Cell compensatory responses of fungi to damage of the cell wall induced by Calcofluor White and Congo Red with emphasis on Sporothrix schenckii and Sporothrix globosa. A review.

The cell wall (CW) of fungi exhibits a complex structure and a characteristic chemical composition consisting almost entirely of interacting crystalline and amorphous polysaccharides. These are synthesized by a number of sugar polymerases and depolymerases encoded by a high proportion of the fungal genome (for instance, 20% in Saccharomyces cerevisiae). These enzymes act in an exquisitely coordinated process to assemble the tridimensional and the functional structure of the wall. Apart from playing a critical role in morphogenesis, cell protection, viability and pathogenesis, the CW represents a potential target for antifungals as most of its constituents do not exist in humans. Chitin, ß-glucans and cellulose are the most frequent crystalline polymers found in the fungal CW. The hexosamine biosynthesis pathway (HBP) is critical for CW elaboration. Also known as the Leloir pathway, this pathway ends with the formation of UDP-N-GlcNAc after four enzymatic steps that start with fructose-6-phosphate and L-glutamine in a short deviation of glycolysis. This activated aminosugar is used for the synthesis of a large variety of biomacromolecules in a vast number of organisms including bacteria, fungi, insects, crustaceans and mammalian cells. The first reaction of the HBP is catalyzed by GlcN-6-P synthase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16), a critical enzyme that has been considered as a potential target for antifungals. The enzyme regulates the amount of cell UDP-N-GlcNAc and in eukaryotes is feedback inhibited by the activated aminosugar and other factors. The native and recombinant forms of GlcN-6-P synthase has been purified and characterized from both prokaryotic and eukaryotic organisms and demonstrated its critical role in CW remodeling and morphogenesis after exposure of some fungi to agents that stress the cell surface by interacting with wall polymers. This review deals with some of the cell compensatory responses of fungi to wall damage induced by Congo Red and Calcofluor White.

Protective effects of curcumin on bleomycin-induced changes in lung glycoproteins.

The present study investigated the therapeutic effect of curcumin on bleomycin (BLM)-induced alterations in glycoprotein components in the fibrotic lungs. Analysis of the bronchoalveolar lavage fluid (BALF) demonstrated increased fibronectin content at 3, 5, 7, and 14 days after BLM administration. Similarly, lung tissue fibronectin content revealed a progressive increase at various times (days 3, 5, 7, 14, and 28) during the development of lung fibrosis. In addition, alveolar macrophage release of fibronectin was also elevated in BLM-treated rats. Analysis of carbohydrate moieties of glycoproteins revealed an increase in total hexose, fucose, sialic acid and hexosamine levels at 7, 14, and 28 days after BLM treatment. Furthermore, the activities of lung glycosidases such as N-acetyl-ß-D-glucosaminidase, ß-glucosidase, ß-galactosidase, and ß-fucosidase in the fibrotic rats were elevated. Importantly, curcumin significantly inhibited the BLM-induced increases in BALF and lung fibronectin levels. Treatment of BLM rats with curcumin dramatically suppressed alveolar macrophage release of fibronectin. Curcumin also inhibited the increases in complex carbohydrates and glycosidases in the fibrotic lungs. These findings suggest that BLM-induced lung fibrosis is associated with accumulation of glycoproteins, and curcumin has the ability to suppress the enhanced deposition of glycoproteins in the fibrotic lung.

Characterising polar compounds using supercritical fluid chromatography-nuclear magnetic resonance spectroscopy (SFC-NMR).

