Journal: Scientific Reports

Article Title: Fungal cell barriers and organelles are disrupted by polyhexamethylene biguanide (PHMB)

doi: 10.1038/s41598-023-29756-w

Figure Lengend Snippet: The effect of time on PHMB associated membrane permeabilisation. Fungi were treated with PHMB [MIC 50 ] and SYTOX Green (8 µM). Fluorescence profiles for each species treated with PHMB are shown, with positive (heat killed; triton x-114) and negative (Terbinafine; untreated) controls. ( A ) S. cerevisiae heat-killed or treated with 1 µg/ml PHMB, 0.84 µg/ml Terbinafine, 1.17 µg/ml Triton x-114 ( B ) C. albicans R1 heat-killed or treated with 1 µg/ml PHMB, 0.84 µg/ml Terbinafine, 1.17 µg/ml Triton x-114 ( C ) F. oxysporum heat-killed or treated with 2 µg/ml PHMB, 3.84 µg/ml Terbinafine, 4.7 µg/ml Triton x-114 ( D ) P. glabrum heat-killed or treated with 2 µg/ml PHMB, 1.7 µg/ml Terbinafine, 2.5 µg/ml Triton x-114.

Article Snippet: In this present study we explore the antifungal mode of action of PHMB against selected fungal species Saccharomyces cerevisiae (S288c; ATCC), Fusarium oxysporum, Penicillium glabrum and Candida albicans R1 as they represent a selection of common pathogenic yeast and filamentous fungi.

Techniques: Membrane, Fluorescence

Journal: Scientific Reports

Article Title: Fungal cell barriers and organelles are disrupted by polyhexamethylene biguanide (PHMB)

doi: 10.1038/s41598-023-29756-w

Figure Lengend Snippet: Fluorescence imaging showing the effect of PHMB on membrane permeability to SYTOX Green. SYTOX Green (8 µM) and varying PHMB concentrations were added to growth medium before 3 h incubation with S. cerevisiae . Live cell images were merged following imaging by phase contrast and green bandpass filter. Scale bar = 10 µm.

Article Snippet: In this present study we explore the antifungal mode of action of PHMB against selected fungal species Saccharomyces cerevisiae (S288c; ATCC), Fusarium oxysporum, Penicillium glabrum and Candida albicans R1 as they represent a selection of common pathogenic yeast and filamentous fungi.

Techniques: Fluorescence, Imaging, Membrane, Permeability, Incubation

Journal: Scientific Reports

Article Title: Fungal cell barriers and organelles are disrupted by polyhexamethylene biguanide (PHMB)

doi: 10.1038/s41598-023-29756-w

Figure Lengend Snippet: Fluorescence imaging showing the reduction of Con A- membrane fluorescence following exposure to increasing concentrations of PHMB. ( A ) S. cerevisiae cultures were treated with PHMB-rhodamine at 4 µg/ml and 8 µg/ml ( B ) C. albicans cultures were treated with 8 µg/ml and 12 µg/ml. Cultures were incubated at room temperature for 4 h and counter-stained with Con A-Alexa Fluor 488. Untreated control = growth media only. Images show quenching of membrane fluorescence intensity with increasing PHMB concentration. Graphs show measured fluorescence intensity of sampled cells (n = 20) at four symmetrical points along the cell membrane and averaged (mean ± SD). Membrane fluorescence was analysed by RM One-way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: In this present study we explore the antifungal mode of action of PHMB against selected fungal species Saccharomyces cerevisiae (S288c; ATCC), Fusarium oxysporum, Penicillium glabrum and Candida albicans R1 as they represent a selection of common pathogenic yeast and filamentous fungi.

Techniques: Fluorescence, Imaging, Membrane, Incubation, Staining, Control, Concentration Assay, Comparison

Journal: Scientific Reports

Article Title: Fungal cell barriers and organelles are disrupted by polyhexamethylene biguanide (PHMB)

doi: 10.1038/s41598-023-29756-w

Figure Lengend Snippet: The effect of PHMB concentration on fungal cell membrane permeabilisation. SYTOX Green (8 µM) and PHMB concentrations were added to fungi (1 × 10 4 cells/ml) in RPMI-1640, 2% glucose. Samples were incubated for 3 h, with fluorescence measurements taken after the incubation period. ( A ) P. glabrum ( B ) F. oxysporum ( C ) C. albicans ( D ) S. cerevisiae . Yellow arrow = MIC 50 concentration. Blue arrow = MIC 90 concentration.

