Complement receptor 3-dependent engagement by Candida glabrata ß-glucan modulates dendritic cells to induce regulatory T-cell expansion.

Candida glabrata is an important pathogen causing invasive infection associated with a high mortality rate. One mechanism that causes the failure of Candida eradication is an increase in regulatory T cells (Treg), which play a major role in immune suppression and promoting Candida pathogenicity. To date, how C. glabrata induces a Treg response remains unclear. Dendritic cells (DCs) recognition of fungi provides the fundamental signal determining the fate of the T-cell response. This study investigated the interplay between C. glabrata and DCs and its effect on Treg induction. We found that C. glabrata ß-glucan was a major component that interacted with DCs and consequently mediated the Treg response. Blocking the binding of C. glabrata ß-glucan to dectin-1 and complement receptor 3 (CR3) showed that CR3 activation in DCs was crucial for the induction of Treg. Furthermore, a ligand-receptor binding assay showed the preferential binding of C. glabrata ß-glucan to CR3. Our data suggest that C. glabrata ß-glucan potentially mediates the Treg response, probably through CR3-dependent activation in DCs. This study contributes new insights into immune modulation by C. glabrata that may lead to a better design of novel immunotherapeutic strategies for invasive C. glabrata infection.

Glycosylation of n-pentenyl glycosides using bromodiethylsulfonium salt as an activator: interception of the glycosyl intermediate by chloride ion transfer.

Utilization of n-pentenyl glycosides (NPGs) in modern carbohydrate synthesis may be hindered by their sluggish activation, which results from reversible halogenation and cyclization processes. Bromodiethylsulfonium bromopentachloroantimonate (BDSB) has been previously shown to be a powerful brominating agent for the cation-π polyene cyclization of less reactive and electron-poor polyenes. This study demonstrates the activation of NPGs using BDSB as a powerful brominating agent. BDSB effectively activates the terminal olefins of NPGs and the reaction proceeds through 5-exo-tet cyclization, offering a rapid and mild approach for glycosylation with a wide range of glycosyl donors, including n-pentenyl mannoside, n-pentenyl galactoside, and n-pentenyl glucoside. The success of this approach derives from the chloride ion transfer from the nonnucleophilic SbCl5Br anion to the glycosyl intermediate, which disrupts the equilibrium and produces a glycosyl chloride intermediate that is smoothly converted to 22 coupling products, with yields ranging from moderate to excellent (49-100%). The ß-selective glycosylation is accomplished when employing NPGs equipped with a neighboring participating group. The practicality of the BDSB-activated glycosylation is demonstrated by a gram-scale synthesis. This study showcases BDSB as a potent activator for NPG glycosylation through the interception of a glycosyl intermediate that diminishes the equilibration during halogenation and 5-exo-tet cyclization.

Surfactant-Assisted Ozonolysis of Alkenes in Water: Mitigation of Frothing Using Coolade as a Low-Foaming Surfactant.

Aqueous-phase ozonolysis in the atmosphere is an important process during cloud and fog formation. Water in the atmosphere acts as both a reaction medium and a reductant during the ozonolysis. Inspired by the atmospheric aqueous-phase ozonolysis, we herein report the ozonolysis of alkenes in water assisted by surfactants. Several types of surfactants, including anionic, cationic, and nonionic surfactants, were investigated. Although most surfactants enhanced the solubility of alkenes in water, they also generated excessive foaming during the ozone bubbling, which led to the loss of products. Mitigation of the frothing was accomplished by using Coolade as a nonionic and low-foaming surfactant. Coolade-assisted ozonolysis of alkenes in water provided the desired carbonyl products in good yields and comparable to those achieved in organic solvents. During the ozonolysis reaction, water molecules trapped within the polyethylene glycol region of Coolade were proposed to intercept the Criegee intermediate to provide a hydroxy hydroperoxide intermediate. Decomposition of the hydroxy hydroperoxide led to formation of the carbonyl product without the need for a reductant typically required for the conventional ozonolysis using organic solvents. This study presents Coolade as an effective surfactant to improve the solubility of alkenes while mitigating frothing during the ozonolysis in water.

Membrane-permeable trehalose improves the freezing ability and developmental competence of in-vitro matured feline oocytes.

