A novel method for studying ergosterol biosynthesis by a cell-free preparation ofAspergillus fumigatusand its inhibition by azole antifungal agents

The developing resistance in fungi has become a key challenge, which is being faced nowadays with the available antifungal agents in the market. Further search for novel compounds from different sources has been explored to meet this problem. The current review describes and highlights recent advancement in the antifungal drug aspects from plant and marine based sources. The current available antifungal agents act on specific targets on the fungal cell wall, like ergosterol synthesis, chitin biosynthesis, sphingolipid synthesis, glucan synthesis etc. We discuss some of the important anti-fungal agents like azole, polyene and allylamine classes that inhibit the ergosterol biosynthesis. Echinocandins inhibit β-1, 3 glucan synthesis in the fungal cell wall. The antifungals poloxins and nikkomycins inhibit fungal cell wall component chitin. Apart from these classes of drugs, several combinatorial therapies have been carried out to treat diseases due to fungal resistance. Recently, many antifungal agents derived from plant and marine sources showed potent activity. The renewed interest in plant and marine derived compounds for the fungal diseases created a new way to treat these resistant strains which are evident from the numerous literature publications in the recent years. Moreover, the compounds derived from both plant and marine sources showed promising results against fungal diseases. Altogether, this review article discusses the current antifungal agents and highlights the plant and marine based compounds as a potential promising antifungal agents.

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Development of a Methodology for Estimating the Ergosterol in Meat Product-Borne Toxigenic Moulds to Evaluate Antifungal Agents

Foods ◽

10.3390/foods10020438 ◽

2021 ◽

Vol 10 (2) ◽

pp. 438

Author(s):

Micaela Álvarez ◽

Alicia Rodríguez ◽

Elena Bermúdez ◽

Elia Roncero ◽

María J. Andrade

Keyword(s):

Antifungal Agents ◽

Limit Of Detection ◽

Meat Product ◽

Meat Products ◽

Ergosterol Biosynthesis ◽

Array Detector ◽

Meat Industry ◽

Ergosterol Content ◽

Validation Parameters ◽

Toxigenic Moulds

Antifungal agents are commonly used in the meat industry to prevent the growth of unwanted moulds, such as toxigenic ones, on dry-cured meat products. For enhancing the application of antifungals, their mode of action must be evaluated. Their effect on the mould ergosterol content is one of the most studied ones, since it is the target site of some commercialised antifungals or of those that are in development. The aim of this study was to develop a methodology for determining how the antifungal agents used in the meat industry work. A method for analysing ergosterol was firstly developed using high-performance liquid chromatography with fluorescence detection coupled to a diode array detector (HPLC-FLD/DAD). The chromatographically optimised conditions (gradient and mobile phases) allowed us to reduce the time per analysis with respect to previously published methods up to 22 min. Withing the six checked extraction methods, method 5, showing the best mean recovery values (99.51%), the shortest retention time (15.8 min), and the lowest standard deviation values (9.92) and working temperature (60 °C), was selected. The limit of detection and limit of quantification were 0.03 and 0.1 µg/mL, respectively. All the validation parameters corroborated the method’s suitability. Finally, its feasibility for evaluating the effect of a commercial antifungal preparation (AP) and different herbs that are frequently added to meat products on the ergosterol content of several toxigenic moulds was studied. Differences at the strain level were obtained in the presence of AP. Moreover, the addition of herbs significantly reduced the ergosterol content in Penicillium nordicum up to 83.91%. The developed methodology is thus suitable for screening the antifungals’ role in altering mould ergosterol biosynthesis before their application in real meat products.

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A novel method for processing adipose-derived stromal stem cells using a closed cell washing concentration device with a hollow fiber membrane module

Biomedical Microdevices ◽

10.1007/s10544-020-00541-0 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Shinji Hayashi ◽

Rieko Yagi ◽

Shuhei Taniguchi ◽

Masami Uji ◽

Hidaka Urano ◽

...

Keyword(s):

Stem Cells ◽

Hollow Fiber ◽

Hollow Fiber Membrane ◽

Membrane Module ◽

Fiber Membrane ◽

Cell Processing ◽

Closed Cell ◽

A Cell ◽

Novel Method ◽

Stromal Stem Cells

AbstractCell-assisted lipotransfer (CAL) is an advanced lipoinjection method that uses autologous lipotransfer with addition of a stromal vascular fraction (SVF) containing adipose-derived stromal stem cells (ASCs). The CAL procedure of manual isolation of cells from fat requires cell processing to be performed in clean environment. To isolate cells from fat without the need for a cell processing center, such as in a procedure in an operation theater, we developed a novel method for processing SVF using a closed cell washing concentration device (CCD) with a hollow fiber membrane module. The CCD consists of a sterilized closed circuit, bags and hollow fiber, semi-automatic device and the device allows removal of >99.97% of collagenase from SVF while maintaining sterility. The number of nucleated cells, ASCs and viability in SVF processed by this method were equivalent to those in SVF processed using conventional manual isolation. Our results suggest that the CCD system is as reliable as manual isolation and may also be useful for CAL. This approach will help in the development of regenerative medicine at clinics without a cell processing center.

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Dark-Field X-Ray Microscopy of Immunogold-Labeled Cells

Microscopy and Microanalysis ◽

10.1017/s1431927696210530 ◽

1996 ◽

Vol 2 (2) ◽

pp. 53-62 ◽

Cited By ~ 3

Author(s):

Henry N. Chapman ◽

Jenny Fu ◽

Chris Jacobsen ◽

Shawn Williams

Keyword(s):

Colloidal Gold ◽

Dark Field Image ◽

Dark Field ◽

X Rays ◽

X Ray ◽

Scanning Transmission ◽

A Cell ◽

Specific Antigens ◽

Genetic Sequences ◽

Novel Method

The methods of immunolabeling make visible the presence of specific antigens, proteins, genetic sequences, or functions of a cell. In this paper we present examples of imaging immunolabels in a scanning transmission x-ray microscope using the novel method of dark-field contrast. Colloidal gold, or silver-enhanced colloidal gold, is used as a label, which strongly scatters x-rays. This leads to a high-contrast dark-field image of the label and reduced radiation dose to the specimen. The x-ray images are compared with electron micrographs of the same labeled, unsectioned, whole cell. It is verified that the dark-field x-ray signal is primarily due to the label and the bright-field x-ray signal, showing absorption due to carbon, is largely unaffected by the label. The label can be well visualized even when it is embedded in or laying behind dense material, such as the cell nucleus. The resolution of the images is measured to be 60 nm, without the need for computer processing. This figure includes the x-ray microscope resolution and the accuracy of the label positioning. The technique should be particularly useful for the study of relatively thick (up to 10 μm), wet, or frozen hydrated specimens.

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An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00640-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6296-6307 ◽

Cited By ~ 20

Author(s):

Petra Keller ◽

Christoph Müller ◽

Isabel Engelhardt ◽

Ekkehard Hiller ◽

Karin Lemuth ◽

...

Keyword(s):

Antifungal Activity ◽

Fungal Infections ◽

Transcriptional Profiling ◽

Benzimidazole Derivative ◽

Benzimidazole Derivatives ◽

Ergosterol Biosynthesis ◽

Screening Assay ◽

Content Type ◽

Life Threatening ◽

A Cell

ABSTRACTFungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, includingCandida glabrataandCandida krusei(species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3β,6α-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3β,6α-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway.

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Magnetic nanoparticles for the measurement of cell mechanics using force-induced remnant magnetization spectroscopy

Nanoscale ◽

10.1039/d0nr01421d ◽

2020 ◽

Vol 12 (27) ◽

pp. 14573-14580

Author(s):

Min Xu ◽

Xueyan Feng ◽

Feng Feng ◽

Hantao Pei ◽

Ruping Liu ◽

...

Keyword(s):

Magnetic Nanoparticles ◽

Cell Lines ◽

Cancer Cell ◽

Cell Mechanics ◽

Magnetic Nanoparticle ◽

Adhesion Force ◽

Force Spectroscopy ◽

Remnant Magnetization ◽

A Cell ◽

Novel Method

Interactions of magnetic nanoparticles with cells were investigated from a cell mechanics perspective, and magnetic nanoparticle-based force spectroscopy was developed as a novel method to measure the adhesion force among various cancer cell lines.

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Pyridines and Pyrimidines Mediating Activity against an Efflux-Negative Strain of Candida albicans through Putative Inhibition of Lanosterol Demethylase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.1.313-318.2004 ◽

2004 ◽

Vol 48 (1) ◽

pp. 313-318 ◽

Cited By ~ 13

Author(s):

Ed T. Buurman ◽

April E. Blodgett ◽

Kenneth G. Hull ◽

Daniel Carcanague

Keyword(s):

Candida Albicans ◽

Antifungal Activity ◽

Ergosterol Biosynthesis ◽

Clear Correlation ◽

Wild Type ◽

Inhibition Of Growth ◽

Ergosterol Synthesis ◽

A Cell ◽

Sterol Profile ◽

Lanosterol Demethylase

ABSTRACT The first step in ergosterol biosynthesis in Saccharomyces cerevisiae consists of the condensation of two acetyl coenzyme A (acetyl-CoA) moieties by acetoacetyl-CoA thiolase, encoded by ERG10. The inhibition of the sterol pathway results in feedback activation of ERG10 transcription. A cell-based reporter assay, in which increased ERG10 transcription results in elevated specific β-galactosidase activity, was used to find novel inhibitors of ergosterol biosynthesis that could serve as chemical starting points for the development of novel antifungal agents. A class of pyridines and pyrimidines identified in this way had no detectable activity against the major fungal pathogen Candida albicans (MICs > 64 μg · ml−1). However, a strain of C. albicans lacking the Cdr1p and Cdr2p efflux pumps was sensitive to the compounds (with MICs ranging from 2 to 64 μg · ml−1), suggesting that they are efficiently removed from wild-type cells. Quantitative analysis of sterol intermediates that accumulated during growth inhibition revealed the accumulation of lanosterol at the expense of ergosterol. Furthermore, a clear correlation was found between the 50% inhibitory concentration at which the sterol profile was altered and the antifungal activity, measured as the MIC. This finding strongly suggests that the inhibition of growth was caused by a reduction in ergosterol synthesis. The compounds described here are a novel class of antifungal pyridines and pyrimidines and the first pyri(mi)dines to be shown to putatively mediate their antifungal activity against C. albicans via lanosterol demethylase.

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Optimization of a dried residue specimen preparation method for quantifying analytes in plutonium metal using WDXRF

Powder Diffraction ◽

10.1154/1.2737463 ◽

2007 ◽

Vol 22 (2) ◽

pp. 152-155 ◽

Cited By ~ 3

Author(s):

Christopher G. Worley ◽

Lisa P. Colletti

Keyword(s):

Thin Films ◽

Preparation Method ◽

Specimen Preparation ◽

Preparation Time ◽

Relative Precision ◽

A Cell ◽

Novel Method ◽

Plutonium Metal

A novel method for preparing thin films was investigated for quantifying gallium and iron in plutonium solutions using WDXRF. This technique was developed to eliminate the potential for radioactive liquid to leak into the spectrometer, decrease specimen preparation time, and minimize waste. Samples were cast in μL quantities onto Kapton, and a surfactant was added to disperse the solution uniformly across the Kapton. After drying the specimens, they were sealed in a cell for analysis. Results to date indicate the method can provide a relative precision of ∼0.5% for gallium and ∼2% for iron, which is more than sufficient for routine sample analyses.

