42 results on '"Chahwan R"'
Search Results
2. Aicardi–Goutieres syndrome: from patients to genes and beyond
- Author
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Chahwan, C and Chahwan, R
- Published
- 2012
- Full Text
- View/download PDF
3. The MutSβ complex is a modulator of p53-driven tumorigenesis through its functions in both DNA double-strand break repair and mismatch repair
- Author
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van Oers, J M M, primary, Edwards, Y, additional, Chahwan, R, additional, Zhang, W, additional, Smith, C, additional, Pechuan, X, additional, Schaetzlein, S, additional, Jin, B, additional, Wang, Y, additional, Bergman, A, additional, Scharff, M D, additional, and Edelmann, W, additional
- Published
- 2013
- Full Text
- View/download PDF
4. PCR-based detection, restriction endonuclease analysis, and transcription of tonB in Haemophilus influenzae and Haemophilus parainfluenzae isolates obtained from children undergoing tonsillectomy and adenoidectomy.
- Author
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Matar, G M, Chahwan, R, Fuleihan, N, Uwaydah, M, and Hadi, U
- Abstract
We developed and evaluated a PCR-based-restriction endonuclease analysis method to detect and analyze the tonB gene of Haemophilus influenzae and Haemophilus parainfluenzae from pediatric patients undergoing tonsillectomy and adenoidectomy. Multiple sites from the same patient, including the surface of adenoids and tonsils, as well as the core of tonsils, were cultured on chocolate agar and identified using standard procedures and the API NH Kit. A total of 55 H. influenzae isolates were recovered from different sites of 20 patients, and 32 H. parainfluenzae isolates were recovered from various sites of 12 patients. DNA was extracted from American Type Culture Collection strains and test isolates by the PureGene kit. Two primers, G1 (21-mer) and G2 (23-mer), were designed by us to amplify by PCR the tonB gene that consists of an 813-bp fragment. A nested PCR using primers T1 (23-mer) and T2 (24-mer) that flank an internal sequence to the gene of the order of 257 bp and restriction endonuclease digestion using XhoI and BglII were done to detect whether heterogeneity within the gene exists between the two species. Reverse transcription-PCR (RT-PCR) was finally done to detect transcription of the gene in both species. Our data have shown that the tonB gene was detected in both species. It is known to encode a virulent protein, TonB, in H. influenzae; however, demonstration of its presence in H. parainfluenzae is novel. Nested-PCR and restriction endonuclease analysis have shown that the tonB gene is apparently structurally the same in both species, with possible differences that may exist in certain H. parainfluenzae isolates. RT-PCR done on selected numbers of H. influenzae and H. parainfluenzae have shown that the tonB gene was transcribed in both species. This shows that the TonB protein, if expressed, may play a different role in the virulence in H. parainfluenzae since it is not needed for heme or heme complexes uptake as with H. influenzae.
- Published
- 2001
- Full Text
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5. The multidimensional nature of epigenetic information and its role in disease
- Author
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Chahwan R, Sn, Wontakal, Sergio Roa, University of Zurich, and Chahwan, Richard
- Subjects
Neoplasms ,570 Life sciences ,biology ,Humans ,RNA ,610 Medicine & health ,2700 General Medicine ,DNA Methylation ,10263 Institute of Experimental Immunology ,Epigenesis, Genetic - Abstract
This year marks the 10th anniversary of the publications that reported the initial human genome sequence. In the historic press conference that announced this landmark accomplishment, it was proclaimed that the genome sequence would "revolutionize the diagnosis, prevention, and treatment of most, if not all, human diseases." However, subsequent work over the past decade has revealed that "complex diseases" are much more intricate than originally thought. Even with the advent of several new powerful technologies, our understanding of the underlying genetic etiologies of most complex and non-Mendelian diseases is far from complete. These results have raised the possibility that the DNA sequence, i.e., genetic information, may not be the only relevant source of information in order to understand the molecular basis of disease. In this review, we assemble evidence that information encoded beyond the DNA sequence, i.e., epigenetic information, may hold the key to a better understanding of various pathological conditions. Unlike the genetic information encoded within the DNA sequence, epigenetic information can be stored in multiple dimensions, such as in the form of DNA modifications, RNA, or protein. Ideas presented here support the view that to better understand the molecular etiology of diseases, we need to gain a better understanding of both the genetic and epigenetic components of biological information. We hence believe that the fast development of genome-wide technologies will facilitate a better understanding of both genetic and epigenetic dimensions of disease.
6. The role of chromatin modifications in genomic stability and immune diversity
- Author
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Reffo, Laura, Phillips, J., and Chahwan, R.
- Abstract
Chromatin architecture is constantly remodelled, to overcome DNA damage, induce immune diversification and ensure gene transcription and replication. If genome remodelling is not accurately regulated, cells can face detrimental consequences, such as apoptosis and transformation, with potential tumorigenesis and premature aging. Global understanding of how chromatin remodelling is regulated and modulated is an ongoing key challenge for the development of potential therapies for all pathological conditions generated by genome instability. AID/APOBECs are powerful DNA mutators, whose activity is fundamental for the establishment of adequate innate and adaptive immune defences. The mechanisms regulating their upstream activity remain elusive. Here, a link between H3K7me2 histone modification and AID activity in B cells has been identified. Downstream of DNA mutators, helicases are ubiquitously involved in the unwinding of DNA double strands, allowing the consequent targeting of other DNA modifying proteins. Their role is therefore pivotal in genome replication, repair and recombination, and understanding their activity is a major goal. Here, a fluorescence based toolkit have been elaborated to visualise and quantify helicases activity on DNA substrates. Among all DNA damages, double strand breaks are particularly dangerous as they can lead to loss of genetic material. A complete elucidation of the different mechanisms of their repair is an ongoing challenge. Here, the putative helicase RuvBL2 was identified as a potential contributor of the homology-directed repair pathway. All these findings provide new important details for the elucidation of the role of DNA metabolizing enzymes and remodellers in the maintenance of genome stability with potential applications to immune diversity.
- Published
- 2021
7. Monitoring chromatin remodelling in fixed and live immune cells using vibrational spectroscopy and microfluidics
- Author
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Morrish, R., Palombo, F., Stone, N., Pagliara, S., and Chahwan, R.
