| 1. |
- Doimo, Mara, et al.
(author)
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Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells.
- 2023
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In: Nucleic acids research. - : Oxford University Press. - 1362-4962 .- 0305-1048. ; 51:14, s. 7392-7408
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Journal article (peer-reviewed)abstract
- Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.
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| 2. |
- Abrahamsson, Sanna, et al.
(author)
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Comparison of online learning designs during the COVID-19 pandemic within bioinformatics courses in higher education
- 2021
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In: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1460-2059. ; 37:Suppl 1
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Journal article (peer-reviewed)abstract
- Motivation: Due to the worldwide COVID-19 pandemic, new strategies had to be adopted to move from classroom-based education to online education, in a very short time. The lack of time to set up these strategies, hindered a proper design of online instructions and delivery of knowledge. Bioinformatics-related training and other onsite practical education, tend to rely on extensive practice, where students and instructors have a face-to-face interaction to improve the learning outcome. For these courses to maintain their high quality when adapted as online courses, different designs need to be tested and the students' perceptions need to be heard. Results: This study focuses on short bioinformatics-related courses for graduate students at the University of Gothenburg, Sweden, which were originally developed for onsite training. Once adapted as online courses, several modifications in their design were tested to obtain the best fitting learning strategy for the students. To improve the online learning experience, we propose a combination of: (i) short synchronized sessions, (ii) extended time for own and group practical work, (iii) recorded live lectures and (iv) increased opportunities for feedback in several formats.
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| 3. |
- Abrahamsson, Sanna, et al.
(author)
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PΨFinder: a practical tool for the identification and visualization of novel pseudogenes in DNA sequencing data
- 2022
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In: BMC Bioinformatics. - : Springer Science and Business Media LLC. - 1471-2105. ; 23
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Journal article (peer-reviewed)abstract
- Background: Processed pseudogenes (PΨgs) are disabled gene copies that are transcribed and may affect expression of paralogous genes. Moreover, their insertion in the genome can disrupt the structure or the regulatory region of a gene, affecting its expression level. These events have been identified as occurring mutations during cancer development, thus being able to identify PΨgs and their location will improve their impact on diagnostic testing, not only in cancer but also in inherited disorders. Results: We have implemented PΨFinder (P-psy-finder), a tool that identifies PΨgs, annotates known ones and predicts their insertion site(s) in the genome. The tool screens alignment files and provides user-friendly summary reports and visualizations. To demonstrate its applicability, we scanned 218 DNA samples from patients screened for hereditary colorectal cancer. We detected 423 PΨgs distributed in 96% of the samples, comprising 7 different parent genes. Among these, we confirmed the well-known insertion site of the SMAD4-PΨg within the last intron of the SCAI gene in one sample. While for the ubiquitous CBX3-PΨg, present in 82.6% of the samples, we found it reversed inserted in the second intron of the C15ORF57 gene. Conclusions: PΨFinder is a tool that can automatically identify novel PΨgs from DNA sequencing data and determine their location in the genome with high sensitivity (95.92%). It generates high quality figures and tables that facilitate the interpretation of the results and can guide the experimental validation. PΨFinder is a complementary analysis to any mutational screening in the identification of disease-causing mutations within cancer and other diseases.
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| 4. |
- Björk, Thomas, et al.
