scholarly journals The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

2012 ◽  
Vol 11 (10) ◽  
pp. 1226-1238 ◽  
Author(s):  
Lorna Gallagher ◽  
Rebecca A. Owens ◽  
Stephen K. Dolan ◽  
Grainne O'Keeffe ◽  
Markus Schrettl ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Aspergillus Fumigatus ◽  
High Performance ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Disulfide Linkages ◽  
Significant Difference ◽  
Culture Supernatants

ABSTRACTThe function of a number of genes in the gliotoxin biosynthetic cluster (gli) inAspergillus fumigatusremains unknown. Here, we demonstrate thatgliKdeletion from two strains ofA. fumigatuscompletely abolished gliotoxin biosynthesis. Furthermore, exogenous H2O2(1 mM), but not gliotoxin, significantly inducedA. fumigatus gliKexpression (P= 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P< 0.001) and H2O2(P< 0.01), unexpectedly, exogenous gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived fromA. fumigatusATCC 26933 significantly inhibited (P< 0.05) the growth of the ΔgliK26933deletion mutant. TheA. fumigatusΔgliK26933mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry withm/z394 to 396. These metabolites (m/z394 to 396) were present at significantly higher levels in the culture supernatants of theA. fumigatusΔgliK26933mutant than in those of the wild type (P= 0.0024 [fold difference, 24] andP= 0.0003 [fold difference, 9.6], respectively) and were absent fromA. fumigatusΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of theA. fumigatusΔgliK26933mutant compared to the wild type (P< 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P= 0.0045) between that ofA. fumigatusATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK46645mutant (31.4 pg/mg mycelium/min), strongly suggesting thatgliKabsence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role forgliKin gliotoxin biosynthesis and reveal new insights into gliotoxin functionality inA. fumigatus.

scholarly journals Determination of acrolein, ethanol, volatile acidity, and copper in different samples of sugarcane spirits

2012 ◽  
Vol 32 (3) ◽  
pp. 568-572 ◽  
Author(s):  
José Masson ◽  
Maria das Graças Cardoso ◽  
Lidiany Mendonça Zacaroni ◽  
Jeancarlo Pereira dos Anjos ◽  
Adelir Aparecida Sackz ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Minas Gerais ◽  
Volatile Acidity ◽  
Legal Limits ◽  
Brazilian Legislation ◽  
Significant Difference ◽  
Average Size

Seventy-one samples of sugarcane spirits from small and average size stills produced in the northern and southern Minas Gerais (Brazil) were analyzed for acrolein using HPLC (High Performance Liquid Chromatography). Ethanol and copper concentrations and volatile acidity were also determined according to methods established by the Ministry of Agriculture, Livestock and Supply (MAPA). A total of 9.85% of the samples tested showed levels of acrolein above the legal limits, while the copper concentrations of 21.00% of the samples and the volatile acidity of 8.85% of the samples were higher than the limits established by the Brazilian legislation. The concentration of acrolein varied from 0 to 21.97 mg.100 mL-1 of ethanol. However, no significant difference at 5% of significance was observed between the samples produced in the northern and southern Minas Gerais. The method used for determination of acrolein in sugarcane spirits involved the formation of a derivative with 2,4-dinitrophenylhydrazine (2,4-DNPH) and subsequent analysis by HPLC.

International Mycotoxin Check Sample Program: Part II. Report on Laboratory Performance for Determination of Aflatoxin M1 in Milk

1982 ◽  
Vol 65 (4) ◽  
pp. 864-868
Author(s):  
Marlin D Friesen ◽  
Liliane Garren
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Aflatoxin M1 ◽  
Check Sample ◽  
Sufficient Data ◽  
Statistical Comparison ◽  
Significant Difference ◽  
Aoac Method

Abstract A sample of aflatoxin M1-contaminated lyophilized cow's milk was analyzed by 80 laboratories in 30 countries. Sufficient data were obtained to permit a statistical comparison of the performance of laboratories using AOAC methods I and II and those using high performance liquid chromatography for quantitation. A significant difference was noted between means for laboratories using AOAC method I as opposed to those using HPLC methods. Overall reproducibility (between- plus within-laboratory precision) was best for laboratories using HPLC methods and poorest for those using AOAC method II.

scholarly journals A VALIDATED REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUS DETERMINATION OF FIVE ANTIEPILEPTIC DRUGS USED IN THE TREATMENT OF LENNOX–GASTAUT SYNDROME IN THEIR PHARMACEUTICAL DOSAGE FORMS

2018 ◽  
Vol 11 (5) ◽  
pp. 167
Author(s):  
Sonia T Hassib ◽  
Hanaa M A Hashem ◽  
Marianne A Mahrouse ◽  
Eman A Mostafa
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Simultaneous Determination ◽  
Antiepileptic Drugs ◽  
High Performance ◽  
Reversed Phase ◽  
Dosage Forms ◽  
Pharmaceutical Dosage Forms ◽  
Significant Difference

