| 1. |
- Bacos, Karl, et al.
(author)
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Type 2 diabetes candidate genes, including PAX5, cause impaired insulin secretion in human pancreatic islets
- 2023
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In: The Journal of clinical investigation. - 0021-9738 .- 1558-8238. ; 133:4
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Journal article (peer-reviewed)abstract
- Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic β-cells. To identify candidates contributing to T2D pathophysiology, we studied human pancreatic islets from ~300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified islet expression changes may predispose to diabetes, as they associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human β-cells based on single-cell RNA-sequencing data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D-SNPs. Mouse knock-out strains demonstrated that T2D-associated candidates regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing β-cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we identified molecular alterations in human pancreatic islets contributing to β-cell dysfunction in T2D pathophysiology.
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| 2. |
- Lindqvist, Andreas, et al.
(author)
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GPR162 is a beta cell CART receptor
- 2023
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In: iScience. - 2589-0042. ; 26:12
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Journal article (peer-reviewed)abstract
- Cocaine and amphetamine-regulated transcript (CART) is expressed in pancreatic islet cells and neuronal elements. We have previously established insulinotropic actions of CART in human and rodent islets. The receptor for CART in the pancreatic beta cells is unidentified. We used RNA sequencing of Cartpt knockdown (KD) INS-1 832/13 cells and identified GPR162 as the most Cartpt-regulated receptor. We therefore tested if GPR162 mediates the effects of CART in beta cells. Binding of CART to GPR162 was established using proximity ligation assay, radioactive binding, and co-immunoprecipitation, and KD of Gpr162 mRNA caused reduced binding. Gpr162 KD cells had blunted CARTp-induced exocytosis, and reduced CARTp-induced insulin secretion. Furthermore, we identified a hitherto undescribed GPR162-dependent role of CART as a regulator of cytoskeletal arrangement. Thus, our findings provide mechanistic insight into the effect of CART on insulin secretion and show that GPR162 is the CART receptor in beta cells.
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| 3. |
- Miskelly, Michael G., et al.
(author)
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RNA sequencing unravels novel L cell constituents and mechanisms of GLP-1 secretion in human gastric bypass-operated intestine
- 2024
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In: Diabetologia. - : Springer. - 0012-186X .- 1432-0428. ; 67:2, s. 356-370
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Journal article (peer-reviewed)abstract
- Aims/hypothesis: Roux-en-Y gastric bypass surgery (RYGB) frequently results in remission of type 2 diabetes as well as exaggerated secretion of glucagon-like peptide-1 (GLP-1). Here, we assessed RYGB-induced transcriptomic alterations in the small intestine and investigated how they were related to the regulation of GLP-1 production and secretion in vitro and in vivo.Methods: Human jejunal samples taken perisurgically and 1 year post RYGB (n=13) were analysed by RNA-seq. Guided by bioinformatics analysis we targeted four genes involved in cholesterol biosynthesis, which we confirmed to be expressed in human L cells, for potential involvement in GLP-1 regulation using siRNAs in GLUTag and STC-1 cells. Gene expression analyses, GLP-1 secretion measurements, intracellular calcium imaging and RNA-seq were performed in vitro. OGTTs were performed in C57BL/6j and iScd1-/- mice and immunohistochemistry and gene expression analyses were performed ex vivo.Results: Gene Ontology (GO) analysis identified cholesterol biosynthesis as being most affected by RYGB. Silencing or chemical inhibition of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in the synthesis of monounsaturated fatty acids, was found to reduce Gcg expression and secretion of GLP-1 by GLUTag and STC-1 cells. Scd1 knockdown also reduced intracellular Ca2+ signalling and membrane depolarisation. Furthermore, Scd1 mRNA expression was found to be regulated by NEFAs but not glucose. RNA-seq of SCD1 inhibitor-treated GLUTag cells identified altered expression of genes implicated in ATP generation and glycolysis. Finally, gene expression and immunohistochemical analysis of the jejunum of the intestine-specific Scd1 knockout mouse model, iScd1-/-, revealed a twofold higher L cell density and a twofold increase in Gcg mRNA expression.Conclusions/interpretation: RYGB caused robust alterations in the jejunal transcriptome, with genes involved in cholesterol biosynthesis being most affected. Our data highlight SCD as an RYGB-regulated L cell constituent that regulates the production and secretion of GLP-1.
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| 4. |
- Ngara, Mtakai, et al.
(author)
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Exploring parasite heterogeneity using single-cell RNA-seq reveals a gene signature among sexual stage Plasmodium falciparum parasites
- 2018
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In: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 371:1, s. 130-138
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Journal article (peer-reviewed)abstract
- The malaria parasite has a complex lifecycle, including several events of differentiation and stage progression, while actively evading immunity in both its mosquito and human hosts. Important parasite gene expression and regulation during these events remain hidden in rare populations of cells. Here, we combine a capillary-based platform for cell isolation with single-cell RNA-sequencing to transcriptionally profile 165 single infected red blood cells (iRBCs) during the intra-erythrocytic developmental cycle (IDC). Unbiased analyses of single-cell data grouped the cells into eight transcriptional states during IDC. Interestingly, we uncovered a gene signature from the single iRBC analyses that can successfully discriminate between developing asexual and sexual stage parasites at cellular resolution, and we verify five, previously undefined, gametocyte stage specific genes. Moreover, we show the capacity of detecting expressed genes from the variable gene families in single parasites, despite the sparse nature of data. In total, the single parasite transcriptomics holds promise for molecular dissection of rare parasite phenotypes throughout the malaria lifecycle.
