| 1. |
- Strunz, B., et al.
(author)
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Continuous human uterine NK cell differentiation in response to endometrial regeneration and pregnancy
- 2021
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In: Science Immunology. - : American Association for the Advancement of Science (AAAS). - 2470-9468. ; 6:56
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Journal article (peer-reviewed)abstract
- Immune cell differentiation is critical for adequate tissue-specific immune responses to occur. Here, we studied differentiation of human uterine natural killer cells (uNK cells). These cells reside in a tissue undergoing constant regeneration and represent the major leukocyte population at the maternal-fetal interface. However, their physiological response during the menstrual cycle and in pregnancy remains elusive. By surface proteome and transcriptome analysis as well as using humanized mice, we identify a differentiation pathway of uNK cells in vitro and in vivo with sequential acquisition of killer cell immunoglobulin-like receptors and CD39. uNK cell differentiation occurred continuously in response to the endometrial regeneration and was driven by interleukin-15. Differentiated uNK cells displayed reduced proliferative capacity and immunomodulatory function including enhanced angiogenic capacity. By studying human uterus transplantation and monozygotic twins, we found that the uNK cell niche could be replenished from circulation and that it was under genetic control. Together, our study uncovers a continuous differentiation pathway of human NK cells in the uterus that is coupled to profound functional changes in response to local tissue regeneration and pregnancy.
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| 2. |
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| 5. |
- Reinius, Henrik, et al.
(author)
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Real-time ventilation and perfusion distributions by electrical impedance tomography during one-lung ventilation with capnothorax
- 2015
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In: Acta Anaesthesiologica Scandinavica. - : Wiley. - 0001-5172 .- 1399-6576. ; 59:3, s. 354-368
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Journal article (peer-reviewed)abstract
- Background: Carbon dioxide insufflation into the pleural cavity, capnothorax, with one-lung ventilation (OLV) may entail respiratory and hemodynamic impairments. We investigated the online physiological effects of OLV/capnothorax by electrical impedance tomography (EIT) in a porcine model mimicking the clinical setting.Methods: Five anesthetized, muscle-relaxed piglets were subjected to first right and then left capnothorax with an intra-pleural pressure of 19cm H2O. The contra-lateral lung was mechanically ventilated with a double-lumen tube at positive end-expiratory pressure 5 and subsequently 10cm H2O. Regional lung perfusion and ventilation were assessed by EIT. Hemodynamics, cerebral tissue oxygenation and lung gas exchange were also measured.Results: During right-sided capnothorax, mixed venous oxygen saturation (P=0.018), as well as a tissue oxygenation index (P=0.038) decreased. There was also an increase in central venous pressure (P=0.006), and a decrease in mean arterial pressure (P=0.045) and cardiac output (P=0.017). During the left-sided capnothorax, the hemodynamic impairment was less than during the right side. EIT revealed that during the first period of OLV/capnothorax, no or very minor ventilation on the right side could be seen (33% vs. 97 +/- 3%, right vs. left, P=0.007), perfusion decreased in the non-ventilated and increased in the ventilated lung (18 +/- 2% vs. 82 +/- 2%, right vs. left, P=0.03). During the second OLV/capnothorax period, a similar distribution of perfusion was seen in the animals with successful separation (84 +/- 4% vs. 16 +/- 4%, right vs. left).Conclusion: EIT detected in real-time dynamic changes in pulmonary ventilation and perfusion distributions. OLV to the left lung with right-sided capnothorax caused a decrease in cardiac output, arterial oxygenation and mixed venous saturation.
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| 6. |
- Smyrlaki, I, et al.
(author)
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Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR
- 2020
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In: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 11:1, s. 4812-
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Journal article (peer-reviewed)abstract
- Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.
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| 9. |
- Vaid, Roshan, et al.
(author)
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Global loss of cellular m(6)A RNA methylation following infection with different SARS-CoV-2 variants
- 2023
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In: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 33:3, s. 299-313
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Journal article (peer-reviewed)abstract
- Insights into host-virus interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics. N-6-Methyladenosine modification (m(6)A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during stress response. Gene expression profiles observed postinfection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m(6)A readers and erasers. We found that infection with SARS-CoV-2 variants causes a loss of m(6)A in cellular RNAs, whereas m(6)A is detected abundantly in viral RNA. METTL3, the m(6)A methyltransferase, shows an unusual cytoplasmic localization postinfection. The B.1.351 variant has a less-pronounced effect on METTL3 localization and loss of m(6)A than did the B.1 and B.1.1.7 variants. We also observed a loss of m(6)A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m(6)A loss is characteristic of SARS-CoV-2-infected cells. Further, transcripts with m(6)A modification are preferentially down-regulated postinfection. Inhibition of the export protein XPO1 results in the restoration of METTL3 localization, recovery of m(6)A on cellular RNA, and increased mRNA expression. Stress granule formation, which is compromised by SARS-CoV-2 infection, is restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m(6)A-dependent manner.