- Subjects
chromatin ,microfluidics ,FTIR ,Raman ,deformability cytometry ,nuclear auxeticity ,antibody diversification ,vibrational spectroscopy - Abstract
Eukaryotic cell growth, maintenance and differentiation relies on the dynamic structure of nuclear chromatin, the macromolecular complex consisting primarily of DNA and histones. Changes to chromatin structure and chemistry may lead to alterations in gene expression, resulting in functional and developmental processes in cells. Additionally, biomechanical properties of the nucleus, which play a role in mechanical signalling pathways, are also affected by chromatin conformation. The regulation and effects of chromatin dynamics in cellular processes have yet to be fully elucidated. Therefore, novel techniques for assessing chemical and mechanical signatures of cells undergoing chromatin changes during cell differentiation at the single cell level have great potential for 1) phenotypic characterisation of single cells for research and clinical purposes and 2) further unravelling the complex coordination of intracellular changes that occur during cell developmental steps and triggering of disease. In this thesis, I have studied chromatin remodelling in immune cells using vibrational spectroscopy and microfluidics. Single cell measurements were conducted through optimisations of experimental and data analysis parameters. Vibrational spectroscopy methods included FTIR spectroscopic imaging and Raman microscopy, both label-free techniques that measure the interaction of light with chemical properties of a sample by interrogating its molecular vibrations. Microfluidics is a technique for manipulating fluids at a submillimetre scale. It was utilised here to enable live cell Raman mapping, as well as for deformability cytometry for assessing mechanical properties of the cell nuclei. To initiate an immune activation, B cells were incubated with a cytokine (CIT) cocktail. The biomechanical property, nuclear auxeticity, was investigated in B cells using deformability cytometry. This property has previously been shown in transitioning embryonic stem (ES) cells, and chromatin decondensation has been determined to hold a regulatory role. Chromatin decondensation was therefore induced in B cells through immune activation (CIT treatment) or Trichostatin A (TSA) treatment. These cells were compared to untreated control cells. A subset of cells for both the CIT and TSA treatment, had auxetic nuclei. No control cells had auxetic nuclei. These results showed nuclear auxeticity in B cells for the first time, and linked it to chromatin decondensation in agreement with previous ES cell data. Using FTIR spectroscopic imaging and Raman microscopy, spectral features associated with chromatin and DNA changes during immune B cell activation were identified. Peak ratios for distinguishing between non-activated and activated immune cells were determined - for FTIR imaging: a DNA-to-protein peak ratio, and for Raman mapping: a peak ratio between two neighbouring peaks, both associated with nucleic acid. Both peak ratios measured the relative change in a peak associated with νs(PO2-), which was therefore shown to be a potential spectral marker for label-free characterisation of immune cells pre- and post activation. The biological origin of the FTIR spectral features was further investigated using additional cell treatments. Chromatin decondensation, intiated through CIT or TSA treatment, gave rise to similar change in the DNA-to-protein peak ratio. This supported the hypothesis that the νs(PO2-) spectral changes can be used to monitor structural changes occurring in chromatin and DNA itself. Finally, the key biological pathways influencing the whole range of Raman spectral differences between non-activated and activated B cells were investigated. Partial least squares (PLS) regression was performed on Raman maps and transcriptomic data. It was determined that a linear correlation exists between the two data types. Transcripts of high importance for this correlation were identified. These included the B cell receptor genes and a number of transcripts of regulatory proteins with known roles in immune activation. Transcripts not previously linked to immune activation were also identified. In summary, novel techniques for phenotypic characterisation of single cells were explored using both chemical and mechanical measurements of B cells undergoing immune activation. Previously unidentified biochemical and biomechanical factors influencing B cell activation were identified. These have added new layers to our understanding of this process and thus revealed potential new research directions. Furthermore, chromatin decondensation and transcriptional changes are key responses during all cell differentiation processes and disease development. Therefore, these experimental approaches have great potential for investigating other cell types and cellular processes.
- Published
- 2021
8. Nucleases and histone acetyltransferases in DNA repair and immune diversity
- Author
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Sheppard, E., Chahwan, R., and Harmer, N.
- Subjects
570 - Abstract
DNA repair mechanisms are essential for genome maintenance and adaptive immunity. A careful balance must be achieved whereby highly accurate and efficient canonical repair protects the genome from accumulating mutations that lead to aging and cancer, and yet mutation and error-prone non-canonical repair is required for generating immune diversity. Immune diversity is achieved within a tightly regulated environment in which mutator proteins are directed to the antibody locus to introduce a swathe of DNA damage. This produces high affinity antibodies that recognise an infinite number of invading pathogens. This process of secondary antibody diversification is dependent on both active transcription and DNA repair. Downstream of histone signalling, DNA repair nucleases are recruited to remove the damaged bases. The structure of damaged regions in the DNA can have very different conformations depending on whether the source of the damage is endogenous or exogenous. Specific DNA nucleases recognise particular DNA substrates and generate DNA intermediates that are repaired in conjunction with polymerases and ligases. Despite their multitude and importance to DNA repair, very few nucleases have been characterised, while the activities of some studied nucleases remain controversial. Conventional techniques for studying DNA nucleases have several disadvantages; they are hazardous, laborious, time-consuming, and capture nuclease activity in a discontinuous manner. Recognising a need for a safer, faster alternative, a fluorescence-based method has been developed for the study of DNA nucleases, nickases and polymerases. Key histone modifications that are known to orchestrate canonical DNA repair have since been discovered to regulate non-canonical repair at the antibody locus. The Kat5 histone lysine acetyltransferase functions highly upstream of DNA repair and promotes active transcription, yet a role for Kat5 in secondary antibody diversification has not yet been established. Using chemical inhibitors to prevent the catalytic activities of Kat5, and the genetic method of an inducible degron system for rapid and reversible downregulation of Kat5, a role for Kat5 in secondary antibody diversification is recognised, and the research contributes to our current understanding of the DNA repair signal transduction pathway.
- Published
- 2019
9. Modular cytosine base editing promotes epigenomic and genomic modifications.
- Author
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Weischedel J, Higgins L, Rogers S, Gramalla-Schmitz A, Wyrzykowska P, Borgoni S, MacCarthy T, and Chahwan R
- Subjects
- CRISPR-Cas Systems, Cytosine chemistry, Epigenomics methods, RNA genetics, CRISPR-Associated Protein 9 metabolism, Cytosine Deaminase genetics, Gene Editing methods
- Abstract
Prokaryotic and eukaryotic adaptive immunity differ considerably. Yet, their fundamental mechanisms of gene editing via Cas9 and activation-induced deaminase (AID), respectively, can be conveniently complimentary. Cas9 is an RNA targeted dual nuclease expressed in several bacterial species. AID is a cytosine deaminase expressed in germinal centre B cells to mediate genomic antibody diversification. AID can also mediate epigenomic reprogramming via active DNA demethylation. It is known that sequence motifs, nucleic acid structures, and associated co-factors affect AID activity. But despite repeated attempts, deciphering AID's intrinsic catalytic activities and harnessing its targeted recruitment to DNA is still intractable. Even recent cytosine base editors are unable to fully recapitulate AID's genomic and epigenomic editing properties. Here, we describe the first instance of a modular AID-based editor that recapitulates the full spectrum of genomic and epigenomic editing activity. Our 'Swiss army knife' toolbox will help better understand AID biology per se as well as improve targeted genomic and epigenomic editing., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
- Full Text
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10. Assessing Extracellular Vesicles in Human Biofluids Using Flow-Based Analyzers.
- Author
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Yim KHW, Krzyzaniak O, Al Hrout A, Peacock B, and Chahwan R
- Subjects
- Humans, Biomarkers metabolism, Flow Cytometry, Extracellular Vesicles metabolism
- Abstract
Extracellular vesicles (EVs) are increasingly being analyzed by flow cytometry. Yet their minuscule size and low refractive index cause the scatter intensity of most EVs to fall below the detection limit of most flow cytometers. A new class of devices, known as spectral flow analyzers, are becoming standards in cell phenotyping studies, largely due to their unique capacity to detect a vast panel of markers with higher sensitivity for light scatter detection. Another class of devices, known as nano-analyzers, provides high-resolution detection of sub-micron-sized particles. Here, the EV phenotyping performance between the Aurora (Cytek) spectral cell analyzer and the NanoFCM (nFCM) nanoflow analyzer are compared. These two devices are specifically chosen given their lead in becoming gold standards in their respective fields. Immune cell-derived EVs remain poorly characterized despite their clinical potential. Therefore, B- and T-cell line-derived EVs and donor-matched human biofluid-derived EVs from plasma, urine, and saliva are used in combination with a panel of established immune markers for this comparative study. A comparative evaluation of both cytometry platforms is performed, discussing their potential and suitability for different applications. It is found that nFCM can accurately i) analyze small EVs (40-200 nm) matching the size accuracy of electron microscopy; ii) measure the concentration of a single EV particle per volume; iii) identify underrepresented EV marker subsets; and iv) provide co-localization of EV surface markers. It can also be shown that human sample biofluids have unique EV marker signatures that can have future clinical relevance. Finally, nFCM and Aurora have their unique strength, preferred fashion of data acquisition, and visualization to fit different research interests., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)
- Published
- 2023
- Full Text
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11. Investigating the tumor-immune microenvironment through extracellular vesicles from frozen patient biopsies and 3D cultures.
