They were then resuspended in water or water containing 75 mM HCl and allowed to grow at room temperature for 1.5 hr. (A) Intracellular ROS accumulation was examined after treatment with 5 μg/ml of dihydrorhodamine 123. (B) Activated caspase-like activities were detected

using a FLICA apoptosis detection kit according to the manufacturer’s specifications. At least three independent cultures were tested and compared. The differences were deemed selleck kinase inhibitor statistically significant by the Student’s t-test (p<0.05) Finally, to better understand the mechanism of cell death at the molecular level, we generated microarray gene expression profiles of S. boulardii cells cultured in an acidic environment. We found that a total of 947 genes were differentially expressed (log2 values greater than 2 or less that −2) of which 470 were up-regulated and 457 down-regulated (Additional file 1). Significantly, functional annotation selleck products revealed that the up-regulated genes were significantly (p<0.0005) over-represented in cell death pathways (Figure 5; Table 1). One of these up-regulated cell death genes, RNY1, encodes a RNase T2 family member that is released from the vacuole into the cytosol during oxidative stress to promote yeast cell death [49]. Since the vacuole is the organelle most responsible for pH homeostasis in yeast [50], this may suggest that a similar mechanism of cell death may be occurring in S. boulardii cells

cultured in an acidic environment. Finally, a significant majority of the other up-regulated cell death genes (80%) were ORFs involved in mitochondrial function, including numerous genes encoding proteins involved in the electron transport chain (Table 1). These microarray results together with our characterization of the cell death phenotype described above suggest that S. boulardii cells undergo PCD when they are cultured in acidic Fluorouracil conditions similar to those found in the stomach.

Figure 5 Functional classification/GO analysis of differentially transcribed genes in S. boulardii cells cultured in 50 mM HCl. Genes showing 2-fold or greater increase (up-regulated) or decrease (down-regulated) in response to an acidic environment were grouped in functional categories. Categories that are significantly enriched relative to the yeast proteome are Lorlatinib marked (*: p<0.05; ***: p<0.0005) Table 1 S. boulardii cell death genes differentially expressed in an acidic environment S. BOULARDII CELL DEATH GENES DIFFERENTIALLY EXPRESSED IN AN ACIDIC ENVIRONMENT MCD1 NMA111 NUC1 TAH18 ATP1 ATP2 ATP7 COR1 COX4 COX5A COX6 COX8 CYT1 INH1 OYE3 PIN3 POR1 QCR2 QCR6 QCR7 QCR8 QCR9 QCR1O RIP1 RNY1 SDH1 SDH2 SDH4 UBX6 Saccharomyces boulardii cell death genes showing 2-fold or greater decrease (underlined) or increase (italics) in response to an acidic environment were identified using the Cytoscape 2.8.3 plugin BiNGO 2.44 after Benjamini & Hochberg false discovery correction for multiple hypothesis testing.