Citronellal Exerts Its Antifungal Activity by Targeting Ergosterol Biosynthesis in Penicillium digitatum

Ergosterol (ERG) is a potential target for the development of antifungal agents against Penicillium digitatum, the pathogen of green mold in citrus fruits. This study examined the mechanism by which citronellal, a typical terpenoid of Cymbopogon nardus essential oil, acts on ergosterol to exhibit its antifungal activity against P. digitatum. We previously reported that citronellal inhibited the growth of P. digitatum with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 1.36 and 2.72 mg/mL, respectively. In citronellal-treated cells, the membrane integrity and ergosterol contents significantly decreased, whereas lanosterol, which serves as a precursor for ergosterol biosynthesis, massively accumulated. Addition of 150 mg/L of exogenous ergosterol decreased the inhibitory rate of citronellal, restoring the ergosterol content and hence the membrane structure to normal levels, and triggered expression of nearly all ERG genes. Based on our findings, we deduce that citronellal damages the cell membrane integrity of P. digitatum by down-regulating the ERG genes responsible for conversion of lanosterol to ergosterol, the key downregulated gene being ERG3, due to the observed accumulation of ergosta-7,22-dienol.

Antifungal Resistance in Candida auris: Molecular Determinants

Since Candida auris integrates strains resistant to multiple antifungals, research has been conducted focused on knowing which molecular mechanisms are involved. This review aims to summarize the results obtained in some of these studies. A search was carried out by consulting websites and online databases. The analysis indicates that most C. auris strains show higher resistance to fluconazole, followed by amphotericin B, and less resistance to 5-fluorocytosine and caspofungin. In C. auris, antifungal resistance to amphotericin B has been linked to an overexpression of several mutated ERG genes that lead to reduced ergosterol levels; fluconazole resistance is mostly explained by mutations identified in the ERG11 gene, as well as a higher number of copies of this gene and the overexpression of efflux pumps. For 5-fluorocytosine, it is hypothesized that the resistance is due to mutations in the FCY2, FCY1, and FUR1 genes. Resistance to caspofungin has been associated with a mutation in the FKS1 gene. Finally, resistance to each antifungal is closely related to the type of clade to which the strain belongs.

Overexpression or Deletion of Ergosterol Biosynthesis Genes Alters Doubling Time, Response to Stress Agents, and Drug Susceptibility inSaccharomyces cerevisiae

ABSTRACTErgosterol (ERG) is a critical sterol in the cell membranes of fungi, and its biosynthesis is tightly regulated by 25 known enzymes along the ERG production pathway. The effects of changes in expression of each ERG biosynthesis enzyme inSaccharomyces cerevisiaewere analyzed by the use of gene deletion or plasmid-borne overexpression constructs. The strains overexpressing the ERG pathway genes were examined for changes in doubling time and responses to a variety of stress agents. In addition, ERG gene overexpression strains and ERG gene deletion strains were tested for alterations in antifungal drug susceptibility. The data show that disruptions in ergosterol biosynthesis regulation can affect a diverse set of cellular processes and can cause numerous phenotypic effects. Some of the phenotypes observed include dramatic increases in doubling times, respiratory deficiencies on glycerol media, cell wall insufficiencies on Congo red media, and disrupted ion homeostasis under iron or calcium starvation conditions. Overexpression or deletion of specific enzymes in the ERG pathway causes altered susceptibilities to a variety of classes of antifungal ergosterol inhibitors, including fluconazole, fenpropimorph, lovastatin, nystatin, amphotericin B, and terbinafine. This analysis of the effect of perturbations to the ERG pathway caused by systematic overexpression of each of the ERG pathway genes contributes significantly to the understanding of the ergosterol biosynthetic pathway and its relationship to stress response and basic biological processes. The data indicate that precise regulation of ERG genes is essential for cellular homeostasis and identify several ERG genes that could be exploited in future antifungal development efforts.IMPORTANCEA common target of antifungal drug treatment is the fungal ergosterol biosynthesis pathway. This report helps to identify ergosterol biosynthesis enzymes that have not previously been appreciated as drug targets. The effects of overexpression of each of the 25 ERG genes inS. cerevisiaewere analyzed in the presence of six stress agents that target essential cellular processes (cell wall biosynthesis, protein translation, respiration, osmotic/ionic stress, and iron and calcium homeostasis), as well as six antifungal inhibitors that target ergosterol biosynthesis. The importance of identifying cell perturbations caused by gene overexpression or deletion is emphasized by the prevalence of gene expression alterations in many pathogenic and drug-resistant clinical isolates. Genes whose altered expression causes the most extensive phenotypic alterations in the presence of stressors or inhibitors have the potential to be drug targets.

