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- Olausson, Josefin, 1983, et al.
(författare)
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Optimization of cerebrospinal fluid microbial DNA metagenomic sequencing diagnostics
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Infection in the central nervous system is a severe condition associated with high morbidity and mortality. Despite ample testing, the majority of encephalitis and meningitis cases remain undiagnosed. Metagenomic sequencing of cerebrospinal fluid has emerged as an unbiased approach to identify rare microbes and novel pathogens. However, several major hurdles remain, including establishment of individual limits of detection, removal of false positives and implementation of universal controls. Twenty-one cerebrospinal fluid samples, in which a known pathogen had been positively identified by available clinical techniques, were subjected to metagenomic DNA sequencing. Fourteen samples contained minute levels of Epstein-Barr virus. The detection threshold for each sample was calculated by using the total leukocyte content in the sample and environmental contaminants found in the bioinformatic classifiers. Virus sequences were detected in all ten samples, in which more than one read was expected according to the calculations. Conversely, no viral reads were detected in seven out of eight samples, in which less than one read was expected according to the calculations. False positive pathogens of computational or environmental origin were readily identified, by using a commonly available cell control. For bacteria, additional filters including a comparison between classifiers removed the remaining false positives and alleviated pathogen identification. Here we show a generalizable method for identification of pathogen species using DNA metagenomic sequencing. The choice of bioinformatic method mainly affected the efficiency of pathogen identification, but not the sensitivity of detection. Identification of pathogens requires multiple filtering steps including read distribution, sequence diversity and complementary verification of pathogen reads.
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| 2. |
- Tian, Yarong, 1989
(författare)
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The transcriptomic landscape of Epstein-Barr virus associated tumors at cellular and single-molecule level
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Epstein-Barr virus (EBV) was the first oncovirus found in humans. Almost all adults worldwide are asymptomatic carriers of EBV. The latent EBV-infection malignifies in approximately 200,000 individuals each year. The risk of developing certain types of EBV-associated cancer is high in specific regions, for example nasopharyngeal carcinoma in Southeast Asia and Burkitt’s lymphoma in Africa. The overall aim of this thesis was to characterize the EBV gene expression patterns in biopsies and elucidate the function of the expressed viral genes. Bulk transcriptome datasets of 615 tumors from four types of known EBV-associated neoplasms and single-cell transcriptome data from 63 nasopharyngeal samples were screened for EBV expression. The most abundant EBV RNA found at both tissue and single-cell levels, were RPMS1 and the novel co-terminating transcripts which we named BAREs. LMP1/BNLF2a/b and LMP2A/B/BNRF1 were expressed to a lesser extent and large differences were observed between individuals. Single-cell sequencing of B-lymphocytes isolated from the peripheral blood of a patient with a high EBV DNA load showed a similar EBV expression profile as the EBV-positive tumors. Moreover, the highly expressed EBV genes RPMS1 and BAREs were subjected to full-length single-molecule sequencing and all isoforms were characterized using our newly developed bioinformatics tool FLAME. Our results show that available EBV cell models inadequately portray primary tumors with regard to the viral gene expression and/or the propensity for reactivation. We developed an in vitro nasopharyngeal pseudostratified epithelium model which could mimic an EBV infection in the nasopharynx. A donor-dependent susceptibility for EBV infection was observed and both latent and lytic EBV expression patterns were detected in cells from a single donor. Single-cell sequencing data analysis could further distinguish that cells in late lytic stage with virus host shutoff were found amongst the suprabasal cells. The single-cell data from peripheral EBV-transformed B-lymphocytes identified that EBV induces proliferative pathways. In nasopharyngeal carcinoma tissue the EBV-transformed epithelial cells exists in a microenvironment with lymphocytic infiltration and interferon. Single-cell characterization of the nasopharyngeal cancer cells identified that the EBV expression of RPMS1 along with the miR-BARTs encoded in the introns promotes immune evasion by downregulation of interferon responsive genes. The findings suggest that EBV contributes to tumorigenesis in two ways, the first is by host cell reprogramming and induction of proliferation by EBNAs and LMP1, and the second is by immune evasion and escape by RPMS1 and BNLF2a.
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