Long non-coding RNAs (lncRNAs) can regulate the transcript levels of genes in the same genomic region. These locally acting lncRNAs have been found deregulated in human disease and some have been ...shown to harbour quantitative trait loci (eQTLs) in autoimmune diseases. However, lncRNAs linked to the transcription of candidate risk genes in loci associated to rheumatoid arthritis (RA) have not yet been identified. The TRAF1 and C5 risk locus shows evidence of multiple eQTLs and transcription of intergenic non-coding sequences. Here, we identified a non-coding transcript (C5T1lncRNA) starting in the 3' untranslated region (UTR) of C5. RA-relevant cell types express C5T1lncRNA and RNA levels are further enhanced by specific immune stimuli. C5T1lncRNA is expressed predominantly in the nucleus and its expression correlates positively with C5 mRNA in various tissues (P=0.001) and in peripheral blood mononuclear cells (P=0.02) indicating transcriptional co-regulation. Knockdown results in a concurrent decrease in C5 mRNA levels but not of other neighbouring genes. Overall, our data show the identification of a novel lncRNA C5T1lncRNA that is fully located in the associated region and influences transcript levels of C5, a gene previously linked to RA pathogenesis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene ...therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Müller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in Crb1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Müller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments.
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•iPSC-derived retinas recapitulate the fetal human Crumbs complex•CRB2 is expressed earlier than CRB1 in fetal or iPSC-derived retina at the SAR•CRB1-RP patient iPSC-derived retinas show disruptions at the OLM•AAV5 infects photoreceptors and Müller glial cells in adult and iPSC-derived retina
Wijnholds and colleagues show that the key Crumbs complex members, CRB1 and CRB2, are recapitulated between human fetal retina and iPSC-derived retinal organoids. CRB2 is expressed earlier than CRB1 in fetal and iPSC-derived human retina. CRB1-RP patient iPSC-derived retinas show a morphological phenotype. In addition, they show that AAV5 infects photoreceptors and Müller glial cells in adult and iPSC-derived human retina.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ...ubiquitously transcribed mScarletI-Geminin (GMMN) (1–110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle. mScarletI fluorescence increased gradually from the S-phase through the M-phase of the cell cycle and was lost at the metaphase-anaphase transition. The resulting hiPSC reporter line generated, which we named ProLiving, is a valuable tool to study cell division and cell cycle characteristics in living hiPSC-derived cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
An induced pluripotent stem cell (iPSC) line, in which a H2B-fluorescent protein fusion is temporally expressed, is a valuable tool to track cells and study cell divisions and apoptosis. To this end ...we introduced a 3rd generation “all-in-one” doxycycline-inducible H2B-mTurquoise2 vector into the AAVS1 locus of PAX3-Venus iPSCs via CRISPR/Cas9. H2B-mTurquoise2 expression is absent but readily induced by doxycycline allowing quantification of cell divisions and imaging of living cells. Besides being a universal reporter in iPSC-based differentiation and toxicity assays, the generated pluripotent and genomically normal LUMCi041-A-2 line is particularly suited to study PAX3-positive stages of development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Innate immune cells, such as monocytes, can adopt a long-lasting pro-inflammatory phenotype, a phenomenon called 'trained immunity'. In trained immunity, increased cytokine levels of genes, like ...interleukin (IL)-6 and tumor necrosis factor (TNF)-α, are observed, which are associated with increased histone 3 lysine 4 trimethylation (H3K4me3) in the promoter region. As systemic IL6 and TNFα levels are increased in rheumatoid arthritis (RA) patients and monocytes are known to be the primary producers of TNFα and IL6, we hypothesized that 'trained immunity' signals may be observed at these genes in monocytes from RA patients. CD14+ monocytes were isolated from untreated RA patients and paired age-matched healthy controls. H3K4me3, mRNA, protein and serum levels of IL6 and TNFα were evaluated by chromatin immunoprecipitation, reverse-transcription quantitative PCR and enzyme-linked immunosorbent assays. Despite elevated serum levels of TNFα and IL6 in the tested RA patients (P<0.05), ex vivo isolated monocytes displayed similar H3K4me3 levels to healthy controls in the promoter region of TNFα and IL6. Concordantly, mRNA and protein levels of IL6 and TNFα were similar before and after lipopolysaccharide stimulation between patients and controls. Together, with the current number of individuals tested we have not detected enhanced trained immunity signals in circulating monocytes from untreated RA patients, despite increased IL6 and TNFα serum levels.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Systemic sclerosis is an autoimmune disease characterized by fibrosis of skin and multiple organs of which the pathogenesis is poorly understood. We studied differentially expressed coding and ...non-coding genes in relation to systemic sclerosis pathogenesis with a specific focus on antisense non-coding RNAs. Skin biopsy−derived RNAs from 14 early systemic sclerosis patients and six healthy individuals were sequenced with ion-torrent and analyzed using DEseq2. Overall, 4,901 genes with a fold change >1.5 and a false discovery rate <5% were detected in patients versus controls. Upregulated genes clustered in immunologic, cell adhesion, and keratin-related processes. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. Sense genes expressed opposite of these antisense genes were also deregulated in 42% of the observed sense−antisense gene pairs. The majority of the antisense genes had a similar effect sizes in an independent North American dataset with three genes (CTBP1-AS2, OTUD6B-AS1, and AGAP2-AS1) exceeding the study-wide Bonferroni-corrected P-value (PBonf < 0.0023, Pcombined = 1.1 × 10−9, 1.4 × 10−8, 1.7 × 10−6, respectively). In this study, we highlight that together with coding genes, (antisense) long non-coding RNAs are deregulated in skin tissue of systemic sclerosis patients suggesting a novel class of genes involved in pathogenesis of systemic sclerosis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, ...necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.