- Author
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Al Hrout A, Levesque MP, and Chahwan R
- Subjects
- Humans, Tumor Microenvironment, Biological Transport, Biopsy, Extracellular Vesicles, Melanoma
- Abstract
Melanomas are highly immunogenic tumors that have been shown to activate the immune response. Nonetheless, a significant portion of melanoma cases are either unresponsive to immunotherapy or relapsed due to acquired resistance. During melanomagenesis, melanoma and immune cells undergo immunomodulatory mechanisms that aid in immune resistance and evasion. The crosstalk within melanoma microenvironment is facilitated through the secretion of soluble factors, growth factors, cytokines, and chemokines. In addition, the release and uptake of secretory vesicles known as extracellular vesicles (EVs) play a key role in shaping the tumor microenvironment (TME). Melanoma-derived EVs have been implicated in immune suppression and escape, promoting tumor progression. In the context of cancer patients, EVs are usually isolated from biofluids such as serum, urine, and saliva. Nonetheless, this approach neglects the fact that biofluid-derived EVs reflect not only the tumor, but also include contributions from different organs and cell types. For that, isolating EVs from tissue samples allows for studying different cell populations resident at the tumor site, such as tumor-infiltrating lymphocytes and their secreted EVs, which play a central anti-tumor role. Herein, we outline the first instance of a method for EV isolation from frozen tissue samples at high purity and sensitivity that can be easily reproduced without the need for complicated isolation methods. Our method of processing the tissue not only circumvents the need for hard-to-acquire freshly isolated tissue samples, but also preserves EV surface proteins which allows for multiplex surface markers profiling. Tissue-derived EVs provide insight into the physiological role of EVs enrichment at tumor sites, which can be overlooked when studying circulating EVs coming from different sources. Tissue-derived EVs could be further characterized in terms of their genomics and proteomics to identify possible mechanisms for regulating the TME. Additionally, identified markers could be correlated to overall patient survival and disease progression for prognostic purposes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Al Hrout, Levesque and Chahwan.)
- Published
- 2023
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12. Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice.
- Author
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Wang S, Lee K, Gray S, Zhang Y, Tang C, Morrish RB, Tosti E, van Oers J, Amin MR, Cohen PE, MacCarthy T, Roa S, Scharff MD, Edelmann W, and Chahwan R
- Subjects
- Animals, B-Lymphocytes, Immunity, Meiosis genetics, Mice, Somatic Hypermutation, Immunoglobulin, DNA Repair, DNA Repair Enzymes genetics, DNA Repair Enzymes metabolism, Exodeoxyribonucleases genetics, Exodeoxyribonucleases metabolism, Neoplasms genetics, Neoplasms immunology
- Abstract
DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1-/- and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1-/- mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1-/- mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1-/- mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1-/- mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1-/- mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2022
- Full Text
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13. Predictive and Prognostic Value of Non-Coding RNA in Breast Cancer.
- Author
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Sobhani N, Chahwan R, Roudi R, Morris R, Volinia S, Chai D, D'Angelo A, and Generali D
- Abstract
For decades since the central dogma, cancer biology research has been focusing on the involvement of genes encoding proteins. It has been not until more recent times that a new molecular class has been discovered, named non-coding RNA (ncRNA), which has been shown to play crucial roles in shaping the activity of cells. An extraordinary number of studies has shown that ncRNAs represent an extensive and prevalent group of RNAs, including both oncogenic or tumor suppressive molecules. Henceforth, various clinical trials involving ncRNAs as extraordinary biomarkers or therapies have started to emerge. In this review, we will focus on the prognostic and diagnostic role of ncRNAs for breast cancer.
- Published
- 2022
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14. Modelling liver cancer microenvironment using a novel 3D culture system.
- Author
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Al Hrout A, Cervantes-Gracia K, Chahwan R, and Amin A
- Subjects
- Cell Line, Tumor, Coculture Techniques, Humans, Spheroids, Cellular metabolism, Tumor Microenvironment, Carcinoma, Hepatocellular pathology, Liver Neoplasms pathology
- Abstract
The tumor microenvironment and its contribution to tumorigenesis has been a focal highlight in recent years. A two-way communication between the tumor and the surrounding microenvironment sustains and contributes to the growth and metastasis of tumors. Progression and metastasis of hepatocellular carcinoma (HCC) have been reported to be exceedingly influenced by diverse microenvironmental cues. In this study, we present a 3D-culture model of liver cancer to better mimic in vivo tumor settings. By creating novel 3D co-culture model that combines free-floating and scaffold-based 3D-culture techniques of liver cancer cells and fibroblasts, we aimed to establish a simple albeit reproducible ex vivo cancer microenvironment model that captures tumor-stroma interactions. The model presented herein exhibited unique gene expression and protein expression profiles when compared to 2D and 3D mono-cultures of liver cancer cells. Our results showed that in vivo like conditions cannot be mimicked by simply growing cancer cells as spheroids, but by co-culturing them with 3D fibroblast with which they were able to crosstalk. This was evident by the upregulation of several pathways involved in HCC, and the increase in secreted factors by co-cultured cancer cells, many of which are also involved in tumor-stroma interactions. Compared to the conventional 2D culture, the proposed model exhibits an increase in the expression of genes associated with development, progression, and poor prognosis of HCC. Our results correlated with an aggressive outcome that better mirrors in vivo HCC, and therefore, a more reliable platform for molecular understanding of HCC., (© 2022. The Author(s).)
- Published
- 2022
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15. Serum extracellular vesicles profiling is associated with COVID-19 progression and immune responses.
- Author
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Yim KHW, Borgoni S, and Chahwan R
- Abstract
Coronavirus disease 2019 (COVID-19) has transformed very quickly into a world pandemic with severe and unexpected consequences on human health. Concerted efforts to generate better diagnostic and prognostic tools have been ongoing. Research, thus far, has primarily focused on the virus itself or the direct immune response to it. Here, we propose extracellular vesicles (EVs) from serum liquid biopsies as a new and unique modality to unify diagnostic and prognostic tools for COVID-19 analyses. EVs are a novel player in intercellular signalling particularly influencing immune responses. We herein show that innate and adaptive immune EVs profiling, together with SARS-CoV-2 Spike S1
+ EVs provide a novel signature for SARS-CoV-2 infection. It also provides a unique ability to associate the co-existence of viral and host cell signatures to monitor affected tissues and severity of the disease progression. And provide a phenotypic insight into COVID-associated EVs., Competing Interests: All authors have declared that no conflict of interest exists., (© 2022 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)- Published
- 2022
- Full Text
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16. Integrative OMICS Data-Driven Procedure Using a Derivatized Meta-Analysis Approach.
