Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: Analysis of FITC-9R uptake in HeLa cells transfected with siRNAs against COPA, SLC4A4, ATP8B3, and CX40.1. (A) FITC-9R uptake of HeLa cells transfected with 4 individual siRNAs that were selected by primary screening. Scale bars = 10 µm. (B) Fluorescence intensity was measured by MetaMorph. Error bars represent SD from three independent experiments.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Transfection, Fluorescence

Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: (A) RNA interference for COPA and SLC4A4. Interference of each gene was executed by two distinct gene-specific siRNAs. HeLa cells were cultured with each siRNA for 72 h. FITC-9R was added 1 h at 37°C before observation, and cells were observed with confocal microscopy. Scale bars = 10 µm. (B) Fluorescence intensity was measured by MetaMorph. Error bars represent SD from three independent experiments.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Cell Culture, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: (A) HeLa cells were transfected with COPA and/or SLC4A4 siRNAs and cultured for 72 h. Then FITC-9R was added to cells and incubated for 1 h at 37°C. Cells were observed with confocal microscopy. Scale bars = 10 µm. (B) Fluorescence intensity was measured by MetaMorph. Error bars represent SD from three independent experiments.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Transfection, Cell Culture, Incubation, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: (A) Confocal images of EGFP-COPA and EGFP-SLC4A4 localization in HeLa cells. Scale bars, 10 µm. Right Panel, high magnification merge images. Scale bars = 2 µm. (B) Confocal microscopy images of double fluorescence imaging show the co-localization of FITC-9R with COPA or SLC4A4 in HeLa cells. HeLa cells identified by DIC (differential interference contrast), and signaling with FITC-9R (9R) (green) were also positive for COPA (red), and co-localization was evident when images were merged (yellow). Similarly, the expression of SLC4A4 co-localized with FITC-9R is shown. pDsRed empty vector was used as a negative control and did not show co-localization with FITC-9R. Scale bars, 20 µm. (C) High magnification images from Fig. 5A. Scale bars = 10 µm.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Confocal Microscopy, Fluorescence, Imaging, Expressing, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: HeLa cells were transfected COPA or SLC4A4 siRNAs, and after 72-9R or FITC-TAT were added and incubated for 1 h at 37°C. Cells were observed with confocal microscopy (A, C). Scale bars = 10 µm. Fluorescence intensity of FITC-9R and FITC-TAT were measured by MetaMorph (B, D). Error bars represent SD from three independent experiments.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Transfection, Incubation, Confocal Microscopy, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Conditions Under Which Glutathione Disrupts the Biofilms and Improves Antibiotic Efficacy of Both ESKAPE and Non-ESKAPE Species

doi: 10.3389/fmicb.2019.02000

Figure Lengend Snippet: Minimum biofilm inhibitory concentration (MBC) of multidrug resistant isolates of Acinetobacter baumannii (MRAB) in the presence of Amikacin.

Article Snippet: Biofilm fluorescence intensity was then determined at Ex 544 nm and Em 590 nm (Tecan infinite M1000 pro, Australia).

Techniques: Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Conditions Under Which Glutathione Disrupts the Biofilms and Improves Antibiotic Efficacy of Both ESKAPE and Non-ESKAPE Species

doi: 10.3389/fmicb.2019.02000

Figure Lengend Snippet: GSH reduces biofilm viability and enhances antibiotic efficiency. GSH showed a concentration-dependent effect in reducing biofilm viability. Ciprofloxacin (1–3 × MIC) significantly reduced biofilm viability of MRSA, MSSA, S. pyogenes , Enterobacter sp., E. coli and K. pneumoniae. For A. baumannii amikacin (1–5 × MIC) significantly reduced biofilm viability when compared to control. 10 mM GSH did not have any effect on biofilm viability, but 30 mM GSH significantly reduced biofilm viability of all bacterial species. A combination of 30 mM GSH and antibiotic of choice further reduced biofilm viability. ∗ P < 0.05 when compared to control, # P < 0.05 when compared to antibiotic at 1 × MIC and ∙ P < 0.05 when compared to 30 mM GSH. Data represent the mean ± SD of n = 4 experiments performed in biological replicate.

Article Snippet: Biofilm fluorescence intensity was then determined at Ex 544 nm and Em 590 nm (Tecan infinite M1000 pro, Australia).

