DMD and 25% of all Holters obtained in patients with BMD. Subgroup analysis of Holters from patients with DMD demonstrated that arrhythmias increased with decreasing ejection fraction regardless of age, but that age was also a significant predictor of arrhythmia development. In conclusion, among patients with DMD or BMD, arrhythmias increase with development of cardiac dysfunction., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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32. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response.
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Gao H, Korn JM, Ferretti S, Monahan JE, Wang Y, Singh M, Zhang C, Schnell C, Yang G, Zhang Y, Balbin OA, Barbe S, Cai H, Casey F, Chatterjee S, Chiang DY, Chuai S, Cogan SM, Collins SD, Dammassa E, Ebel N, Embry M, Green J, Kauffmann A, Kowal C, Leary RJ, Lehar J, Liang Y, Loo A, Lorenzana E, Robert McDonald E 3rd, McLaughlin ME, Merkin J, Meyer R, Naylor TL, Patawaran M, Reddy A, Röelli C, Ruddy DA, Salangsang F, Santacroce F, Singh AP, Tang Y, Tinetto W, Tobler S, Velazquez R, Venkatesan K, Von Arx F, Wang HQ, Wang Z, Wiesmann M, Wyss D, Xu F, Bitter H, Atadja P, Lees E, Hofmann F, Li E, Keen N, Cozens R, Jensen MR, Pryer NK, Williams JA, and Sellers WR
- Subjects
- Animals, Breast Neoplasms drug therapy, Carcinoma drug therapy, Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Pancreatic Ductal drug therapy, Colorectal Neoplasms drug therapy, Disease Models, Animal, Female, Humans, Lung Neoplasms drug therapy, Melanoma drug therapy, Mice, Neoplasm Transplantation, Pancreatic Neoplasms drug therapy, Reproducibility of Results, Skin Neoplasms drug therapy, Stomach Neoplasms drug therapy, Antineoplastic Agents therapeutic use, High-Throughput Screening Assays methods, Neoplasms drug therapy, Xenograft Model Antitumor Assays methods
- Abstract
Profiling candidate therapeutics with limited cancer models during preclinical development hinders predictions of clinical efficacy and identifying factors that underlie heterogeneous patient responses for patient-selection strategies. We established ∼1,000 patient-derived tumor xenograft models (PDXs) with a diverse set of driver mutations. With these PDXs, we performed in vivo compound screens using a 1 × 1 × 1 experimental design (PDX clinical trial or PCT) to assess the population responses to 62 treatments across six indications. We demonstrate both the reproducibility and the clinical translatability of this approach by identifying associations between a genotype and drug response, and established mechanisms of resistance. In addition, our results suggest that PCTs may represent a more accurate approach than cell line models for assessing the clinical potential of some therapeutic modalities. We therefore propose that this experimental paradigm could potentially improve preclinical evaluation of treatment modalities and enhance our ability to predict clinical trial responses.
- Published
- 2015
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33. Loss-of-Function SCN5A Mutations Associated With Sinus Node Dysfunction, Atrial Arrhythmias, and Poor Pacemaker Capture.
- Author
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Chiang DY, Kim JJ, Valdes SO, de la Uz C, Fan Y, Orcutt J, Domino M, Smith M, Wehrens XH, and Miyake CY
- Subjects
- Adolescent, Adult, Arrhythmias, Cardiac physiopathology, Arrhythmias, Cardiac therapy, Child, Child, Preschool, Female, Humans, Infant, Infant, Newborn, Male, Middle Aged, Retrospective Studies, Sick Sinus Syndrome physiopathology, Sick Sinus Syndrome therapy, Arrhythmias, Cardiac genetics, Mutation genetics, NAV1.5 Voltage-Gated Sodium Channel genetics, Pacemaker, Artificial, Sick Sinus Syndrome genetics
- Abstract
Background: Cardiac device implantation can be complicated by inability to adequately place leads because of significant lead capture issues. This study sought to determine whether there are genetic bases that underlie poor lead capture., Methods and Results: Retrospective review of all patients with structurally normal hearts who underwent new device implantation at Texas Children's Hospital between 2009 and 2014 was performed. Patients with inability to capture at 10 V or a final capture threshold ≥3 V at 0.4 ms during implant were analyzed. Among a total of 136 patients (median age, 13 years; range, 3 days to 46 years), 11 patients (8.1%) who underwent dual chamber device implantation had elevated thresholds in the atria (4), ventricle (3), or both chambers (4; atrial-lead threshold, 4.7±4.3 versus 0.7±0.3 V; ventricular-lead, 3.0±3.3 versus 0.7±0.3 V). All 11 patients presented with sinus node dysfunction and 10 had atrial arrhythmias. At implant, inability to find atrial capture was seen in 4 patients. Three demonstrated intermittent complete loss of ventricular capture after implantation: 1 has recurrent syncope, 2 eventually died. Genetic testing performed in 10 demonstrated 7 patients with 6 distinct SCN5A mutations, all predicted to be severe loss-of-function mutations by bioinformatic analyses. In the remaining patients, although putative pathogenic mutations were not found, multiple SCN5A polymorphisms were identified in 2 and a desmin mutation in 1., Conclusions: This study suggests that significant capture issues at implant may be because of loss-of-function SCN5A mutations, providing new insights into SCN5A function. Recognition of this association may be critical for planning device implantation strategies and patient follow-up., (© 2015 American Heart Association, Inc.)