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•CRB1 is associated with retinitis pigmentosa•We made organoids from these patients and studied their biology•The extracellular domains of CRB1 and NOTCH1 interact•Our data link to altered endosomal maturation and increased autophagy
Wijnholds, Buck, and colleagues detect upregulation of the endolysosome-associated gene Wdfy-1 in a Crb1 mouse model. They also show that the extracellular domains of human CRB1 and NOTCH1 interact and levels of WDFY1 increased in human retinal organoids, whereas levels of CRB1, NOTCH1, VPS35, and RAB11A decreased in CRB1-associated retinitis pigmentosa organoids. CRB1 organoids repressed early endosome maturation and increased endosomal degradative compartments.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Case-mix based prospective payment of homecare is being implemented in several countries to work towards more efficient and client-centred homecare. However, existing models can only explain a ...limited part of variance in homecare use, due to their reliance on health- and function-related client data. It is unclear which predictors could improve predictive power of existing case-mix models. The aim of this study was therefore to identify relevant predictors of homecare use by utilizing the expertise of district nurses and health insurers.
We conducted a two-round Delphi-study according to the RAND/UCLA Appropriateness Method. In the first round, participants assessed the relevance of eleven client characteristics that are commonly included in existing case-mix models for predicting homecare use, using a 9-Point Likert scale. Furthermore, participants were also allowed to suggest missing characteristics that they considered relevant. These items were grouped and a selection of the most relevant items was made. In the second round, after an expert panel meeting, participants re-assessed relevance of pre-existing characteristics that were assessed uncertain and of eleven suggested client characteristics. In both rounds, median and inter-quartile ranges were calculated to determine relevance.
Twenty-two participants (16 district nurses and 6 insurers) suggested 53 unique client characteristics (grouped from 142 characteristics initially). In the second round, relevance of the client characteristics was assessed by 12 nurses and 5 health insurers. Of a total of 22 characteristics, 10 client characteristics were assessed as being relevant and 12 as uncertain. None was found irrelevant for predicting homecare use. Most of the client characteristics from the category 'Daily functioning' were assessed as uncertain. Client characteristics in other categories - i.e. 'Physical health status', 'Mental health status and behaviour', 'Health literacy', 'Social environment and network', and 'Other' - were more frequently considered relevant.
According to district nurses and health insurers, homecare use could be predicted better by including other more holistic predictors in case-mix classification, such as on mental functioning and social network. The challenge remains, however, to operationalize the new characteristics and keep stakeholders on board when developing and implementing case-mix classification for homecare prospective payment.
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CEKLJ, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We investigated whether problem-based learning (PBL) can foster conceptual change. Students were randomly assigned to a PBL, lecture-based, or self-study group, all receiving instruction about the ...topic of Newtonian laws. Conceptual change was measured from pre- to immediate post-test (directly after instruction) and from immediate post-test to delayed post-test after one week. Results showed that the PBL-group outperformed both the lecture and the self-study group on the immediate post-test. This result supported the hypothesis that PBL can increase the likelihood of conceptual change. The PBL group also outperformed both other groups at the delayed post-test after one week; the decline in conceptual change from immediate to delayed post-test was similar for all three groups. Findings are discussed in terms of cognitive engagement.
•We investigated whether problem-based learning (PBL) fostered conceptual change (CC).•CC was greater after learning from PBL than from lectures or self-study only.•After one week, this benefit was retained.•The decline in scores from immediate to delayed post-test was similar for all groups.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
•Two siblings with lethal infantile cardiomyopathy had compound mutations in AARS2 gene.•Skin fibroblasts were isolated from both the patients and their mother.•HiPSC lines were successfully derived ...from the skin fibroblasts using replication defective Sendai virus.•HiPSC clones had normal karyotype and pluripotent characteristics.
Combined Oxidative Phosphorylation Deficiency 8 (COXPD8) is an autosomal recessive disorder causing lethal childhood-onset hypertrophic cardiomyopathy. Homozygous or compound heterozygous mutations in the nuclear-encoded mitochondrial alanyl-tRNA synthetase 2 (AARS2) gene underly the pathology. We generated induced pluripotent stem cells (hiPSCs) from two patients carrying the heterozygous compound c.1774 C>T, c.2188 G>A and c.2872 C>T AARS2 mutations, as well as a related healthy control carrying the c.2872 C>T AARS2 mutation. All hiPSC-lines expressed pluripotency markers, maintained a normal karyotype, and differentiated towards the three germ layer derivatives in vitro. These lines can be used to model COXPD8 or mitochondrial dysfunction.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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