Transdifferentiation of erythroblasts to megakaryocytes using FLI1 and ERG transcription factors

SummaryPlatelet transfusion has been widely used to prevent and treat life-threatening thrombocytopenia; however, preparation of a unit of concentrated platelet for transfusion requires at least 4–6 units of whole blood. At present, a platelet unit from a single donor can be prepared using apheresis, but lack of donors is still a major problem. Several approaches to produce platelets from other sources, such as haematopoietic stem cells and pluripotent stem cells, have been attempted but the system is extremely complicated, time-consuming and expensive. We now report a novel and simpler technology to obtain platelets using transdifferentiation of human bone marrow erythroblasts to megakaryocytes with overexpression of the FLI1 and ERG genes. The obtained transdifferentiated erythroblasts (both from CD71+ and GPA+ erythroblast subpopulations) exhibit typical features of megakaryocytes including morphology, expression of specific genes (cMPL and TUBB1) and a marker protein (CD41). They also have the ability to generate megakaryocytic CFU in culture and produce functional platelets, which aggregate with normal human platelets to form a normal-looking clot. Overexpression of FLI1 and ERG genes is sufficient to transdifferentiate erythroblasts to megakaryocytes that can produce functional platelets.

BAALC and EVI1 Prognostic Gene Expression in Adult Acute Myeloid Leukemia Using the Amlprofiler Custom Microarray

Abstract 1420 Background High expression of several genes have been found to be prognostically relevant in AML, such as EVI1, BAALC, MN1 and ERG [1–4]. These studies have mostly reported cutpoints at expression level quartiles with the highest prognostic value. Translation to the clinic remains difficult because a quartile definition cannot be applied to an individual patient. Therefore, from a clinical practice point, standardization has remained an important objective. Aim Using a standardized microarray for gene expression profiling we set out to develop and validate expression cutpoints for the EVI1, BAALC, MN1 and ERG genes that best predict overall survival (OS) for intermediate cytogenetic risk cases, rather than normal karyotype. Methods For training purposes, we employed a cohort of 147 intermediate cytogenetic risk cases [5, 6], and an external dataset of 242 cases [7]. For validation purposes, a cohort of 215 intermediate cytogenetic risk cases was employed [6]. Expression of the EVI1, BAALC, MN1 and ERG genes was measured using a custom microarray (AMLprofiler) with IVD-grade reagents and Affymetrix DX2 equipment. For the BAALC, MN1 and ERG genes, cutpoints were considered from the 10th to 90th percentile in steps of 5%. Optimal cutpoints were selected by performing 100-fold cross validations of the expression data of the training cohort and external cohort. EVI1 has a discontinuous distribution of expression [1]. Because of the low prevalence of high-EVI1 in AML we did not perform cross validation but used the entire training cohort to derive the best cutpoint. Results For BAALC the 30th percentile cutpoint was verified to be clinically relevant in the training and external cohorts (Figure 1). ERG had no significant splits in the training data, and around the 65th percentile in the external cohort (Figure 1). MN1 had a broad range of significant splits around its 30th expression percentile in the training cohort but none in the external cohort (Figure 1). Due to the lack of consistent results across datasets, we have not attempted a validation of ERG and MN1. The best EVI1 cutpoint in the training set was found to be the 6th percentile. This cutpoint turned out to be concordant with a previously developed Q-PCR cutpoint [8, 9]. Validation of the BAALC and EVI1 cutpoints on the independent validation cohort of intermediate cytogenetic risk cases resulted in significantly more favorable OS for cases with low-BAALC expression (HR 0.686, p=0.0205, Figure 2) and worse OS for cases with high-EVI1 expression (HR 2.27, p=0.004, Figure 2). In a multivariable analysis, including age, gender, white blood cell count and, blast percentage in BM, high-EVI1 retained independent prognostic value [HR 2.39, p=.008] while low-BAALC retained independent prognostic value only within the group of low-EVI1 cases [HR 0.602, p=.015]. Conclusion For BAALC and EVI1, but not for MN1 and ERG, clinically relevant cutpoints have been developed that remain independent prognostic factors in intermediate cytogenetic risk AML and which through their incorporation in the AMLprofiler assay can be used for standardized measurements across different laboratories. Disclosures: van Beers: skyline diagnostics: Employment, Equity Ownership, Patents & Royalties. Brand:skyline diagnostics: Employment, Equity Ownership. van Vliet:skyline diagnostics: Employment, Equity Ownership. Vietor:skyline diagnostics: CEO skyline diagnostics Other, Equity Ownership. Valk:skyline diagnostics: Equity Ownership. Lowenberg:skyline diagnostics: CSO skyline diagnostics Other, Equity Ownership.