- Published
- 2015
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34. Expression and function of Kv1.1 potassium channels in human atria from patients with atrial fibrillation.
- Author
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Glasscock E, Voigt N, McCauley MD, Sun Q, Li N, Chiang DY, Zhou XB, Molina CE, Thomas D, Schmidt C, Skapura DG, Noebels JL, Dobrev D, and Wehrens XH
- Subjects
- Aged, Animals, Female, Humans, Immunoblotting, Immunohistochemistry, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Middle Aged, Myocytes, Cardiac metabolism, Patch-Clamp Techniques, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Atrial Fibrillation metabolism, Heart Atria metabolism, Kv1.1 Potassium Channel metabolism
- Abstract
Voltage-gated Kv1.1 channels encoded by the Kcna1 gene are traditionally regarded as being neural-specific with no known expression or intrinsic functional role in the heart. However, recent studies in mice reveal low-level Kv1.1 expression in heart and cardiac abnormalities associated with Kv1.1-deficiency suggesting that the channel may have a previously unrecognized cardiac role. Therefore, this study tests the hypothesis that Kv1.1 channels are associated with arrhythmogenesis and contribute to intrinsic cardiac function. In intra-atrial burst pacing experiments, Kcna1-null mice exhibited increased susceptibility to atrial fibrillation (AF). The atria of Kcna1-null mice showed minimal Kv1 family ion channel remodeling and fibrosis as measured by qRT-PCR and Masson's trichrome histology, respectively. Using RT-PCR, immunocytochemistry, and immunoblotting, KCNA1 mRNA and protein were detected in isolated mouse cardiomyocytes and human atria for the first time. Patients with chronic AF (cAF) showed no changes in KCNA1 mRNA levels relative to controls; however, they exhibited increases in atrial Kv1.1 protein levels, not seen in paroxysmal AF patients. Patch-clamp recordings of isolated human atrial myocytes revealed significant dendrotoxin-K (DTX-K)-sensitive outward current components that were significantly increased in cAF patients, reflecting a contribution by Kv1.1 channels. The concomitant increases in Kv1.1 protein and DTX-K-sensitive currents in atria of cAF patients suggest that the channel contributes to the pathological mechanisms of persistent AF. These findings provide evidence of an intrinsic cardiac role of Kv1.1 channels and indicate that they may contribute to atrial repolarization and AF susceptibility., Competing Interests: Compliance with ethical standards Conflicts of interest None.
- Published
- 2015
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35. Studying clonal dynamics in response to cancer therapy using high-complexity barcoding.
- Author
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Bhang HE, Ruddy DA, Krishnamurthy Radhakrishna V, Caushi JX, Zhao R, Hims MM, Singh AP, Kao I, Rakiec D, Shaw P, Balak M, Raza A, Ackley E, Keen N, Schlabach MR, Palmer M, Leary RJ, Chiang DY, Sellers WR, Michor F, Cooke VG, Korn JM, and Stegmeier F
- Subjects
- Antineoplastic Combined Chemotherapy Protocols therapeutic use, Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Non-Small-Cell Lung genetics, Cell Differentiation, Cell Line, Tumor, Crizotinib, DNA chemistry, DNA, Complementary metabolism, Epithelial-Mesenchymal Transition, Erlotinib Hydrochloride, Fusion Proteins, bcr-abl genetics, Gene Dosage, Gene Library, Humans, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Models, Theoretical, Oligonucleotides genetics, Polymerase Chain Reaction, Proto-Oncogene Proteins c-abl antagonists & inhibitors, Proto-Oncogene Proteins c-met metabolism, Pyrazoles administration & dosage, Pyridines administration & dosage, Quinazolines administration & dosage, Sequence Analysis, RNA, DNA Barcoding, Taxonomic methods, Neoplasms drug therapy, Neoplasms genetics
- Abstract
Resistance to cancer therapies presents a significant clinical challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resolution limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resolution tracking of more than 1 million cancer cells under drug treatment. In two clinically relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quantitative assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.
- Published
- 2015
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36. Identification of microRNA-mRNA dysregulations in paroxysmal atrial fibrillation.
