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  • 1
    In: mBio, American Society for Microbiology, Vol. 11, No. 5 ( 2020-10-27)
    Abstract: Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo . This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
    detail.hit.zdb_id: 2557172-2
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  • 2
    In: Cell Death Discovery, Springer Science and Business Media LLC, Vol. 6, No. 1 ( 2020-01-02)
    Abstract: Melanoma is among the most aggressive cancers due to its tendency to metastasize early. Phenotype switching between a proliferative and an invasive state has been suggested as a critical process for metastasis, though the mechanisms that regulate state transitions are complex and remain poorly understood. Brother of Regulator of Imprinted Sites (BORIS), also known as CCCTC binding factor-Like (CTCFL), is a transcriptional modulator that becomes aberrantly expressed in melanoma. Yet, the role of BORIS in melanoma remains elusive. Here, we show that BORIS is involved in melanoma phenotype switching. Genetic modification of BORIS expression in melanoma cells combined with whole-transcriptome analysis indicated that BORIS expression contributes to an invasion-associated transcriptome. In line with these findings, inducible BORIS overexpression in melanoma cells reduced proliferation and increased migration and invasion, demonstrating that the transcriptional switch is accompanied by a phenotypic switch. Mechanistically, we reveal that BORIS binds near the promoter of transforming growth factor-beta 1 ( TFGB1 ), a well-recognized factor involved in the transition towards an invasive state, which coincided with increased expression of TGFB1 . Overall, our study indicates a pro-invasive role for BORIS in melanoma via transcriptional reprogramming.
    Type of Medium: Online Resource
    ISSN: 2058-7716
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2020
    detail.hit.zdb_id: 2842546-7
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  • 3
    Online Resource
    Online Resource
    Oxford University Press (OUP) ; 2018
    In:  Virus Evolution Vol. 4, No. suppl_1 ( 2018-04-01)
    In: Virus Evolution, Oxford University Press (OUP), Vol. 4, No. suppl_1 ( 2018-04-01)
    Type of Medium: Online Resource
    ISSN: 2057-1577
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2018
    detail.hit.zdb_id: 2818949-8
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  • 4
    In: Thrombosis Research, Elsevier BV, Vol. 210 ( 2022-02), p. 6-11
    Type of Medium: Online Resource
    ISSN: 0049-3848
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2022
    detail.hit.zdb_id: 1500780-7
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  • 5
    In: Journal of the International AIDS Society, Wiley, Vol. 20, No. 1 ( 2017-01)
    Abstract: Introduction : Transmitted drug‐resistance mutations (TDRM) may hamper successful anti‐HIV‐1 therapy and impact future control of the HIV‐1 epidemic. Recently infected, therapy‐naïve individuals are best suited for surveillance of such TDRM. In this study, TDRM, detected by next‐generation sequencing (NGS) were compared to those identified by Sanger‐based population sequencing (SBS) in recently infected HIV‐1 patients. Methods : Historical samples from 80 recently infected HIV‐1 patients, diagnosed between 2000 and 2014, were analysed by MiSeq (NGS) and ABI (SBS). DeepChek‐HIV (ABL) was used for interpretation of the results. Results : Most patients were males (80%); Men who have sex with men (MSM) was the major transmission group (58.8%). Overall, TDRM were detected in 31.3% of patients by NGS and 8.8% by SBS, with SBS TDRM restricted to persons infected with subtype B. All SBS‐detected TDRM were identified by NGS. The prevalence of TDRM impacting protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTI) and non‐nucleoside reverse transcriptase inhibitors (NNRTI) was 11.3, 26.2 7.5%, respectively, in NGS analyses and 0, 3.8 and 5%, respectively, in SBS analyses. More patients with NGS and SBS TDRM were identified in 2008–2014 (37.2% or 13.9%, respectively) compared to 2000–2007 (24.3% or 2.7%, respectively), and a significantly greater number of these patients had multiple NGS TDRM. The most abundant, albeit, minor‐frequency RT TDRM, were the K65R and D67N, while K103N, M184V and T215S were high‐frequency mutations. Minor TDRM did not become a major variant in later samples and did not hinder successful treatment. Conclusions : NGS can replace SBS for mutation detection and allows for the detection of low‐frequency TDRM not identified by SBS. Although rates of TDRM in Israel continued to increase from 2000 to 2014, minor TDRM did not become major species. The need for ongoing surveillance of low‐frequency TDRM should be revisited in a larger study.
