| 1. |
- Kristenson, Linnea, 1991, et al.
(författare)
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Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity
- 2024
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Ingår i: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - 0027-8424 .- 1091-6490. ; 121:15
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Holmqvist, Isak, et al.
(författare)
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FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
- 2021
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Ingår i: Rna. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 27:10, s. 1127-1139
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Tidskriftsartikel (refereegranskat)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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| 3. |
- Hussein, Brwa Ali, 1984, et al.
(författare)
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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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Ingår i: JOURNAL FOR IMMUNOTHERAPY OF CANCER. - 2051-1426. ; 11:8
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Norder, Helene, et al.
(författare)
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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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Ingår i: Genes. - : MDPI AG. - 2073-4425. ; 10:3
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Tidskriftsartikel (refereegranskat)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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| 5. |
- 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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| 6. |
- 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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| 7. |
- Yan, R. X., et al.
(författare)
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Prediction of zinc-binding sites using multiple sequence profiles and machine learning methods
- 2019
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Ingår i: Molecular Omics. - : Royal Society of Chemistry (RSC). - 2515-4184. ; 15:3, s. 205-215
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Tidskriftsartikel (refereegranskat)abstract
- The zinc (Zn2+) cofactor has been proven to be involved in numerous biological mechanisms and the zinc-binding site is recognized as one of the most important post-translation modifications in proteins. Therefore, accurate knowledge of zinc ions in protein structures can provide potential clues for elucidation of protein folding and functions. However, determining zinc-binding residues by experimental means is usually lab-intensive and associated with high cost in most cases. In this context, the development of computational tools for identifying zinc-binding sites is highly desired, especially in the current post-genomic era. In this work, we developed a novel zinc-binding site prediction method by combining several intensively-trained machine learning models. To establish an accurate and generative method, we downloaded all zinc-binding proteins from the Protein Data Bank and prepared a non-redundant dataset. Meanwhile, a well-prepared dataset by other groups was also used. Then, effective and complementary features were extracted from sequences and three-dimensional structures of these proteins. Moreover, several well-designed machine learning models were intensively trained to construct accurate models. To assess the performance, the obtained predictors were stringently benchmarked using the diverse zinc-binding sites. Furthermore, several state-of-the-art in silico methods developed specifically for zinc-binding sites were also evaluated and compared. The results confirmed that our method is very competitive in real world applications and could become a complementary tool to wet lab experiments. To facilitate research in the community, a web server and stand-alone program implementing our method were constructed and are publicly available at http:// bioinformatics. fzu. edu. cn/ znMachine. html. The downloadable program of our method can be easily used for the high-throughput screening of potential zinc-binding sites across proteomes.
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| 8. |
- Ziegler, P., et al.
(författare)
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A primary nasopharyngeal three-dimensional air-liquid interface cell culture model of the pseudostratified epithelium reveals differential donor- and cell type-specific susceptibility to Epstein-Barr virus infection
- 2021
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Ingår i: Plos Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 17:4
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Tidskriftsartikel (refereegranskat)abstract
- Epstein-Barr virus (EBV) is a ubiquitous gamma-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, alpha-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (similar to 0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC. Author summary It has been known for over 50 years that EBV latent infection is associated with NPC. Despite many advances from studies in 2-dimensional cell culture, many aspects of EBV molecular pathogenesis in the nasopharynx remain undefined because the cell types and the biology of the nasopharyngeal epithelium can only be faithfully captured in 3-dimensional cell culture. In the stratified epithelium, cellular differentiation triggers lytic infection but it is not clear to what degree the pseudostratified epithelium is involved. The pseudostratified epithelium is abundant in the lateral wall where the lymphoid-rich fossa of Rosenmuller is located and is a site where NPC tumors most often arises. While the oral epithelium is a site of EBV replication, whether the nasopharyngeal epithelium is a major source of EBV shedding in the nasopharynx is not well defined. Here, we present a 3-dimensional organoid model of the nasopharyngeal pseudostratified epithelium showing that such cells can be infected with EBV in some donor cultures, with examples of both latent and lytic infection. We propose that the cell types of the pseudostratified epithelium should be considered a component of EBV pathogenesis in the nasopharynx and that the difference in donor susceptibility and latent/lytic infection could influence EBV's fitness in the nasopharynx.
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