To detect and characterise compounds in complex matrices, it is often necessary to separate the compound of interest from the matrix before analysis. In our previous work, we have developed the coupling of supercritical fluid chromatography (SFC) with nuclear magnetic resonance (NMR) spectroscopy for the analysis of nonpolar samples [Van Zelst et al., Anal. Chem., 2018, 90, 10457]. In this work, the SFC-NMR setup was successfully adapted to analyse polar samples in complex matrices. In-line SFC-NMR analysis of two N-acetylhexosamine stereoisomers was demonstrated, namely N-acetyl-mannosamine (ManNAc) and N-acetyl-glucosamine (GlcNAc). ManNAc is a metabolite that is present at elevated concentrations in patients suffering from NANS-mediated disease. With our SFC-NMR setup it was possible to distinguish between the polar stereoisomers. Until now, this was not possible with the standard mass-based analysis techniques. The concentrations that are needed in the SFC-NMR setup are currently too high to be able to detect ManNAc in patient samples (1.7 mM vs. 0.7 mM). However, several adaptations to the current setup will make this possible in the future.

Structure and biological activities of a hexosamine-rich cell wall polysaccharide isolated from the probiotic Lactobacillus farciminis.

Lactobacillus farciminis CIP 103136 is a bacterial strain with recognized probiotic properties. However, the mechanisms underlying such properties have only been partially elucidated. In this study, we isolated and purified a cell-wall associated polysaccharide (CWPS), and evaluated its biological role in vitro. The structure of CWPS and responses from stimulation of (i) human macrophage-like THP-1 cells, (ii) human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR2 or TLR4) and (iii) human colonocyte-like T84 intestinal epithelial cells, upon exposure to CWPS were studied. The structure of the purified CWPS from L. farciminis CIP 103136 was analyzed by nuclear magnetic resonance (NMR), MALDI-TOF-TOF MS, and methylation analyses in its native form and following Smith degradation. It was shown to be a novel branched polysaccharide, composed of linear backbone of trisaccharide repeating units of: [→6αGlcpNAc1 → 4ßManpNAc1 → 4ßGlcpNAc1→] highly substituted with single residues of αGlcp, αGalp and αGlcpNAc. Subsequently, the lack of pro- or anti-inflammatory properties of CWPS was established on macrophage-like THP-1 cells. In addition, CWPS failed to modulate cell signaling pathways dependent of TLR2 and TLR4 in transfected HEK-cells. Finally, in T84 cells, CWPS neither influenced intestinal barrier integrity under basal conditions nor prevented TNF-α/IFN-γ cytokine-mediated epithelium impairment.

A quantitative analytical method for valienone and its application in the evaluation of valienone production by a breakthrough microbial process.

Valienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhydrazine (DNPH) in 10 mmol·L-1 H3PO4 at 37 °C for 45 min and the derivatives were separated on Eclipse XDB-C18 (5 µm, 4.6 mm × 150 mm) column at 30 °C eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by computational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5-2 000 µg·mL-1. The intra- and inter-day precisions were 1.1%-2.7% and 1.7%-2.2%, respectively. The absolute recovery of the spiked samples was 97.2%-102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engineered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.

Method for determination of streptomycin and streptidine as markers for streptomycin industrial dregs monitoring in pig and poultry compound feeds.

Antibiotic industrial dregs, generated from the production of antibiotics by fermentation, are banned in China as animal feed additives. Official monitoring programs require the analysis of feeds for possible illegal use of the dregs. A rapid and sensitive method was developed for the simultaneous determination of streptomycin and streptidine as markers for streptomycin industrial dregs in pig and poultry compound feeds. After extraction with 20% aqueous trichloroacetic acid and pH adjustment, sample cleanup was performed by weak cation-exchange solid-phase extraction. UPLC-ESI-MS/MS was carried out using a hydrophilic interaction chromatography(HILIC)column to achieve separation. Quantification required matrix-matched calibrations in a linear range of 50-1000µgkg-1; the calibration curves were linear in this range with coefficients of determination of 0.991 and 0.994 for streptomycin and streptidine, respectively. The method validity parameters-LODs (20µgkg-1) and LOQs (50µgkg-1), recoveries (71-78% and 75-84%, respectively), and relative reproducibility (5.4-9.6%)-satisfy the requirements of routine analysis.

Targeted Ultrasound-Assisted Cancer-Selective Chemical Labeling and Subsequent Cancer Imaging using Click Chemistry.

Metabolic sugar labeling followed by the use of reagent-free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N-azidoacetylmannosamine (Ac4 ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4 ManAz-loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4 ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac4 ManAz-loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies.

A sensitive and efficient method for determination of N-acetylhexosamines and N-acetylneuraminic acid in breast milk and milk-based products by high-performance liquid chromatography via UV detection and mass spectrometry identification.

A sensitive and efficient method of high performance liquid chromatography using 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) as pre-column derivatization reagent coupled with UV detection (HPLC-UV) and online mass spectrometry identification was established for determination of the most common N-Acetylhexosamines (N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-galactosamine (GalNAc)) and N-acetylneuraminic acid (Neu5Ac). In order to obtain the highest liberation level of the three monosaccharides without destruction of Neu5Ac or conversion of GlcNAc/GalNAc to GlcN/GalN in the hydrolysis procedure, the pivotal parameters affecting the liberation of N-acetylhexosamines/Neu5Ac from sample were investigated with response surface methodology (RSM). Under the optimized condition, maximum yield was obtained. The effects of key parameters on derivatization, separation and detection were also investigated. At optimized conditions, three monosaccharides were labeled fast and entirely, and all derivatives exhibited a good baseline resolution and high detection sensitivity. The developed method was linear over the calibration range 0.25-12µM, with R(2)>0.9991. The detection limits of the method were between 0.48 and 2.01pmol. Intra- and inter-day precisions for the three monosaccharides (GlcNAc, GalNAc and Neu5Ac) were found to be in the range of 3.07-4.02% and 3.69-4.67%, respectively. Individual monosaccharide recovery from spiked milk was in the range of 81%-97%. The sensitivity of the method, the facility of the derivatization procedure and the reliability of the hydrolysis conditions suggest the proposed method has a high potential for utilization in routine trace N-acetylhexosamines and Neu5Ac analysis in biological samples.

Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c-di-GMP-dependent regulation.

Elevated levels of the second messenger c-di-GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food-borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface-bound. Secreted carbohydrates represent exclusively cell-wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a ß-1,4-linked N-acetylmannosamine chain decorated with terminal α-1,6-linked galactose. All genes of the pssA-E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS-specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS-mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c-di-GMP-dependent EPS production.

Targeted metabolomics in cultured cells and tissues by mass spectrometry: method development and validation.

Metabolomics is the identification and quantitation of small bio-molecules (metabolites) in biological samples under various environmental and genetic conditions. Mass spectrometry provides the unique opportunity for targeted identification and quantification of known metabolites by selective reaction monitoring (SRM). However, reproducibility of this approach depends on careful consideration of sample preparation, chemical classes, and stability of metabolites to be evaluated. Herein, we introduce and validate a targeted metabolite profiling workflow for cultured cells and tissues by liquid chromatography-triple quadrupole tandem mass spectrometry. The method requires a one-step extraction of water-soluble metabolites and targeted analysis of central metabolites that include glycolysis, amino acids, nucleotides, citric acid cycle, and the hexosamine biosynthetic pathway. The sensitivity, reproducibility and molecular stability of each targeted metabolite were assessed under experimental conditions. Quantitation of metabolites by peak area ratio was linear with a dilution over a 4 fold dynamic range with minimal deviation R(2)=0.98. Inter- and intra-day precision with cells and tissues had an average coefficient of variation <15% for cultured cell lines, and somewhat higher for mouse liver tissues. The method applied in triplicate measurements readily distinguished immortalized cells from malignant cells, as well as mouse littermates based on their hepatic metabolic profiles.

Partial characterization of an exopolysaccharide secreted by a marine bacterium, Vibrio neocaledonicus sp. nov., from New Caledonia.

AIMS: Exopolysaccharides (EPS) are industrially valuable molecules with numerous useful properties. This study describes the techniques used for the identification of a novel Vibrio bacterium and preliminary characterization of its EPS. METHODS AND RESULTS: Bioprospection in marine intertidal areas of New Caledonia followed by screening for EPS producing brought to selection of the isolate NC470. Phylogenetic analysis (biochemical tests, gene sequencing and DNA-DNA relatedness) permitted to identify NC470 as a new member of the Vibrio genus. The EPS was produced in batch fermentation, purified using the ultrafiltration process and analysed by colorimetry, Fourier Transform Infrared spectroscopy, gas chromatography, Nuclear Magnetic Resonance and HPLC-size exclusion chromatography. This EPS exhibits a high N-acetyl-hexosamines and uronic acid content with a low amount of neutral sugar. The molecular mass was 672 × 10(3)  Da. These data are relevant for possible technological exploitation. CONCLUSIONS: We propose the name Vibrio neocaledonicus sp. nov for this isolate NC470, producing an EPS with an unusual sugar composition. Comparison with other known polymers permitted to select applications for this polymer. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to evaluate the marine biodiversity of New Caledonia. It also highlights the biotechnological potential of New Caledonia marine bacteria.

Fluorescent live-cell imaging of metabolically incorporated unnatural cyclopropene-mannosamine derivatives.

Sugar coated: We recently developed methylcyclopropenes as low-molecular-weight tetrazine coupling partners. Here, we demonstrate that methylcyclopropenes can meet the stringent steric demands required for metabolic imaging of unnatural mannosamines on live cells. Using sequential azide-alkyne chemistry, we also demonstrate multicolor imaging of two different metabolically incorporated unnatural sugars.

NMR analysis and site-specific protonation constants of streptomycin.

Streptomycin, the classical aminoglycoside antibiotic, generally considered the most basic drug compound was characterized in terms of protonation macro- and microconstants. ¹H NMR-pH and ¹H-¹³C HSQC-pH titrations were carried out on streptomycin and streptidine, a symmetrical constituent compound of reduced complexity to monitor the proton-binding processes of the basic sites. Accurate, undistorted, electrodeless pH measurement was ensured by a new set of in tube indicators. The microscopic protonation constants of the two guanidino groups of streptomycin were calculated by evaluating the various NMR-pH data and transferring the pair-interactivity parameter from streptidine to streptomycin. Inherent guanidino basicities fall in the range of 13.03-13.39 log k units, which drop to 12.48-12.85 upon protonation of the other site. pH-dependent distribution of the major microspecies and charge-related biological consequences are provided.

High-throughput analysis of hexosamine using a colorimetric method.

A 96-well plate method was developed for analysis of total hexosamine content in biological samples. Four hexosamine monomer derivatives-glucosamine hydrochloride, glucosamine sulfate, galactosamine hydrochloride, and mannosamine hydrochloride-were examined for the linearity of their spectra in the concentration range specified in the assay. The hexosamine concentration analysis range was linear from 0.1 to 1 mM. The quantification of hexosamines from chitin and chitosan upon acid hydrolysis was also tested. Accurate quantification of glucosamine content in chitin and chitosan with different molecular sizes and degrees of acetylation was demonstrated using the new method.

Simultaneous determination of substrate and product in the process of preparation of valienamine by capillary zone electrophoresis.

A simple and rapid CZE method was established for the simultaneous determination of valienamine, acarbose and validamycin A, using a 20-kV CZE with the detection wavelength of 193 nm and 50 mM phosphoric acid-20 mM Tris (pH 5.3) as a running buffer. The calibration curves of valienamine, acarbose, and validamycin A showed a good linear relationship at a concentration range of 5-1000 microg/mL. The detection limits of valienamine, acarbose, and validamycin A were 0.3, 0.6, and 0.6 microg/mL, respectively, and the average recoveries of each of the above were 99.9, 99.5, and 100.3%. The method has been successfully applied for simultaneous determination of substrate and product in the process of preparation of valienamine.

Wound healing activity of flower extract of Calendula officinalis.

The effects of oral and topical application of Calendula officinalis flower extract on excision wounds made in rats were checked. The parameters assessed were the days needed for re-epithelization and percentage of wound closure. The hydroxy proline and hexosamine content in the granuloma tissue of the wound was also measured. The percentage of wound closure was 90.0% in the extract-treated group, whereas the control group showed only 51.1% on the eighth day of wounding (p < .01). The days needed for re-epithelization were 17.7 for the control animals; extract treatment at a dose of 20 or 100 mg/kg b.wt reduced the period to 14 and 13 days, respectively. A significant increase was observed in the hydroxy proline and hexosamine content in the extract-treated group compared with the untreated animals. The data indicate potent wound healing activity ofC. officinalis extract.

Inhibition of lung metastasis in mice by oligonol.

Oligonol is a polyphenol formulation enriched with catechin-type oligomers. As an initial approach to assess the chemopreventive potential of Oligonol, the study investigated the effects of Oligonol on the inhibition of lung metastasis induced by B16F-10 melanoma cells in C57BL/6 mice. Oligonol, which is abundantly found in plants, vegetables and fruits, was found to possess antimetastatic activity against B16F-10 melanoma cells. Continued consumption of Oligonol, even at high doses, for long periods does not seem to cause any toxic symptoms because excess Oligonol is stored in adipose tissue rather than in the liver. However, the mechanism by which Oligonol exerts its antimetastatic activity remains unclear. Further investigations are required to clarify the exact role of Oligonol in such B16F-10 melanoma regulation.

In vivo protective effect of crocetin on benzo(a)pyrene-induced lung cancer in Swiss albino mice.

The objective of this investigation was to determine the efficacy of crocetin in preventing lung tumorigenesis in mice. We evaluated crocetin in Swiss albino mice treated with the tobacco-specific carcinogen benzo(a)pyrene [B(a)P] for their ability to inhibit pulmonary adenoma formation and growth. Male Swiss albino mice (7 weeks old) were given 100 mg/kg B(a)P by i.p. injection, and 4 or 14 weeks later, they were given crocetin 50 mg/kg by i.p. injection 3 days/week. Crocetin (50 mg/kg body weight) reduced proliferating cells by 68% and 45% in 18 and 8 weeks of treatment respectively. The levels of glycoproteins and polyamines were significantly altered in the B(a)P-induced animals than in crocetin treatment groups. The activity of crocetin was more pronounced in the cancer. Taken together, these results indicate that crocetin was capable of inhibiting proliferation cells by inhibiting proliferating cells, glycoprotein and polyamine synthesis.

Studying O-linked protein glycosylations in human plasma.

Recent investigations have implicated aberrant glycosylations in various malignancies, including epithelial ovarian cancer (EOC). The protocol here identifies O-linked carbohydrate patterns in EOC plasma glycoproteins through chemical cleavage and purification of these glycans. Dialyzed plasma is subjected to reductive beta-elimination with alkaline borohydride to release O-linked oligosaccharides from glycoproteins. Enrichment of released glycans, as well as removal of peptide and other contaminants, is followed by carbohydrate pattern analysis with MALDI-FT-ICR-MS.

Improved liquid chromatographic method with pulsed electrochemical detection for the analysis of gentamicin.

The official method for the determination of the composition and related substances of gentamicin prescribed by the European Pharmacopoeia (Ph. Eur.) is liquid chromatography combined with pulsed electrochemical detection (LC-PED). However, this method utilizes a polymer stationary phase which shows rather low efficiency towards the separation of the main gentamicin components. Moreover, the mobile phase contains a lot of non volatile salts: sodium sulphate and sodium octanesulphonate. Following a comparative study, the most performant LC-PED method has been evaluated and validated using a reversed phase C18 column (C18, 250 x 4.6mm ID, 110 A, 5 microm) kept at 35 degrees C with a mobile phase containing volatile ion pairing agents: trifluoroacetic acetic acid (TFA) and pentafluoropropionic acid (PFPA). In addition to the selectivity of the main gentamicin components and its related substances, the method is repeatable, linear and proves to be robust. It is also applicable to a wider number of C18 columns.