- Author
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Chiang DY, Zhang M, Voigt N, Alsina KM, Jakob H, Martin JF, Dobrev D, Wehrens XHT, and Li N
- Subjects
- Aged, Atrial Fibrillation physiopathology, Female, Gene Expression Profiling methods, Humans, Male, Microarray Analysis methods, Middle Aged, Atrial Fibrillation diagnosis, Atrial Fibrillation genetics, MicroRNAs genetics, RNA, Messenger genetics
- Abstract
Background: The molecular mechanisms underlying the early development of atrial fibrillation (AF) remain poorly understood. Emerging evidence suggests that abnormal epigenetic modulation via microRNAs (miRNAs) might be involved in the pathogenesis of paroxysmal AF (pAF)., Objective: To identify key molecular changes associated with pAF, we conducted state-of-the-art transcriptomic studies to identify the abnormal miRNA-mRNA interactions potentially driving AF development., Methods: High-quality total RNA including miRNA was isolated from atrial biopsies of age-matched and sex-matched pAF patients and control patients in sinus rhythm (SR; n=4 per group) and used for RNA-sequencing and miRNA microarray. Results were analyzed bioinformatically and validated using quantitative real-time (qRT)-PCR and 3'UTR luciferase reporter assays., Results: 113 genes and 49 miRNAs were differentially expressed (DE) in pAF versus SR patients. Gene ontology analysis revealed that most of the DE genes were involved in the "gonadotropin releasing hormone receptor pathway" and "p53 pathway". Of these DE genes, bioinformatic analyses identified 23 pairs of putative miRNA-mRNA interactions that were altered in pAF (involving 15 miRNAs and 17 mRNAs). Using qRT-PCR and 3'UTR luciferase reporter assays, the interaction between upregulation of miR-199a-5p and downregulation of FKBP5 was confirmed in samples from pAF patients., Conclusion: Our combined transcriptomic analysis and miRNA microarray study of atrial samples from pAF patients revealed novel pathways and miRNA-mRNA regulations that may be relevant in the development of pAF. Future studies are required to investigate the potential involvement of the gonadotropin releasing hormone receptor and p53 pathways in AF pathogenesis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2015
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37. Promoted decomposition of NOx in automotive diesel-like exhausts by electro-catalytic honeycombs.
- Author
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Huang TJ, Chiang DY, Shih C, Lee CC, Mao CW, and Wang BC
- Subjects
- Air Pollutants analysis, Catalysis, Oxygen analysis, Temperature, Electrochemistry methods, Gasoline, Nitrates analysis, Nitrites analysis, Vehicle Emissions analysis
- Abstract
NO and NO2 (collectively called NOx) are major air pollutants in automotive emissions. More effective and easier treatments of NOx than those achieved by the present methods can offer better protection of human health and higher fuel efficiency that can reduce greenhouse gas emissions. However, currently commercialized technologies for automotive NOx emission control cannot effectively treat diesel-like exhausts with high NOx concentrations. Thus, exhaust gas recirculation (EGR) has been used extensively, which reduces fuel efficiency and increases particulate emission considerably. Our results show that the electro-catalytic honeycomb (ECH) promotes the decomposition of NOx to nitrogen and oxygen, without consuming reagents or other resources. NOx can be converted to nitrogen and oxygen almost completely. The ECHs are shown to effectively remove NOx from gasoline-fueled diesel-like exhausts. A very high NO concentration is preferred in the engine exhaust, especially during engine cold-start. Promoted NOx decomposition (PND) technology for real-world automotive applications is established in this study by using the ECH. With PND, EGR is no longer needed. Diesel-like engines can therefore achieve superior fuel efficiency, and all major automotive pollutants can be easily treated due to high concentration of oxygen in the diesel-like exhausts, leading to zero pollution.
- Published
- 2015
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38. Alterations in the interactome of serine/threonine protein phosphatase type-1 in atrial fibrillation patients.
- Author
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Chiang DY, Lebesgue N, Beavers DL, Alsina KM, Damen JM, Voigt N, Dobrev D, Wehrens XH, and Scholten A
- Subjects
- Animals, Atrial Fibrillation pathology, Humans, Immunohistochemistry, Mass Spectrometry, Mice, Myocytes, Cardiac pathology, Proteomics methods, Atrial Fibrillation metabolism, Myocytes, Cardiac metabolism, Protein Phosphatase 1 biosynthesis
- Abstract
Background: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet current pharmacological treatments are limited. Serine/threonine protein phosphatase type-1 (PP1), a major phosphatase in the heart, consists of a catalytic subunit (PP1c) and a large set of regulatory (R)-subunits that confer localization and substrate specificity to the holoenzyme. Previous studies suggest that PP1 is dysregulated in AF, but the mechanisms are unknown., Objectives: The purpose of this study was to test the hypothesis that PP1 is dysregulated in paroxysmal atrial fibrillation (PAF) at the level of its R-subunits., Methods: Cardiac lysates were coimmunoprecipitated with anti-PP1c antibody followed by mass spectrometry-based, quantitative profiling of associated R-subunits. Subsequently, label-free quantification (LFQ) was used to evaluate altered R-subunit-PP1c interactions in PAF patients. R-subunits with altered binding to PP1c in PAF were further studied using bioinformatics, Western blotting (WB), immunocytochemistry, and coimmunoprecipitation., Results: A total of 135 and 78 putative PP1c interactors were captured from mouse and human cardiac lysates, respectively, including many previously unreported interactors with conserved PP1c docking motifs. Increases in binding were found between PP1c and PPP1R7, cold-shock domain protein A (CSDA), and phosphodiesterase type-5A (PDE5A) in PAF patients, with CSDA and PDE5A being novel interactors validated by bioinformatics, immunocytochemistry, and coimmunoprecipitation. WB confirmed that these increases in binding cannot be ascribed to their changes in global protein expression alone., Conclusions: Subcellular heterogeneity in PP1 activity and downstream protein phosphorylation in AF may be attributed to alterations in PP1c-R-subunit interactions, which impair PP1 targeting to proteins involved in electrical and Ca(2+) remodeling. This represents a novel concept in AF pathogenesis and may provide more specific drug targets for treating AF., (Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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39. Genetic deletion of Rnd3/RhoE results in mouse heart calcium leakage through upregulation of protein kinase A signaling.
