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Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, using genomic and biochemical approaches, we investigated the gene cluster for inositol lipid synthesis in BT using a previously undescribed strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol dihydroceramide, determined the crystal structure of the recombinant BT MIP synthase enzyme and identified the phosphatase responsible for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP-DAG) to phosphatidylinositol (PI-DAG). In vitro, loss of inositol lipid production altered BT capsule expression and antimicrobial peptide resistance. In vivo, loss of inositol lipids decreased bacterial fitness in a gnotobiotic mouse model. We identified a second putative, previously undescribed pathway for bacterial PI-DAG synthesis without a PIP-DAG intermediate, common in Prevotella. Our results indicate that inositol sphingolipid production is widespread in host-associated Bacteroidetes and has implications for symbiosis.

Introduction: Chronic inflammation is a major risk factor for cardiovascular disease in chronic kidney disease (CKD). The underlying mechanisms are incompletely understood, but may be linked to gut dysbiosis. We aim to describe the microbiota-immune interaction in a cohort of pediatric CKD, thus independent of confounding comorbidities frequently seen in adult patients. Methods: We analyzed the fecal microbiome, plasma metabolites and peripheral immune phenotypes in 48 children (normal kidney function (HC, n=10), CKD stage G3-G4 (CKD, n=12), G5 treated by hemodialysis (HD, n=11) or kidney transplantation (KT, n=15)) with a mean age of 10.6 ± 3.8 (mean±SD) years. Results: Children exhibit stage-dependently increased cardiovascular risk as seen by the presence of arterial hypertension despite anti-hypertensive medication. Serum TNF-α (2.87±1.10 (HC); 7.54±2.29 (CKD); 9.63±2.73 (HD); 5.70 ± 1.66 (KT) pg/mL) and sCD14 (1.83±0.28 (HC); 2.88±0.47 (CKD); 2.83±0.69 (HD); 2.22±0.34 (KT) μg/mL) were elevated, indicating inflammation, gut barrier dysfunction and endotoxemia. We observed compositional and functional alterations of the microbiome, including a diminished production of short-chain fatty acids (e.g. propionate 10.43±5.49 (HC); 8.95±4.66 (CKD); 2.75±3.84 (HD); 12.49±4.89 (KT) μM). Plasma metabolite analysis revealed a stage-dependent increase of tryptophan metabolites of bacterial origin (e.g. indoxylsulfate). Serum from HD patients activated the aryl hydrocarbon receptor and stimulated TNF production in monocytes (fold change over HC: 1.80±0.61), corresponding to a pro-inflammatory shift from classical to non-classical and intermediate monocytes. Moreover, unsupervised analysis (FlowSOM) of T cells revealed a loss of mucosa-associated invariant T (MAIT) cells and regulatory T cell subtypes (e.g. CCR6+ and CXCR3+ subsets) in HD patients. Conclusions: In conclusion, gut barrier dysfunction and microbial metabolite imbalance mediate the pro-inflammatory immune phenotype in CKD. These dysbiosis-driven immunological changes are already detectable in children with CKD, in whom comorbidities usually found in adults are absent, highlights the specificity of CKD-related microbiota-immune interaction.

CKD is characterized by a sustained proinflammatory response of the immune system, promoting hypertension and cardiovascular disease. The underlying mechanisms are incompletely understood but may be linked to gut dysbiosis. Dysbiosis has been described in adults with CKD; however, comorbidities limit CKD-specific conclusions.We analyzed the fecal microbiome, metabolites, and immune phenotypes in 48 children (with normal kidney function, CKD stage G3-G4, G5 treated by hemodialysis [HD], or kidney transplantation) with a mean±SD age of 10.6±3.8 years.Serum TNF-Gut barrier dysfunction and microbial metabolite imbalance apparently mediate the proinflammatory immune phenotype, thereby driving the susceptibility to cardiovascular disease. The data highlight the importance of the microbiota-immune axis in CKD, irrespective of confounding comorbidities.

Chronic kidney disease (CKD) is characterized by a sustained pro-inflammatory response. The underlying mechanisms are incompletely understood, but may be linked to gut dysbiosis. Dysbiosis has been described in adults with CKD; however, comorbidities limit CKD-specific conclusions. We analyzed the fecal microbiome, metabolites and immune phenotypes in children at three different CKD stages (G3-G4, G5 (hemodialysis), after kidney transplantation) and healthy controls. Serum TNF-α and sCD14 were stage-dependently elevated, indicating inflammation and gut barrier dysfunction. We observed microbiome alterations in CKD, including a diminished production of short-chain fatty acids. Bacterial tryptophan metabolites were increased in CKD. CKD serum activated the aryl hydrocarbon receptor and stimulated TNF-α production by monocytes, corresponding to a shift towards intermediate/non-classical monocytes. Unsupervised T cell analysis revealed pro-inflammatory shifts in MAIT and Treg cells. Thus, gut barrier dysfunction and microbial metabolites exacerbate inflammation and may therefore contribute to the increased cardiovascular burden in CKD.

