PolyA selected RNA-seq of wild-type human iPSC-derived cortical neurons upon cooling and rewarming
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ABSTRACT: We aim to profile the transcriptomic changes in neurons when responding to cooling, in order to understand the neuroprotective effects of hypothermia. polyA selected RNA-seq were performed from human iPSC-derived cortical neurons under control (37 ºC), cooled (72 h at 32 ºC, day 15-18) and rewarmed (72 h at 32 ºC, day 15-18, followed by 72 h at 37 ºC, day 18-21) conditions.
Project description:C-mannosylation in addition to N- and O-glycosylation is a further but less well studied type of protein glycosylation taking place in the endoplasmic reticulum (ER) characterised by the modification of tryptophan residues with a single mannose effecting protein folding, secretion and/or function. Motivated by an interest in the functional role of C-mannosylation for early developmental processes, we aimed for the functional inactivation of the C mannosyltransferases DPY19L1 and DPY19L3, respectively, and excised parts of their coding sequence from the genome of the hiPSC line CBiPSC2 by applying CRISPR Cas9. To determine the effect of the genomic deletions in DPY19L1 or DPY19L3 on C-mannosylation, TSR2 and TSR3 of human thrombospondin 1 (THBS1) was recombinantly expressed in WT and KO hiPSCs followed by purification an LC-MS analysis. The results are in accordance to our previous findings that DPY19L1 is mainly acting on W1 and W2 whereas DPY19L3 acts on W3 of WxxWxxWxxC motifs of TSRs and proves the functional inactivation of the respective C-mannosyltransferases in the hiPSCs model. A secretome analysis of C mannosyltransferase deficient hiPSCs revealed that secretion of numerous proteins was reduced in the mutants including ADAMTS16, which was previously reported to be essential for optic fissure fusion in zebrafish. In order to analyse C-mannosylation of ADAMTS16, its TSR1 was recombinantly expressed in CHO-K1 WT, DPY19L1 KO and DPY19L3 KO cells, purified and analysed by LC MS analysis. The results revealed that the WSDWSSWSPC motif of TSR1 of ADAMTS16 can be C-mannosylated at all three tryptophan residues. Deletion of DPY19L1 prevented C-mannosylation of the two first tryptophans whereas C-mannosylation of the third tryptophan was not detected in the DPY19L3-mutant.
Project description:We isolated mutants in Arabidopsis with enhanced ambient temperature response. Microarray analysis was performed to understand the extent to which ambient temperature transcriptome is perturbed in the mutants in comparison with the WT at non inductive 12 ºC and after shift to inductive 27 ºC for 2 h and 24 h. We performed global transcript profiling to understand the regulation of of gene expression underlying ambient temperature response. Sterilized wild type Arabidopsis Col-0 as well as the arp6-10 mutant seeds were plated on half-strength MS plates without sugar. Plates were incubated at 4 ºC in dark for 48 hours before transfering to 20 ºC with light. 5 day old seedlings were transfered to 12 ºC incubaor with continuous light for 3 days befroe shifting to 27 ºC. Samples (Whole seedlings) were collected before shifting (0 h) and 2 h and 24 h after shifting to 27 ºC by immediately freezing in liquid nitrogen.
Project description:Osteoarthritis is the most common degenerative joint condition, leading to articular cartilage (AC) degradation, chronic pain and immobility. The lack of appropriate therapies that provide tissue restoration combined with the limited lifespan of joint-replacement implants indicate the need for alternative AC regeneration strategies. Differentiation of human pluripotent stem cells (hPSCs) into AC progenitors may provide a long-term regenerative solution but are still limited due to the continued reliance upon growth factors to recapitulate developmental signalling processes. Recently, TTNPB, a small molecule activator of retinoic acid receptors (RARs), has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. Here, we modified our previous differentiation protocol, by supplementing cells with TTNPB and administering BMP2 at specific times to enhance early development.
Project description:Microarray analysis on Brachypodium distachyon seedlings was performed to determine the response of the transcriptome to changes in ambient temperature, including identification of marker genes that were up-regulated or down-regulated by a moderate increase in growth temperature. Wild-type Brachypodium (Bd21) seedlings were grown on MSR63 media without sucrose (Alves et al. 2009 Nature Protocols vol. 4 pp 638-649) in a short-day photoperiod (14 hr light/ 10 hr dark) at 17 ºC. As the third leaf was emerging, plants were transferred to 12 ºC for 48 hrs. Plants were then maintained at 12 ºC or transferred to one of two temperature treatments: constant 22 ºC or constant 27 ºC. Samples were collected before the shift (0 hr) and at 2 hr and 24 hr after the shift and immediately frozen in liquid nitrogen. For each harvest, two to three replicates were collected that each contained 3 seedlings.