- Author
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Yang X, Wang T, Lin X, Yue X, Wang Q, Wang G, Fu Q, Ai X, Chiang DY, Miyake CY, Wehrens XHT, and Chang J
- Subjects
- Animals, Animals, Newborn, Cells, Cultured, Female, Heart physiology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Rats, Signal Transduction physiology, Up-Regulation physiology, Calcium metabolism, Cyclic AMP-Dependent Protein Kinases biosynthesis, Gene Deletion, Myocytes, Cardiac metabolism, rho GTP-Binding Proteins deficiency, rho GTP-Binding Proteins genetics
- Abstract
Rationale: Rnd3, a small Rho GTPase, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration, and proliferation. The biological function of Rnd3 in the heart remains unexplored., Objective: To define the functional role of the Rnd3 gene in the animal heart and investigate the associated molecular mechanism., Methods and Results: By loss-of-function approaches, we discovered that Rnd3 is involved in calcium regulation in cardiomyocytes. Rnd3-null mice died at the embryonic stage with fetal arrhythmias. The deletion of Rnd3 resulted in severe Ca(2+) leakage through destabilized ryanodine receptor type 2 Ca(2+) release channels. We further found that downregulation of Rnd3 attenuated β2-adrenergic receptor lysosomal targeting and ubiquitination, which in turn resulted in the elevation of β2-adrenergic receptor protein levels leading to the hyperactivation of protein kinase A (PKA) signaling. The PKA activation destabilized ryanodine receptor type 2 channels. This irregular spontaneous Ca(2+) release can be curtailed by PKA inhibitor treatment. Increases in the PKA activity along with elevated cAMP levels were detected in Rnd3-null embryos, in neonatal rat cardiomyocytes, and noncardiac cell lines with Rnd3 knockdown, suggesting a general mechanism for Rnd3-mediated PKA signaling activation. β2-Adrenergic receptor blocker treatment reduced arrhythmia and improved cardiac function., Conclusions: Rnd3 is a novel factor involved in intracellular Ca(2+) homeostasis regulation in the heart. Deficiency of the protein induces ryanodine receptor type 2 dysfunction by a mechanism that attenuates Rnd3-mediated β2-adrenergic receptor ubiquitination, which leads to the activation of PKA signaling. Increased PKA signaling in turn promotes ryanodine receptor type 2 hyperphosphorylation, which contributes to arrhythmogenesis and heart failure., (© 2014 American Heart Association, Inc.)
- Published
- 2015
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40. Loss of microRNA-106b-25 cluster promotes atrial fibrillation by enhancing ryanodine receptor type-2 expression and calcium release.
- Author
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Chiang DY, Kongchan N, Beavers DL, Alsina KM, Voigt N, Neilson JR, Jakob H, Martin JF, Dobrev D, Wehrens XH, and Li N
- Subjects
- 3' Untranslated Regions, Action Potentials, Animals, Atrial Fibrillation genetics, Atrial Fibrillation physiopathology, Calcium Signaling, Case-Control Studies, Disease Models, Animal, Down-Regulation, Gene Knockdown Techniques, Humans, Mice, Knockout, MicroRNAs genetics, Ryanodine Receptor Calcium Release Channel genetics, Time Factors, Transcription, Genetic, Up-Regulation, Atrial Fibrillation metabolism, Calcium metabolism, MicroRNAs metabolism, Myocardium metabolism, Ryanodine Receptor Calcium Release Channel metabolism, Sarcoplasmic Reticulum metabolism
- Abstract
Background: Enhanced sarcoplasmic reticulum Ca(2+)-leak via ryanodine receptor type-2 (RyR2) contributes to the pathogenesis of atrial fibrillation (AF). Recent studies have shown that the level of RyR2 protein is elevated in atria of patients with paroxysmal AF, suggesting that microRNA-mediated post-transcriptional regulation of RyR2 might be an underlying mechanism. Bioinformatic analysis suggests that miR-106b and miR-93, members of the miR-106b-25 cluster, could bind to RyR2-3'-untranslated region and suppress its translation. Thus, we tested the hypothesis that loss of the miR-106b-25 cluster promotes AF via enhanced RyR2-mediated sarcoplasmic reticulum Ca(2+)-leak., Methods and Results: Quantitative real-time polymerase chain reaction showed that the levels of mature miR-106b, miR-93, and miR-25 were lower in atria of patients with paroxysmal AF when compared with patients in sinus rhythm. In vitro assay showed that miR-93 reduced RyR2-3'-untranslated region luciferase activity. Total RyR2 protein in atrial tissue of miR-106b-25(-/-) mice was increased by 42% when compared with wild-type littermates but still maintained a normal subcellular distribution. Ca(2+)-spark frequency and total sarcoplasmic reticulum Ca(2+)-leak were increased in atrial myocytes of miR-106b-25(-/-) mice. Telemetry ECG recordings revealed that miR-106b-25(-/-) mice exhibited more frequent atrial ectopy and were also more susceptible to pacing-induced AF than wild-type littermates. Increased sarcoplasmic reticulum Ca(2+)-release and AF susceptibility in miR-106b-25(-/-) mice were abolished by the RyR2 blocker K201., Conclusions: These results suggest that miR-106b-25 cluster-mediated post-transcriptional regulation of RyR2 is a potential molecular mechanism involved in paroxysmal AF pathogenesis. As such, the miR-106b-25 cluster could be a novel gene-therapy target in AF associated with enhanced RyR2 expression., (© 2014 American Heart Association, Inc.)