 Objective: Lennox–Gastaut syndrome (LGS) is mainly treated with antiepileptic drugs (AEDs) but using one AED is not sufficient to relieve all or even most patients. A combination of agents is usually preferred. In the current study, an isocratic, selective, sensitive, precise, and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous determination of rufinamide (RUF), lamotrigine (LAM), clonazepam (CLO), valproic acid (VAL), and diazepam (DIA) which are commonly used in the management of LGS in their dosage forms using lacosamide as internal standard.Methods: The method depends on using RESTEK C18 column (5 μm, 250 mm × 4.6 mm) and a mobile phase composed of acetonitrile:water (55: 45, v/v), pH = 3.3 adjusted with phosphoric acid. The method was conducted in an isocratic mode with a flow rate of 1ml/min and ultraviolet detection at 210 nm.Results: The linearity range was 2–40 μg/ml for RUF and DIA, 0.5–40 μg/ml for LAM and CLO, and 36–180 μg/ml for VAL.Conclusion: Statistical analysis revealed no significant difference between the results obtained and the official or reported ones for each cited drug. The method is simple to be easily implemented in quality control studies of the mentioned drugs in their pharmaceutical preparations.

scholarly journals Pharmacodynamics of Voriconazole against Wild-Type and Azole-Resistant Aspergillus flavus Isolates in a Nonneutropenic Murine Model of Disseminated Aspergillosis

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Shivaprakash M. Rudramurthy ◽  
Seyedmojtaba Seyedmousavi ◽  
Manpreet Dhaliwal ◽  
Arunaloke Chakrabarti ◽  
Jacques F. Meis ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Murine Model ◽  
Drug Exposure ◽  
Resistant Isolate ◽  
Dependent Manner ◽  
Wild Type ◽  
Time Curve ◽  
Content Type ◽  
Significant Difference ◽  
Susceptible Isolate

ABSTRACT Invasive aspergillosis (IA) due to Aspergillus flavus is associated with high mortality. Although voriconazole (VRC) is widely recommended as the first-line treatment for IA, emergence of azole resistance in Aspergillus spp. is translating to treatment failure. We evaluated the efficacy of voriconazole in a nonneutropenic murine model of disseminated A. flavus infection using two voriconazole-resistant isolates (one harboring the Y319H substitution in the cyp51C gene) and two wild-type isolates without mutations. All isolates exhibited a dose-response relationship, and voriconazole treatment improved mouse survival in a dose-dependent manner. At 40 mg/kg of body weight, 100% efficacy was observed for 1 susceptible isolate and 1 resistant isolate (with mutation), whereas for another susceptible isolate and resistant isolate (without mutation), survival rates were 81% and 72%, respectively. The Hill equation with a variable slope fitted the relationship between the area under the concentration-time curve (AUC)/MIC ratio and 14-day survival well for each strain. An F test showed the 50% effective doses to be significantly different from each other (P = 0.0023). However, contrary to expectation, there was a significant difference in exposure-response relationships between strains, and it appeared that the susceptible strains required a relatively higher exposure than the resistant ones to result in the same treatment effect, the 50% effective pharmacokinetic/pharmacodynamic (PK/PD) index (EI50) required being negatively and log-linearly related to the MIC (P = 0.04). We conclude that the efficacy of voriconazole depended on drug exposure and the voriconazole MIC of the isolates, but lower exposures are required for strains with higher MICs. These findings may have profound significance in clinical practice with respect to dosing and drug choice.

scholarly journals Azole and Amphotericin B MIC Values against Aspergillus fumigatus: High Agreement between Spectrophotometric and Visual Readings Using the EUCAST EDef 9.3.2 Procedure

2020 ◽  
Vol 65 (1) ◽  
pp. e01693-20 ◽  
Author(s):  
Julia Serrano-Lobo ◽  
Ana Gómez ◽  
Waldo Sánchez-Yebra ◽  
Miguel Fajardo ◽  
Belén Lorenzo ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Amphotericin B ◽  
Fungal Growth ◽  
Sensu Stricto ◽  
Resistance Rate ◽  
Wild Type ◽  
Growth Reduction ◽  
High Agreement ◽  
Content Type

ABSTRACTThe EUCAST EDef 9.3.2 procedure recommends visual readings of azole and amphotericin B MICs against Aspergillus spp. Visual determination of MICs may be challenging. In this work, we aim to obtain and compare visual and spectrophotometric MIC readings of azoles and amphotericin B against Aspergillus fumigatussensu lato isolates. A total of 847 A. fumigatussensu lato isolates (A. fumigatus sensu stricto [n = 828] and cryptic species [n = 19]) were tested against amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole using the EUCAST EDef 9.3.2 procedure. Isolates were classified as susceptible or resistant/non-wild type according to the 2020 updated breakpoints. The area of technical uncertainty for the azoles was defined in the updated breakpoints. Visual and spectrophotometric (fungal growth reduction of >95% compared to the control, read at 540 nm) MICs were compared. Essential (±1 2-fold dilution) and categorical agreements were calculated. Overall, high essential (97.1%) and categorical (99.6%) agreements were found. We obtained 100% categorical agreements for amphotericin B, itraconazole, and posaconazole, and consequently, no errors were found. Categorical agreements were 98.7 and 99.3% for voriconazole and isavuconazole, respectively. Most of the misclassifications for voriconazole and isavuconazole were found to be associated with MIC results falling either in the area of technical uncertainty or within one 2-fold dilution above the breakpoint. The resistance rate was slightly lower when the MICs were obtained by spectrophotometric readings. However, all relevant cyp51A mutants were correctly classified as resistant. Spectrophotometric determination of azole and amphotericin B MICs against A. fumigatussensu lato isolates may be a convenient alternative to visual endpoint readings.

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