Terpenoid phenols, including carvacrol, are components of oregano and other plant essential oils that exhibit potent antifungal activity against a wide range of pathogens, including Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa. To gain a mechanistic view of the cellular response to terpenoid phenols, we used Saccharomyces cerevisiae as a model organism and monitored temporal changes in metabolic activity, cytosolic and vacuolar pH, and Ca 2؉ transients. Using a panel of related compounds, we observed dosedependent Ca 2؉ bursts that correlated with antifungal efficacy. Changes in pH were long lasting and followed the Ca 2؉ transients. A vma mutant lacking functional vacuolar H ؉ -ATPase (V-ATPase) and defective in ion homeostasis was hypersensitive to carvacrol toxicity, consistent with a role for ionic disruptions in mediating cell death. Genomic profiling within 15 min of exposure revealed a robust transcriptional response to carvacrol, closely resembling that of calcium stress. Genes involved in alternate metabolic and energy pathways, stress response, autophagy, and drug efflux were prominently upregulated, whereas repressed genes mediated ribosome biogenesis and RNA metabolism. These responses were strongly reminiscent of the effects of rapamycin, the inhibitor of the TOR pathway of nutrient sensing. The results point to the activation of specific signaling pathways downstream of cellular interaction with carvacrol rather than a nonspecific lesion of membranes, as has been previously proposed.While the medicinal properties of herbs have been recognized since ancient times, there has been a resurgence of interest in the antimicrobial properties of botanical extracts. Essential oils have been amply documented to kill a wide range of pathogenic fungi and bacteria, such as Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, including their drug-resistant variants (6,10,21,22). Of the herbal extracts tested, essential oils derived from the genus Oreganum were among the most effective, with an in vitro MIC of 500 ppm against C. albicans (27). Major components of oregano extract, which include the terpenoid phenols carvacrol, thymol, and eugenol, have potent antifungal activity of their own (4, 23, 24). Terpenoid phenols have been shown to be efficacious not only on planktonic cells but also on biofilms of Candida albicans that are resistant to many antifungal drugs. Carvacrol demonstrated the strongest antifungal activity against Candida albicans biofilms, with a MIC of Ͻ0.03% (9). Furthermore, carvacrol was shown to be effective regardless of the maturity of the biofilm. The terpenoid phenols tested were able to inhibit biofilms of several strains of Candida, including C. albicans, C. glabrata, and C. parapsilosis. In addition to their antimycotic, antibacterial, insecticidal, and bioherbicidal properties, essential oils are also well known for their antioxidant characteristics and are used to inhibit lipid peroxidation in preventing food spoilage or as chemoprotective agen...

ABCG2 is a secretory efflux uric acid transporter of the kidney proximal tubule and gut that plays a key role in uric acid excretion. Genetic defect in ABCG2 leads to significant increases in serum urate levels (SUA) causing hyperuricemia and gout. A single common variant, Q141K, is responsible for the majority of ABCG2 associated gout risk. The Q141K mutation is a loss of function mutation associated with a severe reduction in protein abundance and transport function. Previously we hypothesized the Q141K mutation leads to instability in the nucleotide‐binding domain and here we present the specific molecular mechanism of the defect and a proof of concept of its correction using small molecules. We found using a ABCG2 structural model and targeted amino acid substitutions that the Q141K mutations leads to a localized disruption in packing that affects abundance and can be partially rescued with a secondary substitution at H155A. However the use of the signature motif suppressor mutation, G188E, provides full rescue of the Q141K abundance by stabilizing the NBD dimer sandwich, a finding consistent with a Q141K defect in NBD dimer formation. Finally we show that a small molecule, VRT‐325, known to bind directly to the NBD of ABC transporters can rescue both abundance and function of Q141K ABCG2.

Materials/Methods: Eighty-three RMS (embryonal, nZ74; 89%) patients treated from January 2000 and December 2014 were eligible for analysis. Tumor site was in 46 (55%) RMS patients parameningeal (PM), thereof 34 (74%) PM-RMS patients presented with intra-cranial extension. Seventeen (20%) RMS patients presented with orbital location, 10 with urogenital and 10 (13%) with any other location. Median age at the time of PT was 4.5 years (range, 0.8-15.5). All children received systemic chemotherapy according to prospective protocols and 74 (89%) received concomitant chemotherapy. Tumor size was > 5 cm in 41 (49%) cases. The majority (86%) of patients had cN0 disease. Patients had low-intermediate-and high-risk disease in 24%, 63%, and 13% of cases, respectively. Patients were treated using PBS at the scanning gantry by using energy-degraded beams from the 590-MeV cyclotron until 2005 and subsequently the dedicated 250-MeV cyclotron. Single-field uniform dose (SFUD) plans (nZ29) and intensity modulated proton therapy (IMPT) plans (nZ28), as well as combination of both (nZ26) were used. Median total dose delivered was of 54 (range 41.4 e 64.8) Gy(RBE). Results: After a median follow-up time of 55.5 months (range, 0.9-126.3), the cumulative incidence of local failure was 16 (19%). Four (25%) patients presented concomitant distant failures. The estimated 5year actuarial Kaplan-Meier local control rate was 78.5% (95% CI: 69.5-88.5%). For the subgroups of PM-RMS, orbital RMS, Urogenital RMS and other RMS, the estimated LC rates was 67.5%, 93.8%, 100%, and 77.8% respectively (log-rank test PZ0.065). On univariate analysis tumor location, IRS group, stage, COG risk group and tumor size was a significant predictor of local control. Fourteen patients died, all of tumor progression. The estimated 5-year actuarial Kaplan-Meier OS was 80.6% (95% CI: 71.8-90.0). For the subgroups of PM-RMS, orbital RMS and urogenital RMS and other RMS, the estimated OS rates were 69.6%, 100%, 100% and 76.2%, respectively (log-rank test PZ0.039). Grade 3 acute toxicity was observed in 12 (14%) patients. The estimated 5-year incidence of grade 3 non-ocular late toxicity was 3.6% (95% CI 1-12%). No grade 4-5 late toxicity was observed. Two secondary cancers were observed, 1 of which was radiation-induced. Conclusion: PBS PT was well tolerated and led to excellent outcome in children with RMS. Significant predictors for local failure were group/ stage, tumor location and size. Late non-ocular toxicity was minimal. One radiation-induced malignancy was observed.