Techniques: Concentration Assay, Control

Journal: Frontiers in Microbiology

Article Title: Conditions Under Which Glutathione Disrupts the Biofilms and Improves Antibiotic Efficacy of Both ESKAPE and Non-ESKAPE Species

doi: 10.3389/fmicb.2019.02000

Figure Lengend Snippet: Effect of GSH, antibiotics and enzymes on MRAB biofilm biomass and biofilm architecture. (A) Biofilm biomass of MRAB isolates measured using the crystal violet assay showed statistically significant decreases in biomass when treated with amikacin, GSH alone or in combination, compared to untreated control. Amikacin at 1 × MIC reduced biomass to 58–67% whereas at 5 × MIC, biomass was reduced to between 38 and 46% for all isolates. 30 mM GSH decreased biomass to 43–52%, while a combination of GSH + amikacin decreased biomass by 40–45% for all isolates. (B) Both 20 and 40U DNase-I significantly reduced biofilm biomass (56–73% for all MRAB isolates). At higher concentrations of amylase (500 and 1000 μg/ml) and Proteinase K (200 and 500 μg/ml) treatment reduced biofilm biomass by 61–79% and 54–93%, respectively. (C) Biofilm architecture of MRAB-3 imaged using CLSM and complemented with Live/dead bacterial viability stain, showed a marked and distinct type of disruption in biofilm architecture when treated singly with amikacin, GSH, or enzyme, or combinations thereof. In panels (A,B) ∗ P < 0.05 when compared to control and # P < 0.05 when compared to 4 μg/ml amikacin. Data represent the mean ± SD of n = 4 experiments performed in biological replicate.

Article Snippet: Biofilm fluorescence intensity was then determined at Ex 544 nm and Em 590 nm (Tecan infinite M1000 pro, Australia).

Techniques: Crystal Violet Assay, Control, Staining, Disruption

Journal: Frontiers in Microbiology

Article Title: Conditions Under Which Glutathione Disrupts the Biofilms and Improves Antibiotic Efficacy of Both ESKAPE and Non-ESKAPE Species

doi: 10.3389/fmicb.2019.02000

Figure Lengend Snippet: Effect of GSH, GSSG and H 2 O 2 at neutral pH on MRAB biofilm viability. (A) 30 mM GSH (buffered to pH 7) resulted in a small biofilm viability decrease (76–94% viable), while combination with amikacin enhanced the decrease significantly (47–55% viable) when compared to both control and 30 mM GSH (pH 7) alone. (B) 30 mM GSSG alone and in combination with 4 μg/ml amikacin significantly decreased MRAB biofilm viability to 11–14% and 5–6%, respectively, when compared to control. (C,D) Standard H 2 O 2 data used to determine H 2 O 2 production by 10 and 30 mM GSH in 1 × PBS at intrinsic pH, and at buffered neutral pH 7. At intrinsic pH, GSH produced significantly more H 2 O 2 (higher fluorescent intensity) than at its corresponding neutral pH concentration. (E) H 2 O 2 treatment showed variation in MRAB biofilm viability among isolates. When compared to control only, MRAB-1 and MRAB-3 isolates showed statistically significant decreases in biofilm viability at all H 2 O 2 concentrations. However, for MRAB-2 and MRAB-4, biofilm viability was similar to control at all H 2 O 2 concentrations. ∗ P < 0.05 compared to control ∙ P < 0.05 when compared to 30 mM GSH (A) and 30 mM GSSG (B) and # P < 0.05 when compared to all other conditions (D) . Data represent the mean ± SD of n = 4 experiments performed in biological replicate.

Article Snippet: Biofilm fluorescence intensity was then determined at Ex 544 nm and Em 590 nm (Tecan infinite M1000 pro, Australia).

Techniques: Control, Produced, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Conditions Under Which Glutathione Disrupts the Biofilms and Improves Antibiotic Efficacy of Both ESKAPE and Non-ESKAPE Species

doi: 10.3389/fmicb.2019.02000

Figure Lengend Snippet: CFU count of MRAB biofilms after GSH, amikacin and DNase-I treatment: CFU/ml of control, amikacin, DNase-I and GSH of MRAB isolates was log 10 9–10.2, log 10 5.1–8.2, log 10 8.3–10.2 and 5.1–6.5, respectively. Combination treatments comprising: DNase-I + amikacin, GSH + amikacin and GSH + DNase-I on biofilms resulted in a CFU/ml of log 10 4.9–6.2, 3.7–5.3, and 4.6–5.9, respectively. The triple combination of GSH + DNase-I + amikacin decreased the CFU/ml to log 10 4.3–4.9. Data represent the mean ± SD of n = 3 experiments performed in biological replicate. ∗ P < 0.05 compared to control, # P < 0.05 when compared to amikacin vs. GSH + amikacin and ∗∗ P < 0.05 when compared to GSH vs. GSH + amikacin.

Article Snippet: Biofilm fluorescence intensity was then determined at Ex 544 nm and Em 590 nm (Tecan infinite M1000 pro, Australia).

Techniques: Control