Modulation of ERG gene expression in fluconazole-resistant human and animal isolates of Trichophyton verrucosum

AbstractDermatophytes are a group of eukaryotic microorganisms characterized by high capacity to colonize keratinized structures such as the skin, hair, and nails. Over the past years, the incidence of infections caused by zoophilic species, e.g., Trichophyton verrucosum, has been increasing in some parts of the world, especially in Europe. Moreover, the emergence of recalcitrant dermatophytoses and in vitro resistant dermatophytes has become a cause of concern worldwide. Here, we analyzed the mechanisms underlying resistance to fluconazole among clinical isolates of T. verrucosum. Quantitative RT-PCR was carried out to determine the relative expression levels of mRNA transcripts of ERG3, ERG6, and ERG11 genes in the fungal samples using the housekeeping gene GAPDH as a reference. Our results showed that the upregulation of the ERG gene expression is a possible mechanism of resistance to fluconazole in this species. Furthermore, ERG11 is the most statistically significantly overexpressed gene in the pool of fluconazole-resistant T. verrucosum isolates. Additionally, we have demonstrated that exposure to fluconazole increases the levels of expression of ERG genes in fluconazole-resistant isolates of T. verrucosum. In conclusion, this study has shown one of the possible mechanisms of resistance to fluconazole among zoophilic dermatophytes, which involves the maintenance of high levels of expression of ERG genes after drug exposure.

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229 EXPRESSION OF mRNA ENCODING GLYCOLYTIC ENZYMES IN BOVINE CUMULUS CELLS DURING IN VITRO MATURATION: EFFECTS OF TIME AND FSH

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab229 ◽

2010 ◽

Vol 22 (1) ◽

pp. 272

Author(s):

E. S. Caixeta ◽

P. Ripamonte ◽

M. F. Machado ◽

R. B. da Silva ◽

C. Price ◽

...

Keyword(s):

Gene Expression ◽

Expression Pattern ◽

In Vitro Maturation ◽

Housekeeping Gene ◽

Cumulus Cells ◽

Glycolytic Enzymes ◽

Mrna Abundance ◽

Relative Expression ◽

Significant Difference

Mammalian oocytes require pyruvate as an energy source for growth and resumption of meiosis. Because oocytes are not competent to carry out glycolysis, cumulus cells (CC) are responsible for metabolizing glucose into pyruvate and providing it to the oocyte through gap junctions. The understanding of the energetic metabolism of CC in culture conditions might provide basis for the improvement of COC in vitro maturation. The aim of this study was to determine the temporal patterns of mRNA expression of glycolytic enzymes [phosphofructokinase (PFKP), aldolase (ALDOA), triosephosphate isomerase (TPI), enolase (ENO1), pyruvate kinase (PKM2), and lactate dehydrogenase (LDHA)] in bovine CC during COC in vitro maturation with or without FSH. Immature COC (grades 1 and 2) were obtained from 2- to 8-mm follicles from abattoir ovaries (predominantly Bos indicus). Cumulus cells were separated from COC and frozen before (immature group) or after COC culture for 4, 8, 12, 16, and 20 hours with (10 ng/mL) or without FSH. Total RNA was extracted using RNeasy® (Qiagen, Valencia, CA, USA), and 100 ng of RNA was reverse transcribed using oligo dT primers and Omniscript® (Qiagen). Relative expression of target genes was assessed by real-time PCR using bovine-specific primers and Power SYBR green master mix in an ABI Prism® 7300. To select the most stable housekeeping gene for expression normalization, cyclophilin-A (CYC-A), GAPDH, and histone H2AFZ amplification profiles were compared using the geNorm applet for Microsoft Excel (Vandesompele J et al. 2002 Genome Biol. 3, 1-11); the most stable housekeeping gene was CYC-A. Relative expression values were calculated using the AACt method with efficiency correction (Pfaffl MW 2001 Nucleic Acids Res. 29, 2002-2007). Effects of time in culture and of FSH treatment were tested by ANOVA, and groups were compared by Tukey-Kramer Honestly Significant Difference test. Nonparametric analysis was used when data were not normally distributed. Abundance of mRNA of all glycolytic enzymes decreased during in vitro maturation with or without FSH. Expression of PFKP, ALDOA, TPI1, ENO1, and LDHA genes was decreased to around half of the initial value (time 0) by 4 to 8 h of culture (P < 0.05) and did not increase thereafter. A similar expression pattern was observed for PKM2, although mRNA abundance was reduced later in comparison with other enzymes; levels were decreased by 16 (without FSH) to 20 h (with FSH) of culture. The presence of FSH did not alter the overall temporal pattern of gene expression but decreased mRNA abundance for PFKP, ALDOA, and TPI1 at 20, 16 and 16 h of culture, respectively. In conclusion, gene expression of glycolytic enzymes decreased with time during COC in vitro maturation in cattle, and FSH did not have a major influence on this expression pattern. This study was supported by CAPES and FAPESP.

