Journal: Biology

Article Title: Inflammatory Response of Primary Cultured Bovine Mammary Epithelial Cells to Staphylococcus aureus Extracellular Vesicles

doi: 10.3390/biology11030415

Figure Lengend Snippet: Bacterial strains used in the current study.

Article Snippet: Newbould 305 (N305) , ATCC 29740 (American Type Culture Collection, Manassas, VA, USA).

Techniques:

Journal: Biology

Article Title: Inflammatory Response of Primary Cultured Bovine Mammary Epithelial Cells to Staphylococcus aureus Extracellular Vesicles

doi: 10.3390/biology11030415

Figure Lengend Snippet: Transmission electron microscopy (TEM) observations of isolated S. aureus EVs for each mastitis strain Mastidis, M5512VL, USA300, N305 and M5702. Black arrows indicate EVs.

Article Snippet: Newbould 305 (N305) , ATCC 29740 (American Type Culture Collection, Manassas, VA, USA).

Techniques: Transmission Assay, Electron Microscopy, Isolation

Journal: Biology

Article Title: Inflammatory Response of Primary Cultured Bovine Mammary Epithelial Cells to Staphylococcus aureus Extracellular Vesicles

doi: 10.3390/biology11030415

Figure Lengend Snippet: Particle concentration, mean and mode particle diameter and protein concentration were measured in each pellet of S. aureus EVs.

Article Snippet: Newbould 305 (N305) , ATCC 29740 (American Type Culture Collection, Manassas, VA, USA).

Techniques: Concentration Assay, Protein Concentration

Journal: iScience

Article Title: Biological fractionation of lithium isotopes by cellular Na + /H + exchangers unravels fundamental transport mechanisms

doi: 10.1016/j.isci.2023.106887

Figure Lengend Snippet:

Article Snippet: Atomic absorption spectrometry Li Lamp lumina , Perkin elmer , N305-0142.

Techniques: Recombinant, Expressing, Plasmid Preparation, Software, Membrane

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland

doi: 10.3389/fcimb.2018.00277

Figure Lengend Snippet: Bovine S. aureus Newbould 305 (N305) releases EVs in vitro . TEM of S. aureus Newbould 305 (N305) purified EVs after negative staining (A) and selected EVs. (B) Slice through a cryo-electron tomogram obtained from S. aureus N305 EVs. (C) Representative graph of size distribution of S. aureus N305-secreted EVs measured with tunable resistive pulse sensing (TRPS) and nanoparticle tracking analysis (NTA).

Article Snippet: S. aureus N305 (ATCC 29740) was grown in Brain Heart Infusion (BHI) (Difco, pH 7.4) broth at 37°C under vigorous shaking (150 rpm/min).

Techniques: In Vitro, Purification, Negative Staining, Tunable Resistive Pulse Sensing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland

doi: 10.3389/fcimb.2018.00277

Figure Lengend Snippet: Identification and distribution of proteins associated with S. aureus N305-secreted EVs. (A) SDS-PAGE (12%) protein separation. Lanes: MW, Molecular weight standards are indicated on the left (kDa); WC, whole-cell lysates; SP, supernatant; EVs, S. aureus N305 EVs. Proteins from the major Blue Coomassie stained bands that correspond to PdhA, PdhB, PdhC, and PdhD, components of the pyruvate dehydrogenase complex, were identified by LC-MS/MS. (B) Vesicular proteome distribution compared to whole bacterial proteome based on their localization with the predictor PsortB. (C) Vesicular proteome distribution compared to whole bacterial proteome based on their localization with the predictor LipoP. TMH, N-terminal transmembrane helices; SPI and II, signal peptidase I or II; CYT, cytoplasmic proteins. (D) Protein distribution based on their COG annotation (IMG source).

Article Snippet: S. aureus N305 (ATCC 29740) was grown in Brain Heart Infusion (BHI) (Difco, pH 7.4) broth at 37°C under vigorous shaking (150 rpm/min).

Techniques: SDS Page, Molecular Weight, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland

doi: 10.3389/fcimb.2018.00277

Figure Lengend Snippet: S. aureus N305-secreted EVs are not cytotoxic in vitro on MAC-T and PS bovine mammary epithelial cells. Either MAC-T or PS cells were treated with different EVs doses: 0.01, 0.1, 1, and 10 μg for 24 h. DMEM alone was used as mock control. Cellular metabolic activity was evaluated by MTT. The results are shown as the percentage of the control. Data are presented as mean ± SD. Each experiment was done in triplicate. The differences among the groups were assessed by ANOVA. Tukey's Honestly Significant Difference test was applied for comparison of means. No cytotoxic effect of EVs in MAC-T or PS cells was observed after 24 h of treatment. *** P < 0.0005.

Article Snippet: S. aureus N305 (ATCC 29740) was grown in Brain Heart Infusion (BHI) (Difco, pH 7.4) broth at 37°C under vigorous shaking (150 rpm/min).

