222 results on '"Marek Napierala"'
Search Results
2. Correction: Comparative multi-omic analyses of cardiac mitochondrial stress in three mouse models of frataxin deficiency
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Nicole M. Sayles, Jill S. Napierala, Josef Anrather, Nadège Diedhiou, Jixue Li, Marek Napierala, Hélène Puccio, and Giovanni Manfredi
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Medicine ,Pathology ,RB1-214 - Published
- 2024
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3. Comparative multi-omic analyses of cardiac mitochondrial stress in three mouse models of frataxin deficiency
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Nicole M. Sayles, Jill S. Napierala, Josef Anrather, Nadège Diedhiou, Jixue Li, Marek Napierala, Hélène Puccio, and Giovanni Manfredi
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friedreich ataxia ,cardiomyopathy ,integrated stress response ,mitochondria ,frataxin ,mouse model ,Medicine ,Pathology ,RB1-214 - Published
- 2023
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4. Difficulties translating antisense-mediated activation of Frataxin expression from cell culture to mice
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Audrius Kilikevicius, Jun Wang, Xiulong Shen, Frank Rigo, Thahza P. Prakash, Marek Napierala, and David R. Corey
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friedrich’s ataxia ,frataxin ,antisense oligonucleotide ,trinucleotide repeat ,Genetics ,QH426-470 - Abstract
Friedreich’s ataxia (FA) is an inherited neurodegenerative disorder caused by decreased expression of frataxin (FXN) protein. Previous studies have shown that antisense oligonucleotides (ASOs) and single-stranded silencing RNAs can be used to increase expression of frataxin in cultured patient-derived cells. In this study, we investigate the potential for oligonucleotides to increase frataxin expression in a mouse model for FA. After confirming successful in vivo delivery of oligonucleotides using a benchmark gapmer targeting the nuclear noncoding RNA Malat1, we tested anti-FXN oligonucleotides designed to function by various mechanisms. None of these strategies yielded enhanced expression of FXN in the model mice. Our inability to translate activation of FXN expression from cell culture to mice may be due to inadequate potency of our compounds or differences in the molecular mechanisms governing FXN gene repression and activation in FA model mice.
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- 2022
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5. Acute frataxin knockdown in induced pluripotent stem cell-derived cardiomyocytes activates a type I interferon response
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M. Grazia Cotticelli, Shujuan Xia, Rachel Truitt, Nicolai M. Doliba, Andrea V. Rozo, John W. Tobias, Taehee Lee, Justin Chen, Jill S. Napierala, Marek Napierala, Wenli Yang, and Robert B. Wilson
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friedreich ataxia ,cardiomyopathy ,mtdna ,interferon ,innate immunity ,Medicine ,Pathology ,RB1-214 - Published
- 2023
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6. Neurobehavioral deficits of mice expressing a low level of G127V mutant frataxin
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Daniel Fil, Robbie L. Conley, Aamir R. Zuberi, Cathleen M. Lutz, Terry Gemelli, Marek Napierala, and Jill S. Napierala
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Frataxin ,Friedreich's ataxia ,Mitochondria ,Point mutation ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA repeats in intron 1 of FXN, while some are compound heterozygotes with an expanded GAA tract in one allele and a missense or nonsense mutation in the other. A missense mutation, changing a glycine to valine at position 130 (G130V), is prevalent among the clinical variants. We and others have demonstrated that levels of mature FXN protein in FRDA G130V samples are reduced below those detected in samples harboring homozygous repeat expansions. Little is known regarding expression and function of endogenous FXN-G130V protein due to lack of reagents and models that can distinguish the mutant FXN protein from the wild-type FXN produced from the GAA-expanded allele. We aimed to determine the effect of the G130V (murine G127V) mutation on Fxn expression and to define its multi-system impact in vivo. We used CRISPR/Cas9 to introduce the G127V missense mutation in the Fxn coding sequence and generated homozygous mice (FxnG127V/G127V). We also introduced the G127V mutation into a GAA repeat expansion FRDA mouse model (FxnGAA230/KO; KIKO) to generate a compound heterozygous strain (FxnG127V/GAA230). We performed neurobehavioral tests on cohorts of WT and Fxn mutant animals at three-month intervals for one year, and collected tissue samples to analyze molecular changes during that time. The endogenous Fxn G127V protein is detected at much lower levels in all tissues analyzed from FxnG127V/G127V mice compared to age and sex-matched WT mice without differences in Fxn transcript levels. FxnG127V/G127V mice are significantly smaller than WT counterparts, but perform similarly in most neurobehavioral tasks. RNA sequencing analysis revealed reduced expression of genes in oxidative phosphorylation and protein synthesis, underscoring the metabolic consequences in our mouse model expressing extremely low levels of Fxn. Results of these studies provide insight into the unique pathogenic mechanism of the FXN G130V mechanism and the tolerable limit of Fxn/FXN expression in vivo.
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- 2023
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7. Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia
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Dezhen Wang, Elaine S. Ho, M. Grazia Cotticelli, Peining Xu, Jill S. Napierala, Lauren A. Hauser, Marek Napierala, Blanca E. Himes, Robert B. Wilson, David R. Lynch, and Clementina Mesaros
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frataxin ,ceramides ,fatty acids oxidation ,triglycerides ,phospholipids ,lipidomics ,Biochemistry ,QD415-436 - Abstract
Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.
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- 2022
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8. Selected Histone Deacetylase Inhibitors Reverse the Frataxin Transcriptional Defect in a Novel Friedreich’s Ataxia Induced Pluripotent Stem Cell-Derived Neuronal Reporter System
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Anna M. Schreiber, Yanjie Li, Yi-Hsien Chen, Jill S. Napierala, and Marek Napierala
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Friedreich’s ataxia (FRDA) ,reporter cell line ,induced pluripotent stem cells ,neural progenitor cells (NPCs) ,Nanoluciferase ,screening ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Friedreich’s ataxia (FRDA) is a neurodegenerative disorder caused by the expansion of guanine–adenine–adenine repeats within the first intron of the frataxin (FXN) gene. The location and nature of the expansion have been proven to contribute to transcriptional repression of FXN by decreasing the rate of polymerase II (RNA polymerase II) progression and increasing the presence of histone modifications associated with a heterochromatin-like state. Targeting impaired FXN transcription appears as a feasible option for therapeutic intervention, while no cure currently exists. We created a novel reporter cell line containing an FXN-Nanoluciferase (FXN-NLuc) fusion in induced pluripotent stem cells (iPSCs) reprogrammed from the fibroblasts of patients with FRDA, thus allowing quantification of endogenous FXN expression. The use of iPSCs provides the opportunity to differentiate these cells into disease-relevant neural progenitor cells (NPCs). NPCs derived from the FXN-NLuc line responded to treatments with a known FXN inducer, RG109. Results were validated by quantitative PCR and Western blot in multiple FRDA NPC lines. We then screened a commercially available library of compounds consisting of molecules targeting various enzymes and pathways critical for silencing or activation of gene expression. Only selected histone deacetylase inhibitors were capable of partial reactivation of FXN expression. This endogenous, FRDA iPSC-derived reporter can be utilized for high-throughput campaigns performed in cells most relevant to disease pathology in search of FXN transcription activators.
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- 2022
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9. Novel dopamine receptor 3 antagonists inhibit the growth of primary and temozolomide resistant glioblastoma cells.
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Sarah E Williford, Catherine J Libby, Adetokunbo Ayokanmbi, Arphaxad Otamias, Juan J Gordillo, Emily R Gordon, Sara J Cooper, Matthew Redmann, Yanjie Li, Corinne Griguer, Jianhua Zhang, Marek Napierala, Subramaniam Ananthan, and Anita B Hjelmeland
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Medicine ,Science - Abstract
Treatment for the lethal primary adult brain tumor glioblastoma (GBM) includes the chemotherapy temozolomide (TMZ), but TMZ resistance is common and correlates with promoter methylation of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT). To improve treatment of GBMs, including those resistant to TMZ, we explored the potential of targeting dopamine receptor signaling. We found that dopamine receptor 3 (DRD3) is expressed in GBM and is also a previously unexplored target for therapy. We identified novel antagonists of DRD3 that decreased the growth of GBM xenograft-derived neurosphere cultures with minimal toxicity against human astrocytes and/or induced pluripotent stem cell-derived neurons. Among a set of DRD3 antagonists, we identified two compounds, SRI-21979 and SRI-30052, that were brain penetrant and displayed a favorable therapeutic window analysis of The Cancer Genome Atlas data demonstrated that higher levels of DRD3 (but not DRD2 or DRD4) were associated with worse prognosis in primary, MGMT unmethylated tumors. These data suggested that DRD3 antagonists may remain efficacious in TMZ-resistant GBMs. Indeed, SRI-21979, but not haloperidol, significantly reduced the growth of TMZ-resistant GBM cells. Together our data suggest that DRD3 antagonist-based therapies may provide a novel therapeutic option for the treatment of GBM.
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- 2021
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10. Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia
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Daniel Fil, Balu K. Chacko, Robbie Conley, Xiaosen Ouyang, Jianhua Zhang, Victor M. Darley-Usmar, Aamir R. Zuberi, Cathleen M. Lutz, Marek Napierala, and Jill S. Napierala
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friedreich's ataxia ,senescence ,mitochondria ,frataxin ,point mutation ,oxidative stress ,Medicine ,Pathology ,RB1-214 - Abstract
Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA repeat sequences in intron 1 of FXN, whereas a fraction of patients are compound heterozygotes, with a missense or nonsense mutation in one FXN allele and expanded GAAs in the other. A prevalent missense mutation among FRDA patients changes a glycine at position 130 to valine (G130V). Herein, we report generation of the first mouse model harboring an Fxn point mutation. Changing the evolutionarily conserved glycine 127 in mouse Fxn to valine results in a failure-to-thrive phenotype in homozygous animals and a substantially reduced number of offspring. Like G130V in FRDA, the G127V mutation results in a dramatic decrease of Fxn protein without affecting transcript synthesis or splicing. FxnG127V mouse embryonic fibroblasts exhibit significantly reduced proliferation and increased cell senescence. These defects are evident in early passage cells and are exacerbated at later passages. Furthermore, increased frequency of mitochondrial DNA lesions and fragmentation are accompanied by marked amplification of mitochondrial DNA in FxnG127V cells. Bioenergetics analyses demonstrate higher sensitivity and reduced cellular respiration of FxnG127V cells upon alteration of fatty acid availability. Importantly, substitution of FxnWT with FxnG127V is compatible with life, and cellular proliferation defects can be rescued by mitigation of oxidative stress via hypoxia or induction of the NRF2 pathway. We propose FxnG127V cells as a simple and robust model for testing therapeutic approaches for FRDA.