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Variables Affecting In Vivo Performance of High-Capacity Adenovirus Vectors

Journal of Virology ◽

10.1128/jvi.76.4.1600-1609.2002 ◽

2002 ◽

Vol 76 (4) ◽

pp. 1600-1609 ◽

Cited By ~ 44

Author(s):

Gudrun Schiedner ◽

Sabine Hertel ◽

Marion Johnston ◽

Volker Biermann ◽

Volker Dries ◽

...

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

High Capacity ◽

Fold Increase ◽

Expression Cassette ◽

Matrix Attachment Region ◽

Hepatic Expression ◽

Vector Genome

ABSTRACT In high-capacity adenovirus (HC-Ad) vectors the size and/or composition of the vector genome influences vector stability during production and the expression profile following gene transfer. Typically, an HC-Ad vector will contain both a gene or an expression cassette and stuffer DNA that is required to balance the final vector genome to a size of between 27 and 36 kb. To gain an improved understanding of factors that may influence gene expression from HC-Ad vectors, we have generated a series of vectors that carry different combinations of human alpha-1 antitrypsin (hAAT) expression constructs and stuffer DNAs. Expression in vitro did not predict in vivo performance: all vectors expressed hAAT at similar levels when tested in cell culture. Hepatic expression was evaluated following in vivo gene transfer in C57BL/6J mice. hAAT levels obtained from genomic DNA were significantly higher than levels achieved with small cDNA expression cassettes. Expression was independent of the orientation and only marginally influenced by the location of the expression cassette within the vector genome. The use of lambda stuffer DNA resulted in low-level but stable expression for at least 3 months when higher doses were applied. A potential matrix attachment region element was identified within the hAAT gene and caused a 10-fold increase in expression when introduced in an HC-Ad vector genome carrying a phosphoglycerate kinase (pgk) hAAT cDNA construct. We also illustrate the influence of the promoter on anti-hAAT antibody formation in C57BL/6J mice: a human cytomegalovirus but not a pgk promoter resulted in an anti-hAAT antibody response. Thus, the overall design of HC-Ad vectors may significantly influence amounts and duration of gene expression at different levels.

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260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab260 ◽

2007 ◽

Vol 19 (1) ◽

pp. 246

Author(s):

A. Baji Gal ◽

S. Mamo ◽

S. Bodo ◽

A. Dinnyes

Keyword(s):

Gene Expression ◽

Real Time ◽

Standard Error ◽

Real Time Pcr ◽

Housekeeping Gene ◽

Mouse Embryos ◽

Cell Stage ◽

The Mean

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P < 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

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14-3-3- and II/3-10-gene expression as molecular markers to address viability and growth activity of Echinococcus multilocularis metacestodes

Parasitology ◽

10.1017/s0031182005008632 ◽

2005 ◽

Vol 132 (1) ◽

pp. 83-94 ◽

Cited By ~ 26

Author(s):

J. MATSUMOTO ◽

N. MÜLLER ◽

A. HEMPHILL ◽

Y. OKU ◽

M. KAMIYA ◽

...

Keyword(s):

Gene Expression ◽

Molecular Markers ◽

Echinococcus Multilocularis ◽

Expression Profiles ◽

Housekeeping Gene ◽

Wild Type ◽

Expression Levels ◽

Beta Actin ◽

Growth Activity

The present study aimed to search for and characterize parasite molecules, whose expression levels correlate with the viability and growth activity of Echinococcus multilocularis metacestodes. We focused on the expression profiles of 2 parasite-derived genes, 14-3-3 and II/3-10, as putative molecular markers for viability and growth activity of the larval parasite. In experiments in vivo, gene expression levels of 14-3-3 and II/3-10 were relatively quantified by real-time reverse transcription-PCR using a housekeeping gene, beta-actin, as a reference reaction. All three reactions were compared with growth activity of the parasite developing in permissive nu/nu and in non-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels found after 8 days of treatment, which correlated with the kinetics of a housekeeping gene, beta-actin. The conclusion is that 14-3-3, combined with II/3-10, exhibits good potential as a molecular marker to assess viability and growth activity of the parasite.

