C83d26aa-8ccf-42ee-b54c-1275b33aeb46 - samples
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PROVIDER: EGAD00001009667 | EGA |
REPOSITORIES: EGA
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Project description:Male mice on a homogenous C57BL6/J genetic background were sacrificed at 11 to 13 weeks of age by cervical dislocation. Brain, kidneys and liver were immediately harvested, flash frozen in liquid nitrogen and stored at -80°C until use. Animal handling was in accordance with guidelines approved by the European Molecular Biology Laboratory (EMBL). Organ sections (thickness 30 µm) were prepared in a Cryostat (Leica Biosystems, Leica CM3050 S) set to -20 °C and deposited onto SuperFrost glass slides (Carl Roth, H880); the glass slides were pre-cooled to -20°C in the cryostat. ~10 sections were placed on a glass slide, and a total of around 150 sections were prepared for each sample per mouse. Tissue sections on glass slides were then transferred onto metal plates placed on dry ice. The tissue sections were exposed or not to 1 J/cm2 of 254 nm UV light in a XL 1500 UV Spectrolinker (Spectronics Corporation) and subjected to eRIC to determine the RNA-bound proteome. eRIC eluates obtained from the respective organs of two mice were combined per independent experiment. The analyses were conducted in duplicate for each organ studied. For the no crosslink controls, organ sections from four mice were pooled to generate one unique no crosslink eRIC sample per organ studied.
2023-03-02 | PXD032113 | Pride
Project description:In pregnancies involving preeclampsia (PE), there is evidence that the fetal-placental unit is under oxidative stress. Here we examined primary cell lines generated from umbilical cords (UC) delivered by mothers who had either a normal pregnancy or experienced early onset PE to determine whether the two had distinguishable phenotypes. While all UC provided outgrowths when established in 4 % O2, success was less assured for PE cords under ambient (20 % O2) conditions (P < 0.05). Moreover, proliferation rates of established PE lines, although similar to controls in 4 % O2, were significantly lower in 20 % O2. PE lines grown in 4 % O2 were also more susceptible to the pro-oxidant diethylmaleate than control lines, and unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few differentially regulated genes between PE lines and controls in 4 % O2 conditions, but confirmed the more severely stressed phenotype of the PE lines under 20 % O2. We conclude that the primary UC cell lines generated from PE births maintain a susceptibility to oxidative stress that is stable over many cell divisions, but whether the basis of this vulnerability is genetic or epigenetic remains unclear.
2014-08-31 | GSE54400 | GEO
Project description:In pregnancies involving preeclampsia (PE), there is evidence that the fetal-placental unit is under oxidative stress. Here we examined primary cell lines generated from umbilical cords (UC) delivered by mothers who had either a normal pregnancy or experienced early onset PE to determine whether the two had distinguishable phenotypes. While all UC provided outgrowths when established in 4 % O2, success was less assured for PE cords under ambient (20 % O2) conditions (P < 0.05). Moreover, proliferation rates of established PE lines, although similar to controls in 4 % O2, were significantly lower in 20 % O2. PE lines grown in 4 % O2 were also more susceptible to the pro-oxidant diethylmaleate than control lines, and unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few differentially regulated genes between PE lines and controls in 4 % O2 conditions, but confirmed the more severely stressed phenotype of the PE lines under 20 % O2. We conclude that the primary UC cell lines generated from PE births maintain a susceptibility to oxidative stress that is stable over many cell divisions, but whether the basis of this vulnerability is genetic or epigenetic remains unclear. RNA was isolated from early passages (< p5) of UC fibroblast lines (15 PE and 9 CTL lines, grown in T25 flasks) when they reached ~90% confluency in 4 % O2 conditions. In order to collect RNA from cells under 20 % O2, cell lines at either p 4, 5, or 6 were switched from 4 % O2 conditions to 20 % O2 conditions when they were approximately 20 % confluent. When they reached ~90% confluency (generally 2 days in 4 % O2 and 3-4 days in 20 % O2 conditions), medium was removed and RNA STAT60 (I ml; Tel-Test, Friendswood, TX) was immediately added to each T25 flask and RNA extracted by following the manufacturer’s instructions. The samples of RNA were submitted to University Texas Southwestern Medical Center Microarray Core Facility (https://microarray.swmed.edu/) and microarray analysis performed with Illumina HumanHT-12 v4 expression BeadChips. Raw intensity data were background subtracted by using BeadStudio software and analyzed further by GeneSpring 12.6 software (Agilent Technologies Inc., Santa Clara CA), according to the advanced workflow protocol: percentile shift and filter by flags (detected).