(author)
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Alpha 1-antichymotrypsin production in PSA-producing cells is common in prostate cancer but rare in benign prostatic hyperplasia
- 1994
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In: Urology. - : Elsevier BV. - 1527-9995 .- 0090-4295. ; 43:4, s. 427-434
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Journal article (peer-reviewed)abstract
- OBJECTIVE. To investigate the distribution and production of alpha 1-antichymotrypsin (ACT) and prostate-specific antigen (PSA) in benign hyperplastic and malignant prostatic tissue, respectively. METHODS. Using monoclonal anti-ACT and anti-PSA IgGs for immunocytochemistry and alkaline phosphatase conjugated 30-mer oligodeoxynucleotide probes for nonradioactive in situ hybridization, tissue specimens were studied from 15 patients with benign prostatic hyperplasia after transurethral resection of the prostate (TURP) and from 9 patients with bladder cancer who underwent cystoprostatectomy. Cancer specimens were from 23 TURP patients and from ultrasound guided core biopsies in 14 patients. Prostate tumors were graded according to the Gleason system. RESULTS. We found no or only occasional small foci of immunostaining for ACT, and no ACT transcripts in the PSA-producing epithelium in areas with benign nodular hyperplasia. By contrast, a high proportion of cells expressed both ACT and PSA in prostate cancers with low Gleason score, as detected by immunocytochemistry and in situ hybridization. Poorly differentiated tumor cells manifested greater variation in immunostaining for both ACT and PSA. As compared to tumors of low Gleason score, high-score tumors less frequently manifested immunostaining for ACT than for PSA, and less frequently generated hybridization signals for both PSA and ACT transcripts. CONCLUSIONS. A significantly higher proportion of serum PSA has been reported to be complexed to ACT in patients with prostate cancer than in patients with benign prostatic hyperplasia. The presently reported lack of ACT production in PSA-containing BPH nodules may contribute to this by making conditions less optimal for complex formation between PSA and ACT. As opposed to this, production of both ACT and PSA in prostate cancers may enhance the complex formation between PSA and ACT.
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| 5. |
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| 6. |
- Lander, Sanna, et al.
(author)
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Controlling the rate of posolyte degradation in all-quinone aqueous organic redox flow batteries by sulfonated nanocellulose based membranes: The role of crossover and Michael addition
- 2024
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In: Journal of Energy Storage. - : Elsevier BV. - 2352-152X .- 2352-1538. ; 83
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Journal article (peer-reviewed)abstract
- Aqueous organic redox flow battery (AORFB) is a technological route towards the large-scale sustainable energy storage. However, several factors need to be controlled to maintain the AORFB performance. Prevention of posolyte and negolyte cross-contamination in asymmetric AORFBs, one of the main causes of capacity decay, relies on their membranes' ability to prevent migration of the redox-active species between the two electrolytes. The barrier properties are often traded for a reduction in ionic conductivity which is crucial to enable the device operation. Another factor greatly affecting quinone-based AORFBs is the Michael addition reaction (MAR) on the charged posolyte, quinone, which has been identified as a major reason for all-quinone AORFBs performance deterioration. Herein, we investigate deterioration scenarios of an all-quinone AORFB using both experimental and computational methods. The study includes a series of membranes based on sulfonated cellulose nanofibrils and different membrane modifications. The layer-by-layer (LbL) surface modifications, i.e. the incorporation of inorganic materials and the reduction of the pore size of the sulfonated cellulose membranes, were all viable routes to reduce the passive diffusion permeability of membranes which correlated to an increased cycling stability of the battery. The kinetics of MAR on quinone was detected using NMR and its impact on the performance fading was modeled computationally. The localization of MAR close to the membrane, which can be assigned to the surface reactivity, affects the diffusion of MAR reagent and the deterioration dynamics of the present all-quinone AORFB.
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| 7. |
- Lawenius, Lina, et al.
(author)
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Transplantation of gut microbiota from old mice into young healthy mice reduces lean mass but not bone mass
- 2023
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In: Gut Microbes. - 1949-0976. ; 15:1
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Journal article (peer-reviewed)abstract
- Aging is associated with low bone and lean mass as well as alterations in the gut microbiota (GM). In this study, we determined whether the reduced bone mass and relative lean mass observed in old mice could be transferred to healthy young mice by GM transplantation (GMT). GM from old (21-month-old) and young adult (5-month-old) donors was used to colonize germ-free (GF) mice in three separate studies involving still growing 5- or 11-week-old recipients and 17-week-old recipients with minimal bone growth. The GM of the recipient mice was similar to that of the donors, demonstrating successful GMT. GM from old mice did not have statistically significant effects on bone mass or bone strength, but significantly reduced the lean mass percentage of still growing recipient mice when compared with recipients of GM from young adult mice. The levels of propionate in the cecum of mice receiving old donor GM were significantly lower than those in mice receiving young adult donor GM. Bacteroides ovatus was enriched in the microbiota of recipient mice harboring GM from young adult donors. The presence of B. ovatus was not only significantly associated with high lean mass percentage in mice, but also with lean mass adjusted for fat mass in the large human HUNT cohort. In conclusion, GM from old mice reduces lean mass percentage but not bone mass in young, healthy, still growing recipient mice. Future studies are warranted to determine whether GM from young mice improves the musculoskeletal phenotype of frail elderly recipient mice.
