Microbial cyclic lipopeptides are an important class of antifungal compounds with applications in pharmacology and biotechnology. However, the cytotoxicity of many cyclic lipopeptides limits their potential as antifungal drugs. Here we present a structure-activity relationship study on the puwainaphycin/minutissamide (PUW/MIN) family of cyclic lipopeptides isolated from cyanobacteria. PUWs/MINs with variable fatty acid chain lengths differed in the dynamic of their cytotoxic effect despite their similar IC50 after 48 hours (2.8 μM for MIN A and 3.2 μM for PUW F). Furthermore, they exhibited different antifungal potency with the lowest MIC values obtained for MIN A and PUW F against the facultative human pathogen Aspergillus fumigatus (37 μM) and the plant pathogen Alternaria alternata (0.6 μM), respectively. We used a Grignard-reaction with alkylmagnesium halides to lengthen the lipopeptide FA moiety as well as the Steglich esterification on the free hydroxyl substituents to prepare semi-synthetic lipopeptide variants possessing multiple fatty acid tails. Cyclic lipopeptides with extended and branched FA tails showed improved strain-specific antifungal activity against A. fumigatus (MIC = 0.5-3.8 μM) and A. alternata (MIC = 0.1-0.5 μM), but with partial retention of the cytotoxic effect (∼10-20 μM). However, lipopeptides with esterified free hydroxyl groups possessed substantially higher antifungal potencies, especially against A. alternata (MIC = 0.2-0.6 μM), and greatly reduced or abolished cytotoxic activity (>20 μM). Our findings pave the way for a generation of semi-synthetic variants of lipopeptides with improved and selective antifungal activities.
- Publikační typ
- časopisecké články MeSH
Microalgae accumulate bioavailable selenium-containing amino acids (Se-AAs), and these are useful as a food supplement. While this accumulation has been studied in phototrophic algal cultures, little data exists for heterotrophic cultures. We have determined the Se-AAs content, selenium/sulfur (Se/S) substitution rates, and overall Se accumulation balance in photo- and heterotrophic Chlorella cultures. Laboratory trials revealed that heterotrophic cultures tolerate Se doses ∼8-fold higher compared to phototrophic cultures, resulting in a ∼2-3-fold higher Se-AAs content. In large-scale experiments, both cultivation regimes provided comparable Se-AAs content. Outdoor phototrophic cultures accumulated up to 400 μg g-1 of total Se-AAs and exhibited a high level of Se/S substitution (5-10%) with 30-60% organic/total Se embedded in the biomass. A slightly higher content of Se-AAs and ratio of Se/S substitution was obtained for a heterotrophic culture in pilot-scale fermentors. The data presented here shows that heterotrophic Chlorella cultures provide an alternative for Se-enriched biomass production and provides information on Se-AAs content and speciation in different cultivation regimes.
- Klíčová slova
- Chlorella, heterotrophic cultivation, methylselenocysteine, microalgae, phototrophic cultivation, selenium, selenocysteine, selenomethionine,
- MeSH
- aminokyseliny analýza metabolismus MeSH
- biomasa MeSH
- Chlorella klasifikace růst a vývoj metabolismus účinky záření MeSH
- fototrofní procesy MeSH
- heterotrofní procesy MeSH
- mikrořasy chemie růst a vývoj metabolismus účinky záření MeSH
- selen analýza metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aminokyseliny MeSH
- selen MeSH
Selenium (Se) is an indispensable microelement in our diet and health issues resulting from deficiencies are well documented. Se-containing food supplements are available on the market including Se-enriched Chlorella vulgaris (Se-Chlorella) which accumulates Se in the form of Se-amino acids (Se-AAs). Despite its popular uses, data about the bioaccessibility of Se-AAs from Se-Chlorella are completely missing. In the present study, gastrointestinal digestion times were optimized and the in vitro bioaccessibility of Se-AAs in Se-Chlorella, Se-yeast, a commercially available Se-enriched food supplement (Se-supplement) and Se rich foods (Se-foods) were compared. Higher bioaccessibility was found in Se-Chlorella (∼49%) as compared to Se-yeast (∼21%), Se-supplement (∼32%) and Se-foods. The methods used in production of Se-Chlorella biomass were also investigated. We found that disintegration increased bioaccessibility whereas the drying process had no effect. Similarly, temperature treatment by microwave oven also increased bioaccessibility whereas boiling water did not.