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- 2020
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11. Comprehensive analysis of gene expression patterns in Friedreich's ataxia fibroblasts by RNA sequencing reveals altered levels of protein synthesis factors and solute carriers
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Jill Sergesketter Napierala, Yanjie Li, Yue Lu, Kevin Lin, Lauren A. Hauser, David R. Lynch, and Marek Napierala
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Friedreich's ataxia ,Fibroblasts ,Translation ,Solute carriers ,RNA sequencing ,Medicine ,Pathology ,RB1-214 - Abstract
Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease usually caused by large homozygous expansions of GAA repeat sequences in intron 1 of the frataxin (FXN) gene. FRDA patients homozygous for GAA expansions have low FXN mRNA and protein levels when compared with heterozygous carriers or healthy controls. Frataxin is a mitochondrial protein involved in iron–sulfur cluster synthesis, and many FRDA phenotypes result from deficiencies in cellular metabolism due to lowered expression of FXN. Presently, there is no effective treatment for FRDA, and biomarkers to measure therapeutic trial outcomes and/or to gauge disease progression are lacking. Peripheral tissues, including blood cells, buccal cells and skin fibroblasts, can readily be isolated from FRDA patients and used to define molecular hallmarks of disease pathogenesis. For instance, FXN mRNA and protein levels as well as FXN GAA-repeat tract lengths are routinely determined using all of these cell types. However, because these tissues are not directly involved in disease pathogenesis, their relevance as models of the molecular aspects of the disease is yet to be decided. Herein, we conducted unbiased RNA sequencing to profile the transcriptomes of fibroblast cell lines derived from 18 FRDA patients and 17 unaffected control individuals. Bioinformatic analyses revealed significantly upregulated expression of genes encoding plasma membrane solute carrier proteins in FRDA fibroblasts. Conversely, the expression of genes encoding accessory factors and enzymes involved in cytoplasmic and mitochondrial protein synthesis was consistently decreased in FRDA fibroblasts. Finally, comparison of genes differentially expressed in FRDA fibroblasts to three previously published gene expression signatures defined for FRDA blood cells showed substantial overlap between the independent datasets, including correspondingly deficient expression of antioxidant defense genes. Together, these results indicate that gene expression profiling of cells derived from peripheral tissues can, in fact, consistently reveal novel molecular pathways of the disease. When performed on statistically meaningful sample group sizes, unbiased global profiling analyses utilizing peripheral tissues are critical for the discovery and validation of FRDA disease biomarkers.
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- 2017
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12. Excision of the expanded GAA repeats corrects cardiomyopathy phenotypes of iPSC-derived Friedreich's ataxia cardiomyocytes
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Jixue Li, Natalia Rozwadowska, Amanda Clark, Daniel Fil, Jill S. Napierala, and Marek Napierala
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Biology (General) ,QH301-705.5 - Abstract
Friedreich's ataxia is caused by large homozygous, intronic expansions of GAA repeats in the frataxin (FXN) gene, resulting in severe downregulation of its expression. Pathogenic repeats are located in intron one, hence patients express unaffected FXN protein, albeit in low quantities. Although FRDA symptoms typically afflict the nervous system, hypertrophic cardiomyopathy is the predominant cause of death. Our studies were conducted using cardiomyocytes differentiated from induced pluripotent stem cells derived from control individuals, FRDA patients, and isogenic cells corrected by zinc finger nucleases-mediated excision of pathogenic expanded GAA repeats. This correction of the FXN gene removed the primary trigger of the transcription defect, upregulated frataxin expression, reduced pathological lipid accumulation observed in patient cardiomyocytes, and reversed gene expression signatures of FRDA cardiomyocytes. Transcriptome analyses revealed hypertrophy-specific expression signatures unique to FRDA cardiomyocytes, and emphasized similarities between unaffected and ZFN-corrected FRDA cardiomyocytes. Thus, the iPSC-derived FRDA cardiomyocytes exhibit various molecular defects characteristic for cellular models of cardiomyopathy that can be corrected by genome editing of the expanded GAA repeats. These results underscore the utility of genome editing in generating isogenic cellular models of FRDA and the potential of this approach as a future therapy for this disease. Keywords: Friedreich's ataxia, GAA repeats, Genome editing, Isogenic iPSC, Cardiomyocytes, Lipid metabolism
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- 2019
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13. The current state of biomarker research for Friedreich’s ataxia: a report from the 2018 FARA biomarker meeting
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Ian A Blair, Jennifer Farmer, Steven Hersch, Jane Larkindale, David R Lynch, Jill Napierala, Marek Napierala, R Mark Payne, and Sub H Subramony
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biomarkers ,drug development ,Friedreich’s ataxia ,Medicine ,Medicine (General) ,R5-920 - Abstract
The 2018 FARA Biomarker Meeting highlighted the current state of development of biomarkers for Friedreich’s ataxia. A mass spectroscopy assay to sensitively measure mature frataxin (reduction of which is the root cause of disease) is being developed. Biomarkers to monitor neurological disease progression include imaging, electrophysiological measures and measures of nerve function, which may be measured either in serum and/or through imaging-based technologies. Potential pharmacodynamic biomarkers include metabolic and protein biomarkers and markers of nerve damage. Cardiac imaging and serum biomarkers may reflect cardiac disease progression. Considerable progress has been made in the development of biomarkers for various contexts of use, but further work is needed in terms of larger longitudinal multisite studies, and identification of novel biomarkers for additional use cases
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- 2019
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14. Potential biomarker identification for Friedreich's ataxia using overlapping gene expression patterns in patient cells and mouse dorsal root ganglion.
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Marissa Z McMackin, Blythe Durbin-Johnson, Marek Napierala, Jill S Napierala, Luis Ruiz, Eleonora Napoli, Susan Perlman, Cecilia Giulivi, and Gino A Cortopassi
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Medicine ,Science - Abstract
Friedreich's ataxia (FA) is a neurodegenerative disease with no approved therapy that is the result of frataxin deficiency. The identification of human FA blood biomarkers related to disease severity and neuro-pathomechanism could support clinical trials of drug efficacy. To try to identify human biomarkers of neuro-pathomechanistic relevance, we compared the overlapping gene expression changes of primary blood and skin cells of FA patients with changes in the Dorsal Root Ganglion (DRG) of the KIKO FA mouse model. As DRG is the primary site of neurodegeneration in FA, our goal was to identify which changes in blood and skin of FA patients provide a 'window' into the FA neuropathomechanism inside the nervous system. In addition, gene expression in frataxin-deficient neuroglial cells and FA mouse hearts were compared for a total of 5 data sets. The overlap of these changes strongly supports mitochondrial changes, apoptosis and alterations of selenium metabolism. Consistent biomarkers were observed, including three genes of mitochondrial stress (MTIF2, ENO2), apoptosis (DDIT3/CHOP), oxidative stress (PREX1), and selenometabolism (SEPW1). These results prompted our investigation of the GPX1 activity as a marker of selenium and oxidative stress, in which we observed a significant change in FA patients. We believe these lead biomarkers that could be assayed in FA patient blood as indicators of disease severity and progression, and also support the involvement of mitochondria, apoptosis and selenium in the neurodegenerative process.
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- 2019
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15. Stalled DNA Replication Forks at the Endogenous GAA Repeats Drive Repeat Expansion in Friedreich’s Ataxia Cells
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Jeannine Gerhardt, Angela D. Bhalla, Jill Sergesketter Butler, James W. Puckett, Peter B. Dervan, Zev Rosenwaks, and Marek Napierala
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Biology (General) ,QH301-705.5 - Abstract
Friedreich’s ataxia (FRDA) is caused by the expansion of GAA repeats located in the Frataxin (FXN) gene. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing to disease progression. The mechanism leading to repeat expansion and decreased FXN transcription remains unclear. Using single-molecule analysis of replicated DNA, we detected that expanded GAA repeats present a substantial obstacle for the replication machinery at the FXN locus in FRDA cells. Furthermore, aberrant origin activation and lack of a proper stress response to rescue the stalled forks in FRDA cells cause an increase in 3′-5′ progressing forks, which could enhance repeat expansion and hinder FXN transcription by head-on collision with RNA polymerases. Treatment of FRDA cells with GAA-specific polyamides rescues DNA replication fork stalling and alleviates expansion of the GAA repeats, implicating DNA triplexes as a replication impediment and suggesting that fork stalling might be a therapeutic target for FRDA.