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| 8. |
- Martin, William P., et al.
(author)
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Dietary restriction and medical therapy drive PPARα-regulated improvements in early diabetic kidney disease in male rats
- 2022
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In: Clinical science (London, England : 1979). - 1470-8736. ; 136:21, s. 1485-1511
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Journal article (peer-reviewed)abstract
- The attenuation of diabetic kidney disease (DKD) by metabolic surgery is enhanced by pharmacotherapy promoting renal fatty acid oxidation (FAO). Using the Zucker Diabetic Fatty and Zucker Diabetic Sprague Dawley rat models of DKD, we conducted studies to determine if these effects could be replicated with a non-invasive bariatric mimetic intervention. Metabolic control and renal injury were compared in rats undergoing a dietary restriction plus medical therapy protocol (DMT; fenofibrate, liraglutide, metformin, ramipril, and rosuvastatin) and ad libitum-fed controls. The global renal cortical transcriptome and urinary 1H-NMR metabolomic profiles were also compared. Kidney cell type-specific and medication-specific transcriptomic responses were explored through in silico deconvolution. Transcriptomic and metabolomic correlates of improvements in kidney structure were defined using a molecular morphometric approach. The DMT protocol led to ∼20% weight loss, normalized metabolic parameters and was associated with reductions in indices of glomerular and proximal tubular injury. The transcriptomic response to DMT was dominated by changes in fenofibrate- and peroxisome proliferator-activated receptor-α (PPARα)-governed peroxisomal and mitochondrial FAO transcripts localizing to the proximal tubule. DMT induced urinary excretion of PPARα-regulated metabolites involved in nicotinamide metabolism and reversed DKD-associated changes in the urinary excretion of tricarboxylic acid (TCA) cycle intermediates. FAO transcripts and urinary nicotinamide and TCA cycle metabolites were moderately to strongly correlated with improvements in glomerular and proximal tubular injury. Weight loss plus pharmacological PPARα agonism is a promising means of attenuating DKD.
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| 9. |
- Martin, W. P., et al.
(author)
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Medications Activating Tubular Fatty Acid Oxidation Enhance the Protective Effects of Roux-en-Y Gastric Bypass Surgery in a Rat Model of Early Diabetic Kidney Disease
- 2022
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In: Frontiers in Endocrinology. - : Frontiers Media SA. - 1664-2392. ; 12
-
Journal article (peer-reviewed)abstract
- Background: Roux-en-Y gastric bypass surgery (RYGB) improves biochemical and histological parameters of diabetic kidney disease (DKD). Targeted adjunct medical therapy may enhance renoprotection following RYGB. Methods: The effects of RYGB and RYGB plus fenofibrate, metformin, ramipril, and rosuvastatin (RYGB-FMRR) on metabolic control and histological and ultrastructural indices of glomerular and proximal tubular injury were compared in the Zucker Diabetic Sprague Dawley (ZDSD) rat model of DKD. Renal cortical transcriptomic (RNA-sequencing) and urinary metabolomic (1H-NMR spectroscopy) responses were profiled and integrated. Transcripts were assigned to kidney cell types through in silico deconvolution in kidney single-nucleus RNA-sequencing and microdissected tubular epithelial cell proteomics datasets. Medication-specific transcriptomic responses following RYGB-FMRR were explored using a network pharmacology approach. Omic correlates of improvements in structural and ultrastructural indices of renal injury were defined using a molecular morphometric approach. Results: RYGB-FMRR was superior to