Dataset Information

Mock-, SARS-CoV-2-, and UV-SARS-CoV-2-infected human lung microvascular endothelial cells (HL-mECs), at day 1, 2 and 3 p.i.

ABSTRACT: Human lung microvascular endothelial cells (HL-mECs) purchased from Lonza Clonetics (Walkersville, MD, USA) and cultured in EGM-2MV (Lonza) containing 10% (FBS). The proteomic analysis was performed on Mock-, SARS-CoV-2-, and UV-SARS-CoV-2-infected HL-mECs, collected at day 1, 2 and 3 p.i. For each condition, about 2 x 10^6 cells were used.Cells were lysed and proteins were extracted, reduced/alkylated and enzymatically digested using Easy PepTM Mini MS Sample Prep Kit (Thermo Fisher Scientific Rockford, IL, USA). Tryptic and Lys-C digestion was carried out on 100 microg and 70 microg of the two examined preparations, respectively. Following the kit protocol, in less than 3 h and for each examined condition, peptides were generated, cleaned-up to prepare detergent-free samples and resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) for LC-MS/MS analysis. Peptide mixtures were analyzed using Eksigent nanoLC-Ultra 2D System (Eksigent, part of AB SCIEX Dublin, CA, USA) configured in trap-elute mode. Briefly, samples (0.8microg injected) were first loaded on a trap (200 microm x 500 microm ChromXP C18-CL, 3 microm, 120 A) and washed with the loading pump running in isocratic mode with 0.1% formic acid in water for 10 min at a flow of 3 microL/min. The automatic switching of autosampler ten-port valve then eluted the trapped mixture on a nano reversed phase column (75 microm x 15 cm ChromXP C18-CL, 3 microm, 120 A) through a 133 min gradient of eluent B (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. In depth, gradient was: from 5-10% B in 3 min, 10-40% B in 110 min, 40-95% B in 12 min and holding at 95% B for 8 min. The eluted peptides were directly analyzed on a LTQ-OrbitrapXL mass spectrometer (Thermo Fisher Scientific) equipped with a nanospray ion source. The spray capillary voltage was set at 1.7 kV and the ion transfer capillary temperature was held at 220 C. Full MS spectra were recorded over a 400 - 1600 m/z range in positive ion mode, with a resolving power of 60000 (full width at half-maximum) and a scan rate of 2 spectra/s. This step was followed by five low-resolution MS/MS events that were sequentially generated in a data-dependent manner on the top five ions selected from the full MS spectrum (at 35% collision energy), using dynamic exclusion of 0.5 min for MS/MS analysis. Globally, 45 raw data files are uploaded (MOCK: Day 1, n=5, Day 2, n=5, Day 3, n=5; SARS-CoV-2: Day 1, n=5, Day 2, n=5, Day 3, n=5; UV-SARS-CoV-2: Day 1, n=5, Day 2, n=5, Day 3, n=5).

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Pierluigi Mauri Dario Di Silvestre Antonella De Palma

PROVIDER: MSV000086649 | MassIVE | Wed Dec 30 04:47:00 GMT 2020

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Background: Type I interferons (IFNs) are essential to the clearance of viral diseases, in part by initiating upregulation of IFN regulated genes (IRGs). A clear distinction between genes upregulated directly by virus and genes upregulated by secondary IFN production has not been made. Here we investigated the genes regulated by IFN-a2b compared to the genes regulated by SARS-CoV infection in ferrets. Methods: We characterized early host immune responses in peripheral blood and lung necropsies of ferrets injected with IFN-a2b or infected with SARS-CoV/Tor 2 strain, using microarray analysis on the Affymetrix platform. Results: We identified a common IRG signature that was upregulated in both SARS-CoV infected ferrets as well as in ferrets injected with IFN-a2b. We also identified unique patterns of gene expression for leukocyte activation, cell adhesion and complement pathways between IFN-a2b injection and SARS-CoV infection. Conclusions: Our results define the effects of IFN-a2b on the immune system of ferrets highlighting genes regulated by IFN during SARS-CoV infection. We have shown the similarities and differences of top funcional gene groups as well as pathways that play key roles in early immune responses in ferrets in response to IFN-a2b or SARS-CoV. Key words: ferret, gene expression, SARS, interferon. Keywords: time course In experiments with IFN-a2b, for peripheral blood, 15 ferrets were randomly allocated to 3 groups: Day 0, 5 ferrets (no IFN injection), day 1, 6 ferrets (injected), and day 2, 4 ferrets (injected). For lung necropsies of injected ferrets with IFN-a2b, we used 12 ferrets in 3 groups: 4 ferrets, day 0 (no IFN injection), 4 ferrets, day 1 (injected) and 4 ferrets, day 2 (injected). Experimental groups for SARS-CoV infection was as follows: For peripheral blood, 3 and 4 ferrets for day 0 (no infection) and day 2 (infection) respectively. For lung neceropsies, a total of 9 ferrets in 3 groups, each with 3 replicates for day 0 (no infection), day 1 (infection) and day 2 (infection).