- Author
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Cervantes-Gracia K, Chahwan R, and Husi H
- Abstract
The wealth of high-throughput data has opened up new opportunities to analyze and describe biological processes at higher resolution, ultimately leading to a significant acceleration of scientific output using high-throughput data from the different omics layers and the generation of databases to store and report raw datasets. The great variability among the techniques and the heterogeneous methodologies used to produce this data have placed meta-analysis methods as one of the approaches of choice to correlate the resultant large-scale datasets from different research groups. Through multi-study meta-analyses, it is possible to generate results with greater statistical power compared to individual analyses. Gene signatures, biomarkers and pathways that provide new insights of a phenotype of interest have been identified by the analysis of large-scale datasets in several fields of science. However, despite all the efforts, a standardized regulation to report large-scale data and to identify the molecular targets and signaling networks is still lacking. Integrative analyses have also been introduced as complementation and augmentation for meta-analysis methodologies to generate novel hypotheses. Currently, there is no universal method established and the different methods available follow different purposes. Herein we describe a new unifying, scalable and straightforward methodology to meta-analyze different omics outputs, but also to integrate the significant outcomes into novel pathways describing biological processes of interest. The significance of using proper molecular identifiers is highlighted as well as the potential to further correlate molecules from different regulatory levels. To show the methodology's potential, a set of transcriptomic datasets are meta-analyzed as an example., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cervantes-Gracia, Chahwan and Husi.)
- Published
- 2022
- Full Text
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17. Measuring Real-time DNA/RNA Nuclease Activity through Fluorescence.
- Author
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Wyrzykowska P, Rogers S, and Chahwan R
- Abstract
DNA and RNA nucleases are wide-ranging enzymes, taking part in broad cellular processes from DNA repair to immune response control. Growing interest in the mechanisms and activities of newly discovered nucleases inspired us to share the detailed protocol of our nuclease assay ( Sheppard et al. , 2019 ). This easy and inexpensive method can provide data that enables understanding of the molecular mechanism for novel or tested nucleases, from substrate preference and cofactors involved to catalytic rate of reaction., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)
- Published
- 2021
- Full Text
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18. APOBECs orchestrate genomic and epigenomic editing across health and disease.
- Author
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Cervantes-Gracia K, Gramalla-Schmitz A, Weischedel J, and Chahwan R
- Subjects
- Cytidine Deaminase genetics, Cytidine Deaminase metabolism, Genome, Genomics, Retroelements, APOBEC Deaminases genetics, APOBEC Deaminases metabolism, Epigenomics
- Abstract
APOBEC proteins can deaminate cytosine residues in DNA and RNA. This can lead to somatic mutations, DNA breaks, RNA modifications, or DNA demethylation in a selective manner. APOBECs function in various cellular compartments and recognize different nucleic acid motifs and structures. They orchestrate a wide array of genomic and epigenomic modifications, thereby affecting various cellular functions positively or negatively, including immune editing, viral and retroelement restriction, DNA damage responses, DNA demethylation, gene expression, and tissue homeostasis. Furthermore, the cumulative increase in genomic and epigenomic editing with aging could also, at least in part, be attributed to APOBEC function. We synthesize our cumulative understanding of APOBEC activity in a unifying overview and discuss their genomic and epigenomic impact in physiological, pathological, and technological contexts., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2021
- Full Text
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19. Functional Phenotype Flow Cytometry: On Chip Sorting of Individual Cells According to Responses to Stimuli.
- Author
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Nikiforov PO, Hejja B, Chahwan R, Soeller C, Gielen F, and Chimerel C
- Subjects
- Cell Line, Flow Cytometry, Humans, Phenotype, Microfluidics, Receptors, Antigen, B-Cell
- Abstract
The ability to effectively separate and isolate biological cells into specific and well-defined subpopulations is crucial for the advancement of our understanding of cellular heterogeneity and its relevance to living systems. Here is described the development of the functional phenotype flow cytometer (FPFC), a new device designed to separate cells on the basis of their in situ real-time phenotypic responses to stimuli. The FPFC performs a cascade of cell processing steps on a microfluidic platform: introduces biological cells one at a time into a solution of a biological reagent that acts as a stimulus, incubates the cells with the stimulus solution in a flow, and sorts the cells into subpopulations according to their phenotypic responses to the provided stimulus. The presented implementation of the FPFC uses intracellular fluorescence as a readout, incubates cells for 75 s, and operates at a throughput of up to 4 cells min
-1 -resulting in the profiling and sorting of hundreds of cells within a few hours. The design and operation of the FPFC are validated by sorting cells from the human Burkitt's lymphoma cancerous cell line Ramos on the basis of their response to activation of the B cell antigen receptor (BCR) by a targeted monoclonal antibody., (© 2021 The Authors. Advanced Biology published by Wiley-VCH GmbH.)- Published
- 2021
- Full Text
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20. Editorial: Probing the Chromatin Architecture.
- Author
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Palombo F, Pagliara S, Singh A, and Chahwan R
- Abstract
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Published
- 2021
- Full Text
- View/download PDF
21. Single Cell Label-Free Probing of Chromatin Dynamics During B Lymphocyte Maturation.
- Author
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Morrish R, Yim KHW, Pagliara S, Palombo F, Chahwan R, and Stone N
- Abstract
Large-scale intracellular signaling during developmental growth or in response to environmental alterations are largely orchestrated by chromatin within the cell nuclei. Chemical and conformational modifications of the chromatin architecture are critical steps in the regulation of differential gene expression and ultimately cell fate determination. Therefore, establishing chemical properties of the nucleus could provide key markers for phenotypic characterization of cellular processes on a scale of individual cells. Raman microscopy is a sensitive technique that is capable of probing single cell chemical composition-and sub-cellular regions-in a label-free optical manner. As such, it has great potential in both clinical and basic research. However, perceived limitations of Raman spectroscopy such as low signal intensity and the difficulty in linking alterations in vibrational signals directly with ensuing biological effects have hampered advances in the field. Here we use immune B lymphocyte development as a model to assess chromatin and transcriptional changes using confocal Raman microscopy in combination with microfluidic devices and correlative transcriptomics, thereby linking changes in chemical and structural properties to biological outcomes. Live B lymphocytes were assessed before and after maturation. Multivariate analysis was applied to distinguish cellular components within each cell. The spectral differences between non-activated and activated B lymphocytes were then identified, and their correlation with known intracellular biological changes were assessed in comparison to conventional RNA-seq analysis. Our data shows that spectral analysis provides a powerful tool to study gene activation that can complement conventional molecular biology techniques and opens the way for mapping the dynamics in the biochemical makeup of individual cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Morrish, Yim, Pagliara, Palombo, Chahwan and Stone.)
- Published
- 2021
- Full Text
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22. Integrative Analysis of Incongruous Cancer Genomics and Proteomics Datasets.
- Author
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Cervantes-Gracia K, Chahwan R, and Husi H
- Subjects
- Biomarkers, Humans, Systems Biology, Genomics, Neoplasms genetics, Proteomics
- Abstract
Cancer is a complex disease characterized by molecular heterogeneity and the involvement of several cellular mechanisms throughout its evolution and pathogenesis. Despite the great efforts made to untangle these mechanisms, cancer pathophysiology remains far from clear. So far, panels of biomarkers have been reported from high-throughput data generated through different platforms. These biomarkers are primarily focused on one type of coding molecules such as transcripts or proteins, mainly due to the apparent heterogeneity of output data resulting from the use of various techniques specific to the molecular type. Hence, there is a major need to understand how these molecules interact and complement each other to be able to explain the deregulated processes involved. The breadth of large-scale data availability as well as the lack of in-depth analysis of publicly available data has raised concerns and enabled opportunities for new strategies to analyze "Big data" more comprehensively. Here, a new protocol to perform integrative analysis based on a systems biology approach is described. The foundation of the approach relies on groups of datasets from published studies compared within the original described groups and organized in a designated format to allow the integration and cross-comparison among different studies and different platforms. This approach follows an unbiased hypothesis-free methodology that will facilitate the identification of commonalities among different data-set sources, and ultimately map and characterize specific molecular pathways using significantly deregulated molecules. This in turn will generate novel insights about the mechanisms deregulated in complex diseases such as cancer.
