| 1. |
- Holmqvist, Isak, et al.
(author)
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FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
- 2021
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In: Rna. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 27:10, s. 1127-1139
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Journal article (peer-reviewed)abstract
- Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this work-flow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.
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| 2. |
- Hussein, Brwa Ali, 1984, et al.
(author)
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NKG2A gene variant predicts outcome of immunotherapy in AML and modulates the repertoire and function of NK cells
- 2023
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In: JOURNAL FOR IMMUNOTHERAPY OF CANCER. - 2051-1426. ; 11:8
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Journal article (peer-reviewed)abstract
- Background The natural killer (NK) complex (NKC) harbors multiple genes such as KLRC1 (encoding NKG2A) and KLRK1 (encoding NKG2D) that are central to regulation of NK cell function. We aimed at determining to what extent NKC haplotypes impact on NK cell repertoire and function, and whether such gene variants impact on outcome of IL-2-based immunotherapy in acute myeloid leukemia (AML).Methods Genotype status of NKG2D rs1049174 and NKG2A rs1983526 was determined using the TaqMan-Allelic discrimination approach. To dissect the impact of single nucloetide polymorphim (SNP) on NK cell function, we engineered the K562 cell line with CRISPR to be killed in a highly NKG2D-dependent fashion. NK cells were assayed for degranulation, intracellular cytokine production and cytotoxicity using flow cytometry.Results In AML patients receiving immunotherapy, the NKG2A gene variant, rs1983526, was associated with superior leukemia-free survival and overall survival. We observed that superior NK degranulation from individuals with the high-cytotoxicity NKG2D variant was explained by presence of a larger, highly responsive NKG2A(+) subset. Notably, NK cells from donors homozygous for a favorable allele encoding NKG2A mounted stronger cytokine responses when challenged with leukemic cells, and NK cells from AML patients with this genotype displayed higher accumulation of granzyme B during histamine dihydrochloride/IL-2 immunotherapy. Additionally, among AML patients, the NKG2A SNP defined a subset of patients with HLA-B-21 TT with a strikingly favorable outcome.Conclusions The study results imply that a dimorphism in the NKG2A gene is associated with enhanced NK cell effector function and improved outcome of IL-2-based immunotherapy in AML.
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| 3. |
- Kristenson, Linnea, 1991, et al.
(author)
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Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity
- 2024
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In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - 0027-8424 .- 1091-6490. ; 121:15
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Journal article (peer-reviewed)abstract
- Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-gamma signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7 - H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta- subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock - out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM - 3, restored the NK cell ability to eliminate TMEM30A- mutated cells. The key role of the TIM - 3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM - 3 in the realm of cancer immunotherapy.
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| 4. |
- Olausson, Josefin, 1983, et al.
(author)
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Optimization of cerebrospinal fluid microbial DNA metagenomic sequencing diagnostics
- 2022
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1
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Journal article (peer-reviewed)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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| 5. |
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| 6. |
- Muylaert, Isabella, 1965, et al.
(author)
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Replication and Recombination of Herpes Simplex Virus DNA
- 2011
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In: Journal of Biological Chemistry. - 0021-9258. ; 286:18
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Research review (peer-reviewed)abstract
- Replication of Herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of viral proteins: the UL30/42 DNA polymerase, the UL5,8,52 helicase-primase and the UL29 single-strand DNA binding protein ICP8. The replisome is loaded on origins of replication by an initiator protein OBP/UL9. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here we review significant new developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor and the single-strand DNA binding protein, the reconstitution of a functional replisome in vitro, the elucidation of the mechanism for activation of origins of DNA replication, the identification of cellular proteins actively involved in or responding to viral DNA replication and, finally, the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.
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| 7. |
- Nordén, Rickard, 1977, et al.