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Characterizing Transcriptional and Epigenetic Signatures Induced By FLT3-ITD Activation

Blood ◽

10.1182/blood.v124.21.2186.2186 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2186-2186

Author(s):

Barbara Spitzer ◽

Olga A Guryanova ◽

Omar Abdel-Wahab ◽

Nicole Kucine ◽

Mazhar Adli ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Drug Exposure ◽

Chromatin State ◽

Specific Gene ◽

Specific Gene Expression ◽

Flt3 Inhibitor ◽

The Impact

Abstract Molecular studies have shown that specific somatic mutations impact therapeutic response and overall outcome in acute myeloid leukemia (AML) and have informed the development of molecularly targeted therapies. Previous studies have shown that the FLT3-ITD mutant disease allele predicts a poor prognosis in AML. Despite this important insight and the established role of FLT3-ITD mutations in AML pathogenesis, the impact of this mutation on gene regulation has not been extensively investigated. We hypothesized that transcriptional and epigenetic studies using genetically accurate murine models, cell lines, and primary AML samples would allow us to identify how FLT3 activation induces changes in gene expression and chromatin state. To assess the impact of FLT3-ITD associated FLT3 activation on gene expression, we performed RNA-sequencing studies on two FLT3-ITD cell lines (MOLM-13 and MV4-11) in the presence/absence of AC-220, a potent, specific FLT3 inhibitor. We also treated mice expressing a constitutive FLT3-ITD knock-in allele with AC-220 or vehicle, and performed RNA-sequencing on purified granulocyte-macrophage progenitors (GMPs). We assessed the impact of transient (4-12 hours drug treatment) and chronic (10-14 days) FLT3 inhibition on gene expression; we hypothesized that chronic drug exposure would result in more robust FLT3-mutant gene expression changes. In each case, the effects of FLT3-ITD activation/inhibition on gene expression were compared to RNA-seq data from FLT3-ITD mutant patients from TCGA. We first investigated the impact of short-term and chronic drug exposure on FLT3-ITD dependent gene expression in vitro. Comparison of short-term drug and vehicle treated cells revealed 159 differentially expressed (DE) genes (Benjamini-Hochberg false discovery rate (BH FDR) p < 0.05 and an absolute log2 fold change (FC) > 0.8). By contrast, we found that chronic FLT3 inhibition identified 743 DE genes. Comparison between the acutely and chronically treated cell lines revealed overlap of only 19 genes, suggesting important differences between the acute and steady-state effects of FLT3-inhibition. We found more significant overlap between chronic FLT3-inhibitor gene expression and FLT3-ITD specific gene expression in TCGA, demonstrating that long-term drug exposure more robustly delineates mutant-specific effects on gene expression. We next investigated the impact of short and long term FLT3-inhibition on gene expression in vivo. Analysis of DE genes identified 93 genes in the acutely treated mice vs. vehicle, and 274 genes in chronically treated mice (BH FDR p < 0.05 and absolute log2 FC of > 0.5). Only 12 DE genes were shared between these two perturbations compared with vehicle control. We then compared these gene expression signatures to FLT3-ITD specific gene expression from TCGA; we noted a significant inverse correlation between the signatures of chronic FLT3 inhibition in vivo with FLT3-ITD specific gene expression in TCGA (R2=0.47), but no correlation between the gene expression changes of acute FLT3 inhibition and FLT3-ITD TCGA patients (R2=0.09). We next integrated the FLT3 signatures from our in vivo work and TCGA with ChIP-sequencing for H3K4me3 and H3K27me3 in primary samples with FLT3-ITD compared to normal controls. We found that 3.6% of DE genes in our in vivo system, and 7.2% of DE genes in TCGA, had significant changes in H3K4me3 or H3K27me3. The most common alteration in chromatin state observed with FLT3 activation was an increase in H3K4me3 and transcriptional activation, with a smaller set of genes showing increased H3K27me3 and reduced expression, consistent with FLT3-mediated epigenetic repression. Motif analysis showed that DE loci with significant changes in chromatin state were enriched for ELF5, NF-E2, Pu.1, and Bach1 binding sequences, implicating these transcription factors in mediating FLT3-dependent gene expression effects. By studying the global transcriptional changes that occur with chronic, steady-state FLT3 inhibition in in vitro and in vivo systems, we identified a set of gene expression changes characteristic of FLT3-activation. In addition, integrating changes in gene expression and chromatin state allowed us to identify loci with alterations in epigenetic state in the setting of FLT3-ITD associated FLT3 activation, and to identify candidate transcription factors that mediate FLT3-dependent effects on gene expression. Disclosures No relevant conflicts of interest to declare.