RYGB alone with respect to metabolic control, albuminuria, and histological and ultrastructural indices of glomerular injury. RYGB-FMRR reversed DKD-associated changes in mitochondrial morphology in the proximal tubule to a greater extent than RYGB. Attenuation of transcriptomic pathway level activation of pro-fibrotic responses was greater after RYGB-FMRR than RYGB. Fenofibrate was found to be the principal medication effector of gene expression changes following RYGB-FMRR, which led to the transcriptional induction of PPARα-regulated genes that are predominantly expressed in the proximal tubule and which regulate peroxisomal and mitochondrial fatty acid oxidation (FAO). After omics integration, expression of these FAO transcripts positively correlated with urinary levels of PPARα-regulated nicotinamide metabolites and negatively correlated with urinary tricarboxylic acid (TCA) cycle intermediates. Changes in FAO transcripts and nicotinamide and TCA cycle metabolites following RYGB-FMRR correlated strongly with improvements in glomerular and proximal tubular injury. Conclusions: Integrative multi-omic analyses point to PPARα-stimulated FAO in the proximal tubule as a dominant effector of treatment response to combined surgical and medical therapy in experimental DKD. Synergism between RYGB and pharmacological stimulation of FAO represents a promising combinatorial approach to the treatment of DKD in the setting of obesity. Copyright © 2022 Martin, Chuah, Abdelaal, Pedersen, Malmodin, Abrahamsson, Hutter, Godson, Brennan, Fändriks, le Roux and Docherty.
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| 10. |
- 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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| 11. |
- Ringlander, Johan, et al.
(author)
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Deep sequencing of liver explant transcriptomes reveals extensive expression from integrated hepatitis B virus DNA
- 2020
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In: Journal of Viral Hepatitis. - : Wiley. - 1352-0504 .- 1365-2893. ; 27:11, s. 1162-1170
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Journal article (peer-reviewed)abstract
- Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Integration of HBV DNA into the human genome may contribute to oncogenesis and to the production of the hepatitis B surface antigen (HBsAg). Whether integrations contribute to HBsAg levels in the blood is poorly known. Here, we characterize the HBV RNA profile of HBV integrations in liver tissue in patients with chronic HBV infection, with or without concurrent hepatitis D infection, by transcriptome deep sequencing. Transcriptomes were determined in liver tissue by deep sequencing providing 200 million reads per sample. Integration points were identified using a bioinformatic pipeline. Explanted liver tissue from five patients with end-stage liver disease caused by HBV or HBV/HDV was studied along with publicly available transcriptomes from 21 patients. Almost all HBV RNA profiles were devoid of reads in the core and the 3 ' redundancy (nt 1830-1927) regions, and contained a large number of chimeric viral/human reads. Hence, HBV transcripts from integrated HBV DNA rather than from covalently closed circular HBV DNA (cccDNA) predominated in late-stage HBV infection, in particular in cases with hepatitis D virus co-infection. The findings support the suggestion that integrated HBV DNA can be a significant source of HBsAg in humans.
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| 12. |
- Ringlander, Johan, et al.
(author)
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Enrichment Reveals Extensive Integration of Hepatitis B Virus DNA in Hepatitis Delta Virus-Infected Patients
- 2024
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In: JOURNAL OF INFECTIOUS DISEASES. - 0022-1899 .- 1537-6613.