(author)
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Quantification of torque teno virus and Epstein-Barr virus is of limited value for predicting the net state of immunosuppression after lung transplantation
- 2018
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In: Open Forum Infectious Diseases. - : Oxford University Press (OUP). - 2328-8957. ; 5:4
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Journal article (peer-reviewed)abstract
- Background. Major hurdles for survival after lung transplantation are rejections and infectious complications. Adequate methods for monitoring immune suppression status are lacking. Here, we evaluated quantification of torque teno virus (TTV) and Epstein-Barr virus (EBV) as biomarkers for defining the net state of immunosuppression in lung-transplanted patients. Methods. This prospective single-center study included 98 patients followed for 2 years after transplantation. Bacterial infections, fungal infections, viral respiratory infections (VRTI), cytomegalovirus (CMV) viremia, and acute rejections, as well as TTV and EBV levels, were monitored. Results. The levels of torque teno virus DNA increased rapidly after transplantation, likely due to immunosuppressive treatment. A modest increase in levels of Epstein-Barr virus DNA was also observed after transplantation. There were no associations between either TTV or EBV and infectious events or acute rejection, respectively, during follow-up. When Tacrolimus was the main immunosuppressive treatment, TTV DNA levels were significantly elevated 6-24 months after transplantation as compared with Cyclosporine treatment. Conclusions. Although replication of TTV, but not EBV, appears to reflect the functionality of the immune system, depending on the type of immunosuppressive treatment, quantification of TTV or EBV as biomarkers has limited potential for defining the net state of immune suppression.
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| 8. |
- Norder, Helene, et al.
(author)
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High Frequency of Either Altered Pre-Core StartCodon or Weakened Kozak Sequence in the CorePromoter Region in Hepatitis B Virus A1 Strainsfrom Rwanda.
- 2019
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In: Genes. - : MDPI AG. - 2073-4425. ; 10:3
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Journal article (peer-reviewed)abstract
- Hepatitis B virus (HBV) is endemic in Rwanda and is a major etiologic agent for chronic liver disease in the country. In a previous analysis of HBV strains from Rwanda, the S genes of most strains segregated into one single clade of subgenotype, A1. More than half (55%) of the anti-HBe positive individuals were viremic. In this study, 23 complete HBV genomes and the core promoter region (CP) from 18 additional strains were sequenced. Phylogenetic analysis of complete genomes confirmed that most Rwandan strain formed a single unique clade, within subgenotype A1. Strains from 17 of 22 (77%) anti-HBe positive HBV carriers had either mutated the precore start codon (9 strains with either CUG, ACG, UUG, or AAG) or mutations in the Kozak sequence preceding the pre-core start codon (8 strains). These mutually exclusive mutations were also identified in subgenotypes A1 (70/266; 26%), A2 (12/255; 5%), and A3 (26/49; 53%) sequences from the GenBank. The results showed that previous, rarely described HBV variants, expressing little or no HBeAg, are selected in anti-HBe positive subgenotype Al carriers from Rwanda and that mutations reducing HBeAg synthesis might be unique for a particular HBV clade, not just for a specific genotype or subgenotype.
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| 9. |
- Nyström, Kristina, 1977, et al.
(author)
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Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus
- 2018
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In: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 92:19
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Journal article (peer-reviewed)abstract
- A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies. IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.
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| 10. |
- Nyström, Kristina, 1977, et al.
(author)
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Ribavirin: Pharmacology, multiple modes of action and possible future perspectives
- 2019
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In: Future Virology. - : Future Medicine Ltd. - 1746-0794 .- 1746-0808. ; 14, s. 153-160
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Research review (peer-reviewed)abstract
- © 2019 2019 Martin Lagging. Ribavirin is a unique guanosine analog with broad-spectrum activity against many RNA and DNA viruses. In addition to its mutational properties, ribavirin exerts extensive perturbation of cellular and viral gene expression. Furthermore, recent advances indicate that the impact of ribavirin on divergent cellular and viral pathways may be concentration dependent. This review aims at providing an overview of the pharmacology and multiple modes of action of ribavirin as well as pointing to possible novel future uses.
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