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- 2016
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16. Costa Rica – 'Bogate Wybrzeże' = Costa Rica – „Rich Coast'
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Marek Napierala
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turystyka, kostaryka, parki narodowe, tourism, costa rica, national parks. ,Education ,Sports ,GV557-1198.995 ,Medicine - Abstract
Napierała Marek. Costa Rica – “Bogate Wybrzeże” = Costa Rica – „Rich Coast”. Journal of Education, Health and Sport. 2016;6(2):111-121. eISSN 2391-8306. DOI http://dx.doi.org/10.5281/zenodo.46249 http://ojs.ukw.edu.pl/index.php/johs/article/view/3392 https://pbn.nauka.gov.pl/works/715180 The journal has had 7 points in Ministry of Science and Higher Education parametric evaluation. Part B item 755 (23.12.2015). 755 Journal of Education, Health and Sport eISSN 2391-8306 7 © The Author (s) 2016; This article is published with open access at Licensee Open Journal Systems of Kazimierz Wielki University in Bydgoszcz, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 15.02.2016. Revised 18.02.2016. Accepted: 18.02.2016. Costa Rica – “Bogate Wybrzeże” Costa Rica – „Rich Coast” Marek Napierała Uniwersytet Kazimierza Wielkiego w Bydgoszczy Słowa kluczowe: turystyka, Kostaryka, parki narodowe. Key words: tourism, Costa Rica, national parks. Streszczenie Materiał stanowi dokument z obserwacji autora z pobytu w Kostaryce. Pobyt miał miejsce na przełomie 2015 i 2016 roku i był okazją do zwiedzenia miejsc ciekawych w klimacie podrównikowym. Podróżowanie to odwieczne ludzkie marzenie. Treścią wielu wyjazdów staje się odkrywanie nieznanego, szukanie odmienności w klimacie, florze i faunie, kulturze i zwyczajach ludzi. Można tu spotkać wiele gatunków zwierząt jak: małpy, krokodyle, liczne odmiany barwnych papug, kolibrów, tukany, ciekawe gatunki jaszczurek, węży, bogato ubarwione żaby, żółwie czy legwany. Ciekawe są także chrząszcze i wiele gatunków motyli. Kostaryka zostanie w pamięci miejscem fantastycznych krajobrazów, wakacyjny fenomenem. Abstract The material is a document from the observation of the author's stay in Costa Rica. Visit took place at the turn of 2015 and 2016 years and was an opportunity to visit interesting places in equatorial climates. Travelling to the eternal human dream. The content of many trips becomes discovering the unknown, looking for differences in climate, flora and fauna, culture and customs of people. You can meet many animal species as .: monkeys, crocodiles, numerous varieties of colorful parrots, hummingbirds, toucans, interesting species of lizards, snakes, richly colored frogs, turtles and iguanas. Interesting are also many species of beetles and butterflies. Costa Rica is in the memory of the place fantastic landscapes, house phenomenon.
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- 2016
17. Somatic instability of the expanded GAA repeats in Friedreich's ataxia.
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Ashlee Long, Jill S Napierala, Urszula Polak, Lauren Hauser, Arnulf H Koeppen, David R Lynch, and Marek Napierala
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Medicine ,Science - Abstract
Friedreich's ataxia (FRDA) is a genetic neurodegenerative disorder caused by transcriptional silencing of the frataxin gene (FXN) due to expansions of GAA repeats in intron 1. FRDA manifests with multiple symptoms, which may include ataxia, cardiomyopathy and diabetes mellitus. Expanded GAA tracts are genetically unstable, exhibiting both expansions and contractions. GAA length correlates with severity of FRDA symptoms and inversely with age of onset. Thus, tissue-specific somatic instability of long GAA repeats may be implicated in the development of symptoms and disease progression. Herein, we determined the extent of somatic instability of the GAA repeats in heart, cerebral cortex, spinal cord, cerebellar cortex, and pancreatic tissues from 15 FRDA patients. Results demonstrate differences in the lengths of the expanded GAAs among different tissues, with significantly longer GAA tracts detected in heart and pancreas than in other tissues. The expansion bias detected in heart and pancreas may contribute to disease onset and progression, making the mechanism of somatic instability an important target for therapy. Additionally, we detected significant differences in GAA tract lengths between lymphocytes and fibroblast pairs derived from 16 FRDA patients, with longer GAA tracts present in the lymphocytes. This result urges caution in direct comparisons of data obtained in these frequently used FRDA models. Furthermore, we conducted a longitudinal analysis of the GAA repeat length in lymphocytes collected over a span of 7-9 years and demonstrated progressive expansions of the GAAs with maximum gain of approximately 9 repeats per year. Continuous GAA expansions throughout the patient's lifespan, as observed in FRDA lymphocytes, should be considered in clinical trial designs and data interpretation.
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- 2017
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18. Understanding the genetic and molecular pathogenesis of Friedreich’s ataxia through animal and cellular models
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Alain Martelli, Marek Napierala, and Hélène Puccio
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Medicine ,Pathology ,RB1-214 - Abstract
In 1996, a link was identified between Friedreich’s ataxia (FRDA), the most common inherited ataxia in men, and alterations in the gene encoding frataxin (FXN). Initial studies revealed that the disease is caused by a unique, most frequently biallelic, expansion of the GAA sequence in intron 1 of FXN. Since the identification of this link, there has been tremendous progress in understanding frataxin function and the mechanism of FRDA pathology, as well as in developing diagnostics and therapeutic approaches for the disease. These advances were the subject of the 4th International Friedreich’s Ataxia Conference held on 5th–7th May in the Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France. More than 200 scientists gathered from all over the world to present the results of research spanning all areas of investigation into FRDA (including clinical aspects, FRDA pathogenesis, genetics and epigenetics of the disease, development of new models of FRDA, and drug discovery). This review provides an update on the understanding of frataxin function, developments of animal and cellular models of the disease, and recent advances in trying to uncover potential molecules for therapy.
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- 2012
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19. Integrated Photonic True-Time Delay Beamformer for a Ka-band Phased Array Antenna Receiver.
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Vanessa C. Duarte, Joao G. Prata, Carlos Ribeiro, Rogério N. Nogueira, Georg Winzer, Lars Zimmermann, Robert G. Walker, Stephen Clements, Marta Filipowicz, Marek Napierala, Tomasz Nasilowski, Jonathan Crabb, Leontios Stampoulidis, Javad Anzalchi, and Miguel V. Drummond
- Published
- 2018
20. Optical amplifier based on a 7-core fiber for telecommunication satellite purposes.
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Marta Filipowicz, Marek Napierala, M. Murawslii, L. Ostrowski, Lukasz Szostkiewicz, Pawel Mergo, M. Kechagias, J. Farzana, Leontios Stampoulidis, Efstratios Kehayas, and Tomasz Nasilowski
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- 2017
21. Premature transcription termination at the expanded GAA repeats and aberrant alternative polyadenylation contributes to theFrataxintranscriptional deficit in Friedreich’s ataxia
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Yanjie Li, Jixue Li, Jun Wang, Siyuan Zhang, Keith Giles, Thazha P Prakash, Frank Rigo, Jill S Napierala, and Marek Napierala
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Guanine ,Transcription, Genetic ,Adenine ,Gallium ,General Medicine ,Oligonucleotides, Antisense ,Polyadenylation ,Arsenicals ,Friedreich Ataxia ,Iron-Binding Proteins ,Genetics ,Humans ,RNA, Messenger ,Trinucleotide Repeat Expansion ,Molecular Biology ,Genetics (clinical) - Abstract
Frataxin deficiency in Friedreich’s ataxia results from transcriptional downregulation of the FXN gene caused by expansion of the intronic trinucleotide guanine-adenine-adenine (GAA) repeats. We used multiple transcriptomic approaches to determine the molecular mechanism of transcription inhibition caused by long GAAs. We uncovered that transcription of FXN in patient cells is prematurely terminated upstream of the expanded repeats leading to the formation of a novel, truncated and stable RNA. This FXN early terminated transcript (FXN-ett) undergoes alternative, non-productive splicing and does not contribute to the synthesis of functional frataxin. The level the FXN-ett RNA directly correlates with the length of the longer of the two expanded GAA tracts. Targeting GAAs with antisense oligonucleotides or excision of the repeats eliminates the transcription impediment, diminishes expression of the aberrant FXN-ett, while increasing levels of FXN mRNA and frataxin. Non-productive transcription may represent a common phenomenon and attractive therapeutic target in diseases caused by repeat-mediated transcription aberrations.
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- 2022
22. Study on the Sensing Coating of the Optical Fibre CO2 Sensor.
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Karol Wysokinski, Marek Napierala, Tomasz Stanczyk, Stanislaw Lipinski, and Tomasz Nasilowski
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- 2015
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23. Targeting 3′ and 5′ untranslated regions with antisense oligonucleotides to stabilize frataxin mRNA and increase protein expression
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Darshan Parekh, Jill S. Napierala, Jonathan J Cherry, Xiulong Shen, Balkrishen Bhat, Caroline J. Woo, David R. Corey, Jixue Li, David A. Lynch, Jun Wang, Yanjie Li, and Marek Napierala
- Subjects
Untranslated region ,Messenger RNA ,AcademicSubjects/SCI00010 ,RNA Stability ,Gene regulation, Chromatin and Epigenetics ,Intron ,Translation (biology) ,Genetic Therapy ,Biology ,Oligonucleotides, Antisense ,Chromatin ,Cell biology ,Downregulation and upregulation ,Transcription (biology) ,Friedreich Ataxia ,Iron-Binding Proteins ,Genetics ,Frataxin ,biology.protein ,Humans ,RNA, Messenger ,5' Untranslated Regions ,3' Untranslated Regions ,Cells, Cultured - Abstract
Friedreich’s ataxia (FRDA) is a severe multisystem disease caused by transcriptional repression induced by expanded GAA repeats located in intron 1 of the Frataxin (FXN) gene encoding frataxin. FRDA results from decreased levels of frataxin; thus, stabilization of the FXN mRNA already present in patient cells represents an attractive and unexplored therapeutic avenue. In this work, we pursued a novel approach based on oligonucleotide-mediated targeting of FXN mRNA ends to extend its half-life and availability as a template for translation. We demonstrated that oligonucleotides designed to bind to FXN 5′ or 3′ noncoding regions can increase FXN mRNA and protein levels. Simultaneous delivery of oligonucleotides targeting both ends increases efficacy of the treatment. The approach was confirmed in several FRDA fibroblast and induced pluripotent stem cell-derived neuronal progenitor lines. RNA sequencing and single-cell expression analyses confirmed oligonucleotide-mediated FXN mRNA upregulation. Mechanistically, a significant elongation of the FXN mRNA half-life without any changes in chromatin status at the FXN gene was observed upon treatment with end-targeting oligonucleotides, indicating that transcript stabilization is responsible for frataxin upregulation. These results identify a novel approach toward upregulation of steady-state mRNA levels via oligonucleotide-mediated end targeting that may be of significance to any condition resulting from transcription downregulation.