- Published
- 2014
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41. Emerging roles of junctophilin-2 in the heart and implications for cardiac diseases.
- Author
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Beavers DL, Landstrom AP, Chiang DY, and Wehrens XH
- Subjects
- Animals, Calcium metabolism, Excitation Contraction Coupling physiology, Heart Diseases physiopathology, Humans, Heart physiopathology, Heart Diseases metabolism, Intercellular Junctions metabolism, Membrane Proteins metabolism
- Abstract
Cardiomyocytes rely on a highly specialized subcellular architecture to maintain normal cardiac function. In a little over a decade, junctophilin-2 (JPH2) has become recognized as a cardiac structural protein critical in forming junctional membrane complexes (JMCs), which are subcellular domains essential for excitation-contraction coupling within the heart. While initial studies described the structure of JPH2 and its role in anchoring junctional sarcoplasmic reticulum and transverse-tubule (T-tubule) membrane invaginations, recent research has an expanded role of JPH2 in JMC structure and function. For example, JPH2 is necessary for the development of postnatal T-tubule in mammals. It is also critical for the maintenance of the complex JMC architecture and stabilization of local ion channels in mature cardiomyocytes. Loss of this function by mutations or down-regulation of protein expression has been linked to hypertrophic cardiomyopathy, arrhythmias, and progression of disease in failing hearts. In this review, we summarize current views on the roles of JPH2 within the heart and how JPH2 dysregulation may contribute to a variety of cardiac diseases., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)
- Published
- 2014
- Full Text
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42. Impaired local regulation of ryanodine receptor type 2 by protein phosphatase 1 promotes atrial fibrillation.
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Chiang DY, Li N, Wang Q, Alsina KM, Quick AP, Reynolds JO, Wang G, Skapura D, Voigt N, Dobrev D, and Wehrens XH
- Subjects
- Amino Acid Substitution, Animals, Atrial Fibrillation genetics, Calcium Signaling, Disease Models, Animal, Female, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Mutant Strains, Microfilament Proteins deficiency, Microfilament Proteins genetics, Mutant Proteins genetics, Mutant Proteins metabolism, Myocardium metabolism, Nerve Tissue Proteins deficiency, Nerve Tissue Proteins genetics, Phosphorylation, Ryanodine Receptor Calcium Release Channel genetics, Sarcoplasmic Reticulum metabolism, Atrial Fibrillation etiology, Atrial Fibrillation metabolism, Protein Phosphatase 1 metabolism, Ryanodine Receptor Calcium Release Channel metabolism
- Abstract
Aims: Altered Ca(2+) handling in atrial fibrillation (AF) has been associated with dysregulated protein phosphatase 1 (PP1) and subcellular heterogeneities in protein phosphorylation, but the underlying mechanisms remain unclear. This is due to a lack of investigation into the local, rather than global, regulation of PP1 on different subcellular targets such as ryanodine receptor type 2 (RyR2), especially in AF., Methods and Results: We tested the hypothesis that impaired local regulation of PP1 causes RyR2 hyperphosphorylation thereby promoting AF susceptibility. To specifically disrupt PP1's local regulation of RyR2, we used the spinophilin knockout (Sp(-/-)) mice (Mus musculus) since PP1 is targeted to RyR2 via spinophilin. Without spinophilin, the interaction between PP1 and RyR2 was reduced by 64%, while RyR2 phosphorylation was increased by 43% at serine (S)2814 but unchanged at S2808. Lipid bilayer experiments revealed that single RyR2 channels isolated from Sp(-/-) hearts had an increased open probability. Likewise, Ca(2+) spark frequency normalized to sarcoplasmic reticulum Ca(2+) content was also enhanced in Sp(-/-) atrial myocytes, but normalized by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-93 and AIP and also by genetic inhibition of RyR2 S2814 phosphorylation. Finally, Sp(-/-) mice exhibited increased atrial ectopy and susceptibility to pacing-induced AF, both of which were also prevented by the RyR2 S2814A mutation., Conclusion: PP1 regulates RyR2 locally by counteracting CaMKII phosphorylation of RyR2. Decreased local PP1 regulation of RyR2 contributes to RyR2 hyperactivity and promotes AF susceptibility. This represents a novel mechanism for subcellular modulation of calcium channels and may represent a potential drug target of AF., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)
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- 2014
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43. Targeted next generation sequencing identifies clinically actionable mutations in patients with melanoma.
