Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction ...in ergosterol production, an essential fungal membrane sterol. Many
species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. In C. glabrata, these azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the
gene. Here, we generated C. glabrata
mutations that were analogous to azole resistance alleles from C. albicans
. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of
imposed a fitness cost that was not observed with hyperactive
alleles. Crucially, the presence of the DM
allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in
activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.
A crucial limitation in antifungal chemotherapy is the limited number of antifungal drugs currently available. Azole drugs represent the most commonly used chemotherapeutic, and loss of efficacy of ...these drugs is a major risk factor in successful treatment of a variety of fungal diseases.
is a pathogenic yeast that is increasingly found associated with bloodstream infections, a finding likely contributed to by its proclivity to develop azole drug resistance.
often acquires azole resistance via gain-of-function (GOF) mutations in the transcription factor Pdr1. These GOF forms of Pdr1 drive elevated expression of target genes, including the ATP-binding cassette transporter-encoding
locus. GOF alleles of
have been extensively studied, but little is known of how Pdr1 is normally regulated. Here we test the idea that reduction of ergosterol biosynthesis (as occurs in the presence of azole drugs) might trigger activation of Pdr1 function. Using two different means of genetically inhibiting ergosterol biosynthesis, we demonstrated that Pdr1 activity and target gene expression are elevated in the absence of azole drug. Blocks at different points in the ergosterol pathway lead to Pdr1 activation as well as to induction of other genes in this pathway. Delivery of the signal from the ergosterol pathway to Pdr1 involves the transcription factor Upc2A, an
gene regulator. We show that Upc2A binds directly to the
and
promoters. Our studies argue for a physiological link between ergosterol biosynthesis and Pdr1-dependent gene regulation that is not restricted to efflux of azole drugs.
A likely contributor to the increased incidence of non-
candidemias involving
is the ease with which this yeast acquires azole resistance, in large part due to induction of the ATP-binding cassette transporter-encoding gene
Azole drugs lead to induction of Pdr1 transactivation, with a central model being that this factor binds these drugs directly. Here we provide evidence that Pdr1 is activated without azole drugs by the use of genetic means to inhibit expression of azole drug target-encoding gene
These acute reductions in Erg11 levels lead to elevated Pdr1 activity even though no drug is present. A key transcriptional regulator of the
pathway, Upc2A, is shown to directly bind to the
and
promoters. We interpret these data as support for the view that Pdr1 function is responsive to ergosterol biosynthesis and suggest that this connection reveals the normal physiological circuitry in which Pdr1 participates.
Fluconazole is one of the most commonly used antifungals today. A result of this has been the inevitable selection of fluconazole-resistant organisms. This is an especially acute problem in the ...pathogenic yeast
. Elevated minimal inhibitory concentrations for fluconazole in
are frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding gene
. These mutant Pdr1 regulators drive constitutively high expression of target genes like
that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure of
to fluconazole induced expression of both Pdr1 and
, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation, and a hyperactive form of this phosphatase can decrease susceptibility to the echinocandin caspofungin but does not show a similar change for fluconazole susceptibility. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease in
expression and increased fluconazole susceptibility, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin susceptibility together via the control of transcription factor activity.IMPORTANCEDrug-resistant microorganisms are a problem in the treatment of all infectious diseases; this is an especially acute problem with fungi due to the existence of only three major classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeast
, mutant forms of a transcription factor called Pdr1 are commonly associated with decreased fluconazole susceptibility and poor clinical outcomes. Here, we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug susceptibility. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally decreased fluconazole susceptibility. Previous studies showed that calcineurin controls susceptibility to the echinocandin class of antifungal drugs, and our data demonstrate that this protein phosphatase is also required for normal azole drug susceptibility. Calcineurin plays a central role in susceptibility to two of the three major classes of antifungal drugs in
.
The most commonly used antifungal drugs are the azole compounds, which interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires ...resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Analysis of Upc2A genomic binding sites demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. Transcriptomic analyses revealed that, in addition to the well-described ERG genes, a large group of genes encoding components of the translational apparatus along with membrane proteins were responsive to Upc2A. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.
High order plasmonic types by integrating a novel heterogeneous plasmonic and flexible model based on the co-existence of Ag nanospheres (NSs) and Ag nanocubes (NCs) are introduced. The ...point-to-facet type in these hybrid shapes produces surface-enhanced Raman scattering (SERS) signals many-fold larger than in single-plasmonic constructs. A high enhancement factor (EF = 4.6 × 108) in coupled plasmonic particulates allowed SERS-probing at ultralow sample quantities. Then, these plasmonic constructs are anchored onto a flexible polymethyl methacrylate (PMMA)-treated cellulose paper. In addition to strong electromagnetic enhancement, the hydrophobic surface could concentrate target analytes in the hotspot areas, resulting in highly active SERS responses in highly diluted solutions. As a result, the flexible SERS sensing platform exhibits high sensitivity with detection around 10−10 M and point-to-face relative standard deviation (RSD) in one sensor as low as 7.28%, thereby demonstrating good reproducibility. Furthermore, it exhibits perfectly selective detection for trace amounts of interest analytes in a complex solution, significantly enhancing the analyte identification efficiency at nanomolar concentration levels. This study has proven a promising route for an integrated SERS platform with plasmonic nanoconstructs and analyte enrichment as a versatile SERS sensor for highly sensitive, quantitative, selective, and cost-effective SERS detection.