- Published
- 2021
24. Acute frataxin knockdown in induced pluripotent stem cell-derived cardiomyocytes activates a type I interferon response
- Author
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M. Grazia Cotticelli, Shujuan Xia, Rachel Truitt, Nicolai M. Doliba, Andrea V. Rozo, John W. Tobias, Taehee Lee, Justin Chen, Jill S. Napierala, Marek Napierala, Wenli Yang, and Robert B. Wilson
- Subjects
Iron ,Induced Pluripotent Stem Cells ,Neuroscience (miscellaneous) ,Medicine (miscellaneous) ,DNA, Mitochondrial ,Nucleotidyltransferases ,General Biochemistry, Genetics and Molecular Biology ,Mitochondrial Proteins ,Immunology and Microbiology (miscellaneous) ,Friedreich Ataxia ,Iron-Binding Proteins ,Interferon Type I ,Humans ,Myocytes, Cardiac ,Sulfur - Abstract
Friedreich ataxia, the most common hereditary ataxia, is a neuro- and cardio-degenerative disorder caused, in most cases, by decreased expression of the mitochondrial protein frataxin. Cardiomyopathy is the leading cause of premature death. Frataxin functions in the biogenesis of iron-sulfur clusters, which are prosthetic groups that are found in proteins involved in many biological processes. To study the changes associated with decreased frataxin in human cardiomyocytes, we developed a novel isogenic model by acutely knocking down frataxin, post-differentiation, in cardiomyocytes derived from induced pluripotent stem cells (iPSCs). Transcriptome analysis of four biological replicates identified severe mitochondrial dysfunction and a type I interferon response as the pathways most affected by frataxin knockdown. We confirmed that, in iPSC-derived cardiomyocytes, loss of frataxin leads to mitochondrial dysfunction. The type I interferon response was activated in multiple cell types following acute frataxin knockdown and was caused, at least in part, by release of mitochondrial DNA into the cytosol, activating the cGAS-STING sensor pathway.
- Published
- 2022
25. All-Fiber Vector Bending Sensor Based on a Multicore Fiber With Asymmetric Air-Hole Structure
- Author
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Krzysztof Poturaj, Marek Napierala, Tomasz Nasilowski, Krzysztof Markiewicz, Małgorzata Kuklińska, Pawel Mergo, Mariusz Makara, Zbigniew Holdynski, Dawid Budnicki, Itxaso Parola, Grzegorz M. Wojcik, and Lukasz Szostkiewicz
- Subjects
Materials science ,Optical fiber ,business.industry ,Bend radius ,02 engineering and technology ,Bending ,Atomic and Molecular Physics, and Optics ,Electronic mail ,law.invention ,Core (optical fiber) ,Light intensity ,020210 optoelectronics & photonics ,Optics ,law ,Orientation (geometry) ,0202 electrical engineering, electronic engineering, information engineering ,business ,Intensity (heat transfer) - Abstract
In this article we present an all-fiber vector bend sensor by means of a self-fabricated micro-structured multicore optical fiber. The reported solution is based on differential intensity variations of the light transmitted along the cores whose changes are influenced by the bending angle and orientation. The unique asymmetric structure of the air-holes in the optical fiber provides each core with different confinement losses of the fundamental mode depending on the bending radius and orientation, making each of the cores bend-sensitive in a range of at least 80°. It has been experimentally demonstrated that the reported sensor enables the bending angle and orientation to be detected in a full range of 360° without any dead-zones, and the possibility of end point detection with millimeter precision. Additionally, a reconstruction of the bending vector has been carried out theoretically, and a good match can be observed between the experimental and theoretical data.
- Published
- 2020
26. A CRISPR-Cas9, Cre-lox, and Flp-FRT Cascade Strategy for the Precise and Efficient Integration of Exogenous DNA into Cellular Genomes
- Author
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Marek Napierala, Jixue Li, Yanjie Li, Kevin M. Pawlik, and Jill S. Napierala
- Subjects
Genetics ,CRISPR ,Exogenous DNA ,Computational biology ,Biology ,Repeated sequence ,Gene ,Genome ,Recombination ,Selection (genetic algorithm) ,Biotechnology ,Antibiotic resistance genes - Abstract
We describe a protocol for the precise integration of exogenous DNA into user-defined genomic loci in cultured cells. This strategy first introduces a promoter and a lox site to a specific location via a Cas9-induced double-strand break. Second, a gene of interest (GOI) is inserted into the lox site via Cre-lox recombination. Upon correct insertion, a cis-linked antibiotic resistance gene will be expressed from a promoter introduced into the genome in the first step assuring selection for correct integrants. Last, the selection cassette is excised via a Flp-FRT recombination event, leaving a precisely targeted GOI. This method is broadly applicable to any exogenous DNA to be integrated, choice of integration site, and choice of cell type. The most remarkable aspect of this versatile approach, termed "CasPi" (cascaded precise integration), is that it allows for precise genome targeting with large, frequently complex, and repetitive DNA sequences that do not integrate efficiently or at all with current genome targeting methods.
- Published
- 2020
27. A Comprehensive Transcriptome Analysis Identifies FXN and BDNF as Novel Targets of miRNAs in Friedreich’s Ataxia Patients
- Author
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Marek Napierala, Magdalena Jazurek-Ciesiolka, David A. Lynch, Martyna Olga Urbanek-Trzeciak, Lauren A. Hauser, Anna M. Schreiber, Jill S. Napierala, and Julia O. Misiorek
- Subjects
0301 basic medicine ,Ataxia ,microRNA-224-5p ,In silico ,Neuroscience (miscellaneous) ,miRNAseq ,Computational biology ,medicine.disease_cause ,Transcriptome ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Iron-Binding Proteins ,microRNA ,medicine ,Frataxin (FXN) ,Humans ,Gene ,Mutation ,biology ,Brain-Derived Neurotrophic Factor ,Gene Expression Profiling ,Intron ,Fibroblasts ,Friedreich’s ataxia (FRDA) ,RNAseq ,microRNA-10a-5p ,MicroRNAs ,030104 developmental biology ,Neurology ,Friedreich Ataxia ,Frataxin ,biology.protein ,Original Article ,Brain-derived neurotrophic factor (BDNF) ,medicine.symptom ,Trinucleotide Repeat Expansion ,030217 neurology & neurosurgery - Abstract
Friedreich’s ataxia (FRDA) is a genetic neurodegenerative disease that is caused by guanine-adenine-adenine (GAA) nucleotide repeat expansions in the first intron of the frataxin (FXN) gene. Although present in the intron, this mutation leads to a substantial decrease in protein expression. Currently, no effective treatment is available for FRDA, and, in addition to FXN, other targets with therapeutic potential are continuously sought. As miRNAs can regulate the expression of a broad spectrum of genes, are used as biomarkers, and can serve as therapeutic tools, we decided to identify and characterize differentially expressed miRNAs and their targets in FRDA cells compared to unaffected control (CTRL) cells. In this study, we performed an integrated miRNAseq and RNAseq analysis using the same cohort of primary FRDA and CTRL cells. The results of the transcriptome studies were supported by bioinformatic analyses and validated by qRT-PCR. miRNA interactions with target genes were assessed by luciferase assays, qRT-PCR, and immunoblotting. In silico analysis identified the FXN transcript as a target of five miRNAs upregulated in FRDA cells. Further studies confirmed that miRNA-224-5p indeed targets FXN, resulting in decreases in mRNA and protein levels. We also validated the ability of miRNA-10a-5p to bind and regulate the levels of brain-derived neurotrophic factor (BDNF), an important modulator of neuronal growth. We observed a significant decrease in the levels of miRNA-10a-5p and increase in the levels of BDNF upon correction of FRDA cells via zinc-finger nuclease (ZFN)-mediated excision of expanded GAA repeats. Our comprehensive transcriptome analyses identified miRNA-224-5p and miRNA-10a-5p as negative regulators of the FXN and BDNF expression, respectively. These results emphasize not only the importance of miRNAs in the pathogenesis of FRDA but also their potential as therapeutic targets for this disease.
- Published
- 2020
28. Linking Dynamic DNA Secondary Structures to Genome Instability
- Author
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Gabriel Matos-Rodrigues, Niek van Wietmarschen, Wei Wu, Veenu Tripathi, Natasha Koussa, Raphael Pavani, William Nathan, Frida Belinky, Ashraf Mohammed, Marek Napierala, Karen Usdin, Aseem Z. Ansari, Sergei M. Mirkin, and André Nussenzweig
- Abstract
SummaryGenomic double-stranded DNA (dsDNA) becomes single-stranded (ssDNA) during replication, transcription, and DNA repair. ssDNA is therefore believed to be transient, occurring in only a fraction of the genome at a given time, and variable amongst a population of cells. These transiently formed ssDNA segments can also adopt alternative, dynamic DNA conformations, such as cruciform DNA, triplexes, quadruplexes and others. To determine whether there are stable and conserved regions of ssDNA, we utilized our previously developed method S1-END-seq 1 to convert ssDNA to DNA double strand breaks (DSBs), which are then processed for high-throughput sequencing. This approach revealed two predominant dynamic DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulated uniquely in microsatellite unstable human cancer cell lines, and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine (hPu/hPy) mirror repeats common across a variety of human cell lines. Triplex-forming repeats accumulated during replication, blocked DNA synthesis and were hotspots of somatic mutation. In contrast, pathologically expanded (hPu/hPy) repeats in Friedreich’s ataxia patient cells formed a replication-independent and transcription-inducible DNA secondary structure. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.