- Published
- 2021
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- View/download PDF
23. Extracellular Vesicles Orchestrate Immune and Tumor Interaction Networks.
- Author
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Yim KHW, Al Hrout A, Borgoni S, and Chahwan R
- Abstract
Extracellular vesicles (EVs) are emerging as potent and intricate intercellular communication networks. From their first discovery almost forty years ago, several studies have bolstered our understanding of these nano-vesicular structures. EV subpopulations are now characterized by differences in size, surface markers, cargo, and biological effects. Studies have highlighted the importance of EVs in biology and intercellular communication, particularly during immune and tumor interactions. These responses can be equally mediated at the proteomic and epigenomic levels through surface markers or nucleic acid cargo signaling, respectively. Following the exponential growth of EV studies in recent years, we herein synthesize new aspects of the emerging immune-tumor EV-based intercellular communications. We also discuss the potential role of EVs in fundamental immunological processes under physiological conditions, viral infections, and tumorigenic conditions. Finally, we provide insights on the future prospects of immune-tumor EVs and suggest potential avenues for the use of EVs in diagnostics and therapeutics.
- Published
- 2020
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- View/download PDF
24. Single Cell Imaging of Nuclear Architecture Changes.
- Author
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Morrish RB, Hermes M, Metz J, Stone N, Pagliara S, Chahwan R, and Palombo F
- Abstract
The dynamic architecture of chromatin, the macromolecular complex comprised primarily of DNA and histones, is vital for eukaryotic cell growth. Chemical and conformational changes to chromatin are important markers of functional and developmental processes in cells. However, chromatin architecture regulation has not yet been fully elucidated. Therefore, novel approaches to assessing chromatin changes at the single-cell level are required. Here we report the use of FTIR imaging and microfluidic cell-stretcher chips to assess changes to chromatin architecture and its effect on the mechanical properties of the nucleus in immune cells. FTIR imaging enables label-free chemical imaging with subcellular resolution. By optimizing the FTIR methodology and coupling it with cell segmentation analysis approach, we have identified key spectral changes corresponding to changes in DNA levels and chromatin conformation at the single cell level. By further manipulating live single cells using pressure-driven microfluidics, we found that chromatin decondensation - either during general transcriptional activation or during specific immune cell maturation - can ultimately lead to nuclear auxeticity which is a new biological phenomenon recently identified. Taken together our findings demonstrate the tight and, potentially bilateral, link between extra-cellular mechanotransduction and intra-cellular nuclear architecture.
- Published
- 2019
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- View/download PDF
25. A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity.
- Author
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Sheppard EC, Rogers S, Harmer NJ, and Chahwan R
- Subjects
- DNA Repair, DNA, Single-Stranded, DNA-Directed DNA Polymerase, Deoxyribonucleases analysis, Exodeoxyribonucleases, Humans, Kinetics, Phosphoproteins, Ribonucleases analysis, Substrate Specificity, Deoxyribonucleases metabolism, Enzyme Assays methods, Fluorescence, Ribonucleases metabolism
- Abstract
DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.
- Published
- 2019
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- View/download PDF
26. Epigenomic Modifications Mediating Antibody Maturation.
- Author
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Sheppard EC, Morrish RB, Dillon MJ, Leyland R, and Chahwan R
- Subjects
- Animals, Chromatin genetics, Cytidine Deaminase metabolism, DNA Repair, Epigenesis, Genetic, Histones metabolism, Humans, Immune System Diseases genetics, Immunity, Humoral, Neoplasms genetics, RNA, Untranslated genetics, Chromatin metabolism, Immune System Diseases immunology, Immunoglobulin Class Switching, Neoplasms immunology, Somatic Hypermutation, Immunoglobulin
- Abstract
Epigenetic modifications, such as histone modifications, DNA methylation status, and non-coding RNAs (ncRNA), all contribute to antibody maturation during somatic hypermutation (SHM) and class-switch recombination (CSR). Histone modifications alter the chromatin landscape and, together with DNA primary and tertiary structures, they help recruit Activation-Induced Cytidine Deaminase (AID) to the immunoglobulin (Ig) locus. AID is a potent DNA mutator, which catalyzes cytosine-to-uracil deamination on single-stranded DNA to create U:G mismatches. It has been shown that alternate chromatin modifications, in concert with ncRNAs and potentially DNA methylation, regulate AID recruitment and stabilize DNA repair factors. We, hereby, assess the combination of these distinct modifications and discuss how they contribute to initiating differential DNA repair pathways at the Ig locus, which ultimately leads to enhanced antibody-antigen binding affinity (SHM) or antibody isotype switching (CSR). We will also highlight how misregulation of epigenomic regulation during DNA repair can compromise antibody development and lead to a number of immunological syndromes and cancer.
- Published
- 2018
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27. Isotype switching: Mouse IgG3 constant region drives increased affinity for polysaccharide antigens.
- Author
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Harmer NJ and Chahwan R
- Subjects
- Animals, Antibodies, Monoclonal, Mice, Polysaccharides, Immunoglobulin Class Switching, Immunoglobulin G genetics
- Published
- 2016
- Full Text
- View/download PDF
28. Overlapping hotspots in CDRs are critical sites for V region diversification.
- Author
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Wei L, Chahwan R, Wang S, Wang X, Pham PT, Goodman MF, Bergman A, Scharff MD, and MacCarthy T
- Subjects
- Base Sequence, Cell Line, Cytidine Deaminase metabolism, DNA genetics, DNA Primers, Humans, Mutation, Complementarity Determining Regions, Immunoglobulin Variable Region genetics
- Abstract
Activation-induced deaminase (AID) mediates the somatic hypermutation (SHM) of Ig variable (V) regions that is required for the affinity maturation of the antibody response. An intensive analysis of a published database of somatic hypermutations that arose in the IGHV3-23*01 human V region expressed in vivo by human memory B cells revealed that the focus of mutations in complementary determining region (CDR)1 and CDR2 coincided with a combination of overlapping AGCT hotspots, the absence of AID cold spots, and an abundance of polymerase eta hotspots. If the overlapping hotspots in the CDR1 or CDR2 did not undergo mutation, the frequency of mutations throughout the V region was reduced. To model this result, we examined the mutation of the human IGHV3-23*01 biochemically and in the endogenous heavy chain locus of Ramos B cells. Deep sequencing revealed that IGHV3-23*01 in Ramos cells accumulates AID-induced mutations primarily in the AGCT in CDR2, which was also the most frequent site of mutation in vivo. Replacing the overlapping hotspots in CDR1 and CDR2 with neutral or cold motifs resulted in a reduction in mutations within the modified motifs and, to some degree, throughout the V region. In addition, some of the overlapping hotspots in the CDRs were at sites in which replacement mutations could change the structure of the CDR loops. Our analysis suggests that the local sequence environment of the V region, and especially of the CDR1 and CDR2, is highly evolved to recruit mutations to key residues in the CDRs of the IgV region.
- Published
- 2015
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29. Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes.
- Author
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Schaetzlein S, Chahwan R, Avdievich E, Roa S, Wei K, Eoff RL, Sellers RS, Clark AB, Kunkel TA, Scharff MD, and Edelmann W
- Subjects
- Amino Acid Sequence, Amino Acid Substitution genetics, Animals, DNA End-Joining Repair genetics, DNA Mismatch Repair genetics, DNA Repair Enzymes deficiency, DNA Repair Enzymes genetics, Exodeoxyribonucleases deficiency, Exodeoxyribonucleases genetics, Female, Male, Meiosis genetics, Mice, Mice, 129 Strain, Mice, Inbred C57BL, Mice, Knockout, Mice, Mutant Strains, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutant Proteins chemistry, Mutant Proteins genetics, Mutant Proteins metabolism, Sequence Homology, Amino Acid, DNA Repair Enzymes metabolism, Exodeoxyribonucleases metabolism
- Abstract
Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1(EK)) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1(null)) mouse. In contrast to Exo1(null/null) mice, Exo1(EK/EK) mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1(null) mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo.