Techniques: In Vitro, Control, Activity Assay, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland

doi: 10.3389/fcimb.2018.00277

Figure Lengend Snippet: S. aureus N305-secreted EVs induce an immunostimulatory response in vitro on PS bovine mammary epithelial cells. Expression of IL-1β, IL-8, TNF-α, and DEFβ1 by bovine mammary epithelial PS cells shown as fold changes at mRNA level measured by RT-qPCR after 3 h post stimulation with either viable S. aureus N305 cells (N305, green), heat-killed S. aureus N305 cells (N305 HK , yellow), 10 μg of purified staphylococcal lipoteichoic acid (LTA, blue), 10 μg and 20 μg of N305 EVs (EV 10 , white; EV 20 , red). Values were calculated as the mean ± SD obtained from three independent experiments after normalization to mock control DMEM. Asterisks indicate statistical significance as evaluated by one-way analysis of variance (ANOVA). **** P < 0.0001; *** P < 0.0005; ** P < 0.005.

Article Snippet: S. aureus N305 (ATCC 29740) was grown in Brain Heart Infusion (BHI) (Difco, pH 7.4) broth at 37°C under vigorous shaking (150 rpm/min).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Purification, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland

doi: 10.3389/fcimb.2018.00277

Figure Lengend Snippet: Histological consequences of inoculation of S. aureus N305-secreted EVs in murine mammary glands. Left panel: Gross pathology of mammary glands. Representative photographs from dissected mice are shown. Conditions are PBS treatment (PBS) (negative control group), viable S. aureus N305 cells (N305) (positive control group), heat-killed S. aureus N305 cells (N305 HK ) (positive control group), 10 μg of purified staphylococcal lipoteichoic acid (LTA) (positive control group), 1 μg of EVs (EV 1 ) (test group) and 10 μg of EVs (EV 10 ) (test group). Arrows emphasize different morphological and histopathological manifestations in the mammary gland post-treatment: 1, healthy lactating mammary gland; 2, severely inflamed mammary gland with bacterial exudates; 3, slightly inflamed mammary gland; 4, moderately inflamed mammary gland. Macroscopic differences resulting from the different treatments of the mammary glands are clearly visible (e.g., prominent redness and inflammation in the S. aureus N305, LTA and EV 10 groups). Middle and right panels: Representative H&E stained tissue sections of the mammary gland from each group acquired at two magnifications are shown; middle panel: 20x, scale bar = 50 μm; left panel: 40x, scale bar = 20 μm. Arrow labeled 5 highlights the milk secrete in the alveolar lumen; arrows labeled 6 marks red blood cells; arrows labeled 7 marks immune cells in the lumen of alveoli. At 24 h p.i. the PBS group did not show any immune cell influx in the alveolar space, the S. aureus N305 group alveolar lumen had a profound hemorrhage and a stronger immune cell influx compared to the S. aureus N305 HK and LTA groups. The EV 1 and EV 10 groups had a dose-dependent recruitment of immune cells with an influx for EV 10 similar to that observed in the LTA group.

Article Snippet: S. aureus N305 (ATCC 29740) was grown in Brain Heart Infusion (BHI) (Difco, pH 7.4) broth at 37°C under vigorous shaking (150 rpm/min).

Techniques: Negative Control, Positive Control, Purification, Staining, Labeling

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Staphylococcus aureus Extracellular Vesicles Elicit an Immunostimulatory Response in vivo on the Murine Mammary Gland

doi: 10.3389/fcimb.2018.00277

Figure Lengend Snippet: Immunological consequences of inoculation of S. aureus N305-secreted EVs in murine mammary glands. Cytokines were quantified from mammary gland lysates using multiplex immunoassay. Conditions are PBS treatment (PBS, gray) (negative control group), live S. aureus N305 (N305, green) (positive control group), heat-killed S. aureus N305 (N305 HK , yellow) (positive control group), purified staphylococcal lipoteichoic acid (LTA, blue) (positive control group), 1 μg of S. aureus N305-secreted EVs (EV 1 , white) and 10 μg of EVs (EV 10 , red). EVs induced significantly the secretion of MIP-2, MCP-1, KC, RANTES, and BAFF. The induction of MIP-2, KC and MCP-1 secretion was dose-dependent. The secretion of the cytokines IL-1α, IL-1β, IL-6, and IL-17A was only induced by S. aureus N305. The secretion of TNF-α was only induced by LTA. Asterisks indicate statistical significance compared to the negative control (PBS) as evaluated by one-way analysis of variance (ANOVA). **** P < 0.0001; *** P < 0.0005; ** P < 0.005; * P < 0.05.

Article Snippet: S. aureus N305 (ATCC 29740) was grown in Brain Heart Infusion (BHI) (Difco, pH 7.4) broth at 37°C under vigorous shaking (150 rpm/min).

Techniques: Multiplex Assay, Negative Control, Positive Control, Purification