- Published
- 2022
29. Sensitivity tailoring of an all-fiber bend sensor based on a dual-core fiber unbalanced Michelson interferometer
- Author
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Łukasz Szostkiewicz, Pawel Mergo, Krzysztof Markiewicz, Marek Napierala, Itxaso Parola, Krzysztof Wilczyński, Małgorzata Kuklińska, Tomasz Nasilowski, and Dawid Budnicki
- Subjects
Materials science ,Silica fiber ,business.industry ,Michelson interferometer ,Microstructured optical fiber ,Atomic and Molecular Physics, and Optics ,law.invention ,Core (optical fiber) ,Interferometry ,Optics ,Fiber Bragg grating ,law ,Fiber optic sensor ,Fiber ,business - Abstract
In this work, a compact all-fiber bend sensor based on a dual-core microstructured optical fiber has been manufactured and characterized. The sensor relies on the unbalanced Michelson interferometric technique realized by attaching a piece of silica fiber to one of the fiber cores acting as the unbalancing element. Three probes with different lengths of the unbalancing element have been experimentally tested for sensitivity tailoring analysis. Additionally, a theoretical model has been developed for comparison and verification of the results. Good linear behavior of the spectral shift with bend has been measured and it has been proven that the sensitivity of the sensor depends on the length of the unbalancing element and the orientation of the cores. Higher sensitivities are achieved for shorter lengths of the unbalancing element and orientation of the core axis parallel to the bend direction. The highest sensitivity reported is 9.97 pm/µm for the case of 34 µm of unbalancing element and orientation of 0 degrees.
- Published
- 2021
30. S1-END-seq reveals DNA secondary structures in human cells
- Author
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Gabriel Matos-Rodrigues, Niek van Wietmarschen, Wei Wu, Veenu Tripathi, Natasha C. Koussa, Raphael Pavani, William J. Nathan, Elsa Callen, Frida Belinky, Ashraf Mohammed, Marek Napierala, Karen Usdin, Aseem Z. Ansari, Sergei M. Mirkin, and André Nussenzweig
- Subjects
DNA Replication ,DNA, Cruciform ,Nylons ,Humans ,Nucleic Acid Conformation ,DNA Breaks, Double-Stranded ,DNA ,Cell Biology ,Molecular Biology - Abstract
DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA)
- Published
- 2022
31. Cellular pathophysiology of Friedreich's ataxia cardiomyopathy
- Author
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Marek Napierala, Jarmon G Lees, Mirella Dottori, Shiang Y. Lim, and Alice Pébay
- Subjects
Pathology ,medicine.medical_specialty ,Ataxia ,Heart disease ,business.industry ,Induced Pluripotent Stem Cells ,Cardiomyopathy ,Inflammation ,medicine.disease ,Pathophysiology ,Pathogenesis ,Autonomic nervous system ,Fibrosis ,Friedreich Ataxia ,Iron-Binding Proteins ,medicine ,Humans ,Myocytes, Cardiac ,medicine.symptom ,Cardiology and Cardiovascular Medicine ,business ,Cardiomyopathies - Abstract
Friedreich's ataxia (FRDA) is a hereditary neuromuscular disorder. Cardiomyopathy is the leading cause of premature death in FRDA. FRDA cardiomyopathy is a complex and progressive disease with no cure or treatment to slow its progression. At the cellular level, cardiomyocyte hypertrophy, apoptosis and fibrosis contribute to the cardiac pathology. However, the heart is composed of multiple cell types and several clinical studies have reported the involvement of cardiac non-myocytes such as vascular cells, autonomic neurons, and inflammatory cells in the pathogenesis of FRDA cardiomyopathy. In fact, several of the cardiac pathologies associated with FRDA including cardiomyocyte necrosis, fibrosis, and arrhythmia, could be contributed to by a diseased vasculature and autonomic dysfunction. Here, we review available evidence regarding the current understanding of cellular mechanisms for and the involvement of cardiac non-myocytes in the pathogenesis of FRDA cardiomyopathy.
- Published
- 2021
32. Reverse Phase Protein Array Reveals Correlation of Retinoic Acid Metabolism With Cardiomyopathy in Friedreich's Ataxia
- Author
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Lauren A. Hauser, Marek Napierala, Amanda Clark, Jennifer M. Farmer, Kimal Rajapakshe, David A. Lynch, Peining Xu, Clementina Mesaros, Dean P. Edwards, Yu-Yun Chen, Ian A. Blair, Shixia Huang, Cristian Coarfa, and Jill S. Napierala
- Subjects
Male ,Proteomics ,Retinoic acid ,Biochemistry ,Analytical Chemistry ,chemistry.chemical_compound ,reverse phase protein array ,Iron-Binding Proteins ,Gene expression ,RA, retinoic acid ,Cells, Cultured ,ROL, retinol ,0303 health sciences ,frataxin ,biology ,LDH, lactate dehydrogenase ,030302 biochemistry & molecular biology ,PDT, population doubling time ,Reverse phase protein lysate microarray ,Middle Aged ,DE, differentially expressed ,Aldehyde Oxidoreductases ,H2O2, hydrogen peroxide ,CTRL, control ,Biomarker (medicine) ,biomarker ,Female ,medicine.symptom ,Cardiomyopathies ,Adult ,IRB, Institutional Review Board ,Ataxia ,Adolescent ,retinoid metabolism ,ALDH1A3, aldehyde dehydrogenase family 1 member A3 ,Protein Array Analysis ,UAB, University of Alabama at Birmingham ,GAA, guanine–adenine–adenine ,CHOP, Children's Hospital of Philadelphia ,03 medical and health sciences ,Retinoids ,Young Adult ,FRDA, Friedreich's ataxia ,medicine ,Humans ,RAL, retinal ,Molecular Biology ,030304 developmental biology ,AOX1, aldehyde oxidase 1 ,Aged ,RPPA, reverse phase protein array ,HL, hearing loss ,Research ,Friedreich's ataxia ,FARS, Functional Assessment Rating Scale ,FC, fold change ,Fibroblasts ,Molecular biology ,Fold change ,CMP, cardiomyopathy ,FXN, frataxin ,chemistry ,ISTD, internal standard ,Friedreich Ataxia ,BCM, Baylor College of Medicine ,Frataxin ,biology.protein ,integrins ,Biomarkers - Abstract
Identifying biomarkers is important for assessment of disease progression, prediction of symptom development, and determination of treatment effectiveness. While unbiased analyses of differential gene expression using next-generation sequencing methods are now routinely conducted, proteomics studies are more challenging because of traditional methods predominantly being low throughput and offering a limited dynamic range for simultaneous detection of hundreds of proteins that drastically differ in their intracellular abundance. We utilized a sensitive and high-throughput proteomic technique, reverse phase protein array (RPPA), to attain protein expression profiles of primary fibroblasts obtained from patients with Friedreich's ataxia (FRDA) and unaffected controls (CTRLs). The RPPA was designed to detect 217 proteins or phosphorylated proteins by individual antibody, and the specificity of each antibody was validated prior to the experiment. Among 62 fibroblast samples (44 FRDA and 18 CTRLs) analyzed, 30 proteins/phosphoproteins were significantly changed in FRDA fibroblasts compared with CTRL cells (p < 0.05), mostly representing signaling molecules and metabolic enzymes. As expected, frataxin was significantly downregulated in FRDA samples, thus serving as an internal CTRL for assay integrity. Extensive bioinformatics analyses were conducted to correlate differentially expressed proteins with critical disease parameters (e.g., selected symptoms, age of onset, guanine–adenine–adenine sizes, frataxin levels, and Functional Assessment Rating Scale scores). Members of the integrin family of proteins specifically associated with hearing loss in FRDA. Also, RPPA data, combined with results of transcriptome profiling, uncovered defects in the retinoic acid metabolism pathway in FRDA samples. Moreover, expression of aldehyde dehydrogenase family 1 member A3 differed significantly between cardiomyopathy-positive and cardiomyopathy-negative FRDA cohorts, demonstrating that metabolites such as retinol, retinal, or retinoic acid could become potential predictive biomarkers of cardiac presentation in FRDA., Graphical abstract, Highlights • RPPA analyzing Friedreich's ataxia cells reveals changed expression of 30 proteins. • Integrin expression correlates with hearing loss in Friedreich's ataxia samples. • Retinol levels are lower in serum of patients with Friedreich's ataxia than controls., In Brief We conducted a sensitive and high-throughput reverse phase protein array to attain protein expression profiles of primary fibroblasts from patients with Friedreich's ataxia (FRDA) and unaffected controls using a pool of 217 validated antibodies. Our extensive bioinformatics analyses correlated differentially expressed (DE) proteins with critical disease parameters. Expression levels of several integrin proteins specifically associated with hearing loss in FRDA. Also, reverse phase protein array data integrated with transcriptome data uncovered defects in retinoic acid metabolism in FRDA samples.
- Published
- 2021
33. Efficient electroporation of neuronal cells using synthetic oligonucleotides: identifying duplex RNA and antisense oligonucleotide activators of human frataxin expression
- Author
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Marek Napierala, Jennifer N. Putman, Frank Rigo, Yanjie Li, Thahza P. Prakash, Sharon Beasley, Xiulong Shen, and David R. Corey
- Subjects
Induced Pluripotent Stem Cells ,Oligonucleotides ,Gene Expression ,Article ,Cell Line ,03 medical and health sciences ,Iron-Binding Proteins ,Humans ,Progenitor cell ,Induced pluripotent stem cell ,Molecular Biology ,030304 developmental biology ,Neurons ,Regulation of gene expression ,0303 health sciences ,biology ,Oligonucleotide ,Electroporation ,030302 biochemistry & molecular biology ,RNA ,Fibroblasts ,Oligonucleotides, Antisense ,Cell biology ,Friedreich Ataxia ,Cell culture ,Frataxin ,biology.protein - Abstract
Oligonucleotide drugs are experiencing greater success in the clinic, encouraging the initiation of new projects. Resources are insufficient to develop every potentially important project, and persuasive experimental data using cell lines close to disease target tissue is needed to prioritize candidates. Friedreich's ataxia (FRDA) is a devastating and currently incurable disease caused by insufficient expression of the enzyme frataxin (FXN). We have previously shown that synthetic nucleic acids can activate FXN expression in human patient-derived fibroblast cells. We chose to further test these compounds in induced pluripotent stem cell-derived neuronal progenitor cells (iPSC-NPCs). Here we describe methods to deliver oligonucleotides and duplex RNAs into iPSC-NPCs using electroporation. Activation of FXN expression is potent, easily reproducible, and potencies parallel those determined using patient-derived fibroblast cells. A duplex RNA and several antisense oligonucleotides (ASOs) with different combinations of 2′-methoxyethyl (2′-MOE), 2′-fluoro (2′-F), and constrained ethyl (cEt) were active, providing multiple starting points for further development and highlighting improved potency as an important goal for preclinical development. Our data support the conclusion that ASO-mediated activation of FXN is a feasible approach for treating FRDA and that electroporation is a robust method for introducing ASOs to modulate gene expressions in neuronal cells.