2014-08-31 | E-GEOD-54400 | biostudies-arrayexpress
Project description:A549 cells were treated for 8 or 20 h using 20 ng/mL brefeldin A (BFA), 5 µM golgicide A (GCA), 10 µM monensin (MON) or the appropriate vehicle control (BFA/EtOH, MON/EtOH, GCA/DMSO).
2017-12-18 | E-MTAB-5627 | biostudies-arrayexpress
Project description:The cfMethyl-Seq libraries of the serial plasma cfDNA samples from the four NSCLC patients were constructed following the standard protocol. 10 ng of cfDNA in the volume of 25 µl was used as input material. 5’-end dephosphorylation was done with 3 µl 10xCutSmart buffer and 2 µl quick CIP from NEB (Ipswich, MA) at 37 oC for 30 min then heat-inactivated at 80 oC for 5 min. The 3’-end blocking was done with 0.5 µl 10xCutSmart buffer, 3 µl 2.5 mM CoCl2, 1 µl terminal transferase (all from NEB), and 0.5 µl 1 mM ddGTP at 37 oC for 2 h followed by 75 oC for 20 min. The mixture was then purified with 2x AmpureXP beads (Beckman Coulter, Indianapolis, IN) and eluted in 21.5 µl RT-PCR grade water (Thermo-Fisher, Waltham, MA). Restriction digestion was done with 2.5 µl 10xCutSmart buffer and 1 µl MspI (NEB) for 18 h at 37  oC and 20 min at 65 oC . 0.5 µl 10xCutSmart buffer, 0.3 µl dACGTP mixture (100 mM dATP, 10 mM dCTP, 10 mM dGTP), 1 µl Klenow (exo-, 5U/µl, NEB) and 2.6 µl RT-PCR water, 0.6 µl 50 mM DTT (ThermoFisher) was added to the mixture for end repair and A-overhang addition with the program 30  oC for 20 min, 37 oC for 1 h and 75 oC for 20 min. Adapter ligation was then performed with 1 µl 10xThermoFisher HC T4 ligase buffer, 0.4 µl 100 mM ATP (ThermoFisher), 0.2 µl 50 mM DTT, 1 µl ThermoFisher HC T4 DNA ligase (30 Weiss Unit/µl), 5 ng home-made duplex UMI adapter with all the cytosines methylated (protocol adopted from Kennedy et al.) at 16 oC for 20 h and 65 oC for 20 min. Bisulfite conversion of the adapter-ligated product was carried out with QIAGEN EpiTect plus DNA bisulfite kit following their protocol for two rounds of conversion. The converted product was purified with Qiagen MinElute spin column and eluted with 20 µl RT-PCR water. PCR amplification was done using the NEBNext Multiplex Oligos for Illumina (2.5 µl of universal and index primer each) and 25 µl KAPA HiFi HotStart Uracil+ ReadyMix (Roche) with the following cycling conditions: 98  oC for 45 s, 15 cycles of 98  oC for 15 s, 60  oC for 30 s and 72  oC for 30 s, followed by a final extension at 72  oC for 5 min. The PCR product was purified with 1x AmpureXP beads and eluted with 30 µl EB buffer. DNA concentration was measured by Qubit 1xdsDNA HS assay. 5% TBE-UREA PAGE and bioanalyzer assay was performed as quality control on each library before sequencing.