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Ergosterol Biosynthesis Inhibitors Become Fungicidal when Combined with Calcineurin Inhibitors against Candida albicans, Candida glabrata, and Candida krusei

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.3.956-964.2003 ◽

2003 ◽

Vol 47 (3) ◽

pp. 956-964 ◽

Cited By ~ 170

Author(s):

Chiatogu Onyewu ◽

Jill R. Blankenship ◽

Maurizio Del Poeta ◽

Joseph Heitman

Keyword(s):

Candida Albicans ◽

Antifungal Agents ◽

Calcineurin Inhibitors ◽

Therapeutic Window ◽

Ergosterol Biosynthesis ◽

Drug Synergism ◽

Wild Type ◽

Ergosterol Biosynthesis Inhibitors ◽

Biosynthesis Inhibitors ◽

Mutant Strains

ABSTRACT Azoles target the ergosterol biosynthetic enzyme lanosterol 14α-demethylase and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity. Unfortunately, azoles are generally fungistatic and resistance to fluconazole is emerging in several fungal pathogens. We recently established that the protein phosphatase calcineurin allows survival of Candida albicans during the membrane stress exerted by azoles. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles, resulting in potent fungicidal activity, and mutant strains lacking calcineurin are markedly hypersensitive to azoles. Here we establish that drugs targeting other enzymes in the ergosterol biosynthetic pathway (terbinafine and fenpropimorph) also exhibit dramatic synergistic antifungal activity against wild-type C. albicans when used in conjunction with CsA and FK506. Similarly, C. albicans mutant strains lacking calcineurin B are markedly hypersensitive to terbinafine and fenpropimorph. The FK506 binding protein FKBP12 is required for FK506 synergism with ergosterol biosynthesis inhibitors, and a calcineurin mutation that confers FK506 resistance abolishes drug synergism. Additionally, we provide evidence of drug synergy between the nonimmunosuppressive FK506 analog L-685,818 and fenpropimorph or terbinafine against wild-type C. albicans. These drug combinations also exert synergistic effects against two other Candida species, C. glabrata and C. krusei, which are known for intrinsic or rapidly acquired resistance to azoles. These studies demonstrate that the activity of non-azole antifungal agents that target ergosterol biosynthesis can be enhanced by inhibition of the calcineurin signaling pathway, extending their spectrum of action and providing an alternative approach by which to overcome antifungal drug resistance.

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Ergosterol biosynthesis inhibition: a target for antifungal agents.

Acta Biochimica Polonica ◽

10.18388/abp.1995_4900 ◽

1995 ◽

Vol 42 (4) ◽

pp. 465-479 ◽

Cited By ~ 30

Author(s):

K Barrett-Bee ◽

G Dixon

Keyword(s):

Crucial Role ◽

Biosynthetic Pathway ◽

Antifungal Agents ◽

Growth Conditions ◽

Ergosterol Biosynthesis ◽

Squalene Epoxidase ◽

Great Success ◽

Different Types ◽

Combination Studies

The isoprenoid sterols play a crucial role in the viability of all fungi; those unable to synthesise ergosterol because of inhibition, growth conditions or mutation must take it up from the environment. A range of compound types have been discovered which interfere with the biosynthetic pathway from acetate to ergosterol and these compounds have antifungal actions. Inhibition of several of the steps has yielded agents which have been used with great success as medical and agrochemical agents. The most important biosynthetic steps that have been exploited are inhibition of squalene epoxidase, (the allylamines and tolnaftate) C14 demethylation (the azoles), delta 7,8 isomerase and delta 14 reductase which are inhibited by the morpholines. Recent research has shown that inhibition of C24 methyltransferase and C4 demethylation also yield antifungal agents. Combination studies demonstrate that synergy between agents of different types can be measured. Fungicidal effects were observed when a combination of two fungistatic agents was used.

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