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 mu M for MIN A and 3.2 mu M 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 mu M) and the plant pathogen Alternaria alternata (0.6 mu M), 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 mu M) and A. alternata (MIC = 0.1-0.5 mu M), but with partial retention of the cytotoxic effect (similar to 10-20 mu M). However, lipopeptides with esterified free hydroxyl groups possessed substantially higher antifungal potencies, especially against A. alternata (MIC = 0.2-0.6 mu M), and greatly reduced or abolished cytotoxic activity (>20 mu M). Our findings pave the way for a generation of semi-synthetic variants of lipopeptides with improved and selective antifungal activities.

Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, using genomic and biochemical approaches, we investigated the gene cluster for inositol lipid synthesis in BT using a previously undescribed strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol dihydroceramide, determined the crystal structure of the recombinant BT MIP synthase enzyme and identified the phosphatase responsible for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP-DAG) to phosphatidylinositol (PI-DAG). In vitro, loss of inositol lipid production altered BT capsule expression and antimicrobial peptide resistance. In vivo, loss of inositol lipids decreased bacterial fitness in a gnotobiotic mouse model. We identified a second putative, previously undescribed pathway for bacterial PI-DAG synthesis without a PIP-DAG intermediate, common in Prevotella. Our results indicate that inositol sphingolipid production is widespread in host-associated Bacteroidetes and has implications for symbiosis.

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.

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.

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

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

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, litt...

Acrobic acid is key substance in the human metabolism and the rapid and accurate determination in food is of a great interest. Ascorbic acid is an electroactive compound, however poorly responded on the bare carbon paste electrodes. In this paper, brilliant cresyl blue and multi-walled carbon nanotubes were used for the modification of carbon paste electrode. Brilliant cresyl blue acts as a mediator improving the transition of electrons, whereas multiwalled carbon nanotubes increased the surface of the electrode. Both brilliant cresyl blue and multiwalled carbon nanotubes were added directly to the composite material. The electrochemical behavior of modified electode was determined in electrolyte at various pH, and the effect of the scan rate was also performed. It was shown that the electrochemical process on the surface of the modified carbon paste electrode was diffusion-controlled. The resulted modified carbon paste electrode showed a good electrocatalytic activity towards the oxidation of ascorbic acid at a reduced overpotential of +100 mV descreasing the risk of interferences. A linear response of the ascorbic acid oxidation current measured by the amperometry in the range of 0.1 - 350 µmol.L-1 was obtained applying the sensor for the standard solution. The limit of detection and limit of quantification was found to be 0.05 and 0.15 µmol.L-1, respectively. The novel method was applied for the determination of ascorbic acid in pharmaceutical vitamin preparation and fruit juice, and the results were in good agreement with the standard HPLC method. The presented modification of carbon paste electrode is suitable for the fast, sensitive and very accurate determination of ascorbic acid in fruit juices and pharmaceutical preparation.

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

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.

Vu D.L., Ertek B., Dilgin Y., Cervenka L. (2015): Voltammetric determination of tannic acid in beverages using pencil graphite electrode. Czech J. Food Sci., 33: 72-76. The pretreated pencil graphite electrode (Pre-PGE) prepared by a chronoamperometry technique was applied for the determination of tannic acid using anodic stripping differential pulse voltammetry. The currents obtained from voltammetry measurements at optimum conditions were linearly correlated with the concentration of tannic acid. Calibration curve was obtained for tannic acid in the concentration range of 5.0-500 × 10 -9 mol/l. The limit of detec - tion was found to be 1.5 × 10 -9 mol/l. The content of tannic acid in beverage samples determined with Pre-PGE was in good agreement with that obtained by the standard spectrophotometric method.

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&ndash, 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&ndash, 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 &micro, g/mL, respectively, and weak antibacterial activity against Bacillus subtilis with a MIC value of 37.5 &micro, g/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.

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.

Proper setting of cultivation conditions is essential for production of high-value compounds in microbial biotechnology. The present study characterizes photoautotrophic growth and capacity to accumulate the antiproliferative secondary metabolite Nocuolin A (NoA) in cyanobacterium Nostoc sp. CCAP 1453/38. As the cyanobacterial culture was found to be non-axenic, the bacteria accompanying the culture were characterized, then the growth demands and NoA production in the Nostoc-bacterial consortium were determined, and finally an axenic strain was prepared. For the purposes of growth characterization, the culture was maintained in a quasi-continuous regime under various light intensities, temperatures, and inorganic carbon concentrations in a small-scale laboratory photobioreactor. The maximum biomass growth rate obtained was 0.10 h−1 (doubling time Dt = 6.93 h). Following optimal growth conditions were identified: temperature of 35 °C, light intensity 600 μmol(photons) m−2 s−1, and 2500 ppm CO2 in the sparging gas. As the temperature optima for the biomass production and for NoA accumulation differed, biphasic cultivation for maximal NoA yield was designed, leading to a three times more effective cultivation procedure compared to batch culture maintained at a temperature optimal for NoA production. The increased NoA accumulation at reduced temperature that correlated with enhanced expression of NoA biosynthetic genes after the temperature shift suggested its regulation occurs at the expression level. It has further been shown that NoA production is reduced in axenic culture, which indicates that it is also triggered by presence of bacteria. This study shows an example of how a biphasic cultivation mode with different temperatures can be used in high-value compound production processes. It also brings direct evidence that cyanobacterial strain axenization can lead to a rapid decrease in production of valuable compounds and that non-axenic strains may be considered more suitable for retrieval and initial production of novel pharmaceutical leads.

Lutein is a natural carotenoid with health promoting properties whose commercial source is the yellow Marigold flower, but its production is limited by climate, planting area and high labor costs. The microalga Chlorella vulgaris is a promising lutein source; however, no commercial lutein production uses microalgae. In the present study, the lower phase (LP4) of the biphasic solvent system composed of n-heptane–ethanol–water (5:4:1.5, v/v/v) was used both as a solvent for microalgae biomass extraction and as a mobile phase for lutein isolation using high performance counter-current chromatography (HPCCC). The ultrasound-assisted extraction of biomass with LP4 for 30 min led to an extract with a lutein content (3.20 mg/g dried biomass) that was higher than those obtained by the reference solvents tetrahydrofuran and dichloromethane. A multi-injection HPCCC method was developed for lutein isolation, where LP4 was pumped as the mobile phase at a flow rate of 8 mL/min, whereas its upper phase was the stationary phase. In total, 2 g of C. vulgaris extract were processed affording 60 mg of lutein (92% purity), which was further cleaned up by gel permeation chromatography yielding 50 mg of lutein (97% purity). The chemical identity of the purified compound was confirmed by mass spectrometry and absorption spectroscopy in comparison with an authentic standard. The developed HPCCC method may serve as a reference for promoting the development of a new production model of microalgae-based lutein.

In this paper, a silica-gel-modified carbon paste electrode (Si-gel/CPE) was used to determine the anti-cancer drug emodin by anodic stripping differential pulse voltammetry (ASDPV). The effects of the silica-gel content, the pH of the supporting electrolyte, and the scan rate on the oxidation current of emodin were investigated. The oxidation currents of emodin obtained from ASDPV measurements were linearly correlated with the concentration in the range of 5.0 × 10-9 to 300.0 × 10-9 mol L-1. The limit of detection was determined to be 1.5 × 10-9 mol L-1. The current method was successfully applied to determine emodin in a knotweed root sample, with recovery rate of 92.5% to 98.3%.

This study investigates a new approach for the amperometric determination of sulfide using a hematoxylin multiwalled carbon nanotubes modified carbon paste electrode (HM-MWCNTs/CPE). The experimental results show that HM-MWCNTs/CPE significantly enhances the electrocatalytic activity towards sulfide oxidation. Cyclic voltammetric studies show that the peak potential of sulfide shifted from +400 mV at unmodified CPE to +175 mV at HM-MWCNTs/CPE. The currents obtained from amperometric measurements at optimum conditions were linearly correlated with the concentration of sulfide. The calibration curve was obtained for sulfide concentrations in the range of 0.5–150×10−6 mol L−1. The detection limit was found to be 0.2×10−6 mol L−1 for the amperometric method. The proposed method was successfully applied to a river water sample in Pardubice, Czech Republic.

The attachment of Staphylococcus epidermidis to the surface of carbon paste electrode (CPE) by applying positive potentials (50 - 600 mV) with regard of various buffer pH, cultivation time and solid-medium type was studied. The attachment process was analyzed by measuring the electric current derived from the dye (amido black) adsorbed on the vacant areas of CPE after attachment of microbial cells. The pH was not identified as a single main factor affecting the attachment (p > 0.05), however further insight revealed that the potentials applied had different effects on the microbial cells attachment. Both cultivation time and solid-medium type significantly affected the microbial attachment. Generally, increase of cultivation time up to 168 h resulted in increase of adhesion. Applying potentials 300, 400 and 200 mV resulted in the highest attachment process for bacteria cultivated for 24, 48 and 168 h, respectively. S. epidermidis cultivated on the blood agar and Baird-Parker agar plates showed the higher extent of attachment.

This study investigates a new approach for the electrocatalytic determination of sulfide in a flow injection analysis (FIA) system using a Prussian blue modified glassy carbon electrode (PB/GCE). The results from experiments show that PB/GCE significantly enhances the electrocatalytic activity towards sulfide oxidation. A homemade flow electrochemical cell was used to perform the electrocatalytic determination of sulfide in the FIA system. The currents obtained from amperometric measurements in the HA system at optimum conditions (carrier solution, pH 8.0; Britton-Robinson buffer solution containing 0.1 M KCl; flow rate, 1.4 mL/min; transmission tubing length, 10 cm; injection volume, 100 mu L; constant applied potential, +150 mV vs. Ag/AgCl/KClsat) were linearly correlated with the sulfide concentration. A calibration curve was obtained for sulfide concentrations in the range of 0.5 - 100 mu M. The detection limit was found to be 0.3 mu M for the amperometric method. The proposed method was successfully applied to wastewater sample. Finally, results from sulfide measurements by PB/GCE were in good agreement with those obtained from the spectrophotometric method.