| EGAD00001010881 | EGA
Project description:Periodontitis (PD) as a chronic inflammatory disease instigated by periodontal pathogenic bacteria and mediated by the host immune response, resulting in the destruction of supporting periodontal tissue and tooth loss in adults. Constructing an injectable delivery system that allows for sustained drug release within periodontal pockets to continuously eliminate pathogenic bacteria, reduce periodontal inflammation, and promote periodontal tissue regeneration is an effective strategy for periodontitis treatment. Here, we explore the therapeutic potential and underlying mechanisms of curcumin-loaded Pickering emulsion (PE-CUR) in periodontitis treatment. The Pickering emulsion formulation offers an effective delivery system, enhancing curcumin's bioavailability and therapeutic efficacy. In vitro studies, we demonstrate that the PE-CUR exhibit excellent biocompatibility and anti-inflammatory property by elimination of reactive oxygen species (ROS). In addition, PE-CUR significantly eliminate key periodontal pathogens, including Porphyromonas gingivalis (P. gingivalis) and Staphylococcus aureus (S. aureus). In a rat model of periodontitis, PE-CUR significantly attenuates periodontal tissue inflammation and alveolar bone loss, indicating a protective effect against periodontal tissue destruction. Mechanistically, PE-CUR promotes the expression of anti-inflammatory factors and inhibits the release of pro-inflammatory factors by regulating macrophage polarization from M1 to M2 and modulating the MAPK and PI3K-AKT signaling pathway. Furthermore, PE-CUR decreases the formation of osteoclasts as well as stimulates the activation and differentiation of osteoblasts successively, thereby ameliorating the periodontal inflammation and promoting the alveolar bone regeneration. Overall, our findings elucidate the therapeutic mechanisms of PE-CUR in periodontitis, suggesting its potential as a promising treatment strategy warranting further clinical investigation.
2024-08-16 | GSE274645 | GEO
Project description:As part of a wider project to assess the impact of ultrasound on in vitro plant growth, this paper aimed to determine whether the application of piezoelectric ultrasound (PE-US) would induce changes to the transcriptome of in vitro potato (Solanum tuberosum L.). After exposing explants (single-node segments with a single leaf) to PE-US (35 kHz; 70 W) for 20 min, the effect of this stressor was determined at 0 h, 24 h, 48 h, 1 w and 4 w to assess the possible immediate and residual effects of PE-US on the potato transcriptome.
2020-03-03 | GSE135294 | GEO
Project description:Preeclampsia (PE) is a major health problem in pregnancy, and miRNAs play a role in the pathophysiology of PE. However, the expression of circulating miRNAs was not analyzed in the second trimester of pregnancy, a period of major relevance to identify predictive biomarkers for PE. Therefore, we examined the expression profiles of 84 circulating miRNAs using PCR-array in plasma collected between 20 and 25 weeks of gestation from pregnant who subsequently developed PE and from those who remained healthy along pregnancy randomly selected from a prospective cohort of pregnant. miR-204-5p (P=0.0082) and other 22 miRNAs were upregulated (fold change > 2.0, while miR-24-3p, miR-22-3p, miR-143-3p, and miR-376c-3p had fold change > 4.0) and six miRNAs were downregulated (fold change < 0.5) in plasma from pregnant who developed PE. Target genes for differentially expressed miRNAs were extracted from the miRTarBase of experimentally validated miRNA-target interactions and used in gene set functional enrichment and REACTOME pathway analysis. The 20 pathways with the lowest false discovery rate for the upregulated and downregulated miRNAs are related to pathophysiology of PE, such as apoptosis, angiogenesis, and signal transduction, including signaling by receptor tyrosine kinases, VEGF and TGFB family members. Moreover, we explored the 37 target genes and pathways for miR-204-5p, including MMP-9, MALAT1, TGFBR2, and SIRT1. The circulating miRNAs differentially expressed in the second trimester of pregnancy are related to target genes and pathways of pathophysiological relevance during the early development of PE, and our findings provide for potential circulating predictive biomarkers in PE. For the PCR array, total RNA isolated was converted into cDNA using the miScript II RT Kit (Qiagen®, Leusden, Netherlands), according to the manufacturer’s instructions
