Journal: Anaerobe

Article Title: A 3D intestinal tissue model supports Clostridioides difficile germination, colonization, toxin production and epithelial damage

doi: 10.1016/j.anaerobe.2018.02.006

Figure Lengend Snippet: Uninfected 3D scaffolds (A and D) or 2D transwells (G and J), NT C. difficile infected 3D scaffolds (B and E) or 2D transwells (H and K) and UK1 C. difficile infected 3D scaffolds (C and F) or 2D transwells (I and L) were fixed at 4hrs (SC: A–C and TW: G–I), and 48 hrs (SC: D–F and TW: J–L) and immunostained with antibody against ZO-1 (Green) and with DAPI (Blue). Samples were visualized by confocal microscopy. Representative images of 3D scaffolds and 2D transwells are shown at 20× magnification and 40× magnfication, respectively. White arrows indicate intact tight junctions. The extent of tight junction loss (M–N) and monolayer disruption (O–P) was quantified by three individuals who scored blinded samples. The scores were averaged for each sample and scores for each infection condition at each time point were averaged. The average and SD for tight junction loss (M–N) and monolayer disruption (O–P) are presented. Statistical significance was calculated using the Kruskal-Wallis test followed by Dunn’s post-test. Only statistically significant comparisons are shown (* p= 0.03, **p= 0.01, ***p=0.003,****p= 0.0009).

Article Snippet: Confocal 3D maximum projection images were assembled with a NIS-Elements AR software package (ver 4.20.01, Nikon) and ImageJ.

Techniques: Infection, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Real-time imaging of yeast cells reveals several distinct mechanisms of curing of the [URE3] prion

doi: 10.1074/jbc.M117.809079

Figure Lengend Snippet: Characterization of the aggregates formed by 1660[URE3] overexpressing Btn2, Hsp42, or Cur1. A, the fluorescence recovery after photobleaching of aggregates present in 1660[URE3] yeast overexpressing Btn2, Cur1, and Hsp42. A minimum of five aggregates were photobleached for each sample on the Nikon A1R confocal microscope. The data were normalized by setting the initial fluorescence value to 1.0. The average value and a standard deviation are plotted for each time point. Photobleaching experiments were performed on the Nikon A1R confocal microscope. B, the time course of curing of 1660[URE3] by overexpression of Btn2–RFP, Cur1–RFP, and Hsp42–Cherry. The data represent the averages and standard deviation obtained from three independent experiments. C, colocalization of GFP–Ure2 with Btn2, Hsp42, and Cur1. Btn2–RFP or Hsp42–Cherry was overexpressed for two generations. Cur1–RFP was overexpressed for five generations. The asterisk shows colocalization of Ure2–GFP aggregate with Cur1–RFP. The maximum Z-stack projections of confocal images obtained on the Nikon A1R confocal microscope.

Article Snippet: The maximum Z -stack projections of confocal images obtained on the Nikon A1R confocal microscope.

Techniques: Fluorescence, Microscopy, Standard Deviation, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Real-time imaging of yeast cells reveals several distinct mechanisms of curing of the [URE3] prion

doi: 10.1074/jbc.M117.809079

Figure Lengend Snippet: Mechanism of curing [URE3] by Cur1 and Cur1–NLS. A, the curing of 1660[URE3] yeast was measured as a function of generation time in yeast overexpressing RFP-labeled Cur1–NLS or in yeast coexpressing Sis1 and RFP-labeled Cur1 or in yeast coexpressing Sis1 and RFP-labeled Cur1–NLS. The dashed line is the data shown in Fig. 3B for the curing of 1660[URE3] by overexpression of RFP-labeled Cur1. The Sis1 and the RFP-labeled Cur1 constructs were both overexpressed from the GAL1 promoter. B, colocalization of Sis-GFP with RFP-labeled Cur1 or Cur1–NLS. The cells were imaged after five generations in galactose medium to induce expression of the different Cur1 constructs. C, the intensity of GFP-labeled Sis1 in the nucleus compared with its intensity in the cytoplasm was measured in vector control cells or cells overexpressing either RFP-labeled Cur1 or Cur1 NLS. After fixing the cells with 4% paraformaldehyde, the nucleus was stained with 4′,6′-diamino-2-phenylindole to delineate the nucleus. The ImageJ program was used to measure the GFP fluorescence intensity in the nuclear and cytoplasmic compartments. A minimum of 30 cells were measured for each condition. The average values and standard deviation are plotted for each condition. D, images of 1660[URE3] yeast overexpressing RFP-labeled Cur1–NLS for five generations. The maximum Z-stack projections of confocal images were obtained on the Nikon A1R confocal microscope.

Article Snippet: The maximum Z -stack projections of confocal images obtained on the Nikon A1R confocal microscope.

Techniques: Labeling, Over Expression, Construct, Expressing, Plasmid Preparation, Staining, Fluorescence, Standard Deviation, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Real-time imaging of yeast cells reveals several distinct mechanisms of curing of the [URE3] prion

doi: 10.1074/jbc.M117.809079

Figure Lengend Snippet: The curing of 1660[URE3] by other molecular chaperones. A, the rate of curing of 1660[URE3] was measured using the red/white colony assay for yeast overexpressing Ydj1, Yjd1(D36N), DnaJB6, Hsp104, or coexpressing Hsp104 and DnaJB6. All proteins were overexpressed from the GAL1 promoter. The data points represent the averages and standard deviation obtained from three independent experiments. B, images of 1660[URE3] overexpressing either Ydj1 or DnaJB6 for either two generations or four generations. The maximum Z-stack projections of confocal images were obtained on the Zeiss 880 confocal microscope. C, images of 1660[URE3] prior to overexpression and overexpressing Hsp104 for nine generations from the GAL1 promoter. The images are maximized projections of Z-stack confocal images obtained on the Nikon A1R confocal microscope.

Article Snippet: The maximum Z -stack projections of confocal images obtained on the Nikon A1R confocal microscope.

Techniques: Colony Assay, Standard Deviation, Microscopy, Over Expression