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Journal article (peer-reviewed)abstract
- Background. Hepatitis B virus (HBV) DNA may become integrated into the human genome of infected human hepatocytes. Expression of integrations can produce the surface antigen (HBsAg) that is required for synthesis of hepatitis D virus (HDV) particles and the abundant subviral particles in the blood of HBV- and HDV-infected subjects. Knowledge about the extent and variation of HBV integrations and impact on chronic HDV is still limited. Methods. We investigated 50 pieces of liver explant tissue from 5 patients with hepatitis D-induced cirrhosis, using a deep-sequencing strategy targeting HBV RNA. Results. We found that integrations were abundant and highly expressed, with large variation in the number of integration-derived (HBV/human chimeric) reads, both between and within patients. The median number of unique integrations for each patient correlated with serum levels of HBsAg. However, most of the HBV reads represented a few predominant integrations. Conclusions. The results suggest that HBV DNA integrates in a large proportion of hepatocytes, and that the HBsAg output from these integrations vary >100-fold depending on clone size and expression rate. A small proportion of the integrations seems to determine the serum levels of HBsAg and HDV RNA in HBV/HDV coinfected patients with liver cirrhosis.
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| 13. |
- Scherer, D., et al.
(author)
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RNA Sequencing of Hepatobiliary Cancer Cell Lines: Data and Applications to Mutational and Transcriptomic Profiling.
- 2020
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In: Cancers. - : MDPI AG. - 2072-6694. ; 12:9
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Journal article (peer-reviewed)abstract
- Cancer cell lines allow the identification of clinically relevant alterations and the prediction of drug response. However, sequencing data for hepatobiliary cancer cell lines in general, and particularly gallbladder cancer (GBC), are sparse. Here, we apply RNA sequencing to characterize 10 GBC, eight hepatocellular carcinoma, and five cholangiocarcinoma (CCA) cell lines. RNA extraction, quality control, library preparation, sequencing, and pre-processing of sequencing data were implemented using state-of-the-art techniques. Public data from the MSK-IMPACT database and a large cohort of Japanese biliary tract cancer patients were used to illustrate the usage of the released data. The total number of exonic mutations varied from 7207 for the cell line NOZ to 9760 for HuCCT1. Researchers planning experiments that require TP53 mutations could use the cell lines NOZ, OCUG-1, SNU308, or YoMi. Mz-Cha-1 showed mutations in ATM, SNU308 presented SMAD4 mutations, and the only investigated cell line that showed ARID1A mutations was GB-d1. SNU478 was the cell line with the global gene expression pattern most similar to GBC, intrahepatic CCA, and extrahepatic CCA. EGFR, KMT2D, and KMT2C generally presented a higher expression in the investigated cell lines than in Japanese primary GBC tumors. We provide the scientific community with detailed mutation and gene expression data, together with three showcase applications, with the aim of facilitating the design of future in vitro cell culture assays for research on hepatobiliary cancer.
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| 14. |
- Tierens, Anne, et al.
(author)
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Residual disease detected by flow cytometry is an independent predictor of survival in childhood acute myeloid leukaemia; results of the NOPHO-AML 2004 study
- 2016
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In: British Journal of Haematology. - : Wiley. - 0007-1048 .- 1365-2141. ; 174:4, s. 600-609
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Journal article (peer-reviewed)abstract
- Early response after induction is a prognostic factor for disease outcome in childhood acute myeloid leukaemia (AML). Residual disease (RD) detection by multiparameter flow cytometry (MFC) was performed at day 15 and before consolidation therapy in 101 patients enrolled in the Nordic Society of Paediatric Haemato-Oncology AML 2004 study. A multicentre laboratory approach to RD analysis was used. Event-free survival (EFS) and overall survival (OS) was significantly different in patients with and without RD at both time points, using a 0.1% RD cut-off level. RD-negative and -positive patients after first induction showed a 5-year EFS of 65.7% and 22.7%, respectively (P < 0.001) and an OS of 77.6% (P = 0.025) and 51.8%. RD-negative and -positive patients at start of consolidation therapy had a 5-year EFS of 57.7% and 11.7%, respectively (P < 0001) and an OS of 786% and 2811%) (P < 0001). In multivariate analysis only RD was significantly correlated with survival. RD before consolidation therapy was the strongest independent prognostic factor for EFS [hazard ratio (HR): 5.0; 95% confidence interval (CI): 1.9-133] and OS (HR: 7.0; 95% CI: 20-245). In conclusion, RD before consolidation therapy identifies patients at high risk of relapse in need of intensified treatment. In addition, RD detection can be performed in a multicentre setting and can be implemented in future trials.