- Published
- 2013
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- View/download PDF
30. RNF8 links nucleosomal and cytoskeletal ubiquitylation of higher order protein structures.
- Author
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Chahwan R, Gravel S, Matsusaka T, and Jackson SP
- Subjects
- Humans, Cell Cycle Proteins metabolism, DNA-Binding Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism, Schizosaccharomyces, Schizosaccharomyces pombe Proteins metabolism
- Published
- 2013
- Full Text
- View/download PDF
31. Dma/RNF8 proteins are evolutionarily conserved E3 ubiquitin ligases that target septins.
- Author
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Chahwan R, Gravel S, Matsusaka T, and Jackson SP
- Subjects
- Cell Cycle, Cell Cycle Proteins genetics, Cell Division, Cell Line, Tumor, Centrosome metabolism, DNA-Binding Proteins genetics, HeLa Cells, Humans, Mitosis, RNA Interference, RNA, Small Interfering, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Schizosaccharomyces pombe Proteins genetics, Septins metabolism, Ubiquitin-Protein Ligases, Ubiquitination, Cell Cycle Proteins metabolism, DNA-Binding Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism, Schizosaccharomyces cytology, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins metabolism
- Abstract
The budding yeast proteins Dma1 and Dma2 are members of the unique FHA-RING domain protein family and are linked to mitotic regulation and septin organization by ill-defined mechanisms. We show that Dma2 has ubiquitin ligase activity, and that septins Shs1 and Cdc11 are likely direct in vivo targets. We further propose that human RNF8, rather than Chfr, is the mammalian Dma homolog. As in yeast, RNF8 localizes to the centrosomes and cell division sites and promotes ubiquitylation of the septin SEPT7, whose depletion increases cell division anomalies. Together, these findings reveal evolutionary and functional conservation of Dma proteins, and suggest that RNF8 maintains genome stability through independent, yet analogous, nuclear and cytoplasmic ubiquitylation activities.
- Published
- 2013
- Full Text
- View/download PDF
32. AIDing antibody diversity by error-prone mismatch repair.
- Author
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Chahwan R, Edelmann W, Scharff MD, and Roa S
- Subjects
- Animals, Cytidine Deaminase genetics, Deamination, Humans, Mutation, Ubiquitination, Antibody Diversity, Cytidine Deaminase immunology, DNA Mismatch Repair
- Abstract
The creation of a highly diverse antibody repertoire requires the synergistic activity of a DNA mutator, known as activation-induced deaminase (AID), coupled with an error-prone repair process that recognizes the DNA mismatch catalyzed by AID. Instead of facilitating the canonical error-free response, which generally occurs throughout the genome, DNA mismatch repair (MMR) participates in an error-prone repair mode that promotes A:T mutagenesis and double-strand breaks at the immunoglobulin (Ig) genes. As such, MMR is capable of compounding the mutation frequency of AID activity as well as broadening the spectrum of base mutations; thereby increasing the efficiency of antibody maturation. We here review the current understanding of this MMR-mediated process and describe how the MMR signaling cascade downstream of AID diverges in a locus dependent manner and even within the Ig locus itself to differentially promote somatic hypermutation (SHM) and class switch recombination (CSR) in B cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
33. The ATPase activity of MLH1 is required to orchestrate DNA double-strand breaks and end processing during class switch recombination.
- Author
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Chahwan R, van Oers JM, Avdievich E, Zhao C, Edelmann W, Scharff MD, and Roa S
- Subjects
- Animals, B-Lymphocytes immunology, DNA End-Joining Repair, DNA Repair, Mice, MutL Protein Homolog 1, Somatic Hypermutation, Immunoglobulin, Adaptor Proteins, Signal Transducing physiology, Adenosine Triphosphatases metabolism, DNA Breaks, Double-Stranded, Immunoglobulin Class Switching genetics, Nuclear Proteins physiology, Recombination, Genetic
- Abstract
Antibody diversification through somatic hypermutation (SHM) and class switch recombination (CSR) are similarly initiated in B cells with the generation of U:G mismatches by activation-induced cytidine deaminase but differ in their subsequent mutagenic consequences. Although SHM relies on the generation of nondeleterious point mutations, CSR depends on the production of DNA double-strand breaks (DSBs) and their adequate recombination through nonhomologous end joining (NHEJ). MLH1, an ATPase member of the mismatch repair (MMR) machinery, is emerging as a likely regulator of whether a U:G mismatch progresses toward mutation or DSB formation. We conducted experiments on cancer modeled ATPase-deficient MLH1G67R knockin mice to determine the function that the ATPase domain of MLH1 mediates in SHM and CSR. Mlh1(GR/GR) mice displayed a significant decrease in CSR, mainly attributed to a reduction in the generation of DSBs and diminished accumulation of 53BP1 at the immunoglobulin switch regions. However, SHM was normal in these mice, which distinguishes MLH1 from upstream members of the MMR pathway and suggests a very specific role of its ATPase-dependent functions during CSR. In addition, we show that the residual switching events still taking place in Mlh1(GR/GR) mice display unique features, suggesting a role for the ATPase activity of MLH1 beyond the activation of the endonuclease functions of its MMR partner PMS2. A preference for switch junctions with longer microhomologies in Mlh1(GR/GR) mice suggests that through its ATPase activity, MLH1 also has an impact in DNA end processing, favoring canonical NHEJ downstream of the DSB. Collectively, our study shows that the ATPase domain of MLH1 is important to transmit the CSR signaling cascade both upstream and downstream of the generation of DSBs.
- Published
- 2012
- Full Text
- View/download PDF
34. Mismatch-mediated error prone repair at the immunoglobulin genes.
- Author
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Chahwan R, Edelmann W, Scharff MD, and Roa S
- Subjects
- Animals, Antibodies immunology, Cytidine Deaminase metabolism, DNA Repair Enzymes genetics, DNA-Binding Proteins genetics, Humans, Mice, Mice, Knockout, Mutation, DNA Mismatch Repair, DNA Repair, Genes, Immunoglobulin
- Abstract
The generation of effective antibodies depends upon somatic hypermutation (SHM) and class-switch recombination (CSR) of antibody genes by activation induced cytidine deaminase (AID) and the subsequent recruitment of error prone base excision and mismatch repair. While AID initiates and is required for SHM, more than half of the base changes that accumulate in V regions are not due to the direct deamination of dC to dU by AID, but rather arise through the recruitment of the mismatch repair complex (MMR) to the U:G mismatch created by AID and the subsequent perversion of mismatch repair from a high fidelity process to one that is very error prone. In addition, the generation of double-strand breaks (DSBs) is essential during CSR, and the resolution of AID-generated mismatches by MMR to promote such DSBs is critical for the efficiency of the process. While a great deal has been learned about how AID and MMR cause hypermutations and DSBs, it is still unclear how the error prone aspect of these processes is largely restricted to antibody genes. The use of knockout models and mice expressing mismatch repair proteins with separation-of-function point mutations have been decisive in gaining a better understanding of the roles of each of the major MMR proteins and providing further insight into how mutation and repair are coordinated. Here, we review the cascade of MMR factors and repair signals that are diverted from their canonical error free role and hijacked by B cells to promote genetic diversification of the Ig locus. This error prone process involves AID as the inducer of enzymatically-mediated DNA mismatches, and a plethora of downstream MMR factors acting as sensors, adaptors and effectors of a complex and tightly regulated process from much of which is not yet well understood., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