2021-03-15 | GSE168860 | GEO
Project description:The HH16/17 chicken proepicardium (PE) gives rise to the embryonic epicardium (Epi) which significantly contributes formation of the coronary vasculature during cardiac development. In contrast to explanted Epi cells, explanted PE cells can undergo differentiation into a cardiac myocyte phenotype. In order to assess which genes are associated with PE differentiation into distinc cellular lineages, two interconnented microarray gene-expression series were performed. 1: Gene-expression profiles at 12, 24, 36, 48, 60, 72 and 120 hours during cardiac myocyte differentiation from including the HH16/17 PE (t0h) were determined by hybridizing Cy3 and Cy5 labelled amplified RNA to the ArkGen 20K oligo arrays in a 2-color looped experiment design, i.e., hybridization of successive time-points per array, including dye swaps, resulting in four technical replicates for each time point. 2: Gene expression changes during normal embryonic Epi maturation were assess by hybridizing Epi from stages HH25, HH29, HH32 and HH37 in all possible pair-wise combinations, including dye-swaps, leading to 6 replicates per time point. To allow for valid comparisons between the Epi and untreated PE differentiation, these two array series were connected via hybridization of both Epi stage HH25 and HH29 with the PE explant at 48 hours in culture, with dye swaps. Keywords: time course, cardiac myocyte differentiation, embryonic coronary vessel formation, cell type comparison, mRNA expression
2010-12-21 | GSE13923 | GEO
Project description:We hypothesized that the potential ability of the 18 kDa mitochondrial translocator protein (TSPO) to regulate gene expression may underlie its numerous reported functions, ranging from mitochondrial activity till mental disorders. Therefore, we applied microarray analysis of gene expression to U118MG glioblastoma cells. Within 1 hr the TSPO ligand PK 11195 induced changes in expression of immediate early genes and transcription factors. After 24 hrs primarily gene expression for enzymes and other proteins is changed. The associated gene products are known to affect various cellular functions: programmed cell death, cell cycle, viability, proliferation, migration, differentiation, development, tumorigenesis, and inflammatory / immune responses. Our microscopic studies showed intense TSPO mitochondrial labeling but no TSPO signal in the cell nuclei. Thus, it appears that the TSPO is part of the retrograde mitochondrial-nuclear signaling pathway for modulation of gene expression and thereby may exert its numerous effects on cell function, phenotype, and disease. U118MG cells (1.255 à 106) were seeded in Petri dishes (i.e. 28.5 à 103 cells / cm2) and allowed to proliferate for 3 days in full medium. Experiments for gene expression assayed with microarray typically consisted of three experimental groups and a vehicle control group (n = 3 for each group). Serum deprived medium was applied for 24 hrs, in the experimental groups ending with the inclusion a choice of various PK 11195 exposures i.e. either 1 hr, 3 hrs, or 24 hrs. Exposure to PK 11195 implies serum deprived medium with 1% alcohol (vehicle) and PK 11195 (25 µM final concentration) for the required time period. 25 µM of PK 11195 is the optimal concentration for these types of experiments with U118MG cells (Kugler et al., 2008). The cells were collected by trypsinization, washed by centrifugation in phosphate buffered saline (PBS) (400 à g, 5 min), and lysed [RLT lysis buffer provided with the RNeasy Mini Kit diluted with β-mercaptoethanol (1 : 100)], according to the manufacturer's instructions. Lysates were stored at -70oC.
2016-02-18 | E-GEOD-77998 | biostudies-arrayexpress