Article Snippet: In this present study we explore the antifungal mode of action of PHMB against selected fungal species Saccharomyces cerevisiae (S288c; ATCC), Fusarium oxysporum, Penicillium glabrum and Candida albicans R1 as they represent a selection of common pathogenic yeast and filamentous fungi.

Techniques: Concentration Assay, Membrane, Incubation, Fluorescence

Journal: Scientific Reports

Article Title: Fungal cell barriers and organelles are disrupted by polyhexamethylene biguanide (PHMB)

doi: 10.1038/s41598-023-29756-w

Figure Lengend Snippet: PHMB uptake rate by F. oxysporum , S. cerevisiae and C. albicans . The uptake was calculated to be 0.04 µg/ml min −1 , 0.03 µg/ml min −1 , 0.05 µg/ml min −1 respectively at concentrations of 8 µg/ml and above. ( A ) F. oxysporum , ( B ) C. albicans , (C) S. cerevisiae . Red line = MIC 90 concentration , Yellow line = MIC 50 concentration, Blue line = linear regression.

Article Snippet: In this present study we explore the antifungal mode of action of PHMB against selected fungal species Saccharomyces cerevisiae (S288c; ATCC), Fusarium oxysporum, Penicillium glabrum and Candida albicans R1 as they represent a selection of common pathogenic yeast and filamentous fungi.

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Fungal cell barriers and organelles are disrupted by polyhexamethylene biguanide (PHMB)

doi: 10.1038/s41598-023-29756-w

Figure Lengend Snippet: Confocal imaging of S. cerevisiae incubated with PHMB. S. cerevisiae were treated with PHMB-rhodamine (4 µg/ml) for 4 h at room temp. Cells were counterstained with DAPI and Con A-Alexa Fluor 488 and imaged by confocal microscopy. Top panels: Confocal images before (left) and after (right) image processing by SRRF. Bottom-left panel: Cross-sectional view of confocal Z-stacks of S. cerevisiae (59 slices). Images show PHMB-rhodamine accumulation within the cytosol and co-localisation with the nucleus (DAPI). The graph confirms high intracellular accumulation of PHMB-rhodamine.

Article Snippet: In this present study we explore the antifungal mode of action of PHMB against selected fungal species Saccharomyces cerevisiae (S288c; ATCC), Fusarium oxysporum, Penicillium glabrum and Candida albicans R1 as they represent a selection of common pathogenic yeast and filamentous fungi.

Techniques: Imaging, Incubation, Confocal Microscopy

Journal: bioRxiv

Article Title: Analysis of ageing-dependent thiol oxidation reveals early oxidation of proteins involved in core proteostasis functions

doi: 10.1101/2023.05.08.539783

Figure Lengend Snippet: (A) Simplified scheme of the experimental design and data analysis. Yeast cells grown in a complete supplement mixture (CSM) medium with 2% glucose were collected during the mid-logarithmic growth (Log) and four consecutive time points upon entry into the stationary phase. Entry into the stationary phase on day 0 marks the start of yeast chronological ageing. Collected samples were subjected to chemical labelling of reversibly oxidized thiols using the OxICAT technique followed by tryptic digestion for LC-MS/MS analysis. Identified peptides were quantified and the proportion of reversibly oxidized cysteine residues (% oxidation) was calculated. The resulting “yeast OxiAge” dataset was compared to data on age-dependent oxidation in other species. A web application was developed containing a comprehensive database of evolutionarily conserved proteome redox modifications during ageing. Upper panel, samples collection; lower panel, samples and data processing. (B) Lifespan curve of chronologically aged wild-type yeast S. cerevisiae , strain YPH499. Samples were collected on indicated days after entry into the stationary growth phase. Cell viability was determined with propidium iodide (PI) staining and the percentage of dead (stained) cells was determined by FACS. At least two biological replicates per time point were analysed. Data are presented as mean ± SD. (C) Pairwise comparison of in vivo cysteine modification during proliferation (Log) and different stages of chronological ageing (days 0, 3, 6, and 9) from the “yeast OxiAge” dataset (imputed values). Difference in % oxidation between time points is shown on the x-axis; -log 10 p -value (two-tailed Welch’s t -test) is shown on the y-axis.; grey horizontal line, p -value = 0.05. Grey dots, peptides of non-significant change in % oxidation or change of less than 7%; blue dots, peptides of a significant decrease in oxidation of more than 7%; red dots, peptides of a significant increase in oxidation of more than 7%. (D) Distribution of the in vivo oxidation status of 3,178 unique Cys-peptides containing reversible-oxidized cysteine residues from the “yeast OxiAge” dataset (non-imputed values). Peptides from each time point were classified into four oxidation groups based on the average % oxidation. Unique peptides per group were counted for each time point.