35. The multidimensional nature of epigenetic information and its role in disease.
- Author
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Chahwan R, Wontakal SN, and Roa S
- Subjects
- DNA Methylation, Humans, Neoplasms genetics, RNA genetics, Epigenesis, Genetic
- Abstract
This year marks the 10th anniversary of the publications that reported the initial human genome sequence. In the historic press conference that announced this landmark accomplishment, it was proclaimed that the genome sequence would "revolutionize the diagnosis, prevention, and treatment of most, if not all, human diseases." However, subsequent work over the past decade has revealed that "complex diseases" are much more intricate than originally thought. Even with the advent of several new powerful technologies, our understanding of the underlying genetic etiologies of most complex and non-Mendelian diseases is far from complete. These results have raised the possibility that the DNA sequence, i.e., genetic information, may not be the only relevant source of information in order to understand the molecular basis of disease. In this review, we assemble evidence that information encoded beyond the DNA sequence, i.e., epigenetic information, may hold the key to a better understanding of various pathological conditions. Unlike the genetic information encoded within the DNA sequence, epigenetic information can be stored in multiple dimensions, such as in the form of DNA modifications, RNA, or protein. Ideas presented here support the view that to better understand the molecular etiology of diseases, we need to gain a better understanding of both the genetic and epigenetic components of biological information. We hence believe that the fast development of genome-wide technologies will facilitate a better understanding of both genetic and epigenetic dimensions of disease.
- Published
- 2011
36. Crosstalk between genetic and epigenetic information through cytosine deamination.
- Author
-
Chahwan R, Wontakal SN, and Roa S
- Subjects
- Animals, Biological Evolution, Deamination, Humans, Cytosine metabolism, Cytosine Deaminase metabolism, Epigenesis, Genetic
- Abstract
Decades of work have elucidated the existence of two forms of heritable information, namely genetic and epigenetic, which are collectively referred to as the 'dual inheritance'. The underlying mechanisms behind these two modes of inheritance have so far remained distinct. Cytosine deaminases, such as activation-induced cytidine deaminase (AID) and other members of the APOBEC family, have been implicated both in genetic variation of somatic cells and in epigenetic remodeling of germ and pluripotent cells. We hereby synthesize these seemingly dissociated functions into one coherent model, and further suggest that cytosine deaminases, particularly AID, might have a broader influence by modulating epigenetic information in somatic or cancer cells, as well as by triggering genetic variation in germ and pluripotent cells through mutation followed by natural selection. We therefore propose that the AID/APOBEC family of deaminases are likely to have acted as drivers throughout vertebrate evolution., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
37. The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination.
- Author
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Ramachandran S, Chahwan R, Nepal RM, Frieder D, Panier S, Roa S, Zaheen A, Durocher D, Scharff MD, and Martin A
- Subjects
- Animals, Cell Line, Cytidine Deaminase metabolism, Humans, Immunoglobulin A genetics, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Mice, Reverse Transcriptase Polymerase Chain Reaction, Tumor Suppressor p53-Binding Protein 1, Ubiquitin-Protein Ligases metabolism, Ubiquitination genetics, DNA-Binding Proteins genetics, Recombination, Genetic, Ubiquitin-Protein Ligases genetics
- Abstract
An effective immune response requires B cells to produce several classes of antibodies through the process of class switch recombination (CSR). Activation-induced cytidine deaminase initiates CSR by deaminating deoxycytidines at switch regions within the Ig locus. This activity leads to double-stranded DNA break formation at the donor and recipient switch regions that are subsequently synapsed and ligated in a 53BP1-dependent process that remains poorly understood. The DNA damage response E3 ubiquitin ligases RNF8 and RNF168 were recently shown to facilitate recruitment of 53BP1 to sites of DNA damage. Here we show that the ubiquitination pathway mediated by RNF8 and RNF168 plays an integral part in CSR. Using the CH12F3-2 mouse B cell line that undergoes CSR to IgA at high rates, we demonstrate that knockdown of RNF8, RNF168, and 53BP1 leads to a significant decrease in CSR. We also show that 53BP1-deficient CH12F3-2 cells are protected from apoptosis mediated by the MDM2 inhibitor Nutlin-3. In contrast, deficiency in either E3 ubiquitin ligase does not protect cells from Nutlin-3-mediated apoptosis, indicating that RNF8 and RNF168 do not regulate all functions of 53BP1.
- Published
- 2010
- Full Text
- View/download PDF
38. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase.
- Author
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Kolas NK, Chapman JR, Nakada S, Ylanko J, Chahwan R, Sweeney FD, Panier S, Mendez M, Wildenhain J, Thomson TM, Pelletier L, Jackson SP, and Durocher D
- Subjects
- Adaptor Proteins, Signal Transducing, Amino Acid Motifs, Amino Acid Sequence, Ataxia Telangiectasia Mutated Proteins, BRCA1 Protein metabolism, Cell Cycle Proteins metabolism, Cell Line, Tumor, DNA Repair, DNA-Binding Proteins chemistry, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins metabolism, Molecular Sequence Data, Nuclear Proteins chemistry, Nuclear Proteins metabolism, Phosphorylation, Protein Serine-Threonine Kinases metabolism, Protein Structure, Tertiary, RNA, Small Interfering, Trans-Activators chemistry, Trans-Activators metabolism, Tumor Suppressor Proteins metabolism, Tumor Suppressor p53-Binding Protein 1, Ubiquitin metabolism, Ubiquitin-Conjugating Enzymes metabolism, Ubiquitination, Cell Nucleus Structures genetics, DNA Breaks, Double-Stranded, DNA-Binding Proteins metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.
- Published
- 2007
- Full Text
- View/download PDF
39. Involvement of mammalian Mus81 in genome integrity and tumor suppression.
- Author
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McPherson JP, Lemmers B, Chahwan R, Pamidi A, Migon E, Matysiak-Zablocki E, Moynahan ME, Essers J, Hanada K, Poonepalli A, Sanchez-Sweatman O, Khokha R, Kanaar R, Jasin M, Hande MP, and Hakem R
- Subjects
- Alleles, Animals, Chromosome Aberrations, DNA Damage, Embryo, Mammalian cytology, Embryonic and Fetal Development, Gamma Rays, Gene Targeting, Genetic Predisposition to Disease, Heterozygote, Lymphoma etiology, Lymphoma genetics, Lymphoma pathology, Meiosis, Mice, Mitomycin pharmacology, Neoplasms etiology, Recombination, Genetic, Saccharomyces cerevisiae Proteins, Sister Chromatid Exchange, Stem Cells, T-Lymphocytes physiology, DNA-Binding Proteins genetics, DNA-Binding Proteins physiology, Endonucleases, Genome, Genomic Instability, Neoplasms genetics
- Abstract
Mus81-Eme1 endonuclease has been implicated in the rescue of stalled replication forks and the resolution of meiotic recombination intermediates in yeast. We used gene targeting to study the physiological requirements of Mus81 in mammals. Mus81-/- mice are viable and fertile, which indicates that mammalian Mus81 is not essential for recombination processes associated with meiosis. Mus81-deficient mice and cells were hypersensitive to the DNA cross-linking agent mitomycin C but not to gamma-irradiation. Remarkably, both homozygous Mus81-/- and heterozygous Mus81+/- mice exhibited a similar susceptibility to spontaneous chromosomal damage and a profound and equivalent predisposition to lymphomas and other cancers. These studies demonstrate a critical role for the proper biallelic expression of the mammalian Mus81 in the maintenance of genomic integrity and tumor suppression.
