Dataset Information

Human T-cells EVs LC-MSMS - Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo

ABSTRACT: As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation vs. apoptosis induction). Morphology and size were analyzed by electron microscopy and nanoparticle tracking analysis. The protein content of these vesicles was analyzed by bidimensional gel electrophoresis followed by mass spec and western blot.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, T Cell

SUBMITTER: Christine Tucher

LAB HEAD: Dr. Martin Schiller

PROVIDER: PXD009074 | Pride | 2018-03-15

REPOSITORIES: Pride

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Publications

Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo.

Tucher Christine C Bode Konrad K Schiller Petra P Claßen Laura L Birr Carolin C Souto-Carneiro Maria Margarida MM Blank Norbert N Lorenz Hanns-Martin HM Schiller Martin M

Frontiers in immunology 20180315

Extracellular vesicles (EVs) are released from nearly all mammalian cells and different EV populations have been described. Microvesicles represent large EVs (LEVs) released from the cellular surface, while exosomes are small EVs (SEVs) released from an intracellular compartment. As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation Vs. apoptosis induction). We could cle ...[more]

PMID: 29599781

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Project description:Data analysis. Spot detection and matching were performed with a comparative cross analysis of all the gels using DeCyder software v.6.5 (GE Healthcare). 178 spots were selected based on 1.15-fold for protein ratio cut-off, allowing for the appearance of the spots in 23 out of 28 gels (69 out of 84 total images). Data from 95 spots were submitted. 93 spots were identified with high confidence. Spot picking and Trypsin digestion. The spots of interest were picked up by Ettan Spot Picker (GE Healthcare) based on the in-gel analysis and spot picking design by DeCyder software. The gel spots were washed a few times then digested in-gel with modified porcine trypsin protease (Promega, Fitchburg, WI). The digested tryptic peptides were desalted using a Zip-tip C18 (Millipore, Billerica, MA). Peptides were eluted from the Zip-tip with 0.5 uL of matrix solution (alpha-cyano-4-hydroxycinnamic acid, 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid, 25mM ammonium bicarbonate) and spotted on a MALDI plate. Mass Spectrometry. MALDI-TOF MS and TOF/TOF tandem MS/MS were performed on AB SCIEX TOF/TOF 5800 System (AB SCIEX). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each of the 7-10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions). Database search. Both the resulting peptide mass and the associated fragmentation spectra were submitted to GPS Explorer workstation equipped with MASCOT search engine (Matrix Science, Boston, MA) to search the Swiss-Prot database. Searches were performed without constraining protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine residues, and with one missed cleavage also allowed in the search parameters. Candidates with either protein score C.I.% or Ion C.I.% greater than 95 were considered significant. When multiple IDs were significant for a given spot, the selection was made by evaluating apparent molecular weight, isoelectric point, the location of the spot in the gel, and the presence of strips of multiple protein isoforms in the adjacent spots.

Project description:The experiment was carried out during two vegetation seasons from 25th of April to 10th of September. Two Polish potato cultivars, the drought-tolerant Gwiazda and drought-sensitive Oberon were used. Selected tubers with transverse diameters of 3 - 4 cm were pre-sprouted for 2 weeks before planting. Plants were grown in a vegetation hall in pots filled with a thin layer of gravel on the bottom and the universal vegetable soil substrate ‘Hollas’ (Agaris Polska Ltd., Poland) produced from peat with the addition of chalk at a pH range of 5.5-6.5. Additionally, in phase 20 of the BBCH-scale of plant development, MIS-3 (Intermag) fertilizer was applied. Water content (WC) in volumetric basis in soil pots was measured according Black (1965). Weather conditions during the years of study were monitored using a Weather Campbell Station (Campbell Scientific Inc.) located in close proximity, and a thermohygrograph placed between pots. Meteorological data of air temperature, the photosynthetically active radiation, and humidity were comparable in the years of study and favorable for potato development. Three weeks after the initiation of the tuberisation phase (56 DAP), plants were divided into 3 groups, each consisting of 6 plants. The first group of plants was subjected to soil drought (remained without irrigation, day/night temperature 22°C/18°C), the second one to high temperature (day/night temperature 38°C/25°C) and the third one was watered according to needs and at optimal temperature (control plants, day/night temperature 22°C/18°C). Stress application lasted 14 days and finished at 70 DAP. During this period, plants were placed in phytotron equipped with six Hortilux Schreder Lamps with Philips light bulbs of 1600 W each. Air humidity was in the range 65-70%. WC under 14 days of soil drought reached 30% (v/v), and remained 80% (v/v) in control and high temperature conditions. During the recovery period, after 14 days of stress treatment, WC reached control levels. Plant root material for proteomic research was collected on the 14th day of stress treatment (70 DAP).

Project description:Although somatic cell nuclear transfer (SCNT) cloning is more efficient in bovine than in all other species tested so far, there is a high rate of pregnancy failure that has been linked to structural and functional abnormalities of the placenta. We tested the hypothesis that these changes may originate from disturbed embryo-maternal interactions in the pre-implantation period. Therefore, we evaluated the transcriptome response of the endometrium to SCNT embryos (produced from five different donor cell cultures) as compared to embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (Day 8) and transferred to recipients. The recipients were slaughtered at day 18 of pregnancy and the uterus was recovered. Pregnancy was verified by the presence of at least one normally developed embryo. Transcriptome profiling of endometrium samples using a custom cDNA microarray covering transcripts expressed in the endometrium and/or oviduct epithelium revealed 58 transcripts that were differently abundant between endometrium samples from SCNT vs. IVF pregnancies. Prominent examples are NR2F2 (encoding the orphan nuclear receptor COUP-TFII) and GJA1 (encoding connexin 43). Both transcripts are known to play important roles in placentation and were significantly less abundant in endometrium from SCNT vs. IVF pregnancies. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication already in the pre- or peri-implantation period. Endometrium transcriptome profiles may serve as a novel readout to evaluate SCNT embryos for their ability to induce pregnancy with a functional placenta. Keywords: response to different embryos Nineteen German Fleckvieh (Simmental) heifers were slaughtered at day 18 of pregnancy. Cycle-synchronized recipient heifers received either IVP or SCNT embryos at day 7 of the estrous cycle. Animals were slaughtered at day 18. Endometrial (intercaruncular) tissue samples were obtained from 10 pregnant animals after transfer of IVP embryos and from 9 pregnant animals after transfer of SCNT embryos.

Dataset Information

Human T-cells EVs LC-MSMS - Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo

Publications

Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo.

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