Antifungal amphiphilic aminoglycoside K20: bioactivities and mechanism of action

K20 is a novel amphiphilic antifungal aminoglycoside that is synthetically derived from the antibiotic kanamycin A. Reported here are investigations of K20′s antimicrobial activities, cytotoxicity, and fungicidal mechanism of action. In vitro growth inhibitory activities against a variety of human and plant pathogenic yeasts, filamentous fungi, and bacteria were determined using microbroth dilution assays and time-kill curve analyses, and hemolytic and animal cell cytotoxic activities were determined. Effects on Cryptococcus neoformans H-99 infectivity were determined with a preventive murine lung infection model. The antifungal mechanism of action was studied using intact fungal cells, yeast lipid mutants, and small unilamellar lipid vesicles. K20 exhibited broad-spectrum in vitro antifungal activities but not antibacterial activities. Pulmonary, single dose-administration of K20 reduced C. neoformans lung infection rates 4-fold compared to controls. Hemolysis and half-maximal cytotoxicities of mammalian cells occurred at concentrations that were 10 to 32-fold higher than fungicidal MICs. With fluorescein isothiocyanate (FITC), 20–25 mg/L K20 caused staining of \u3e95% of C. neoformans and Fusarium graminearum cells and at 31.3 mg/L caused rapid leakage (30–80% in 15 min) of calcein from preloaded small unilamellar lipid vesicles. K20 appears to be a broad-spectrum fungicide, capable of reducing the infectivity of C. neoformans, and exhibits low hemolytic activity and mammalian cell toxicity. It perturbs the plasma membrane by mechanisms that are lipid modulated. K20 is a novel amphiphilic aminoglycoside amenable to scalable production and a potential lead antifungal for therapeutic and crop protection applications

Antifungal Activities of 4”,6”-Disubstituted Amphiphilic Kanamycins

Amphiphilic kanamycins derived from the classic antibiotic kanamycin have attracted interest due to their novel bioactivities beyond inhibition of bacteria. In this study, the recently described 4″,6″-diaryl amphiphilic kanamycins reported as inhibitors of connexin were examined for their antifungal activities. Nearly all 4″,6″-diaryl amphiphilic kanamycins tested had antifungal activities comparable to those of 4″,6″-dialkyl amphiphilic kanamycins, reported previously against several fungal strains. The minimal growth inhibitory concentrations (MICs) correlated with the degree of amphiphilicity (cLogD) of the di-substituted amphiphilic kanamycins. Using the fluorogenic dyes, SYTOXTM Green and propidium iodide, the most active compounds at the corresponding MICs or at 2×MICs caused biphasic dye fluorescence increases over time with intact cells. Further lowering the concentrations to half MICs caused first-order dye fluorescence increases. Interestingly, 4×MIC or 8×MIC levels resulted in fluorescence suppression that did not correlate with the MIC and plasma membrane permeabilization. The results show that 4″,6″-diaryl amphiphilic kanamycins are antifungal and that amphiphilicity parameter cLogD is useful for the design of the most membrane-active versions. A cautionary limitation of fluorescence suppression was revealed when using fluorogenic dyes to measure cell-permeation mechanisms with these antifungals at high concentrations. Finally, 4″,6″-diaryl amphiphilic kanamycins elevate the production of cellular reactive oxygen species as other reported amphiphilic kanamycins

Estudi per a la transformació d'un taller de tecnologia en un espai "maker" d'un institut de secundària

Organitzacions de nivell internacional, posen de manifest una educació desfasada, incapaç de donar resposta a les necessitats laborals presents i de futur (World Economic Forum, 2017). El present document planteja el moviment "Maker" com a eina per millorar habilitats, com el pensament crític i creatiu, la resolució de problemes i competències de l'àmbit STEAM i Digital, alineades amb les necessitats laborals. També es recull l'estat de l'art del moviment "Maker" i quines són les bases del moviment i la seva filosofia. S'analitzen els espais "Maker" en entorns escolars, les possibilitats formatives, la planificació per a la implantació i els avantatges i inconvenients. Finalment es plantegen tres activitats didàctiques, una lligada al taller tradicional (construcció amb eines) i les altres dues a espais "Makers" (impressió 3D i talladora làser). Comparar les activitats, permet mostrar com els espais "Maker" tenen una clara tendència a treballar sobre l'àmbit digital i en menor grau també sobre l'àmbit matemàtic i el personal i social

HPLC-MS analysis of cowpea extracts for destruxin (DTX) production.

<p>(A) Analysis of not colonized (free of fungus) plants (negative control); (B) plants endophytically colonized by <i>Metarhizium robertsii</i> ARSEF 2575; and (C) not-colonized plants spiked with DTX standards (positive control). The cowpea seeds, both fungus-inoculated and control (not colonized) were incubated on moist filter paper under optimal light (16L∶8D) and temperature (25°C) conditions for 12 days at which time the germlings had developed roots, stems, cotyledons and two true leaves. DTXs were extracted from entire plants using methanol 100% and SPE-C18 cartridges.</p

Time course of <i>in vitro</i> production of DTXs A, B, and E by <i>Metarhizium anisopliae</i> s.l. ARSEF 759.

<p>Destruxin concentrations in supernatants of submerged liquid cultures were determined by quantitative HPLC-UV analysis of the major components, viz., DTXs A, B and E. Values are expressed in mg DTXs per g dry weight mycelium.</p

Mean mortality (%) ± standard error of <i>Tenebrio molitor</i> larvae 5 days after treatment, and <i>Galleria mellonella</i> 3 days after treatment.

<p>Bioassays were performed 3 times (using two replicates for each isolate) under controlled conditions (27°C), using new batches of larvae and conidia in each bioassay. Controls were treated with Tween 80 (0.01%) solution. Means followed by the same letter in a column do not differ statistically (<i>P</i> ≥ 0.05) (Kruskal-Wallis test followed by Student-Newman-Keuls).</p

<i>Metarhizium</i> spp. isolates used in this study, including their hosts and origins (state and country).

<p>* USDA-ARS Collection of Entomopathogenic Fungal Cultures, Ithaca, NY.</p><p>Identifications were provided September 2012 by curator of ARSEF* Richard Humber.</p

Time course of <i>in vitro</i> production of DTXs A, B, and E by <i>Metarhizium robertsii</i> ARSEF 2575.

<p>Destruxin concentrations in supernatant of submerged liquid cultures were determined by quantitative HPLC analysis of the major components, viz., DTXs A, B and E. Values are expressed in mg DTXs per g dry weight mycelium.</p