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Jeck WR, Parker J, Carson CC, Shields JM, Sambade MJ, Peters EC, Burd CE, Thomas NE, Chiang DY, Liu W, Eberhard DA, Ollila D, Grilley-Olson J, Moschos S, Neil Hayes D, and Sharpless NE
- Subjects
- Cell Line, Tumor, Female, Humans, Male, High-Throughput Nucleotide Sequencing, Melanoma genetics, Mutation, Neoplasm Proteins genetics
- Abstract
Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here, we describe a NextGen sequencing approach to fully analyzing 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high-throughput sequencing and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard-of-care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)
- Published
- 2014
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44. Mutations of isocitrate dehydrogenase 1 and 2 in intrahepatic cholangiocarcinoma.
- Author
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Grassian AR, Pagliarini R, and Chiang DY
- Subjects
- Bile Duct Neoplasms pathology, Cell Differentiation genetics, Cholangiocarcinoma pathology, DNA Methylation, Glutarates metabolism, Humans, Mutation, Prolyl Hydroxylases metabolism, Signal Transduction genetics, Bile Duct Neoplasms genetics, Bile Ducts, Intrahepatic, Cholangiocarcinoma genetics, Isocitrate Dehydrogenase genetics
- Abstract
Purpose of Review: Exome sequencing studies have recently expanded the genetic characterization of intrahepatic cholangiocarcinomas. Among a number of novel genes, isocitrate dehydrogenase (IDH) is recurrently mutated in intrahepatic cholangiocarcinomas. We review the effects of these mutations on several biochemical pathways, as well as potential changes to downstream signaling pathways., Recent Findings: Hotspot mutations in IDH isoforms 1 or 2 occur in approximately 15% of intrahepatic cholangiocarcinomas. These mutations result in elevated levels of an oncometabolite, 2-hydroxyglutarate, which is associated with higher DNA CpG methylation and altered histone methylation that accompany a block in cellular differentiation. Exploratory studies have suggested additional phenotypes associated with IDH1/2 mutations., Summary: Tumors with IDH1 or IDH2 mutations may represent a distinct subtype of cholangiocarcinomas. Further studies are required to elucidate the exact role that mutant IDH1/2 and 2-hydroxyglutarate play in tumorigenesis, and what are the best strategies to target these tumor types.
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- 2014
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45. Ryanodine receptor-mediated calcium leak drives progressive development of an atrial fibrillation substrate in a transgenic mouse model.
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Li N, Chiang DY, Wang S, Wang Q, Sun L, Voigt N, Respress JL, Ather S, Skapura DG, Jordan VK, Horrigan FT, Schmitz W, Müller FU, Valderrabano M, Nattel S, Dobrev D, and Wehrens XHT
- Subjects
- Age Factors, Animals, Atrial Fibrillation genetics, Atrial Fibrillation physiopathology, Calcium-Calmodulin-Dependent Protein Kinase Type 2 metabolism, Disease Models, Animal, Disease Progression, Female, Heart Conduction System metabolism, Heart Conduction System physiopathology, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Myocytes, Cardiac metabolism, Atrial Fibrillation metabolism, Calcium metabolism, Ryanodine Receptor Calcium Release Channel metabolism, Sarcoplasmic Reticulum metabolism
- Abstract
Background: The progression of atrial fibrillation (AF) from paroxysmal to persistent forms remains a major clinical challenge. Abnormal sarcoplasmic reticulum (SR) Ca(2+) leak via the ryanodine receptor type 2 (RyR2) has been observed as a source of ectopic activity in various AF models. However, its potential role in progression to long-lasting spontaneous AF (sAF) has never been tested. This study was designed to test the hypothesis that enhanced RyR2-mediated Ca(2+) release underlies the development of a substrate for sAF and to elucidate the underlying mechanisms., Methods and Results: CREM-IbΔC-X transgenic (CREM) mice developed age-dependent progression from spontaneous atrial ectopy to paroxysmal and eventually long-lasting AF. The development of sAF in CREM mice was preceded by enhanced diastolic Ca(2+) release, atrial enlargement, and marked conduction abnormalities. Genetic inhibition of Ca(2+)/calmodulin-dependent protein kinase II-mediated RyR2-S2814 phosphorylation in CREM mice normalized open probability of RyR2 channels and SR Ca(2+) release, delayed the development of spontaneous atrial ectopy, fully prevented sAF, suppressed atrial dilation, and forestalled atrial conduction abnormalities. Hyperactive RyR2 channels directly stimulated the Ca(2+)-dependent hypertrophic pathway nuclear factor of activated T cell/Rcan1-4, suggesting a role for the nuclear factor of activated T cell/Rcan1-4 system in the development of a substrate for long-lasting AF in CREM mice., Conclusions: RyR2-mediated SR Ca(2+) leak directly underlies the development of a substrate for sAF in CREM mice, the first demonstration of a molecular mechanism underlying AF progression and sAF substrate development in an experimental model. Our work demonstrates that the role of abnormal diastolic Ca(2+) release in AF may not be restricted to the generation of atrial ectopy but extends to the development of atrial remodeling underlying the AF substrate.