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| 15. |
- Torinsson Naluai, Åsa, 1968, et al.
(author)
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Transcriptomics unravels molecular changes associated with cilia and COVID-19 in chronic rhinosinusitis with nasal polyps
- 2023
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13:1
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Journal article (peer-reviewed)abstract
- Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common upper respiratory tract complication where the pathogenesis is largely unknown. Herein, we investigated the transcriptome profile in nasal mucosa biopsies of CRSwNP patients and healthy individuals. We further integrated the transcriptomics data with genes located in chromosomal regions containing genome-wide significant gene variants for COVID-19. Among the most significantly upregulated genes in polyp mucosa were CCL18, CLEC4G, CCL13 and SLC9A3. Pathways involving "Ciliated epithelial cells" were the most differentially expressed molecular pathways when polyp mucosa and non-polyp mucosa from the same patient was compared. Natural killer T-cell (NKT) and viral pathways were the most statistically significant pathways in the mucosa of CRSwNP patients compared with those of healthy control individuals. Upregulated genes in polyp mucosa, located within the genome-wide associated regions of COVID-19, included LZTFL1, CCR9, SLC6A20, IFNAR1, IFNAR2 and IL10RB. Interestingly, the second most over-expressed gene in our study, CLEC4G, has been shown to bind directly to SARS-CoV-2 spike's N-terminal domain and mediate its entry and infection. Our results on altered expression of genes related to cilia and viruses point to the de-regulation of viral defenses in CRSwNP patients, and may give clues to future intervention strategies.
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| 16. |
- Wikström, Tove, et al.
(author)
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Microbial and human transcriptome in vaginal fluid at midgestation: Association with spontaneous preterm delivery.
- 2022
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In: Clinical and translational medicine. - : Wiley. - 2001-1326. ; 12:9
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Journal article (peer-reviewed)abstract
- Intrauterine infection and inflammation caused by microbial transfer from the vagina are believed to be important factors causing spontaneous preterm delivery (PTD). Multiple studies have examined the relationship between the cervicovaginal microbiome and spontaneous PTD with divergent results. Most studies have applied a DNA-based assessment, providing information on the microbial composition but not transcriptional activity. A transcriptomic approach was applied to investigate differences in the active vaginal microbiome and human transcriptome at midgestation between women delivering spontaneously preterm versus those delivering at term.Vaginal swabs were collected in women with a singleton pregnancy at 18 + 0 to 20 + 6 gestational weeks. For each case of spontaneous PTD (delivery<37 + 0weeks) two term controls were randomized (39 + 0 to 40 + 6weeks). Vaginal specimens were subject to sequencing of both human and microbial RNA. Microbial reads were taxonomically classified using Kraken2 and RefSeq as a reference. Statistical analyses were performed using DESeq2. GSEA and HUMAnN3 were used for pathway analyses.We found 17 human genes to be differentially expressed (false discovery rate, FDR<0.05) in the preterm group (n =48) compared to the term group (n =96). Gene expression of kallikrein-2 (KLK2), KLK3 and four isoforms of metallothioneins 1 (MT1s) was higher in the preterm group (FDR<0.05). We found 11 individual bacterial species to be differentially expressed (FDR<0.05), most with a low occurrence. No statistically significant differences in bacterial load, diversity or microbial community state types were found between the groups.In our mainly white population, primarily bacterial species of low occurrence were differentially expressed at midgestation in women who delivered preterm versus at term. However, the expression of specific human transcripts including KLK2, KLK3 and several isoforms of MT1s was higher in preterm cases. This is of interest, because these genes may be involved in critical inflammatory pathways associated with spontaneous PTD.
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| 17. |
- Ziegler, P., et al.
(author)
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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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In: Plos Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 17:4
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Journal article (peer-reviewed)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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