Project description:Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection causes an immune-mediated disease. We have recently shown that SARS-CoV-induced epithelial Calu-3 cytokines could exacerbate and dampen host inflammatory and T cell responses, respectively, through modulating the functions of macrophages and dendritic cells, thereby suggesting that not only are lung epithelial cells the primary cells of SARS-CoV infection, but they also involve in initiating and orchestrating the host innate and adaptive immunity. Comprehensive evaluation of the complex epithelial signaling to SARS-CoV is, thus, crucial for paving the way to better understand SARS pathogenesis and develop the innovative therapeutics against SARS. Here, based on the microarray-based functional genomics, we reported that 2B4 cells, a clonal derivative of Calu-3 cells, elicited a temporal and spatial activation of nuclear factor (NF)kappaB, activator protein (AP)-1 (ATF2/c-Jun), and interferon regulatory factor (IRF)-3/-7 at 12-, 24-, and 48-hrs post infection (p.i.), respectively, resulting in the activation of many antiviral genes, including interferon (IFN)-Î², -Î»s, SARS-related inflammatory mediators, and various IFN-stimulated genes (ISGs). While elevated responses of IFN-Î² and IFN-Î»s were not detected until 48-hrs p.i., as a consequence of a delayed IRF-3/-7 activation, we showed, for the first time, that both types of IFNs exerted previously under-described non-redundant, complementary, and/or synergistic effects on the epithelial defense against SARS-CoV. Collectively, our results highlight the molecular mechanisms of the sequential activation of virus- and IFN-dependent signaling of lung epithelial cells against SARS-CoV and identify novel cellular targets for future studies, aiming at advancing strategies against SARS. DNA microarrays were performed in the Molecular Genomics (MG) Core Facilities at UTMB. The detailed information about the procedures of microarray analysis has been posted on the MG Core Facilities website (genomics@scms.utmb.edu). To characterize the dynamic, spatial, and temporal changes of the gene expression induced by SARS-CoV, confluent 2B4 cells grown in T-75 flasks were infected with SARS-CoV (MOI=0.1) or remained uninfected (as control) for 12, 24, and 48hrs. Because 2B4 cells were also permissive to the productive infection of Dhori virus (DHOV), a member of the Orthomyxoviridae family within the Thogotovirus genus, resulting in robust responses of IFNs and other pro-inflammatory mediators, we also established parallel cultures of DHOV-infected 2B4 cells (MOI=0.1) for the comparative analysis of global gene expression elicited by SARS-CoV- versus DHOV-infected 2B4 cells. To meet the minimal number required for application of statistical algorithms, we performed the study in triplicate at each time point for mock-, SARS-CoV-, and DHOV-infected cultures, yielding a total of 27 arrays. Briefly, supernatants were harvested from differentially treated cultures at 12-, 24-, and 48-hrs p.i. for subsequent analyses of viral yields and cytokine profiling, whereas the cells were subjected to total RNA extraction by using an RNAqueous-4PCR kit and following the protocol recommended by the manufacturer (Ambion, Austin, TX). Purified RNA samples were sent to our core facility for conversion to cDNA, biotin-labeled, and hybridized to 27 Affymetrix Human Genome U133 Plus 2.0 âGene Chipsâ each of which contained 54,675 probe set identifiers representing more than ~47,400 transcripts that identify ~38,500 well-characterized genes, and various internal controls (Affymetrix, Santa Clara, CA). Mock-infected cells were compared to cells infected with SARS-CoV or DHOV at each time point.

Dataset Information

Mock-, SARS-CoV-2-, and UV-SARS-CoV-2-infected human lung microvascular endothelial cells (HL-mECs), at day 1, 2 and 3 p.i.

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure