Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis

A liquid chromatography/mass spectrometry (LC/MS) method for separation and characterization of ergosterol biosynthetic precursors was developed to study the effect of Posaconazole on sterol biosynthesis in fungi. Ergosterol biosynthetic precursors were characterized from their electron ionization mass spectra acquired by a normal-phase chromatography, particle beam LC/MS method. Fragment ions resulting from cleavage across the D-ring and an abundant M - 15 fragment ion were diagnostic for methyl substitution at C-4 and C-14. Comparison of the sterol profile in control and treated Candida albicans incubations showed depletion of ergosterol and accumulation of C-4 and C-14 methyl-substituted sterols following treatment with Posaconazole. These C-4 and C-14 methyl sterols are known to be incapable of sustaining cell growth. The results demonstrate that Posaconazole exerts its antifungal activity by inhibition of ergosterol biosynthesis. Furthermore, Posaconazole appears to disrupt ergosterol biosynthesis by inhibition of lanosterol 14alpha-demethylase.     

Compatibility and fungal degradation of poly[(R)-3-hydroxybutyrate]/aliphatic copolyester blend

Poly[(R)-3-hydroxybutyrate] (PHB) was blended with an aliphatic copolyester, which was synthesized by the esterification of adipic acid, ethylene glycol, and lactic acid. The blend showed a single T_g, which varied systematically but convexly upwards with the composition. The growth rate of PHB spherulites, the crystallization temperature, and the equilibrium melting temperature of the blend were decreased as the amount of the copolyester was increased. Therefore, the blend system was determined to be compatible. However, the degree of crystallinity, and the enthalpies of crystallization and fusion of PHB in the blend remained almost constant, regardless of the compositional change, although the crystallization rate was decreased upon blending. No chemical change such as transesterification was observed as a result of the blending, yet there was a slight change in the crystalline morphology of PHB. The rate of fungal degradation was lowered with an increase in the copolyester content of the blend. © 1996 John Wiley & Sons, Inc.     

Secondary Metabolites,including a New 5,6-Dihydropyran-2-One,Produced by the Fungus Diplodia corticola. Aphicidal Activity of the Main Metabolite,Sphaeropsidin A

An undescribed 5,6-dihydropyran-2-one, namely diplopyrone C, was isolated and characterized from the cultures of an isolate of the fungus Diplodia corticola recovered from Quercus suber in Algeria. The structure and relative stereostructure of (5S,6S,7Z,9S,10S)-5-hydroxy-6-(2-(3-methyloxiran-2-yl)vinyl)-5,6-dihydro-2H-pyran-2-one were assigned essentially based on NMR and MS data. Furthermore, ten known compounds were isolated and identified in the same cultures. The most abundant product, the tetracyclic pimarane diterpene sphaeropsidin A, was tested for insecticidal effects against the model sucking aphid, Acyrthosiphon pisum. Results showed a toxic dose-dependent oral activity of sphaeropsidin A, with an LC₅₀ of 9.64 mM.     

Increased synthesis of ajmalicine and catharanthine by cell suspension cultures ofCatharanthus roseus in response to fungal culture-filtrates

The ammonium sulfate-precipitated fraction from mycelia and culture-filtrates and the crude, cell-free culture filtrates from the growth medium of the fungiChrysosporium palmorum, Eurotium rubrum, Micromucor isabellina, andPythium aphanidermatum when aseptically added to cell suspensions ofCantharanthus roseus caused a rapid and dramatic increase in indole alkaloid biosynthesis. Up to 400 μg/L ajmalicine and 600 μg/L catharanthine were detected in C.roseus cell suspension grown in the presence of theM. isabellina fungal culture filtrate for 3 d. Untreated cells produced only trace levels of ajmalicine and catharanthine per liter of cell suspension after 15 d of culture.     

Fungistatic activity of Zanthoxylum rhoifolium Lam. bark extracts against fungal plant pathogens and investigation on mechanism of action in Botrytis cinerea

Plant-derived compounds are emerging as an alternative choice to synthetic fungicides. Chloroform–methanol extract, obtained from the bark of Zanthoxylum rhoifolium, a member of Rutaceae, showed a fungistatic effect on Botrytis cinerea, Sclerotinia sclerotiorum, Alternaria alternata, Colletotrichum gloeosporioides and Clonostachys rosea, when added to the growth medium at different concentrations. A fraction obtained by gel separation and containing the alkaloid O-Methylcapaurine showed significant fungistatic effect against B. cinerea and S. sclerotiorum, two of the most destructive phytopathogenic fungi. The underlying mechanism of such an inhibition was further investigated in B. cinerea, a fungus highly prone to develop fungicide resistance, by analysing the expression levels of a set of genes (BcatrB, P450, CYP51 and TOR). O-Methylcapaurine inhibited the expression of all the analysed genes. In particular, the expression of BcatrB gene, encoding a membrane drug transporter involved in the resistance to a wide range of xenobiotic compounds, was strongly inhibited (91%).     

SERS as a valuable tool for detection and treatment follow‐up of fungal infection in mice lungs: use of Amphotericin B and its nanoencapsulation onto magnetic nanoparticles

This study reports on the use of surface‐enhanced Raman scattering (SERS) detection and follow‐up treatment of Paracoccidioides brasiliensis (Pb) infection in lung's mice. We also reports on the introduction of a new drug carrier system [nanoparticle‐Amphotericin B (NP‐AmB)], comprising magnetic NP surface functionalized with AmB, and its use in the treatment of infected and non‐infected mice. SERS was successfully used to monitor the efficacy of the mice's treatment using the new NP‐AmB, while free AmB (F‐AmB), considering the current drug of choice for treatment of Pb infection, was also used and taken as reference for the treatment. We found SERS provides a robust platform to discriminate infected lung tissues from non‐infected ones based on fingerprints assessed via SERS spectra and focused on the redox state of heme groups present in the collected biological material. Finally, SERS data reported in this study indicated that the new NP‐AmB formulation provides similar clinical response as the F‐AmB, although incorporating 40% lower content of AmB and administered in a time interval schedule (every 72 h) three times longer than F‐AmB (every 24 h). Copyright © 2013 John Wiley & Sons, Ltd.     

阵列式光纤光谱仪的小麦霉变在线检测

小麦是我国战略性储藏粮食品种，但小麦易受霉菌感染而发生霉变，影响其食用安全。加强小麦有害霉菌侵染程度的早期检测是控制其危害的需要措施。然而，现有的平板计数和荧光染色等检测方法，普遍存在前处理繁杂、时效性差等不足。故此，拟运用阵列式光纤光谱仪结合化学计量学方法，建立霉变小麦的实时在线检测方法，并为进一步开发粮食品质与安全在线检测装备提供参考。小麦样品经辐照灭菌后分别接种五种谷物中常见有害霉菌：串珠镰刀菌83227、层出镰刀菌195647、雪腐镰刀菌3.503、寄生曲霉3.3950和赭曲霉3.3486，并置于28 ℃和85%相对湿度环境中储藏7 d以加速霉变。在样品储藏的第0，1，3，5和7 d，运用阵列式光纤光谱仪和漫反射探头在线采集样品的漫反射特征光谱，并依据国标平板计数法测定样品菌落总数水平。光谱采集步骤为：在线检测平台上，设置样品运动速度0.15 m·s-1和光谱仪积分时间20 ms，采集波段为600~1 600 nm，重复测量3次。然后，首先对小麦原始光谱进行平滑、多元散射校正和导数变换等预处理以消除光谱噪音；随后，运用主成分分析(PCA)对受不同霉变程度(储藏阶段)的小麦样品进行区分；最后，利用线性判别分析(LDA)和偏最小二乘回归分析(PLSR)建立小麦有害霉菌侵染程度的定性定量分析模型。小麦在储藏期内经历未霉变、开始霉变和严重霉变三个阶段。原始和二阶微分光谱显示霉菌侵染引起小麦光谱特征发生显著改变，PCA结果表明不同储藏阶段(霉变程度)小麦样品存在一定分离趋势。利用前十个主成分得分建立的LDA判别模型，对不同霉变程度小麦样品的识别率达90.0%以上。结果表明：小麦样品菌落总数的PLSR定量预测模型的预测决定系数(R2_p)为0.859 2，预测均方根误差(RMSEP)为0.401 Log CFU·g-1，相对分析偏差(RPD)达2.65。应用阵列式光纤光谱仪结合计量学方法在线评估小麦霉变具有一定可行性。下一步研究中，应扩大样品量，补充自然霉变及受更多代表性霉菌侵染的小麦样品，以不断增强模型的鲁棒性和方法的适用性。