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| 13. |
- Chen, G, et al.
(author)
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Single-cell analyses of X Chromosome inactivation dynamics and pluripotency during differentiation
- 2016
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In: Genome research. - : Cold Spring Harbor Laboratory. - 1549-5469 .- 1088-9051. ; 26:10, s. 1342-1354
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Journal article (peer-reviewed)abstract
- Pluripotency, differentiation, and X Chromosome inactivation (XCI) are key aspects of embryonic development. However, the underlying relationship and mechanisms among these processes remain unclear. Here, we systematically dissected these features along developmental progression using mouse embryonic stem cells (mESCs) and single-cell RNA sequencing with allelic resolution. We found that mESCs grown in a ground state 2i condition displayed transcriptomic profiles diffused from preimplantation mouse embryonic cells, whereas EpiStem cells closely resembled the post-implantation epiblast. Sex-related gene expression varied greatly across distinct developmental states. We also identified novel markers that were highly enriched in each developmental state. Moreover, we revealed that several novel pathways, including PluriNetWork and Focal Adhesion, were responsible for the delayed progression of female EpiStem cells. Importantly, we “digitalized” XCI progression using allelic expression of active and inactive X Chromosomes and surprisingly found that XCI states exhibited profound variability in each developmental state, including the 2i condition. XCI progression was not tightly synchronized with loss of pluripotency and increase of differentiation at the single-cell level, although these processes were globally correlated. In addition, highly expressed genes, including core pluripotency factors, were in general biallelically expressed. Taken together, our study sheds light on the dynamics of XCI progression and the asynchronicity between pluripotency, differentiation, and XCI.
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| 17. |
- Dyrdak, R., et al.
(author)
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Outbreak of enterovirus D68 of the new B3 lineage in Stockholm, Sweden, August to September 2016
- 2016
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In: Eurosurveillance. - 1025-496X .- 1560-7917. ; 21:46, s. 5-10
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Journal article (peer-reviewed)abstract
- We report an enterovirus D68 ( EV-D68) outbreak in Stockholm Sweden in 2016. Between 22 August and 25 September EV-D68 was detected in 74/ 495 respiratory samples analysed at the Karolinska University Hospital. During the peak week, 30/ 91 ( 33%) samples were EV-D68 positive. Viral protein ( VP) P4/ VP2 sequencing revealed that cases were caused by B3 lineage strains. Forty-four ( 59%) EV-D68-positive patients were children aged = 5 years. Ten patients had severe respiratory or neurological symptoms and one died. We report an outbreak of enterovirus D68 ( EV-D68) infections in Stockholm, Sweden in late August and September of 2016 caused by the newly described B3 lineage [1].
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| 18. |
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| 19. |
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| 20. |
- Johnsson, P, et al.
(author)
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Transcriptional kinetics and molecular functions of long noncoding RNAs
- 2022
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In: Nature genetics. - : Springer Science and Business Media LLC. - 1546-1718 .- 1061-4036. ; 54:43, s. 306-
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Journal article (peer-reviewed)abstract
- An increasing number of long noncoding RNAs (lncRNAs) have experimentally confirmed functions, yet little is known about their transcriptional dynamics and it is challenging to determine their regulatory effects. Here, we used allele-sensitive single-cell RNA sequencing to demonstrate that, compared to messenger RNAs, lncRNAs have twice as long duration between two transcriptional bursts. Additionally, we observed increased cell-to-cell variability in lncRNA expression due to lower frequency bursting producing larger numbers of RNA molecules. Exploiting heterogeneity in asynchronously growing cells, we identified and experimentally validated lncRNAs with cell state-specific functions involved in cell cycle progression and apoptosis. Finally, we identified cis-functioning lncRNAs and showed that knockdown of these lncRNAs modulated the nearby protein-coding gene’s transcriptional burst frequency or size. In summary, we identified distinct transcriptional regulation of lncRNAs and demonstrated a role for lncRNAs in the regulation of mRNA transcriptional bursting.
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| 21. |
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| 22. |
- Larsson, AJM, et al.
(author)
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Transcriptional bursts explain autosomal random monoallelic expression and affect allelic imbalance
- 2021
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In: PLoS computational biology. - : Public Library of Science (PLoS). - 1553-7358. ; 17:3, s. e1008772-
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Journal article (peer-reviewed)abstract
- Transcriptional bursts render substantial biological noise in cellular transcriptomes. Here, we investigated the theoretical extent of allelic expression resulting from transcriptional bursting and how it compared to the amount biallelic, monoallelic and allele-biased expression observed in single-cell RNA-sequencing (scRNA-seq) data. We found that transcriptional bursting can explain the allelic expression patterns observed in single cells, including the frequent observations of autosomal monoallelic gene expression. Importantly, we identified that the burst frequency largely determined the fraction of cells with monoallelic expression, whereas the burst size had little effect on monoallelic observations. The high consistency between the bursting model predictions and scRNA-seq observations made it possible to assess the heterogeneity of a group of cells as their deviation in allelic observations from the expected. Finally, both burst frequency and size contributed to allelic imbalance observations and reinforced that studies of allelic imbalance can be confounded from the inherent noise in transcriptional bursting. Altogether, we demonstrate that allele-level transcriptional bursting renders widespread, although predictable, amounts of monoallelic and biallelic expression in single cells and cell populations.