Introduction
VE1 is a monoclonal antibody detecting mutant BRAF V600E protein by immunohistochemistry (IHC) with a high concordance rate with molecular analysis in many cancers. Materials and ...methods: BRAF V600E mutation was assessed on 94 pediatric LCH patients using sequencing analysis and VE1 immunohistochemistry with stringent and lenient-scoring criteria. Results: BRAF V600E mutation exon 15 was detected by sequencing in 47.9% of LCH cases. BRAF V600E mutation rate in multiple-system LCH was 65.2%, significantly higher than in single-system LCH (p = .001). VE1 assays showed 35.6% sensitivity, 75.5% specificity (Stringent criteria), and 91.1% sensitivity, 35.7% specificity (Lenient criteria). Conclusions: The proportion of BRAF V600E mutational status was relatively high and related to high-risk LCH. Molecular assays for BRAF mutation detection are preferred in LCH lesions. VE1 is not ready as an alternative option for LCH BRAF testing.
Micropropagation has proven to be an effective method for large-scale plant production in a short time and a useful tool for plant breeding. Microbial contamination is one of the most difficult ...micropropagation challenges, resulting in reduced plant quality and loss of valuable stocks. Therefore, sterilization of culture media is a critical step in plant micropropagation. However, sterilized media might reduce the activity of plant growth regulators and nutritional components of culture media. The sterilization effects of silver nanoparticles (AgNP) on the growth of explants and culture media were examined. The treatment with 250 ppm AgNP for 15 to 20 min of 4-wk-old
ex vitro
leaves proved optimal for controlling the contamination. Furthermore, the Murashige and Skoog medium containing 4 ppm AgNP resulted in 100% medium disinfection (no contamination) after 4 wk of culture. The plantlets obtained from non-sterilized MS medium (NoM) containing 4 ppm AgNP and 4 g L
−1
agar gave similar results as the control medium with 8 g L
−1
agar and the absence of AgNP. Large scale culture systems using NoM in large plastic containers of two different sizes (NoM1 and NoM2) could produce quality plantlets. Chrysanthemum plantlets in the NoM1 system showed higher antioxidant enzyme activities of ascorbate peroxidase and superoxide dismutase than plantlets in the autoclaved medium. Furthermore, the plantlets from NoM were better acclimatized under greenhouse conditions than those from the autoclaved medium (AuM) system. The developmental stages (flower buds and blooming time) of NoM1 and NoM2 plantlets, were 1 wk earlier than those from the AuM system. The successful use of AgNP as a sterilizer and as a component of culture media would reduce the cost of micropropagation and improve plants' quality.
The sterilization effectiveness of copper nanoparticles (CuNPs) replaced common disinfectants such as mercury chloride (HgCl
2
) or calcium hypochlorite (Ca(ClO)
2
) on different explants ...(petioles—P, inflorescence—F and stem nodes—S) of ex vitro tuberous begonias (
Begonia × tuberhybrida
Voss) was investigated. Subsequently, the P, F and S explants were cut transversely into thin cell layer (tTCL) approximately 1 mm in length (P-tTCL, F-tTCL and S-tTCL) and cultured on SE induction medium in order to investigate the effect of disinfectants on somatic embryogenesis, somatic embryo morphology, antioxidant activity (Catalase—CAT and Ascorbate Peroxidase—APX), and carbohydrate content (sugar and starch) of somatic embryos and their subsequent growth. The results showed that CuNPs can replace HgCl
2
and Ca(ClO)
2
and are effective in surface disinfection of P, F and S explants. CuNPs enhanced embyrogenic callus and somatic embryogenesis of treated explants compared to those on HgCl
2
and Ca(ClO)
2
. CuNP-treated F-tTCL and S-tTCL explants recorded the highest number of somatic embryos (38.00–36.67 embryos, respectively) as well as the percentage of somatic embryos in cotyledon-shape (47.37–49.09%, respectively). Moreover, the antioxidant enzyme activity (CAT and APX) and starch content of somatic embryo clusters derived from CuNPs-treated F-tTCL and S-tTCL explants were higher and the sugar content was lower as compared to those sterilizated with HgCl
2
or Ca(ClO)
2
. Plantlets derived from different different disinfectant treatments showed no difference in morphology, in vitro rooting and acclimatization stages in the greenhouse.
Key message
Surface disinfection efficiency of CuNPs on ex vitro begonia explants compared to those of common disinfectants.
CuNPs effected on somatic embryogenesis, antioxidant activity, carbohydrate content.
Langerhans cell histiocytosis (LCH) is more common in children than adults and involves many organs. In children, the BRAF V600E mutation is associated with recurrent and high-risk LCH.
We collected ...paraffin blocks of 94 pediatric LCH patients to detect BRAF V600E mutation by sequencing. The relationship between BRAF V600E status and clinicopathological parameters were also critically analyzed.
BRAF V600E mutation exon 15 was detected in 45 cases (47.9%). Multiple systems LCH showed a significantly higher BRAF V600E mutation rate than a single system (p=.001). No statistical significance was evident for other clinical characteristics such as age, sex, location, risk organs involvement, and CD1a expression.
In Vietnamese LCH children, the proportion of BRAF V600E mutational status was relatively high and related to multiple systems.
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