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- 2014
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46. Cellular and molecular mechanisms of atrial arrhythmogenesis in patients with paroxysmal atrial fibrillation.
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Voigt N, Heijman J, Wang Q, Chiang DY, Li N, Karck M, Wehrens XHT, Nattel S, and Dobrev D
- Subjects
- Aged, Arrhythmias, Cardiac physiopathology, Atrial Appendage pathology, Atrial Appendage physiopathology, Calcium-Binding Proteins physiology, Case-Control Studies, Cells, Cultured, Computer Simulation, Female, Humans, Male, Models, Cardiovascular, Myocytes, Cardiac pathology, Myocytes, Cardiac physiology, Patch-Clamp Techniques, Ryanodine Receptor Calcium Release Channel physiology, Sarcoplasmic Reticulum Calcium-Transporting ATPases physiology, Sodium-Calcium Exchanger physiology, Atrial Fibrillation physiopathology, Calcium physiology, Calcium Signaling physiology, Heart Atria physiopathology, Membrane Potentials physiology, Sarcoplasmic Reticulum physiology
- Abstract
Background: Electrical, structural, and Ca2+ -handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca2+ -release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca2+ -release events in paroxysmal AF (pAF) are unknown., Method and Results: Right-atrial appendages from control sinus rhythm patients or patients with pAF (last episode a median of 10-20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca2+ current, and Na+ /Ca2+ -exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca2+ leak and incidence of delayed after-depolarizations in pAF. Ca2+ -transient amplitude and sarcoplasmic reticulum Ca2+ load (caffeine-induced Ca2+ -transient amplitude, integrated Na+/Ca2+ -exchange current) were larger in pAF. Ca2+ -transient decay was faster in pAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting increased SERCA2a function. In agreement, phosphorylation (inactivation) of the SERCA2a-inhibitor protein phospholamban was increased in pAF. Ryanodine receptor fractional phosphorylation was unaltered in pAF, whereas ryanodine receptor expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both ryanodine receptor dysregulation and enhanced SERCA2a activity promote increased sarcoplasmic reticulum Ca2+ leak and sarcoplasmic reticulum Ca2+ -release events, causing delayed after-depolarizations/triggered activity in pAF., Conclusions: Increased diastolic sarcoplasmic reticulum Ca2+ leak and related delayed after-depolarizations/triggered activity promote cellular arrhythmogenesis in pAF patients. Biochemical, functional, and modeling studies point to a combination of increased sarcoplasmic reticulum Ca2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.
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- 2014
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47. A coregulatory network of NR2F1 and microRNA-140.
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Chiang DY, Cuthbertson DW, Ruiz FR, Li N, and Pereira FA
- Subjects
- 3' Untranslated Regions physiology, Animals, COUP Transcription Factor I genetics, Down-Regulation physiology, Ear, Inner cytology, Humans, Kruppel-Like Transcription Factors biosynthesis, Kruppel-Like Transcription Factors genetics, Mice, Mice, Knockout, MicroRNAs genetics, COUP Transcription Factor I metabolism, Ear, Inner metabolism, Gene Regulatory Networks physiology, MicroRNAs metabolism
- Abstract
Background: Both nuclear receptor subfamily 2 group F member 1 (NR2F1) and microRNAs (miRNAs) have been shown to play critical roles in the developing and functional inner ear. Based on previous studies suggesting interplay between NR2F1 and miRNAs, we investigated the coregulation between NR2F1 and miRNAs to better understand the regulatory mechanisms of inner ear development and functional maturation., Results: Using a bioinformatic approach, we identified 11 potential miRNAs that might coregulate target genes with NR2F1 and analyzed their targets and potential roles in physiology and disease. We selected 6 miRNAs to analyze using quantitative real-time (qRT) -PCR and found that miR-140 is significantly down-regulated by 4.5-fold (P=0.004) in the inner ear of NR2F1 knockout (Nr2f1(-/-)) mice compared to wild-type littermates but is unchanged in the brain. Based on this, we performed chromatin-immunoprecipitation followed by qRT-PCR and confirmed that NR2F1 directly binds and regulates both miR-140 and Klf9 in vivo. Furthermore, we performed luciferase reporter assay and showed that miR-140 mimic directly regulates KLF9-3'UTR, thereby establishing and validating an example coregulatory network involving NR2F1, miR-140, and Klf9., Conclusions: We have described and experimentally validated a novel tissue-dependent coregulatory network for NR2F1, miR-140, and Klf9 in the inner ear and we propose the existence of many such coregulatory networks important for both inner ear development and function.
