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| 3. |
- Polinati, P. P., et al.
(författare)
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Patient-specific induced pluripotent stem cell-derived RPE cells : Understanding the pathogenesis of retinopathy in long-chain 3-hydroxyacyl-CoA dehydrogenase deiciency
- 2015
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Ingår i: Investigative Ophthalmology and Visual Science. - : Lippincott-Raven Publishers. - 0146-0404 .- 1552-5783. ; 56:5, s. 3371-3382
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Retinopathy is an important manifestation of trifunctional protein (TFP) deficiencies but not of other defects of fatty acid oxidation. The common homozygous mutation in the TFP α-subunit gene HADHA (hydroxyacyl-CoA dehydrogenase), c.1528G>C, affects the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity of TFP and blindness in infancy. The pathogenesis of the retinopathy is unknown. This study aimed to utilize human induced pluripotent stem cell (hiPSC) technology to create a disease model for the disorder, and to derive clues for retinopathy pathogenesis.Methods: We implemented hiPSC technology to generate LCHAD deficiency (LCHADD) patient-specific retinal pigment epithelial (RPE) monolayers. These patient and control RPEs were extensively characterized for function and structure, as well as for lipid composition by mass spectrometry.Results: The hiPSC-derived RPE monolayers of patients and controls were functional, as they both were able to phagocytose the photoreceptor outer segments in vitro. Interestingly, the patient RPEs had intense cytoplasmic neutral lipid accumulation, and lipidomic analysis revealed an increased triglyceride accumulation. Further, patient RPEs were small and irregular in shape, and their tight junctions were disorganized. Their ultrastructure showed decreased pigmentation, few melanosomes, and more melanolysosomes.Conclusions: We demonstrate that the RPE cell model reveals novel early pathogenic changes in LCHADD retinopathy, with robust lipid accumulation, inefficient pigmentation that is evident soon after differentiation, and a defect in forming tight junctions inducing apoptosis. We propose that LCHADD-RPEs are an important model for mitochondrial TFP retinopathy, and that their early pathogenic changes contribute to infantile blindness of LCHADD.
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| 5. |
- Elbasani, E, et al.
(författare)
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Kaposi's Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter
- 2020
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 12:9
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Tidskriftsartikel (refereegranskat)abstract
- CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus–host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.
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| 10. |
- Otonkoski, T, et al.
(författare)
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Physical exercise-induced hyperinsulinemic hypoglycemia is an autosomal-dominant trait characterized by abnormal pyruvate-induced insulin release
- 2003
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 52:1, s. 199-204
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Tidskriftsartikel (refereegranskat)abstract
- We have identified patients in whom strenuous physical exercise leads to hypoglycemia caused by inappropriate insulin release (exercise-induced hyperinsulinism [EIHI]). The aim of the present study was to test the hypothesis that the increased levels of lactate and/or pyruvate during anaerobic exercise would trigger the aberrant insulin secretion in these patients. A total of 12 patients (8 women and 4 men from two families) were diagnosed with EIHI, based on hypoglycemia and a more than threefold increase in plasma insulin induced by a 10-min bicycle exercise test. The mode of inheritance was autosomal dominant in these families. The acute response of insulin release to a bolus of intravenous pyruvate (13.9 mmol/1.73 m2) was studied in the patients and eight healthy control subjects. Insulin secretion did not respond to the pyruvate bolus in healthy control subjects. However, all EIHI patients responded to pyruvate, displaying a brisk increase in plasma insulin. The 1 + 3-min peak response was 5.6-fold in the patients and 0.9-fold in the control subjects (P < 0.001). To test the hypothesis that the pathogenesis of EIHI would involve monocarboxylate transport or metabolism in the β-cell, we sequenced the genes encoding the known monocarboxylate transporter proteins and tested the transport of pyruvate into patient fibroblasts. The results revealed normal coding sequences and pyruvate transport. In conclusion, EIHI represents a new autosomal-dominant hyperinsulinemia syndrome that may be more common than has been realized. The pyruvate test provides a simple, safe, and specific diagnostic test for this condition.
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| 12. |
- Sepponen, K., et al.
(författare)
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Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells
- 2022
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Ingår i: Differentiation. - : Elsevier BV. - 0301-4681. ; 128:Nov-Dec, s. 83-100
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Tidskriftsartikel (refereegranskat)abstract
- Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans. © 2022 The Authors
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| 16. |
- Ylipaasto, P, et al.
(författare)
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Enterovirus infection in human pancreatic islet cells, islet tropism in vivo and receptor involvement in cultured islet beta cells.
