Journal: PLoS Biology

Article Title: The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

doi: 10.1371/journal.pbio.1002083

Figure Lengend Snippet: (A) Cultures of AJY3711 ( P GAL - 3xHA-DHR1 ) expressing untagged WT Dhr1 (pAJ3082), WT Dhr1–13myc (pAJ2311), or dhr1 K420A - 13myc (pAJ3081) were shifted to glucose media for 6 h to deplete 3xHA-Dhr1. RNA was prepared from whole cell extracts (Input) or immunoprecipitated samples (IP) and separated by electrophoresis through agarose/formaldehyde gels or denaturing polyacrylamide gels for the A0-A1 fragment and U3. RNAs were detected by Northern blotting using probes specific to A2-A3 (AJO603), D-A2 (AJO130), A0-A1 (AJO1850), and U3 (AJO1686). (B) Table of proteins identified by MS in the Dhr1 K420A particle. Only proteins with at least three peptide-spectrum matches are listed. (C) The CPK (red), 18S rRNA (cyan), and r-proteins identified in the Dhr1 K420A particle (3B) are shown in orange in the structure of the mature S . cerevisiae 40S subunit (left). For comparison the proteins missing from the Dhr1 K420A particle are shown in yellow on the right. The Dhr1 K420A particle likely adopts a more open conformation in the absence of r-proteins.

Article Snippet: Resulting spectra were searched against the UniProtKB YEAST Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FASTA using Sequest HT in the Proteome Discoverer v1.4 software (Thermo Scientific).

Techniques: Expressing, Immunoprecipitation, Electrophoresis, Northern Blot, Comparison

Journal: PLoS Biology

Article Title: The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

doi: 10.1371/journal.pbio.1002083

Figure Lengend Snippet: (A) The parental and a Dhr1-HTP tagged strain were subjected to the CRAC protocol (see ), cross-linked RNA was partially digested, radioactively labeled, ligated to linkers, and after nickel purification resolved on a 4%–12% NuPAGE gel. Protein-RNA complex was transferred to nitrocellulose and RNA was extracted from the regions indicated by a red dashed box. (B) Dhr1 preferentially cross-links to U3. Reads from Dhr1 ( n = 2) and a negative control CRAC experiment were mapped to the 2008 S . cerevisiae genomic reference sequence and mapped reads were assigned to genomic features. The histogram shows the average percentage of all mapped reads that contained box C/D and (C) box H/ACA snoRNA sequences. Note that only a very small fraction of the reads from the control experiment mapped to snoRNAs. (D) Dhr1 preferentially cross-links to the 5′ end of the U3. Plotted is the average read distribution frequency over the U3A (snR17A) gene generated from two Dhr1 CRAC datasets. A schematic representation of the U3 gene and functional sequence elements are indicated below the plot. (E) Same as in (D) but for nucleotide substitutions. The secondary structure of the U3 was adopted from Granneman and colleagues and generated using VARNA ( http://varna.lri.fr ). The coloring indicates the frequency by which the nucleotide was substituted. Additional supporting data are provided in .

Article Snippet: Resulting spectra were searched against the UniProtKB YEAST Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FASTA using Sequest HT in the Proteome Discoverer v1.4 software (Thermo Scientific).

Techniques: Labeling, Purification, Negative Control, Sequencing, Control, Generated, Functional 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: 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: Open Life Sciences

Article Title: Effect of selenium and methods of protein extraction on the proteomic profile of Saccharomyces yeast

doi: 10.1515/biol-2022-0496

Figure Lengend Snippet: Protein extraction from Saccharomyces cerevisiae ATCC MYA-2200

Article Snippet: 8 , GAPDH 3 , P00359 , Saccharomyces cerevisiae (strain ATCC 204508/S288c) , 108 , 19 , 35,838 , 6.46 , C, O , 55 , 0.324 , 0.21.

Techniques: Protein Extraction, Protein Concentration, Extraction

Journal: Open Life Sciences

Article Title: Effect of selenium and methods of protein extraction on the proteomic profile of Saccharomyces yeast

doi: 10.1515/biol-2022-0496

Figure Lengend Snippet: Image of a 2D PAGE experiment demonstrating electrophoretic separation of proteins extracted from S. cerevisiae ATCC MYA-2200 cells cultured in the absence (a), presence of 20 mg Se 4+ /L (b), fused image showing protein spot patterns from the experiment (c). Linear wide range gradients of immobilised pH (pH 3–10) in the first dimension and on 12% SDS-PAGE for the second dimension. The spots marked with small arrows indicate proteins with altered expression under the influence of selenium.

Article Snippet: 8 , GAPDH 3 , P00359 , Saccharomyces cerevisiae (strain ATCC 204508/S288c) , 108 , 19 , 35,838 , 6.46 , C, O , 55 , 0.324 , 0.21.

Techniques: Cell Culture, SDS Page, Expressing

Journal: Open Life Sciences

Article Title: Effect of selenium and methods of protein extraction on the proteomic profile of Saccharomyces yeast

doi: 10.1515/biol-2022-0496

Figure Lengend Snippet: Representatives of 2-DE gel spots in response to selenium stress in the S. cerevisiae ATCC MYA-2200 yeast using mass spectrometry

Article Snippet: 8 , GAPDH 3 , P00359 , Saccharomyces cerevisiae (strain ATCC 204508/S288c) , 108 , 19 , 35,838 , 6.46 , C, O , 55 , 0.324 , 0.21.

Techniques:

Journal: Open Life Sciences

Article Title: Effect of selenium and methods of protein extraction on the proteomic profile of Saccharomyces yeast

doi: 10.1515/biol-2022-0496

Figure Lengend Snippet: Differentially expressed proteins in response to selenium stress in the S. cerevisiae ATCC MYA-2200

Article Snippet: 8 , GAPDH 3 , P00359 , Saccharomyces cerevisiae (strain ATCC 204508/S288c) , 108 , 19 , 35,838 , 6.46 , C, O , 55 , 0.324 , 0.21.

Techniques: Control