- Published
- 2004
- Full Text
- View/download PDF
40. Eme1 is involved in DNA damage processing and maintenance of genomic stability in mammalian cells.
- Author
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Abraham J, Lemmers B, Hande MP, Moynahan ME, Chahwan C, Ciccia A, Essers J, Hanada K, Chahwan R, Khaw AK, McPherson P, Shehabeldin A, Laister R, Arrowsmith C, Kanaar R, West SC, Jasin M, and Hakem R
- Subjects
- Amino Acid Sequence, Animals, Chromosomal Instability, DNA Damage, DNA-Binding Proteins metabolism, Endodeoxyribonucleases genetics, Endonucleases genetics, Endonucleases metabolism, Humans, Mice, Molecular Sequence Data, Saccharomyces cerevisiae Proteins, Schizosaccharomyces pombe Proteins genetics, Sister Chromatid Exchange, Stem Cells, DNA Repair physiology, Endodeoxyribonucleases metabolism, Genomic Instability
- Abstract
Yeast and human Eme1 protein, in complex with Mus81, constitute an endonuclease that cleaves branched DNA structures, especially those arising during stalled DNA replication. We identified mouse Eme1, and show that it interacts with Mus81 to form a complex that preferentially cleaves 3'-flap structures and replication forks rather than Holliday junctions in vitro. We demonstrate that Eme1-/- embryonic stem (ES) cells are hypersensitive to the DNA cross-linking agents mitomycin C and cisplatin, but only mildly sensitive to ionizing radiation, UV radiation and hydroxyurea treatment. Mammalian Eme1 is not required for the resolution of DNA intermediates that arise during homologous recombination processes such as gene targeting, gene conversion and sister chromatid exchange (SCE). Unlike Blm-deficient ES cells, increased SCE was seen only following induced DNA damage in Eme1-deficient cells. Most importantly, Eme1 deficiency led to spontaneous genomic instability. These results reveal that mammalian Eme1 plays a key role in DNA repair and the maintenance of genome integrity.
- Published
- 2003
- Full Text
- View/download PDF
41. DNA-based subtypes and antimicrobial susceptibility profiles of Haemophilus influenzae and Haemophilus parainfluenzae isolated from different tonsillar sites of children undergoing tonsillectomy and/or adenoidectomy.
- Author
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Matar GM, Chahwan R, Fuleihan N, Uwaydah M, El-Hajj M, and Hadi U
- Subjects
- Adenoidectomy, Adolescent, Child, Child, Preschool, Female, Haemophilus influenzae genetics, Haemophilus influenzae isolation & purification, Haemophilus parainfluenzae genetics, Haemophilus parainfluenzae isolation & purification, Humans, Hypertrophy, Male, Microbial Sensitivity Tests, Tonsillectomy, Tonsillitis microbiology, Tonsillitis pathology, Anti-Bacterial Agents pharmacology, DNA, Bacterial analysis, Drug Resistance, Bacterial, Haemophilus influenzae drug effects, Haemophilus parainfluenzae drug effects
- Abstract
We did a comparative analysis between DNA-based subtypes and antimicrobial susceptibility profiles on Haemophilus influenzae and Haemophilus parainfluenzae, isolated from multiple tonsillar sites per individual from patients with chronic recurrent tonsillitis and/or tonsillar idiopathic hypertrophy and undergoing tonsillectomy and/or adenoidectomy. A total of eighty-eight Haemophilus isolates were obtained aseptically from the surface and core of tonsils and/or adenoids of 32 out of 60 patients and identified at the species level by the X and V factors and the API NH Kit. The H. influenzae and H. parainfluenzae isolates as well as ATCC strains were tested for antimicrobial susceptibility using a panel of antimicrobial agents. Random amplified polymorphic DNA (RAPD) was done on extracted DNA from all Haemophilus isolates and ATCC strains, using one 10 mer and one 18 mer primers to subtype the two species. Antimicrobial susceptibility testing data have shown a variation in generated susceptibility patterns to tested antimicrobial agents among H. influenzae and H. parainfluenzae isolates. This variation was demonstrated too among isolates obtained from different tonsillar sites (core and surface) in a single patient. RAPD analysis identified 58/88 (66%) different RAPD patterns. Variations in RAPD patterns among H. influenzae and H. parainfluenzae were also observed in isolates obtained from different tonsillar sites of the same individual. A correlation between RAPD patterns and antimicrobial susceptibility data, have shown: 1) the predominance of one strain (RAPD pattern) of either Haemophilus species among isolated organisms per patient, and exhibiting different antimicrobial susceptibility profiles or 2) the existence of multiple strains (RAPD patterns) of either Haemophilis species per patient, and showing either a single or multiple antimicrobial susceptibility profile(s). These observations question the validity of swab cultures obtained from a single tonsillar site per patient, for detection, identification and determination of antimicrobial profiles of the etiology of tonsillitis, since swab specimens taken from only one site may or may not reflect the etiology of infection.
- Published
- 2002
42. PCR-based detection, restriction endonuclease analysis, and transcription of tonB in Haemophilus influenzae and Haemophilus parainfluenzae isolates obtained from children undergoing tonsillectomy and adenoidectomy.
- Author
-
Matar GM, Chahwan R, Fuleihan N, Uwaydah M, and Hadi U
- Subjects
- Adenoidectomy, Child, Haemophilus influenzae genetics, Humans, Reverse Transcriptase Polymerase Chain Reaction, Tonsillectomy, Tonsillitis surgery, Transcription, Genetic, Bacterial Proteins genetics, Haemophilus Infections diagnosis, Haemophilus influenzae isolation & purification, Membrane Proteins genetics, Restriction Mapping methods, Tonsillitis diagnosis
- Abstract
We developed and evaluated a PCR-based-restriction endonuclease analysis method to detect and analyze the tonB gene of Haemophilus influenzae and Haemophilus parainfluenzae from pediatric patients undergoing tonsillectomy and adenoidectomy. Multiple sites from the same patient, including the surface of adenoids and tonsils, as well as the core of tonsils, were cultured on chocolate agar and identified using standard procedures and the API NH Kit. A total of 55 H. influenzae isolates were recovered from different sites of 20 patients, and 32 H. parainfluenzae isolates were recovered from various sites of 12 patients. DNA was extracted from American Type Culture Collection strains and test isolates by the PureGene kit. Two primers, G1 (21-mer) and G2 (23-mer), were designed by us to amplify by PCR the tonB gene that consists of an 813-bp fragment. A nested PCR using primers T1 (23-mer) and T2 (24-mer) that flank an internal sequence to the gene of the order of 257 bp and restriction endonuclease digestion using XhoI and BglII were done to detect whether heterogeneity within the gene exists between the two species. Reverse transcription-PCR (RT-PCR) was finally done to detect transcription of the gene in both species. Our data have shown that the tonB gene was detected in both species. It is known to encode a virulent protein, TonB, in H. influenzae; however, demonstration of its presence in H. parainfluenzae is novel. Nested-PCR and restriction endonuclease analysis have shown that the tonB gene is apparently structurally the same in both species, with possible differences that may exist in certain H. parainfluenzae isolates. RT-PCR done on selected numbers of H. influenzae and H. parainfluenzae have shown that the tonB gene was transcribed in both species. This shows that the TonB protein, if expressed, may play a different role in the virulence in H. parainfluenzae since it is not needed for heme or heme complexes uptake as with H. influenzae.
- Published
- 2001
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