- Published
- 2019
34. Therapeutic Prospects for Friedreich’s Ataxia
- Author
-
Siyuan Zhang, Jill S. Napierala, and Marek Napierala
- Subjects
0301 basic medicine ,Ataxia ,Mitochondrion ,Toxicology ,Bioinformatics ,Article ,03 medical and health sciences ,0302 clinical medicine ,Iron-Binding Proteins ,medicine ,Animals ,Humans ,Mitochondrial protein ,Pharmacology ,biology ,business.industry ,Disease progression ,Mitochondria ,030104 developmental biology ,Friedreich Ataxia ,Disease Progression ,Frataxin ,biology.protein ,medicine.symptom ,business ,030217 neurology & neurosurgery - Abstract
Friedreich's ataxia (FRDA) is a progressive disease affecting multiple organs that is caused by systemic insufficiency of the mitochondrial protein frataxin. Current therapeutic strategies aim to elevate frataxin levels and/or alleviate the consequences of frataxin deficiency. Recent significant advances in the FRDA therapeutic pipeline are bringing patients closer to a cure.
- Published
- 2019
35. Intermodal measurements in few-mode fibers with phase- sensitive OTDR
- Author
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Luc Thévenaz, Jakub Kaczorowski, Tomasz Nasilowski, Lukasz Szostkiewicz, Krzysztof Markiewicz, Alejandro Dominguez-Lopez, Zhisheng Yang, and Marek Napierala
- Subjects
Amplified spontaneous emission ,Optical fiber ,Materials science ,Phase sensitive ,business.industry ,Measure (physics) ,Mode (statistics) ,Physics::Optics ,Optical time-domain reflectometer ,law.invention ,Optics ,law ,Fiber ,business ,Effective refractive index - Abstract
We experimentally verify the possibility of measuring the difference in effective refractive index between higher-order-modes using a phase-sensitive OTDR, indicating that it is possible to measure the average difference over long fiber spans.
- Published
- 2021
36. A CRISPR-Cas9, Cre
- Author
-
Jixue, Li, Yanjie, Li, Kevin M, Pawlik, Jill S, Napierala, and Marek, Napierala
- Subjects
Gene Editing ,Recombination, Genetic ,Genome ,Genetic Techniques ,Integrases ,Genetic Vectors ,Research Articles: Expanding the CRISPR Toolbox ,Clustered Regularly Interspaced Short Palindromic Repeats ,DNA ,CRISPR-Cas Systems ,Promoter Regions, Genetic - Abstract
We describe a protocol for the precise integration of exogenous DNA into user-defined genomic loci in cultured cells. This strategy first introduces a promoter and a lox site to a specific location via a Cas9-induced double-strand break. Second, a gene of interest (GOI) is inserted into the lox site via Cre-lox recombination. Upon correct insertion, a cis-linked antibiotic resistance gene will be expressed from a promoter introduced into the genome in the first step assuring selection for correct integrants. Last, the selection cassette is excised via a Flp-FRT recombination event, leaving a precisely targeted GOI. This method is broadly applicable to any exogenous DNA to be integrated, choice of integration site, and choice of cell type. The most remarkable aspect of this versatile approach, termed “CasPi” (cascaded precise integration), is that it allows for precise genome targeting with large, frequently complex, and repetitive DNA sequences that do not integrate efficiently or at all with current genome targeting methods.
- Published
- 2020
37. Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy
- Author
-
Aravind Asokan, Jixue Li, Marek Napierala, Yanjie Li, Trevor J. Gonzalez, Jill S. Napierala, and Jun Wang
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Ataxia ,Genetic enhancement ,Induced Pluripotent Stem Cells ,Biology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Transcription (biology) ,Iron-Binding Proteins ,Genetics ,medicine ,Animals ,Humans ,Myocytes, Cardiac ,Regulatory Elements, Transcriptional ,Transcription control ,Promoter Regions, Genetic ,Induced pluripotent stem cell ,Molecular Biology ,Gene ,Research Articles ,030304 developmental biology ,Neurons ,0303 health sciences ,Correction ,nutritional and metabolic diseases ,Cell Differentiation ,Genetic Therapy ,Dependovirus ,Cell biology ,Gene Expression Regulation ,Friedreich Ataxia ,030220 oncology & carcinogenesis ,Frataxin ,biology.protein ,Molecular Medicine ,medicine.symptom ,Plasmids - Abstract
Friedreich's ataxia (FRDA) is the most common inherited form of ataxia in humans. It is caused by severe downregulation of frataxin (FXN) expression instigated by hyperexpansion of the GAA repeats located in intron 1 of the FXN gene. Despite numerous studies focused on identifying compounds capable of stimulating FXN expression, current knowledge regarding cis-regulatory elements involved in FXN gene expression is lacking. Using a combination of episomal and genome-integrated constructs, we defined a minimal endogenous promoter sequence required to efficiently drive FXN expression in human cells. We generated 19 constructs varying in length of the DNA sequences upstream and downstream of the ATG start codon. Using transient transfection, we evaluated the capability of these constructs to drive FXN expression. These analyses allowed us to identify a region of the gene indispensable for FXN expression. Subsequently, selected constructs containing the FXN expression control regions of varying lengths were site specifically integrated into the genome of HEK293T and human-induced pluripotent stem cells (iPSCs). FXN expression was detected in iPSCs and persisted after differentiation to neuronal and cardiac cells, indicating lineage independent function of defined regulatory DNA sequences. Finally, based on these results, we generated AAV encoding miniFXN genes and demonstrated in vivo FXN expression in mice. Results of these studies identified FXN sequences necessary to express FXN in human and mouse cells and provided rationale for potential use of endogenous FXN sequence in gene therapy strategies for FRDA.
- Published
- 2020
38. Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia
- Author
-
Victor M. Darley-Usmar, Daniel Fil, Xiaosen Ouyang, Jianhua Zhang, Jill S. Napierala, Balu K. Chacko, Marek Napierala, Cathleen M. Lutz, Robbie L. Conley, and Aamir Zuberi
- Subjects
Senescence ,NF-E2-Related Factor 2 ,Nonsense mutation ,Neuroscience (miscellaneous) ,Medicine (miscellaneous) ,lcsh:Medicine ,Mice, Transgenic ,Biology ,Mitochondrion ,Compound heterozygosity ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Immunology and Microbiology (miscellaneous) ,Iron-Binding Proteins ,medicine ,lcsh:Pathology ,Missense mutation ,Animals ,Genetic Predisposition to Disease ,Cellular Senescence ,Cell Proliferation ,Mutation ,Point mutation ,Frataxin ,lcsh:R ,Fatty Acids ,Friedreich's ataxia ,Fibroblasts ,Cell biology ,Mitochondria ,Mice, Inbred C57BL ,Disease Models, Animal ,Oxidative Stress ,Phenotype ,Friedreich Ataxia ,biology.protein ,Energy Metabolism ,lcsh:RB1-214 ,Research Article - Abstract
Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA repeat sequences in intron 1 of FXN, whereas a fraction of patients are compound heterozygotes, with a missense or nonsense mutation in one FXN allele and expanded GAAs in the other. A prevalent missense mutation among FRDA patients changes a glycine at position 130 to valine (G130V). Herein, we report generation of the first mouse model harboring an Fxn point mutation. Changing the evolutionarily conserved glycine 127 in mouse Fxn to valine results in a failure-to-thrive phenotype in homozygous animals and a substantially reduced number of offspring. Like G130V in FRDA, the G127V mutation results in a dramatic decrease of Fxn protein without affecting transcript synthesis or splicing. FxnG127V mouse embryonic fibroblasts exhibit significantly reduced proliferation and increased cell senescence. These defects are evident in early passage cells and are exacerbated at later passages. Furthermore, increased frequency of mitochondrial DNA lesions and fragmentation are accompanied by marked amplification of mitochondrial DNA in FxnG127V cells. Bioenergetics analyses demonstrate higher sensitivity and reduced cellular respiration of FxnG127V cells upon alteration of fatty acid availability. Importantly, substitution of FxnWT with FxnG127V is compatible with life, and cellular proliferation defects can be rescued by mitigation of oxidative stress via hypoxia or induction of the NRF2 pathway. We propose FxnG127V cells as a simple and robust model for testing therapeutic approaches for FRDA., Summary: Sole expression of the mutant frataxin G127V protein is sufficient for viability of the first Friedreich's ataxia point mutation mouse model, but induces premature senescence in isolated cells.