Project description:C-mannosylation is a modification of tryptophan residues with a single mannose effecting protein folding, secretion and/or function. To date, only few proteins have been proven to be C-mannosylated and studies aiming at global assessment of protein C-mannosylation from cells or tissues are scarce. In order to interrogate the C-mannosylome of human induced pluripotent stem cells (hiPSCs), we made use of the common finding that C-mannosylation is important for protein secretion. Thus we compared the secretomes of C-mannosyltransferase-deficient DPY19L1 and DPY19L3 mutants to their parental wild-type hiPSCs by mass-spectrometry-based quantitative proteomics. Secretion of numerous proteins was reduced in the mutants.
Project description:Cardiovascular diseases are a major cause of life-threatening burden around the world. The heart has a very low regeneration capacity and donor organs for transplantation are scarce. Therefore regeneration of lost myocardium with stem cell-derived cardiomyocytes (CMs) provides an attractive strategy for heart repair. Human pluripotent stem cells (hPSCs) can be efficiently differentiated in vitro into CMs but the molecular mechanisms behind this process are still not fully understood. In particular identification of secreted autocrine and/or paracrine factors that function as important extrinsic signals remained elusive because the mass spectrometry (MS)-based identification of secreted proteins from cell culture supernatants is impeded by high levels of albumin present in common differentiation media. Thus we established an albumin-free cardiomyogenic differentiation medium and performed secretomics at seven different time points during in vitro differentiation. This analysis led to the identification of 4832 proteins with 1802 being significantly altered during differentiation and 431 of these were annotated as secreted according to gene ontology. Bioinformatics revealed enrichment of extrinsic Wnt pathway-related proteins 3 days upon induction of differentiation and of extracellular matrix proteins in the resulting CMs. Numerous extrinsic components of Wnt, Activin A, Nodal, TGFβ, BMP or FGF signaling pathways were quantitatively assessed during differentiation. Notably, the abundance of pathway agonists was generally lower compared to the respective antagonists but their curves of progression over timer were rather similar. We hypothesize that Activin A, Nodal and TGFβ signaling are turned down shortly upon initiation of cardiac differentiation whereas BMP signaling is switched on. Wnt and FGF signaling peaks between d0 and d3 of differentiation and interestingly, Activin A and TGFβ signaling seem to be reactivated at the cardiac progenitor stages and/or in early CMs.
Project description:253 genes showed more than 4-fold up- or down-regulated expressions by poly (I:C) at 15 ºC and 25 ºC. Fish were treated with poly(I:C) and reared at 15 ºC and 25 ºC and isolation of total RNA from spleen of the fish at 3 and 24 hours after the treatment.
Project description:In this study, we investigated the effects of ethanol exposure on cardiomyocyte differentiation by treating the cells with various concentrations of ethanol. The results indicated that ethanol exposure during hiPSC-CM differentiation reduced cell viability, cell proliferation, and cardiomyocyte yield, and caused mitochondrial dysfunction, redox imbalance, and changes of proteomic profiles.
Project description:Microarray analysis of Aspergillus niger under conditions with differing combinations of carbon source, nitrogen source, nitrogen concentration, and culture pH Fermentor cultures were grown in minimal medium (MM) at a constant temperature of 30 ± 0.5 ºC and with differing combinations of carbon source (either 277.5 mM glucose or 333.0 mM xylose), nitrogen source (NH4Cl or NaNO3) and nitrogen concentration (4x: 282.4 mM; 8x: 564.8 mM), and pH (pH4 or pH5) of the medium (M. Braaksma, A.K. Smilde, M.J. van der Werf, P.J. Punt, submitted for publication). At different time points samples were collected, quenched immediately in methanol at -45 ºC and centrifuged at -20 ºC to remove supernatant. Part of the biomass was frozen into liquid nitrogen and stored at -80 ºC for microarray analysis. For each of the 16 culture conditions one sample was selected for microarray analysis; samples were collected either around the time point carbon source depleted or a considerable time (~24 h) after carbon souce depletion. In addition some technical duplicates were included. 20 samples were analyzed from 16 different fermentation conditions. The fermentation conditions were varied according to a full factorial design of four factors tested at two levels. From one fermentation two different time samples were analyzed, from another fermentation four samples, two technical replicates of two different time samples, were analyzed. Samples were collected either around the time point carbon source depleted or a considerable time (~24 h) after carbon souce depletion.
Project description:About 200 ml liquid culture (OD730 = 0.4) was centrifuged at 4 000 g, 4 ºC, for 10 min, and the supernatant containing the extracellular proteins was collected. The supernatant was filtered through 0.45 µm pore size filters to remove remaining cell debris, and the proteins in the filtrate were further concentrated by ultrafiltration using centrifugal filters with a molecular weight cut-off of 5 kDa (Millipore). The concentrated extracellular medium was precipitated with 10% trichloroacetic acid at 4 ºC overnight and then dissolved in 50 mM ammonium bicarbonate