Journal: eLife

Article Title: The RAM signaling pathway links morphology, thermotolerance, and CO 2 tolerance in the global fungal pathogen Cryptococcus neoformans

doi: 10.7554/eLife.82563

Figure Lengend Snippet: ( A ) H99 cells were plated onto RPMI solid medium buffered to either pH 6 or pH 7. Fluconazole containing E-test strips were placed onto the lawn of H99 cells, and the plates were incubated at 37°C in ambient air or in 5% CO 2 . The larger the halo surrounding the E-strip, the more sensitive the cells are to fluconazole. The intercept value of the halo with the E-strip is the minimal inhibitory concentration. ( B ) Cells of the previously identified CO 2 -tolerant H99 and CO 2 -sensitive A7-35-23 were serial diluted, spotted onto RPMI media buffered to pH 6 or pH 7, and incubated at 37°C in ambient air or in 5% CO 2 .

Article Snippet: CO 2 levels were controlled by a VWR CO 2 incubator or by a Pro-CO 2 controller (Biospherix, Lacona, NY, USA).

Techniques: Incubation, Stripping Membranes, Concentration Assay

Journal: eLife

Article Title: The RAM signaling pathway links morphology, thermotolerance, and CO 2 tolerance in the global fungal pathogen Cryptococcus neoformans

doi: 10.7554/eLife.82563

Figure Lengend Snippet: ( A ). The clinical reference strain H99 and environmental strains A7-35-23 and A1-38-2 were grown overnight in yeast peptone dextrose (YPD), serially diluted, and spotted onto solid YPD media plates. Photographs were taken 2 days after incubation in the indicated condition. ( B ) This serial dilution spotting assay was similarly performed for H99 and the mutants indicated. Two independent overexpression transformants for each mutant background were included as biological replicates. ( C ) Heatmap showing normalized total RNA counts of NanoString targets in H99 and cbk1Δ cultured at either ambient or 5% CO 2 , red indicates higher and blue indicates lower transcript abundance. ( D ) Volcano plot showing significantly differentially expressed transcripts (p-value of <0.05) in the cbk1Δ compared to H99 in the 5% CO 2 condition. ( E ) Serial dilution spotting assay of H99 and four of the mutants found in the deletion set screening to be CO 2 sensitive which also correspond to significantly downregulated genes shown in the volcano plot.

Article Snippet: CO 2 levels were controlled by a VWR CO 2 incubator or by a Pro-CO 2 controller (Biospherix, Lacona, NY, USA).

Techniques: Incubation, Serial Dilution, Spotting Assay, Over Expression, Mutagenesis, Cell Culture

Journal: eLife

Article Title: The RAM signaling pathway links morphology, thermotolerance, and CO 2 tolerance in the global fungal pathogen Cryptococcus neoformans

doi: 10.7554/eLife.82563

Figure Lengend Snippet: ( A and B ) The strains above were grown overnight in yeast peptone dextrose (YPD), serially diluted, and spotted onto the indicated solid media. YPD +2% congo red was used to assay cell wall stress tolerance. YPD +1 μg/mL FK506 was used to assay growth under calcineurin inhibition. YPD +4 mM H 2 O 2 was used to assay oxidative stress tolerance. L-DOPA media was used to assay melaninization. RPMI media buffered with MOPS(3-(N-Morpholino)propanesulfonate) was used to test tolerance to 37°C or 37°C+5% CO 2 at pH 6 and pH 7.4. Photographs were taken 2 days of incubation. Christensen Urea Agar was used to assay urease activity, indicated by change in media coloration from yellow to pink. ( C ) The cells incubated for 2 days on RPMI pH 7.4, 37°C+5% CO 2 , were stained with India Ink and observed under the microscope to check capsule size. ( D ) Cells were grown overnight in liquid culture and then diluted to the concentration of OD 600 =0.01. Equal volumes of the indicated MAT a and MATα cells were mixed, and 3 μL was spotted onto V8 pH 5.0 solid medium. After incubation at 22°C for 2 weeks in the dark, mating colonies were examined with a SZX16 stereoscope (Olympus).

Article Snippet: CO 2 levels were controlled by a VWR CO 2 incubator or by a Pro-CO 2 controller (Biospherix, Lacona, NY, USA).

Techniques: Inhibition, Incubation, Activity Assay, Staining, Microscopy, Concentration Assay

Journal: eLife

Article Title: The RAM signaling pathway links morphology, thermotolerance, and CO 2 tolerance in the global fungal pathogen Cryptococcus neoformans

doi: 10.7554/eLife.82563

Figure Lengend Snippet: Heatmap showing normalized total RNA counts of NanoString targets in H99, cbk1Δ , sup1 , and sup2 cultured at either ambient or 5% CO 2 . Red indicates higher and blue indicates lower transcript abundance.

Article Snippet: CO 2 levels were controlled by a VWR CO 2 incubator or by a Pro-CO 2 controller (Biospherix, Lacona, NY, USA).

Techniques: Cell Culture

Journal: Scientific Reports

Article Title: Elevated CO 2 regulates the Wnt signaling pathway in mammals, Drosophila melanogaster and Caenorhabditis elegans

doi: 10.1038/s41598-019-54683-0

Figure Lengend Snippet: Validation of mouse multi-tissue microarray analysis. ( a,b) Validation in mouse tissues. Fzd9 , Wnt4 , Wnt7a , and Wnt8b expression in the lung (n = 6–7) ( a ) and diaphragm skeletal muscle (n = 4–5) ( b ) from mice exposed to normoxic hypercapnia for 7 days. NC, normocapnia; HC, hypercapnia. ( c,d) Validation in mouse lung and skeletal muscle cells. Fzd9 and Wnt7a expressions in MLE-12 cells (Ctrl, n = 22–23; 20%CO 2 , n = 5 per group), ASM cells (Ctrl, n = 22–23; 20%CO 2 , n = 5 per group) ( c ), or C2C12 myoblast (Ctrl, n = 18–19; 20%CO 2 , n = 4–5 per group) or myotube (Ctrl, n = 14–15; 20%CO 2 , n = 3–4 per group) ( d ) exposed to high CO 2 for up to 24 hours (c) or 6 hours (d). Ctrl, control conditions. All values are represented as mean with error bars shown as the 95% confidence interval. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test or one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The atmosphere of the C-Chamber was controlled with a PRO CO 2 carbon dioxide controller (BioSpherix).

Techniques: Microarray, Expressing, Control, Two Tailed Test

Journal: Scientific Reports

Article Title: Elevated CO 2 regulates the Wnt signaling pathway in mammals, Drosophila melanogaster and Caenorhabditis elegans

doi: 10.1038/s41598-019-54683-0

Figure Lengend Snippet: Validation of the transcriptomic datasets of hypercapnia in a human bronchial cell line and invertebrates. ( a) FZD9 and WNT7a expression in BEAS-2B cells exposed to high CO 2 for up to 12 hours (Ctrl, n = 20–23; 20%CO 2 , n = 5–6 per group). Ctrl, control conditions. ( b) Fz and wg expression in Drosophila S2 cells exposed to high CO 2 for up to 30 min (Ctrl, n = 14–15; 20%CO 2 , n = 4–5 per group). All values are represented as mean with error bars shown as the 95% confidence interval. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The atmosphere of the C-Chamber was controlled with a PRO CO 2 carbon dioxide controller (BioSpherix).

Techniques: Expressing, Control