- Published
- 2020
39. Progress towards drug discovery for Friedreich's Ataxia: Identifying synthetic oligonucleotides that more potently activate expression of human frataxin protein
- Author
-
David R. Corey, Yanjie Li, Johnathan Wong, Marek Napierala, Thahza P. Prakash, Frank Rigo, and Xiulong Shen
- Subjects
Clinical Biochemistry ,Pharmaceutical Science ,01 natural sciences ,Biochemistry ,Article ,chemistry.chemical_compound ,Structure-Activity Relationship ,Iron-Binding Proteins ,Drug Discovery ,Humans ,Molecular Biology ,Gene ,Cells, Cultured ,Regulation of gene expression ,biology ,Dose-Response Relationship, Drug ,Molecular Structure ,010405 organic chemistry ,Chemistry ,Drug discovery ,Oligonucleotide ,Organic Chemistry ,RNA ,Oligonucleotides, Antisense ,Non-coding RNA ,0104 chemical sciences ,Cell biology ,010404 medicinal & biomolecular chemistry ,Friedreich Ataxia ,Frataxin ,biology.protein ,Molecular Medicine ,DNA - Abstract
Friedreich’s Ataxia (FRDA) is an incurable genetic disease caused by an expanded trinucleotide AAG repeat within intronic RNA of the frataxin (FXN) gene. We have previously demonstrated that synthetic antisense oligonucleotides or duplex RNAs that are complementary to the expanded repeat can activate expression of FXN and return levels of FXN protein to near normal. The potency of these compounds, however, was too low to encourage vigorous pre-clinical development. We now report testing of “gapmer” oligonucleotides consisting of a central DNA portion flanked by chemically modified RNA that increases binding affinity. We find that gapmer antisense oligonucleotides are several fold more potent activators of FXN expression relative to previously tested compounds. The potency of FXN activation is similar to a potent benchmark gapmer targeting the nuclear noncoding RNA MALAT-1, suggesting that our approach has potential for developing more effective compounds to regulate FXN expression in vivo.
- Published
- 2020
40. Dual-core all-fiber integrated immunosensor for detection of protein antigens
- Author
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Karol Wysokiński, Magdalena Staniszewska, Dawid Budnicki, Łukasz Ostrowski, Marek Napierala, Janusz Fidelus, Tomasz Nasilowski, Łukasz Szostkiewicz, Michal Murawski, and Marcin Staniszewski
- Subjects
Microprobe ,Materials science ,Optical fiber ,Light ,Biomedical Engineering ,Biophysics ,Polarization-maintaining optical fiber ,Biosensing Techniques ,02 engineering and technology ,01 natural sciences ,law.invention ,010309 optics ,law ,0103 physical sciences ,Electrochemistry ,Astronomical interferometer ,Fiber Optic Technology ,Fiber ,Optical Fibers ,Spectrometer ,business.industry ,Temperature ,Optical Devices ,Michelson interferometer ,General Medicine ,021001 nanoscience & nanotechnology ,Refractometry ,Interferometry ,Spectrophotometry ,Immunoglobulin G ,Optoelectronics ,0210 nano-technology ,business ,Biotechnology - Abstract
An optical fiber interferometric microprobe for detection of specific proteins is presented in this paper. The microprobe is an all-fiber device, which is based on Michelson interferometer configuration, which allows for detection of protein antigens in an analyzed solution thanks to antibodies immobilized on the sensor surface. The interferometer is made of dual core fiber and has a precisely formed arm length difference, achieved by splicing a fragment of polarization maintaining fiber to one of the cores. An all-fiber configuration of the sensor decreases substantially cross-sensitivities to temperature and deformation in comparison to other optical fiber interferometers. Reported sensor has a sensing region on the tip of the interferometer and therefore may be used for point measurements in medicine. The immunosensor and optical measuring system are designed to utilize the most common broadband light sources that operate at a central wavelength of 1.55 µm. The results show that it is possible to detect a protein antigen present in a solution by using an all-fiber interferometer coated with specific antibodies. The resulting peak shift can reaches 0.6 nm, which is sufficient to be measured by an optical spectrum analyzer or a spectrometer. A model allowing for estimation of the value of lower limit of detection for such sensors has been elaborated. The elaborated detection system may act as a framework for detection of various antigens and thus it can find future applications in medical diagnostics.
- Published
- 2018
41. DDIS-24. DECREASE IN GLIOBLASTOMA GROWTH IN VITRO WITH TREATMENT OF NOVEL ANALOGS OF GLUCOSE TRANSPORTER INHIBITORS
- Author
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Gloria A. Benavides, Arphaxad Otamias, Sarah E. Scott, Sixue Zhang, Omar Moukha-Chafiq, Victor M. Darley-Usmar, Vibha Pathak, Yanjie Li, Corinne E. Augelli-Szafran, Marek Napierala, Anita B. Hjelmeland, Wei Zhang, Sajina Gc, Catherine J. Libby, and Anh Nhat Tran
- Subjects
Cancer Research ,Oncology ,Chemistry ,Drug Discovery ,Glucose transporter ,medicine ,Neurology (clinical) ,Pharmacology ,medicine.disease ,In vitro ,Glioblastoma - Abstract
Despite available treatments including surgical resection, radiation and chemotherapy, glioblastoma (GBM) is incurable with rapid recurrence and low median survival rate of just fourteen months. Development of more effective treatments is difficult due to the highly heterogeneous nature of GBM. One aspect of that heterogeneity involves brain tumor initiating cells (BTICs) that have a stem cell-like ability to self-renew. BTICs can readily alter their metabolism and survive in low nutrient environments due in part to increased GLUT3 expression. We believe that the higher expression of GLUT3 in cancer cells compared to non-tumor cells makes it a therapeutic target, although the potential for toxicity must be considered. In recently accepted studies by Libby et al., we reported on two novel GLUT inhibitors identified by structure based virtual screening (SBVS) using a GLUT3 homology model. We are creating a structure-activity relationship profile and seek to increase the potency, selectivity and stability of the GLUT inhibitors. In this study we have tested a number of novel analogs and identified three that have maintained efficacy against BTICs in vitro. Importantly, these compounds display minimal toxicity against human astrocytes. The novel derivatives have increased stability compared to the lead compounds and are efficacious in the nanomolar range. In the future, we intend to utilize our anti-GLUT compounds alone and in combination with radio- and chemotherapy with the hope of clinical translation.
- Published
- 2019
42. Optical power 1 × 7 splitter based on multicore fiber technology
- Author
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Łukasz Szostkiewicz, Tomasz Nasilowski, Anna Pytel, Marek Napierala, Łukasz Ostrowski, Michal Murawski, and Pawel Mergo
- Subjects
PHOSFOS ,Multi-mode optical fiber ,Computer science ,02 engineering and technology ,Microstructured optical fiber ,021001 nanoscience & nanotechnology ,01 natural sciences ,Atomic and Molecular Physics, and Optics ,Electronic, Optical and Magnetic Materials ,Fiber-optic communication ,010309 optics ,Control and Systems Engineering ,Splitter ,0103 physical sciences ,Electronic engineering ,Fiber optic splitter ,Electrical and Electronic Engineering ,0210 nano-technology ,Plastic optical fiber ,Instrumentation ,Photonic-crystal fiber - Abstract
Multicore and microstructured fibers open a new door for designing all-fiber telecom components. In this article we propose a design of an optical power splitter based on the phenomenon of power coupling in the tapered splice between a single-core (SMF-28) and a seven core fiber (MCF-7), which was originally developed for spatial division multiplexing telecommunication systems. Comprehensive numerical analysis is presented and backed up with an experimental demonstration.
- Published
- 2017
43. Three fold symmetric microstructured fibers for customized sub-nanosecond supercontinuum generation
- Author
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Zbyszek Holdynski, Tomasz Nasilowski, Marek Napierala, Michalina Jozwik, Lukasz Szostkiwicz, and Pawel Mergo
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Materials science ,business.industry ,Single-mode optical fiber ,Nonlinear optics ,02 engineering and technology ,Microstructured optical fiber ,Nanosecond ,021001 nanoscience & nanotechnology ,Cladding (fiber optics) ,01 natural sciences ,Atomic and Molecular Physics, and Optics ,Electronic, Optical and Magnetic Materials ,Supercontinuum ,010309 optics ,Wavelength ,Optics ,0103 physical sciences ,Broadband ,Electrical and Electronic Engineering ,Physical and Theoretical Chemistry ,0210 nano-technology ,business - Abstract
We present three fold symmetric microstructured fibers and their application potential for customized single mode supercontinuum generation. In our microstructured fibers we simultaneously achieve high nonlinearity and single mode operation by increasing three cladding air holes, which are next to the core. Selectively modified microstructures allow a large degree of freedom in the dispersion profile design. With the use of sub nanosecond pump source at the wavelength of 1064 nm we show, that the designed microstructure provides various ways of generating a unique flat and broadband spectrum.
- Published
- 2017
44. Passive higher order mode filter for 850 nm multimode fiber transmission
- Author
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L. Ostrowski, Lukasz Chorchos, Lukasz Szostkiewicz, T. Nasilowski, Marek Napierala, B. Bienkowska, and Jaroslaw P. Turkiewicz
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Mode volume ,Multi-mode optical fiber ,Materials science ,business.industry ,Plastic-clad silica fiber ,02 engineering and technology ,Condensed Matter Physics ,01 natural sciences ,Graded-index fiber ,Atomic and Molecular Physics, and Optics ,Electronic, Optical and Magnetic Materials ,010309 optics ,020210 optoelectronics & photonics ,Optics ,0103 physical sciences ,0202 electrical engineering, electronic engineering, information engineering ,Dispersion-shifted fiber ,Electrical and Electronic Engineering ,Plastic optical fiber ,business ,Hard-clad silica optical fiber ,Photonic-crystal fiber - Published
- 2017
45. Alleviating GAA Repeat Induced Transcriptional Silencing of the Friedreich's Ataxia Gene During Somatic Cell Reprogramming
- Author
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Marek Napierala, Yanjie Li, Urszula Polak, and Jill Sergesketter Butler
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0301 basic medicine ,Transcription, Genetic ,Somatic cell ,Induced Pluripotent Stem Cells ,Epigenesis, Genetic ,03 medical and health sciences ,0302 clinical medicine ,Original Research Reports ,Iron-Binding Proteins ,Humans ,Gene silencing ,Cell Lineage ,Gene Silencing ,Epigenetics ,Induced pluripotent stem cell ,Cells, Cultured ,Neurons ,Genetics ,biology ,Cell Differentiation ,Cell Biology ,Hematology ,Epigenome ,Fibroblasts ,Cellular Reprogramming ,Chromatin ,Cell biology ,030104 developmental biology ,Friedreich Ataxia ,Genetic Loci ,DNA methylation ,Frataxin ,biology.protein ,Butyric Acid ,Tranylcypromine ,Trinucleotide Repeat Expansion ,Reprogramming ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene, which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs, the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming, modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds, sodium butyrate (NaB) and Parnate, led to an increase in FXN expression and correction of repressive marks at the FXN locus, which persisted for several passages. However, prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore, we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together, these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications, thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA.