Overexpression of BAALC and MN1 Predict Worse Outcome in De Novo AML with Partial Tandem Duplication of MLL.

Abstract 1002 Poster Board I-24 Background and purpose: Overexpression of BAALC, MN1, or ERG gene has been described to have adverse impact on the outcome of acute myeloid leukemia (AML) with normal karyotype. The majority of patients with partial tandem duplication of MLL gene (MLL-PTD) had normal karyotypes. The prognostic relevance of overexpression of these genes in MLL-PTD AML was not clear. Aims: We aimed to (1) measure the mRNA expression levels of FLT3, BAALC, FHIT, MN1, and ERG genes in AML patients with MLL-PTD (2) compare the expression levels of these genes with normal controls, and (3) determine their prognostic significance. Patients and methods: Bone marrow samples from 93 de novo AML patients with MLL-PTD at diagnosis were analyzed. MLL-PTD was screened by Southern blot analysis or reverse transcriptase polymerase chain reaction (RT-PCR) then confirmed by real-time quantitative PCR (RQ-PCR). RQ-PCR assay with TaqMan probe was performed to measure the expression of FLT3, BAALC, FHIT, MN1, and ERG genes in MLL-PTD AML as well as in 34 normal controls for comparison. The expression levels of target genes were calculated as the copy number of each gene normalized to the copy number of ABL control gene (NCN). Positive and negative controls as well as standard curve constructs were included in each assay. Mutational analysis was performed by DNA/cDNA PCR followed by GeneScan analysis for detection of internal tandem duplication of FLT3 gene (FLT3/ITD). The expression levels of each target genes were dichotomized at the median value to low and high expression groups. The event-free and overall survivals (EFS and OS) were compared between the 2 groups. Results: The expression levels of mRNAs for FLT3, BAALC, FHIT, MN1, and ERG genes at diagnosis in MLL-PTD AML and 34 normal controls are shown in Table. MLL-PTD patients had significantly higher expression levels of FLT3, BAALC, MN1, and ERG compared to normal controls. The expression of FHIT was also higher than that of controls, but did not reach statistic significance. FLT3/ITD was present in 26 of 52 patients (50%). The expression levels of the above genes were not different between patients with FLT3/ITD and those without. The median age of the entire cohort was 56 years. The median EFS and OS of the 52 patients who received standard induction chemotherapy were 5.8 and 11.4 months, respectively. The complete remission (CR) rate was higher in the low expression group than that of high expression group for BAALC (P = 0.011). The CR rate was not significantly different between low and high expression groups for FLT3, FHIT, MN1, or ERG, and between FLT3/ITD(+) and (-) groups. There were no significant difference in EFS or OS between patients with FLT3/ITD and those without. Patients with high expression of BAALC had a significantly shorter survival than those with low expression group; the median EFS was 10.3 mons (95% CI: 5.9-14.7 mons) vs. 0 mon, P = 0.044 and the median OS was 16.4 mons (95% CI: 8.3-25.5 mons) vs. 10.9 mons (95% CI: 6.5-15.3 mons), P = 0.031. Patients with high expression of MN1 also had a poor outcome compared with low expression group; the median EFS was 10.9 mons (95% CI: 0-28.3 mons) vs. 4.1 mons (95% CI: 0-11.7mons), P = 0.002 and the median OS was 29.7 mons (95% CI: 0-70.7mons) vs.11.0 mons (95% CI: 10.7-11.3 mons), P = 0.024. The EFS and OS were not significantly different between low and high expression groups for FLT3, BAALC, FHIT, and ERG. Conclusions: Our results showed that MLL-PTD was associated with overexpression of FLT3, BAALC, MN1, and ERG. The patients with lower expression level of BAALC had a higher CR rate and patients with overexpression of BAALC or MN1 had poor EFS and OS. FLT3/ITD had no prognostic impact. Supported by grants NSC97-2314-B-182 -011-MY3, NSC96-2314-B-195-006-MY3, and MMH-E-96009. Disclosures: No relevant conflicts of interest to declare.