Article Snippet: For each cluster, a custom analysis was performed using as an input species yeast S. cerevisiae and as a background cysteine-containing proteins according to amino acid sequences provided from the yeast proteome (yeast proteome retrieved on 14.03.2022 from UniProtKB for strain ATCC 204508 / S288c, Proteome ID UP000002311).

Techniques: Liquid Chromatography with Mass Spectroscopy, Staining, Comparison, In Vivo, Modification, Two Tailed Test

Journal: bioRxiv

Article Title: Analysis of ageing-dependent thiol oxidation reveals early oxidation of proteins involved in core proteostasis functions

doi: 10.1101/2023.05.08.539783

Figure Lengend Snippet: (A) Venn diagram of common proteins found oxidized during ageing in different species. Purple, S. cerevisiae strain YPH499 (“yeast OxiAge”, this study); blue, S. cerevisiae strain DBY746 (Brandes et al .); turquoise, C. elegans strain N2 (Knoefler et al .); green, D. melanogaster strain white Dahomey (Menger et al .); yellow, M. musculus strain C57BL/6 (“Oximouse”, Xiao et al .). (B-H) Pairwise comparison of commonly oxidized proteins (B, D, F, G) and cysteine residues (C, E, H) between species. Orthologs were compared to the whole “yeast OxiAge” dataset (“All clusters”) and each cluster A-D individually. Green, unique proteins from “yeast OxiAge” dataset (B, D, F, G); turquoise, unique cysteine residues from “yeast OxiAge” dataset (C, E, H); blue, unique proteins or cysteine residues from yeast Brandes et al . dataset; brown, unique proteins or cysteine residues from worm Knoefler et al . dataset; grey, unique proteins from fruit fly Menger et al . dataset; pale green, unique proteins or cysteine residues from mice Xiao et al . dataset. (I) Heatmap of average % oxidation of Cys-peptides from yeast Brandes et al . dataset for commonly oxidized cysteine residues with the “yeast OxiAge” dataset. Only peptides from clusters A-D of “yeast OxiAge” and peptides with increased oxidation in Brandes et al . are shown. Peptides are presented in alphabetical order and sorted based on the biological process. (J) Heatmap of average % oxidation of Cys-peptides from the “Oximouse” dataset for commonly oxidized cysteine residues with the “yeast OxiAge” dataset. Only peptides from clusters A-D of “yeast OxiAge” and peptides with increased oxidation of > 5% in “Oximouse” are shown. Peptides are presented in alphabetical order and sorted based on the biological process. The difference in average % oxidation between old and young mice is shown in a single-colour warm palette heatmap.

Article Snippet: For each cluster, a custom analysis was performed using as an input species yeast S. cerevisiae and as a background cysteine-containing proteins according to amino acid sequences provided from the yeast proteome (yeast proteome retrieved on 14.03.2022 from UniProtKB for strain ATCC 204508 / S288c, Proteome ID UP000002311).

Techniques: Comparison

Journal: bioRxiv

Article Title: Analysis of ageing-dependent thiol oxidation reveals early oxidation of proteins involved in core proteostasis functions

doi: 10.1101/2023.05.08.539783

Figure Lengend Snippet: (A) Design of the MySQL database containing information about the average % oxidation of aged proteome of S. cerevisiae (this study and Brandes et al .), C. elegans (Knoefler et al .), D. melanogaster (Menger et al .), and M. musculus (Xiao et al .). Unfiltered data was used to allow the user to perform custom filtering based on individual criteria. The database consists of 14 main tables divided into four groups (grey squares) based on function. Schemes of the tables contain the table name (blue background), the number of records (rows) within a table (sea-green background), and column names within each table. Column names starting with “ID” indicate the identification number referencing a record in another table.

Article Snippet: For each cluster, a custom analysis was performed using as an input species yeast S. cerevisiae and as a background cysteine-containing proteins according to amino acid sequences provided from the yeast proteome (yeast proteome retrieved on 14.03.2022 from UniProtKB for strain ATCC 204508 / S288c, Proteome ID UP000002311).

Techniques: