Browsing by Author "Fisher, Randall"

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    DNA damage response of haematopoietic stem and progenitor cells to high-LET neutron irradiation
    (Nature, 2021-10) Engelbrecht, Monique; Ndimba, Roya; De Kock, Maryna; Miles, Xanthene; Nair, Shankari; Fisher, Randall; Du Plessis, Peter; Bolcaen, Julie; Botha, Matthys Hendrik; Zwanepoel, Elbie; Sioen, Simon; Baeyens, Ans; Nieto-Camero, Jaime; De Kock, Evan; Vandevoorde, Charlot
    The radiosensitivity of haematopoietic stem and progenitor cells (HSPCs) to neutron radiation remains largely underexplored, notwithstanding their potential role as target cells for radiation-induced leukemogenesis. New insights are required for radiation protection purposes, particularly for aviation, space missions, nuclear accidents and even particle therapy. In this study, HSPCs (CD34+CD38+ cells) were isolated from umbilical cord blood and irradiated with 60Co γ-rays (photons) and high energy p(66)/Be(40) neutrons. At 2 h post-irradiation, a significantly higher number of 1.28 ± 0.12 γ-H2AX foci/cell was observed after 0.5 Gy neutrons compared to 0.84 ± 0.14 foci/cell for photons, but this decreased to similar levels for both radiation qualities after 18 h. However, a significant difference in late apoptosis was observed with Annexin-V+/PI+ assay between photon and neutron irradiation at 18 h, 43.17 ± 6.10% versus 55.55 ± 4.87%, respectively. A significant increase in MN frequency was observed after both 0.5 and 1 Gy neutron irradiation compared to photons illustrating higher levels of neutron-induced cytogenetic damage, while there was no difference in the nuclear division index between both radiation qualities. The results point towards a higher induction of DNA damage after neutron irradiation in HSPCs followed by error-prone DNA repair, which contributes to genomic instability and a higher risk of leukemogenesis.
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    Next generation sequencing demonstrates minor variant HIV drug resistance mutations
    (Stellenbosch : Stellenbosch University, 2016-03) Fisher, Randall; Van Zyl, Gert; Engelbrecht, Susan; Preiser, Wolfgang; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
    ENGLISH ABSTRACT: Introduction: The South African public sector antiretroviral therapy (ART) roll-out has been associated with emergence of HIV drug resistance (HIVDR) in therapy-naïve and treated patients. These drug-resistant viral strains are archived in viral reservoirs and may persist as minority variants when outgrown by fitter wild-type strains, in the absence of sufficient drug pressure. Although minority drug resistant variants may predict failure, they are not readily detectable with PCR and Sanger sequencing when constituting less than 20% of the viral population. Next generation sequencing (NGS) allows the detection of minor drug resistant variants and investigation of linkage of HIVDR mutations when sufficient read-length is obtained. Various NGS platforms, available in South Africa, offer per-sample cost reductions when sufficient numbers of samples are pooled. However, targeted resequencing on NGS platforms requires template enrichment by PCR that can result in PCR-induced error and template re-sampling error. Resampling error is a result of random error or a primer selection bias and is more pronounced when using long fusion primers for NGS amplicon sequencing. Methods: As data were limited on minor HIVDR variants in infants who became infected despite the Western Cape Prevention of Mother-to-Child HIV Transmission (PMTCT) regimen and in adults with virological failure without major protease inhibitor (PI)-resistance mutations on a PI-based regimen, we performed NGS in these populations. In the first cross-sectional study we sample minority variants from seven adults who failed PI-based therapy due to poor adherence. Samples were enriched for NGS with a nested fusion primer PCR and minor variants were identified on the 454 Life Sciences, FLX Titanium platform (Connecticut, USA). In the second cross-sectional study we sampled viral species from 15 PMTCT-failed infants. Nested PCR amplicons were size-fragmented and ligated to platform-specific sequencing adaptors. The sequence library was sequenced in parallel on the Ion Torrent Personal Genome Machine (PGM) (Life Technologies, California, USA) and the Illumina MiSeq (California, USA) and was validated by clonal sequencing. In study three, we attempted to characterise resampling error when using fusion primers and correct it by partitioning PCR reactions in emulsion. We compared the 454 NGS sequence yields when using “open” or emulsified PCR for template enrichment and investigated the effect of varying degrees of primer-template mismatches when the sampled population consisted of mixtures of plasmids. Findings: In the first study, NGS improved the drug resistance mutation detection in five of the seven patients although no majority variants with PI-resistance were identified. We concluded that limited or intermittent drug pressure resulted in insufficient selection for major drug-resistant variants to emerge. In study two, NGS conducted on the PGM and MiSeq improved the drug resistance detection in 15 PMTCT-failed infants. Although amplicon sequencing using fusion primers allows the most efficient use of NGS coverage, it is prone to PCR resampling error depending on the degree of template-to-primer mismatches. In study three, emulsion PCR was able to reduce but not correct this resampling error.