- Klíčová slova
- Bioaccessibility, Gas chromatography atmospheric pressure chemical ionization high resolution mass spectrometry (GC-APCI-HRMS), Methanesulfonic acid (PubChem CID: 6395), Methylselenocysteine (PubChem CID: 147004), Selenium enriched Chlorella vulgaris, Selenium enriched food supplement, Selenium rich foods, Selenized yeast, Selenoamino acids, Selenocysteine (PubChem CID: 6326983), Selenomethionine (PubChem CID: 15103),
- MeSH
- biomasa MeSH
- Chlorella vulgaris metabolismus MeSH
- fortifikované potraviny analýza MeSH
- hmotnostní spektrometrie metody MeSH
- limita detekce MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí metody MeSH
- potravní doplňky analýza MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- selen chemie metabolismus MeSH
- selenocystein analýza metabolismus MeSH
- selenomethionin analýza metabolismus MeSH
- teplota MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- selen MeSH
- selenocystein MeSH
- selenomethionin MeSH
Muscotoxins are cyanobacterial cyclic lipopeptides with potential applications in biomedicine and biotechnology. In this study, Desmonostoc muscorum CCALA125 strain extracts were enriched by polymeric resin treatment, and subjected to HPCCC affording three cyclic lipopeptides (1⁻3), which were further repurified by semi-preparative HPLC, affording 1, 2, and 3, with a purity of 86%, 92%, and 90%, respectively. The chemical identities of 2⁻3 were determined as muscotoxins A and B, respectively, by comparison with previously reported ESI-HRMS/MS data, whereas 1 was determined as a novel muscotoxin variant (muscotoxin C) using NMR and ESI-HRMS/MS data. Owing to the high yield (50 mg), compound 2 was broadly screened for its antimicrobial potential exhibiting a strong antifungal activity against Alternaria alternata, Monographella cucumerina, and Aspergillus fumigatus, with minimum inhibitory concentration (MIC) values of 0.58, 2.34, and 2.34 µg/mL; respectively, and weak antibacterial activity against Bacillus subtilis with a MIC value of 37.5 µg/mL. Compounds 1 and 3 were tested only against the plant pathogenic fungus Sclerotinia sclerotiorum due to their low yield, displaying a moderate antifungal activity. The developed chromatographic method proved to be an efficient tool for obtaining muscotoxins with potent antifungal properties.
- Klíčová slova
- antimicrobial activity, cyanobacteria, cyclic lipopeptides (CLPs), high performance countercurrent chromatography (HPCCC), muscotoxins, γ-methylproline,
- MeSH
- antiinfekční látky chemie izolace a purifikace farmakologie MeSH
- Ascomycota účinky léků MeSH
- Bacillus subtilis účinky léků MeSH
- bakteriální toxiny chemie izolace a purifikace farmakologie MeSH
- cyklické peptidy chemie farmakologie MeSH
- lipopeptidy chemie farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- sinice metabolismus MeSH
- syntetické pryskyřice chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antiinfekční látky MeSH
- bakteriální toxiny MeSH
- cyklické peptidy MeSH
- lipopeptidy MeSH
- syntetické pryskyřice MeSH
Benthic cyanobacteria recognized as producers of natural products, including cyanotoxins, have been neglected for systematic toxicological studies. Thus, we have performed a broad study investigating cyanotoxin potential of 311 non-planktic nostocacean representatives combining molecular and chemical analyses. Out of these, a single strain Nostoc sp. Treb K1/5, was identified as a new microcystin producer. Microcystins [Asp3]MC-YR, [Asp3]MC-FR, [Asp3]MC-HtyR and Ala-Leu/Ile-Asp-Arg-Adda-Glu-Mdha are reported for the first time from the genus Nostoc. All the studied strains were also analyzed for the occurrence of nodularins, cylindrospermopsin and (homo)anatoxin-a, yet no novel producer has been discovered. Our findings indicate rare occurrence of the common cyanotoxins in non-planktic nostocaceae which is in contrast with frequent reports of cyanotoxin producers among phylogenetically closely related planktic cyanobacteria.
- Klíčová slova
- Anabaena, Cyanotoxins, Heterocytous cyanobacteria, Microcystin, Non-planktic Nostocaceae, Nostoc,
- MeSH
- fylogeneze MeSH
- mikrocystiny chemie metabolismus MeSH
- Nostoc genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- mikrocystiny MeSH
Quantification of selenated amino-acids currently relies on methods employing inductively coupled plasma mass spectrometry (ICP-MS). Although very accurate, these methods do not allow the simultaneous determination of standard amino-acids, hampering the comparison of the content of selenated versus non-selenated species such as methionine (Met) and selenomethionine (SeMet). This paper reports two approaches for the simultaneous quantification of Met and SeMet. In the first approach, standard enzymatic hydrolysis employing Protease XIV was applied for the preparation of samples. The second approach utilized methanesulfonic acid (MA) for the hydrolysis of samples, either in a reflux system or in a microwave oven, followed by derivatization with diethyl ethoxymethylenemalonate. The prepared samples were then analyzed by multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS). Both approaches provided platforms for the accurate determination of selenium/sulfur substitution rate in Met. Moreover the second approach also provided accurate simultaneous quantification of Met and SeMet with a low limit of detection, low limit of quantification and wide linearity range, comparable to the commonly used gas chromatography mass spectrometry (GC-MS) method or ICP-MS. The novel method was validated using certified reference material in conjunction with the GC-MS reference method.
- Klíčová slova
- Diethyl ethoxymethylenemalonate, Liquid chromatography mass spectrometry, Methanesulfonic acid, Methionine, Protease XIV, Selenomethionine,
- MeSH
- Chlorella vulgaris metabolismus MeSH
- limita detekce MeSH
- lineární modely MeSH
- malonáty MeSH
- methansulfonáty MeSH
- methionin analýza metabolismus MeSH
- pronasa MeSH
- reprodukovatelnost výsledků MeSH
- selenomethionin analýza metabolismus MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- diethyl (ethoxymethylene)malonate MeSH Prohlížeč
- malonáty MeSH
- methanesulfonic acid MeSH Prohlížeč
- methansulfonáty MeSH
- methionin MeSH
- pronasa MeSH
- selenomethionin MeSH
In this work we present a simple and cost-effective approach for the determination of selenium species in algae and yeast biomass, based on a combination of thin-layer chromatography (TLC) with diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). Extraction of freeze-dried biomass was performed in 4M methanesulphonic acid and the selenium species were vaporized from cellulose TLC plates employing a continuous-wave infrared diode laser with power up to 4 W using a simple laboratory-built apparatus. Selenomethionine and selenocysteine were quantified with limits of detection 3 μg L-1 in a Se-enriched microalgae Chlorella vulgaris and yeast certified reference material SELM-1. Results delivered by TLC-DLTV ICP MS were consistent with those obtained by a routine coupling of high-performance liquid chromatography (HPLC) to ICP MS. In addition, the TLC approach is capable of analyzing extract containing even undiluted crude hydrolysates that could damage HPLC columns.
- Klíčová slova
- Chlorella vulgaris algae, Diode laser thermal vaporization (DLTV), Inductively coupled plasma mass spectrometry (ICP MS), Liquid chromatography (LC), Selenium speciation (selenomethionine, selenocysteine), Thin-layer chromatography (TLC),
- MeSH
- chemické techniky analytické ekonomika metody MeSH
- Chlorella vulgaris chemie MeSH
- chromatografie na tenké vrstvě * MeSH
- hmotnostní spektrometrie * přístrojové vybavení MeSH
- lasery polovodičové MeSH
- Saccharomyces cerevisiae chemie MeSH
- selenocystein analýza MeSH
- selenomethionin analýza MeSH
- spektrální analýza MeSH
- volatilizace MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- selenocystein MeSH
- selenomethionin MeSH
Early diagnosis and treatment of multiple sclerosis (MS) in the initial stages of the disease can significantly retard its progression. The aim of the present study was to identify changes in the cerebrospinal fluid proteome in patients with relapsing-remitting MS and clinically isolated MS syndrome who are at high risk of developing MS (case group) compared to healthy population (control) in order to identify potential new markers, which could ultimately aid in early diagnosis of MS. The protein concentrations of each of the 11 case and 15 control samples were determined using a bicinchoninic acid assay. Nanoscale liquid chromatography coupled with tandem mass spectrometry was used for protein identification. Proteomics data were processed using the Perseus software suite and R. The results were filtered using the Benjamini-Hochberg procedure for the false discovery rate (FDR) correction (FDR<0.05). The results showed that, 26 proteins were significantly dysregulated in case samples compared to the controls. Nine proteins were found to be significantly less abundant in case samples, while the abundance of 17 proteins was significantly increased in case samples compared to controls. Three of the proteins were previously linked to RR MS, including immunoglobulin (Ig) γ-1 chain C region, Ig heavy chain V-III region BRO and Ig κ chain C region. Three proteins that were uniquely expressed in patients with RR MS were identified and these proteins may serve as prognostic biomarkers for identifying patients with a high risk of developing RR MS.
- Klíčová slova
- biomarkers, cerebrospinal fluid, clinically isolated syndrome, multiple sclerosis, proteins, proteomic analysis,
- Publikační typ
- časopisecké články MeSH
In this paper, a pencil graphite electrode was pretreated using chronoamperometry technique in phosphate buffer solution (pH=7.0) for sensitive determination of quercetin. Oxidation of quercetin was investigated using pretreated pencil graphite electrode and anodic stripping differential pulse voltammetry. Under optimal conditions, the anodic current of quercetin exhibited linear response to its concentration in the range from 0.001 to 1.5 µmol/L with the limit of detection of 0.3·10-3 µmol/L. The proposed method was successfully applied for the determination of quercetin in cranberry and blackcurrant juices with recovery rate from 93.2 to 94.7%. Solid-phase extraction was found to be necessary prior to voltammetric determination of quercetin in fruit juice samples using pretreated pencil graphite electrode.
- Klíčová slova
- fruit juices, pencil graphite electrode, quercetin, voltammetry,
- Publikační typ
- časopisecké články MeSH