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| 23. |
- Lentini, A, et al.
(author)
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Elastic dosage compensation by X-chromosome upregulation
- 2022
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In: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1, s. 1854-
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Journal article (peer-reviewed)abstract
- X-chromosome inactivation and X-upregulation are the fundamental modes of chromosome-wide gene regulation that collectively achieve dosage compensation in mammals, but the regulatory link between the two remains elusive and the X-upregulation dynamics are unknown. Here, we use allele-resolved single-cell RNA-seq combined with chromatin accessibility profiling and finely dissect their separate effects on RNA levels during mouse development. Surprisingly, we uncover that X-upregulation elastically tunes expression dosage in a sex- and lineage-specific manner, and moreover along varying degrees of X-inactivation progression. Male blastomeres achieve X-upregulation upon zygotic genome activation while females experience two distinct waves of upregulation, upon imprinted and random X-inactivation; and ablation of Xist impedes female X-upregulation. Female cells carrying two active X chromosomes lack upregulation, yet their collective RNA output exceeds that of a single hyperactive allele. Importantly, this conflicts the conventional dosage compensation model in which naïve female cells are initially subject to biallelic X-upregulation followed by X-inactivation of one allele to correct the X dosage. Together, our study provides key insights to the chain of events of dosage compensation, explaining how transcript copy numbers can remain remarkably stable across developmental windows wherein severe dose imbalance would otherwise be experienced by the cell.
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| 24. |
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| 26. |
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| 27. |
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| 28. |
- Picelli, S, et al.
(author)
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Tn5 transposase and tagmentation procedures for massively scaled sequencing projects
- 2014
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In: Genome research. - : Cold Spring Harbor Laboratory. - 1549-5469 .- 1088-9051. ; 24:12, s. 2033-2040
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Journal article (peer-reviewed)abstract
- Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large-scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from subpicogram amounts of cDNA. The comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, because naked Tn5 can be annealed to any oligonucleotide of choice, for example, molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enable innovation in sequencing-based applications.
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| 29. |
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| 32. |
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| 33. |
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| 34. |
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| 35. |
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| 36. |
- Vaid, R, et al.
(author)
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Global loss of cellular m6A RNA methylation following infection with different SARS-CoV-2 variants
- 2023
-
In: Genome research. - : Cold Spring Harbor Laboratory. - 1549-5469 .- 1088-9051. ; 33:3, s. 299-313
-
Journal article (peer-reviewed)abstract
- Insights into host–virus interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics.N6-Methyladenosine modification (m6A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during stress response. Gene expression profiles observed postinfection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m6A readers and erasers. We found that infection with SARS-CoV-2 variants causes a loss of m6A in cellular RNAs, whereas m6A is detected abundantly in viral RNA. METTL3, the m6A methyltransferase, shows an unusual cytoplasmic localization postinfection. The B.1.351 variant has a less-pronounced effect on METTL3 localization and loss of m6A than did the B.1 and B.1.1.7 variants. We also observed a loss of m6A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m6A loss is characteristic of SARS-CoV-2-infected cells. Further, transcripts with m6A modification are preferentially down-regulated postinfection. Inhibition of the export protein XPO1 results in the restoration of METTL3 localization, recovery of m6A on cellular RNA, and increased mRNA expression. Stress granule formation, which is compromised by SARS-CoV-2 infection, is restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m6A-dependent manner.
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| 37. |
- Vertesy, A, et al.
(author)
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Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells
- 2018
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In: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9:1, s. 1873-
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Journal article (peer-reviewed)abstract
- In contrast to mouse, human female germ cells develop asynchronously. Germ cells transition to meiosis, erase genomic imprints, and reactivate the X chromosome. It is unknown if these events all appear asynchronously, and how they relate to each other. Here we combine exome sequencing of human fetal and maternal tissues with single-cell RNA-sequencing of five donors. We reconstruct full parental haplotypes and quantify changes in parental allele-specific expression, genome-wide. First we distinguish primordial germ cells (PGC), pre-meiotic, and meiotic transcriptional stages. Next we demonstrate that germ cells from various stages monoallelically express imprinted genes and confirm this by methylation patterns. Finally, we show that roughly 30% of the PGCs are still reactivating their inactive X chromosome and that this is related to transcriptional stage rather than fetal age. Altogether, we uncover the complexity and cell-to-cell heterogeneity of transcriptional and epigenetic remodeling in female human germ cells.
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