    Type of Medium: Online Resource
    ISSN: 1758-2652 , 1758-2652
    Language: English
    Publisher: Wiley
    Publication Date: 2017
    detail.hit.zdb_id: 2467110-1
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  • 6
    In: Antiviral Therapy, SAGE Publications, Vol. 22, No. 5 ( 2017-07), p. 431-441
    Abstract: Direct-acting antiviral (DAA) treatment regimens and response rates of patients with HCV genotype-1 (GT1) are currently considered subtype-dependent. Identification of clinically relevant resistance-associated substitutions (RASs) in the NS3 and NS5A proteins at baseline and in DAA failures, may also impact clinical decisions. Methods In a multicentre cohort study ( n=308), NS3 or NS5B sequencing ( n=248) was used to discriminate between GT1 subtypes. The correlation between baseline NS3 and NS5A RASs on the 12-week sustained virological response (SVR12) rates of 160 of the patients treated with second-generation DAAs was also assessed. Post-treatment resistance analysis was performed on samples from 58 patients exhibiting DAA virological failure. Results GT1a, GT1b and GT1d subtypes were identified in 23.0%, 75.4% and 1.2% of tested samples. GT1b was most prevalent (97.7%, 128/131) among patients born in the former Soviet Union. The Q80K NS3 RAS was identified in 17.5% (10/57) of the GT1a carriers, most of whom were Israeli-born. NS3 and NS5A baseline RASs showed a negligible correlation with SVR12 rates. Treatment-emergent RASs were observed among 8.9% (4/45) and 76.9% (10/13) of first- and second-generation DAA failures, respectively, with D168V/E (NS3), Y93H and L31M (NS5A) being the most prevalent mutations. Conclusions NS3 sequencing analysis can successfully discriminate between GT1 subtypes and identify NS3 amino acid substitutions. While pre-treatment NS3 and NS5A RASs marginally affect second-generation DAA SVR12 rates, post-treatment resistance analysis should be considered prior to re-therapy.
    Type of Medium: Online Resource
    ISSN: 1359-6535 , 2040-2058
    Language: English
    Publisher: SAGE Publications
    Publication Date: 2017
    detail.hit.zdb_id: 2118396-X
    SSG: 15,3
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  • 7
    In: Pigment Cell & Melanoma Research, Wiley, Vol. 36, No. 3-4 ( 2023-05), p. 299-313
    Abstract: Melanoma is the deadliest form of skin cancer, due to its tendency to metastasize early. Brother of regulator of imprinted sites (BORIS), also known as CCCTC binding factor‐like (CTCFL), is a transcription regulator that becomes ectopically expressed in melanoma. We recently showed that BORIS contributes to melanoma phenotype switching by altering the gene expression program of melanoma cells from an intermediate melanocytic state toward a more mesenchymal‐like state. However, the mechanism underlying this transcriptional switch remains unclear. Here, ATAC‐seq was used to study BORIS‐mediated chromatin accessibility alterations in melanoma cells harboring an intermediate melanocytic state. The gene set that gained promoter accessibility, following ectopic BORIS expression, showed enrichment for biological processes associated with melanoma invasion, while promoters of genes associated with proliferation showed reduced accessibility. Integration of ATAC‐seq and RNA‐seq data demonstrated that increased chromatin accessibility was associated with transcriptional upregulation of genes involved in tumor progression processes, and the aberrant activation of oncogenic transcription factors, while reduced chromatin accessibility and downregulated genes were associated with repressed activity of tumor suppressors and proliferation factors. Together, these findings indicate that BORIS mediates transcriptional reprogramming in melanoma cells by altering chromatin accessibility and gene expression, shifting the cellular transcription landscape of melanoma cells toward a mesenchymal‐like genetic signature.
    Type of Medium: Online Resource
    ISSN: 1755-1471 , 1755-148X
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2023
    detail.hit.zdb_id: 2425880-5
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