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New Reference Genes for qRT-PCR Analysis as a Potential Target for Identification of Trichophyton verrucosum in Different Culture Conditions

Pathogens ◽

10.3390/pathogens10111361 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1361

Author(s):

Sebastian Gnat ◽

Dominik Łagowski ◽

Aneta Nowakiewicz ◽

Aleksandra Trościańczyk ◽

Mariusz Dyląg

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Housekeeping Gene ◽

Culture Conditions ◽

Pcr Analysis ◽

Trichophyton Verrucosum ◽

Diagnostic Laboratory ◽

Reliable Technique ◽

Qrt Pcr ◽

Diagnostic Difficulties

Dermatophytes are a group of filamentous fungi infecting skin, hair, and nails that raise great diagnostic difficulties. qRT-PCR is a reliable technique for quantifying gene expression with increasingly frequent use in mycological diagnostics. Knowledge of genes and molecular markers with potential to be used in the identification of dermatophytes is of great importance for the development of this branch of diagnostics. In this article, the suitability of six candidate reference genes (TUBB, ACTB, ADPRF, RPL2, SDHA, and EEF1A1) was investigated for gene expression analysis in the dermatophyte Trichophyton verrucosum, which was cultured in various mycological media that are commonly used in a diagnostic laboratory, i.e., Sabouraud, potato dextrose, and keratin-supplemented MM-Cove. The different culture conditions are extremely important factors for the growth and physiology of dermatophytes. Gene expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Regarding the stability of expression, SDHA was the most stable housekeeping gene; hence, this gene is recommended for future qRT-PCR studies on T. verrucosum strains. These results allow us to conclude that the SDHA gene can be an additional good candidate as an identification target in the qRT-PCR technique.

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In vitro chemosensitivity against enzastaurin correlates with gene expression of IL8 and GSK3-beta

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.13046 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 13046-13046 ◽

Cited By ~ 2

Author(s):

O. Oberschmidt ◽

U. Eismann ◽

M. M. Lahn ◽

J. Fleeth ◽

F. Lüdtke ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Data ◽

Drug Exposure ◽

Human Tumor ◽

Rna Isolation ◽

Expression Data ◽

Gene Expressions ◽

In Vitro Chemosensitivity ◽

Pkc Β

13046 Background: Enzastaurin (E) is an active antitumoral agent which selectively inhibits the β-isoform of protein kinase C (PKC-β). The compound blocks the enzyme’s ATP-binding site and signal transmission is abrogated resulting in the inhibition of neovascularization. The aim of the present study was to correlate gene expression with in vitro chemosensitivity of freshly explanted human tumor specimens. Such correlations in tumors taken directly from patients will help to rationally design subsequent clinical trials. Methods: Soft-agar colony forming assays were performed on freshly biopsied tumor cells exposed to various concentrations of E. Corresponding pieces of tumor specimens were shock-frozen and prepared for RNA isolation and cDNA generation followed by multiplex real-time PCR experiments. Gene expression data were correlated against cloning assay results. Results: Gene expression data of PKC-β1, PKC-β2, IL8RA, IL8RB, IL8, GSK3-β, and TGF-β were correlated against in vitro chemosensitivity pattern of E from 66 samples. After 1h-drug exposure gene expressions in sensitive versus resistant specimens were statistically significant with p = 0.013 for IL8 [median copy number (mcn): 1881 vs. 694; n = 66] and p = 0.012 for GSK3-beta (mcn: 1.6 vs. 7.0; n = 66). No correlation was detected for PKC-β1, PKC-β2, IL8RA, and IL8RB. Detection of TGF-β failed in most samples. Conclusions: Low expression of GSK3-β and high expression of IL8 correlate statistically significantly with increased in vitro sensitivity to E in freshly explanted human tumors. These findings may help direct further clinical development of this compound. No significant financial relationships to disclose.

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