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| 5. |
- Ngara, Mtakai, et al.
(author)
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Lessons from single-cell RNA sequencing of human islets
- 2022
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In: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 65:8, s. 1241-1250
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Research review (peer-reviewed)abstract
- Islet dysfunction is central in type 2 diabetes and full-blown type 2 diabetes develops first when the beta cells lose their ability to secrete adequate amounts of insulin in response to raised plasma glucose. Several mechanisms behind beta cell dysfunction have been put forward but many important questions still remain. Furthermore, our understanding of the contribution of each islet cell type in type 2 diabetes pathophysiology has been limited by technical boundaries. Closing this knowledge gap will lead to a leap forward in our understanding of the islet as an organ and potentially lead to improved treatments. The development of single-cell RNA sequencing (scRNAseq) has led to a breakthrough for characterising the transcriptome of each islet cell type and several important observations on the regulation of cell-type-specific gene expression have been made. When it comes to identifying type 2 diabetes disease mechanisms, the outcome is still limited. Several studies have identified differentially expressed genes, although there is very limited consensus between the studies. As with all new techniques, scRNAseq has limitations; in addition to being extremely expensive, genes expressed at low levels may not be detected, noise may not be appropriately filtered and selection biases for certain cell types are at hand. Furthermore, recent advances suggest that commonly used computational tools may be suboptimal for analysis of scRNAseq data in small-scale studies. Fortunately, development of new computational tools holds promise for harnessing the full potential of scRNAseq data. Here we summarise how scRNAseq has contributed to increasing the understanding of various aspects of islet biology as well as type 2 diabetes disease mechanisms. We also focus on challenges that remain and propose steps to promote the utilisation of the full potential of scRNAseq in this area. Graphical abstract: [Figure not available: see fulltext.].
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| 6. |
- Ngara, Mtakai
(author)
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Single-cell RNA sequencing for subtype discovery in Plasmodium falciparum and mammalian cells
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Since the dawn of massively parallel sequencing technologies in mid-2000s their utility in profiling the expression of genes in a genome-wide fashion has matured and progressed from cell populations to individual cells. In particular, single-cell RNA sequencing (scRNA-seq) has impacted numerous domains in life sciences and hold immense promise in biology and medicine. Indeed, it has become realistic to chart the complete set of cell types and states in multicellular organisms, and projects have started to map out cell types in humans (i.e. the Human Cell Atlas project) and model organsims. In this thesis, I present the application of scRNA-seq to infectious disease and cancer as well as a computational assessment of the general possibilities and limitations of scRNA-seq for enumerating cell types and states de novo. In Paper I, we describe the ability of scRNA-seq to profile transcriptomes from individual malaria-causing P. falciparum parasites. We reveal heterogeneity even among synchronized cultures of parasites during their red blood cell life cycle. Moreover, we identify a subset of sexually differentiated P. falciparum with a distinct gene signature, likely important for parasite transmission that may be exploited for the design of transmission- blocking drugs and/or vaccines. In Paper II, I present a computational strategy to identify the magnitude of biological gene expression differences needed for accurate inference of cell identities using scRNA-seq. Interestingly, rather large differences are needed for proper cell state discrimination, irrespective of scRNA-seq protocol, implying that large number of cell states may escape detection. In Paper III, we used scRNA-seq and bulk RNA-seq to characterize the molecular programs during the later stages of lung metastasis. We demonstrate that a transition from epithelial to mesenchymal cell characteristics occurs in cancer cells during metastasis, and that the mesenchymal properties are maintained during metastasis growth extending over a week. In Paper IV we performed transcriptome analyses on stem and progenitor populations in myelodysplastic syndrome (MDS) patients. We provide evidence that the MDS stem cells and the progenitors have distinct transcriptome. Altogether, this thesis expands the applications of scRNA-seq towards parasite biology and cancer metastasis and we provide valuable insights into the abilities of current scRNA-seq technologies in mapping cell states in an unbiased fashion.
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| 7. |
- Woll, Petter S, et al.
(author)
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Myelodysplastic Syndromes Are Propagated by Rare and Distinct Human Cancer Stem Cells In Vivo.
- 2014
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In: Cancer Cell. - : Elsevier BV. - 1878-3686 .- 1535-6108. ; 25:6, s. 794-808
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Journal article (peer-reviewed)abstract
- Evidence for distinct human cancer stem cells (CSCs) remains contentious and the degree to which different cancer cells contribute to propagating malignancies in patients remains unexplored. In low- to intermediate-risk myelodysplastic syndromes (MDS), we establish the existence of rare multipotent MDS stem cells (MDS-SCs), and their hierarchical relationship to lineage-restricted MDS progenitors. All identified somatically acquired genetic lesions were backtracked to distinct MDS-SCs, establishing their distinct MDS-propagating function in vivo. In isolated del(5q)-MDS, acquisition of del(5q) preceded diverse recurrent driver mutations. Sequential analysis in del(5q)-MDS revealed genetic evolution in MDS-SCs and MDS-progenitors prior to leukemic transformation. These findings provide definitive evidence for rare human MDS-SCs in vivo, with extensive implications for the targeting of the cells required and sufficient for MDS-propagation.
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