- Published
- 2013
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48. Identification of driver genes in hepatocellular carcinoma by exome sequencing.
- Author
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Cleary SP, Jeck WR, Zhao X, Chen K, Selitsky SR, Savich GL, Tan TX, Wu MC, Getz G, Lawrence MS, Parker JS, Li J, Powers S, Kim H, Fischer S, Guindi M, Ghanekar A, and Chiang DY
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Female, Histone-Lysine N-Methyltransferase, Humans, Intracellular Signaling Peptides and Proteins genetics, Kelch-Like ECH-Associated Protein 1, Male, Middle Aged, Mutation, Myeloid-Lymphoid Leukemia Protein genetics, NF-E2-Related Factor 2 genetics, Sequence Analysis, DNA, Carcinoma, Hepatocellular genetics, Exome, Liver Neoplasms genetics
- Abstract
Unlabelled: Genetic alterations in specific driver genes lead to disruption of cellular pathways and are critical events in the instigation and progression of hepatocellular carcinoma (HCC). As a prerequisite for individualized cancer treatment, we sought to characterize the landscape of recurrent somatic mutations in HCC. We performed whole-exome sequencing on 87 HCCs and matched normal adjacent tissues to an average coverage of 59×. The overall mutation rate was roughly two mutations per Mb, with a median of 45 nonsynonymous mutations that altered the amino acid sequence (range, 2-381). We found recurrent mutations in several genes with high transcript levels: TP53 (18%); CTNNB1 (10%); KEAP1 (8%); C16orf62 (8%); MLL4 (7%); and RAC2 (5%). Significantly affected gene families include the nucleotide-binding domain and leucine-rich repeat-containing family, calcium channel subunits, and histone methyltransferases. In particular, the MLL family of methyltransferases for histone H3 lysine 4 were mutated in 20% of tumors., Conclusion: The NFE2L2-KEAP1 and MLL pathways are recurrently mutated in multiple cohorts of HCC., (© 2013 by the American Association for the Study of Liver Diseases.)
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- 2013
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49. Junctophilin-2 is necessary for T-tubule maturation during mouse heart development.
- Author
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Reynolds JO, Chiang DY, Wang W, Beavers DL, Dixit SS, Skapura DG, Landstrom AP, Song LS, Ackerman MJ, and Wehrens XH
- Subjects
- Animals, Calcium metabolism, Heart growth & development, Heart Failure etiology, Mice, Mice, Inbred C57BL, RNA, Small Interfering genetics, Heart embryology, Membrane Proteins physiology, Myocytes, Cardiac cytology, Sarcolemma physiology
- Abstract
Aims: Transverse tubules (TTs) provide the basic subcellular structures that facilitate excitation-contraction (EC) coupling, the essential process that underlies normal cardiac contractility. Previous studies have shown that TTs develop within the first few weeks of life in mammals but the molecular determinants of this development have remained elusive. This study aims to elucidate the role of junctophilin-2 (JPH2), a junctional membrane complex protein, in the maturation of TTs in cardiomyocytes., Methods and Results: Using a novel cardiac-specific short-hairpin-RNA-mediated JPH2 knockdown mouse model (Mus musculus; αMHC-shJPH2), we assessed the effects of the loss of JPH2 on the maturation of the ventricular TT structure. Between embryonic day (E) 10.5 and postnatal day (P) 10, JPH2 mRNA and protein levels were reduced by >70% in αMHC-shJPH2 mice. At P8 and P10, knockdown of JPH2 significantly inhibited the maturation of TTs, while expression levels of other genes implicated in TT development remained mostly unchanged. At the same time, intracellular Ca(2+) handling was disrupted in ventricular myocytes from αMHC- shJPH2 mice, which developed heart failure by P10 marked by reduced ejection fraction, ventricular dilation, and premature death. In contrast, JPH2 transgenic mice exhibited accelerated TT maturation by P8., Conclusion: Our findings suggest that JPH2 is necessary for TT maturation during postnatal cardiac development in mice. In particular, JPH2 may be critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum, thereby enabling the maturation of the TT network.
- Published
- 2013
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50. Chronic exercise: a contributing factor to atrial fibrillation?
- Author
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Wehrens XH, Chiang DY, and Li N
- Subjects
- Animals, Atrial Fibrillation physiopathology, Exercise Test methods, Physical Conditioning, Animal methods, Physical Conditioning, Animal physiology, Physical Endurance physiology
- Published
- 2013
- Full Text
- View/download PDF
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