- 2004
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Ingår i: Diabetologia. - Berlin : Springer verlag. - 0012-186X .- 1432-0428. ; 47:2, s. 225-239
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Tidskriftsartikel (refereegranskat)abstract
- AIMS/HYPOTHESIS: It is thought that enterovirus infections cause beta-cell damage and contribute to the development of Type 1 diabetes by replicating in the pancreatic islets. We sought evidence for this through autopsy studies and by investigating known enterovirus receptors in cultured human islets. METHODS: Autopsy pancreases from 12 newborn infants who died of fulminant coxsackievirus infections and from 65 Type 1 diabetic patients were studied for presence of enteroviral ribonucleic acid by in situ hybridisation. Forty non-diabetic control pancreases were included in the study. The expression and role of receptor candidates in cultured human islets were investigated with receptor-specific antibodies using immunocytochemistry and functional assays. RESULTS: Enterovirus-positive islet cells were found in some of both autopsy specimen collections, but not in control pancreases. No infected cells were seen in exocrine tissue. The cell surface molecules, poliovirus receptor and integrin alphavbeta3, which act as enterovirus receptors in established cell lines, were expressed in beta cells. Antibodies to poliovirus receptor, human coxsackievirus and adenovirus receptor and integrin alphavbeta3 protected islets and beta cells from adverse effects of poliovirus, coxsackie B viruses, and several of the arginine-glycine-aspartic acid motifs containing enteroviruses and human parechovirus 1 respectively. No evidence was found for expression of the decay-accelerating factor which acts as a receptor for several islet-cell-replicating echoviruses in established cell lines. CONCLUSIONS/INTERPRETATION: The results show a definite islet-cell tropism of enteroviruses in the human pancreas. Some enteroviruses seem to use previously identified cell surface molecules as receptors in beta cells, whereas the identity of receptors used by other enteroviruses remains unknown.
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| 18. |
- Asplund, Olof, et al.
(författare)
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Islet Gene View-a tool to facilitate islet research
- 2022
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Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12
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Tidskriftsartikel (refereegranskat)abstract
- Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (GCG, 56%), amylin (IAPP, 52%), insulin (INS, 44%), and somatostatin (SST, 24%). Inhibition of two DEGs, UNC5D and SERPINE2, impaired glucose-stimulated insulin secretion and impacted cell survival in a human beta-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D.
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| 21. |
- Christesen, Henrik B. T., et al.
(författare)
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Rapid genetic analysis in congenital hyperinsulinism
- 2007
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Ingår i: Hormone Research. - : S. Karger AG. - 0301-0163. ; 67:4, s. 184-188
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Tidskriftsartikel (refereegranskat)abstract
- Backgound: In severe, medically unresponsive congenital hyperinsulinism (CHI), the histological differentiation of focal versus diffuse disease is vital, since the surgical management is completely different. Genetic analysis may help in the differential diagnosis, as focal CHI is associated with a paternal germline ABCC8 or KCNJ11 mutation and a focal loss of maternal chromosome 11p15, whereas a maternal mutation, or homozygous/compound heterozygous ABCC8 and KCNJ11 mutations predict diffuse-type disease. However, genotyping usually takes too long to be helpful in the absence of a founder mutation. Methods: In 4 patients, a rapid genetic analysis of the ABBC8 and KCNJ11 genes was performed within 2 weeks on request prior to the decision of pancreatic surgery. Results: Two patients had no mutations, rendering the genetic analysis non-informative. Peroperative multiple biopsies showed diffuse disease. One patient had a paternal KCNJ11 mutation and focal disease confirmed by positron emission tomography scan and biopsies. One patient had a de novo heterozygous ABBC8 mutation and unexplained diffuse disease confirmed by positron emission tomography scan and biopsies. Conclusion: A rapid analysis of the entire ABBC8 and KCNJ11 genes should not stand alone in the preoperative assessment of patients with CHI, except for the case of maternal, or homozygous/compound heterozygous disease-causing mutations. Copyright (c) 2007 S. Karger AG, Basel.
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| 22. |
- Dash, SN, et al.
(författare)
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sept7b is required for the differentiation of pancreatic endocrine progenitors
- 2016
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6, s. 24992-
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Tidskriftsartikel (refereegranskat)abstract
- Protection or restoration of pancreatic β-cell mass as a therapeutic treatment for type 1 diabetes requires understanding of the mechanisms that drive the specification and development of pancreatic endocrine cells. Septins are filamentous small GTPases that function in the regulation of cell division, cytoskeletal organization and membrane remodeling and are involved in various tissue-specific developmental processes. However, their role in pancreatic endocrine cell differentiation remains unknown. Here we show by functional manipulation techniques in transgenic zebrafish lines that suppression of sept7b, the zebrafish ortholog of human SEPT7, profoundly increases the number of endocrine progenitors but limits their differentiation, leading to reduction in β- and α-cell mass. Furthermore, we discovered that shh (sonic hedgehog) expression in the endoderm, essential for the development of pancreatic progenitors of the dorsal pancreatic bud, is absent in larvae depleted of sept7b. We also discovered that sept7b is important for the differentiation of ventral pancreatic bud-derived cells: sept7b-depleted larvae exhibit downregulation of Notch receptors notch1a and notch1b and show precocious differentiation of NeuroD-positive endocrine cells in the intrapancreatic duct and gut epithelium. Collectively, this study provides a novel insight into the development of pancreatic endocrine progenitors, revealing an essential role for sept7b in endocrine progenitor differentiation.
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| 24. |
- Flanagan, Sarah E, et al.
(författare)
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Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease.
- 2014
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Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1546-1718 .- 1061-4036. ; 46:8, s. 812-814
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Tidskriftsartikel (refereegranskat)abstract
- Monogenic causes of autoimmunity provide key insights into the complex regulation of the immune system. We report a new monogenic cause of autoimmunity resulting from de novo germline activating STAT3 mutations in five individuals with a spectrum of early-onset autoimmune disease, including type 1 diabetes. These findings emphasize the critical role of STAT3 in autoimmune disease and contrast with the germline inactivating STAT3 mutations that result in hyper IgE syndrome.
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