- Published
- 2016
46. Excision of the expanded GAA repeats corrects cardiomyopathy phenotypes of iPSC-derived Friedreich's ataxia cardiomyocytes
- Author
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Marek Napierala, Natalia Rozwadowska, Amanda Clark, Daniel Fil, Jixue Li, and Jill S. Napierala
- Subjects
0301 basic medicine ,Ataxia ,Induced Pluripotent Stem Cells ,Cardiomyopathy ,Down-Regulation ,Biology ,Perilipin-5 ,Article ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Iron-Binding Proteins ,medicine ,Humans ,Myocytes, Cardiac ,Induced pluripotent stem cell ,Gene ,lcsh:QH301-705.5 ,Zinc finger ,Gene Editing ,Principal Component Analysis ,Gene Expression Profiling ,Cell Differentiation ,Cell Biology ,General Medicine ,Lipid Droplets ,medicine.disease ,Phenotype ,Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ,Cell biology ,Up-Regulation ,030104 developmental biology ,lcsh:Biology (General) ,Friedreich Ataxia ,Frataxin ,biology.protein ,RNA, Long Noncoding ,medicine.symptom ,Trinucleotide Repeat Expansion ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Friedreich's ataxia is caused by large homozygous, intronic expansions of GAA repeats in the frataxin (FXN) gene, resulting in severe downregulation of its expression. Pathogenic repeats are located in intron one, hence patients express unaffected FXN protein, albeit in low quantities. Although FRDA symptoms typically afflict the nervous system, hypertrophic cardiomyopathy is the predominant cause of death. Our studies were conducted using cardiomyocytes differentiated from induced pluripotent stem cells derived from control individuals, FRDA patients, and isogenic cells corrected by zinc finger nucleases-mediated excision of pathogenic expanded GAA repeats. This correction of the FXN gene removed the primary trigger of the transcription defect, upregulated frataxin expression, reduced pathological lipid accumulation observed in patient cardiomyocytes, and reversed gene expression signatures of FRDA cardiomyocytes. Transcriptome analyses revealed hypertrophy-specific expression signatures unique to FRDA cardiomyocytes, and emphasized similarities between unaffected and ZFN-corrected FRDA cardiomyocytes. Thus, the iPSC-derived FRDA cardiomyocytes exhibit various molecular defects characteristic for cellular models of cardiomyopathy that can be corrected by genome editing of the expanded GAA repeats. These results underscore the utility of genome editing in generating isogenic cellular models of FRDA and the potential of this approach as a future therapy for this disease. Keywords: Friedreich's ataxia, GAA repeats, Genome editing, Isogenic iPSC, Cardiomyocytes, Lipid metabolism
- Published
- 2019
47. Modular and smooth introduction of photonics in high-throughput communication satellites – perspective of project BEACON
- Author
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Vanessa C. Duarte, Stephen Clements, Georg Winzer, Jonathan Crabb, João G. Prata, Lars Zimmermann, Robert Walker, Leontios Stampoulidis, Marek Napierala, Marta Filipowicz, Miguel V. Drummond, Tomasz Nasilowski, Javad Anzalchi, and Rogério N. Nogueira
- Subjects
Computer architecture ,business.industry ,Computer science ,Perspective (graphical) ,Communications satellite ,Photonics ,Modular design ,business ,Throughput (business) - Published
- 2019
48. The current state of biomarker research for Friedreich’s ataxia: a report from the 2018 FARA biomarker meeting
- Author
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David A. Lynch, Steven M. Hersch, R. Mark Payne, Jane Larkindale, Ian A. Blair, S. H. Subramony, Jill S. Napierala, Marek Napierala, and Jennifer M. Farmer
- Subjects
0301 basic medicine ,Oncology ,medicine.medical_specialty ,Ataxia ,Protein biomarkers ,Friedreich’s ataxia ,Disease ,03 medical and health sciences ,0302 clinical medicine ,Serum biomarkers ,Internal medicine ,medicine ,biology ,business.industry ,Disease progression ,biomarkers ,drug development ,030104 developmental biology ,Drug development ,030220 oncology & carcinogenesis ,Perspective ,Frataxin ,biology.protein ,Biomarker (medicine) ,medicine.symptom ,business ,Biotechnology - Abstract
The 2018 FARA Biomarker Meeting highlighted the current state of development of biomarkers for Friedreich’s ataxia. A mass spectroscopy assay to sensitively measure mature frataxin (reduction of which is the root cause of disease) is being developed. Biomarkers to monitor neurological disease progression include imaging, electrophysiological measures and measures of nerve function, which may be measured either in serum and/or through imaging-based technologies. Potential pharmacodynamic biomarkers include metabolic and protein biomarkers and markers of nerve damage. Cardiac imaging and serum biomarkers may reflect cardiac disease progression. Considerable progress has been made in the development of biomarkers for various contexts of use, but further work is needed in terms of larger longitudinal multisite studies, and identification of novel biomarkers for additional use cases, Lay abstract Biomarkers are characteristics that can be objectively measured, evaluated and used as indicators of disease progression or the effect of a therapy. Friedreich’s ataxia is a progressive multisystem neuromuscular disease with no treatment. Current clinical measures cannot robustly detect disease progression in less than a year, meaning that clinical trials are long and drug development is slow. The Friedreich’s Ataxia Research Alliance and the scientific community are looking for biomarkers that show change in shorter time frames that can accelerate drug development. The 2018 FARA Biomarker Meeting summarized the exciting findings that represent the current state of the field.
- Published
- 2019
49. Modular coherent photonic-aided payload receiver for communications satellites
- Author
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Marek Napierala, Rogério N. Nogueira, Tomasz Nasilowski, Leontios Stampoulidis, Robert Walker, João G. Prata, Marta Filipowicz, Carlos F. Ribeiro, Javad Anzalchi, Miguel V. Drummond, Jonathan Crabb, Marios Kechagias, Vanessa C. Duarte, Stephen Clements, Georg Winzer, and Lars Zimmermann
- Subjects
0301 basic medicine ,Bridging (networking) ,Computer science ,Science ,General Physics and Astronomy ,02 engineering and technology ,7. Clean energy ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,lcsh:Science ,Digital divide ,Multidisciplinary ,business.industry ,Payload ,Integrated optics ,General Chemistry ,Modular design ,021001 nanoscience & nanotechnology ,030104 developmental biology ,Microwave photonics ,Communications satellite ,lcsh:Q ,Photonics ,0210 nano-technology ,business ,Computer hardware - Abstract
Ubiquitous satellite communications are in a leading position for bridging the digital divide. Fulfilling such a mission will require satellite services on par with fibre services, both in bandwidth and cost. Achieving such a performance requires a new generation of communications payloads powered by large-scale processors, enabling a dynamic allocation of hundreds of beams with a total capacity beyond 1 Tbit s−1. The fact that the scale of the processor is proportional to the wavelength of its signals has made photonics a key technology for its implementation. However, one last challenge hinders the introduction of photonics: while large-scale processors demand a modular implementation, coherency among signals must be preserved using simple methods. Here, we demonstrate a coherent photonic-aided receiver meeting such demands. This work shows that a modular and coherent photonic-aided payload is feasible, making way to an extensive introduction of photonics in next generation communications satellites., Satellite communications shall only thrive if being able to compete with fibre in both bandwidth and cost. Here, the authors demonstrate that a coherent and yet modular photonic-aided payload is feasible, paving the way for a new generation of communications satellites with photonics at their core.
- Published
- 2019
50. Progress in understanding Friedreich's ataxia using human induced pluripotent stem cells
- Author
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Jill S. Napierala, Julia O. Misiorek, Marek Napierala, and Anna M. Schreiber
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Ataxia ,biology ,business.industry ,Health Policy ,nutritional and metabolic diseases ,Article ,Multisystem disease ,03 medical and health sciences ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Frataxin ,biology.protein ,medicine ,Cancer research ,Pharmacology (medical) ,medicine.symptom ,Human Induced Pluripotent Stem Cells ,business ,Induced pluripotent stem cell ,Pharmacology, Toxicology and Pharmaceutics (miscellaneous) ,030217 neurology & neurosurgery - Abstract
INTRODUCTION: Friedreich’s ataxia (FRDA) is an autosomal recessive multisystem disease mainly affecting the peripheral and central nervous systems, and heart. FRDA is caused by a GAA repeat expansion in the first intron of the frataxin (FXN) gene, that leads to reduced expression of FXN mRNA and frataxin protein. Neuronal and cardiac cells are primary targets of frataxin deficiency and generating models via differentiation of induced pluripotent stem cells (iPSCs) into these cell types is essential for progress towards developing therapies for FRDA. AREAS COVERED: This review is focused on modeling FRDA using human iPSCs and various iPSC-differentiated cell types. We emphasized the importance of patient and corrected isogenic cell line pairs to minimize effects caused by biological variability between individuals. EXPERT OPINION: The versatility of iPSC-derived cellular models of FRDA is advantageous for developing new therapeutic strategies, and rigorous testing in such models will be critical for approval of the first treatment for FRDA. Creating a well-characterized and diverse set of iPSC lines, including appropriate isogenic controls, will facilitate achieving this goal. Also, improvement of differentiation protocols, especially towards proprioceptive sensory neurons and organoid generation, is necessary to utilize the full potential of iPSC technology in the drug discovery process.
- Published
- 2019
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