Oocytes are highly sensitive to cryopreservation, which frequently results in an irreversible loss of developmental competence. We examined the effect of membrane-permeable trehalose on the freezing ability of feline oocytes matured in vitro. In Experiment 1, intracellular trehalose (trehalose hexaacetate; Tre-(OAc)6) was synthesized from trehalose precursor and subjected to spectroscopic characterization. The membrane permeability of the Tre-(OAc)6 was investigated by incubating oocytes with different concentrations of Tre-(OAc)6 (3, 15, and 30 mM). Optimum concentration and the toxicity of Tre-(OAc)6 were assessed in Experiment 2. The effects of Tre-(OAc)6 on freezing ability in terms of apoptotic gene expression and developmental competence of in-vitro matured oocytes were examined in Experiments 3 and 4, respectively. The Tre-(OAc)6 permeated into the ooplasm of cat oocytes in a dose- and time-dependent manner. The highest concentration of intracellular trehalose was detected when the oocytes were incubated for 24 h with 30 mM Tre-(OAc)6. For the toxicity test, incubation of oocytes with 3 mM Tre-(OAc)6 for 24 h did not affect maturation rate and embryo development. However, high doses of Tre-(OAc)6 (15 and 30 mM) significantly reduced maturation and fertilization rates (p < 0.05). In addition, frozen-thawed oocytes treated with 3 mM Tre-(OAc)6 significantly upregulated anti-apoptotic (BCL-2) gene expression compared with the control (0 mM) and other Tre-(OAc)6 concentrations (15 and 30 mM). Oocyte maturation in the presence of 3 mM Tre-(OAc)6 prior to cryopreservation significantly improved oocyte developmental competence in terms of cleavage and blastocyst rates when compared with the control group (p < 0.05). Our results lead us to infer that increasing the levels of intracellular trehalose by Tre-(OAc)6 during oocyte maturation improves the freezing ability of feline oocytes, albeit at specific concentrations.

Surfactant-mediated thioglycosylation of 1-hydroxy sugars in water.

Thioglycosides are an important class of sugars, since they can be used as non-ionic biosurfactants, biomimetic glycosides, and building blocks for carbohydrate synthesis. Previously, Brønsted- or Lewis-acid-catalyzed dehydrative glycosylations between a 1-hydroxy sugar and a thiol have been reported to yield open-chain dithioacetal sugars as the major products instead of the desired thioglycosides. These dithioacetal sugars are by-products derived from the endocyclic bond cleavage of the thioglycosides. Herein, we report dehydrative glycosylation in water mediated by a Brønsted acid-surfactant combined catalyst (BASC). Glycosylations between 1-hydroxy furanosyl/pyranosyl sugars and primary, secondary, and tertiary aliphatic/aromatic thiols in the presence of dodecyl benzenesulfonic acid (DBSA) provided the thioglycoside products in moderate to good yields. Microwave irradiation led to improvements in the yields and a shortening of the reaction time. Remarkably, open-chain dithioacetal sugars were not detected in the DBSA-mediated glycosylations in water. This method is a simple, convenient, and rapid approach to produce a library of thioglycosides without the requirement of anhydrous conditions. Moreover, this work also provides an excellent example of complementary reactivity profiles of glycosylation in organic solvents and water.

Interaction Between Dendritic Cells and Candida krusei ß-Glucan Partially Depends on Dectin-1 and It Promotes High IL-10 Production by T Cells.

Host-Candida interaction has been broadly studied during Candida albicans infection, with a progressive shift in focus toward non-albicans Candida species. C. krusei is an emerging multidrug resistant pathogen causing rising morbidity and mortality worldwide. Therefore, understanding the interplay between the host immune system and C. krusei is critically important. Candia cell wall ß-glucans play significant roles in the induction of host protective immune responses. However, it remains unclear how C. krusei ß-glucan impacts dendritic cell (DC) responses. In this study, we investigated DC maturation and function in response to ß-glucans isolated from the cell walls of C. albicans, C. tropicalis, and C. krusei. These three distinct Candida ß-glucans had differential effects on expression of the DC marker, CD11c, and on DC maturation. Furthermore, bone-marrow derived DCs (BMDCs) showed enhanced cytokine responses characterized by substantial interleukin (IL)-10 production following C. krusei ß-glucan stimulation. BMDCs stimulated with C. krusei ß-glucan augmented IL-10 production by T cells in tandem with increased IL-10 production by BMDCs. Inhibition of dectin-1 ligation demonstrated that the interactions between dectin-1 on DCs and cell wall ß-glucans varied depending on the Candida species. The effects of C. krusei ß-glucan were partially dependent on dectin-1, and this dependence, in part, led to distinct DC responses. Our study provides new insights into immune regulation by C. krusei cell wall components. These data may be of use in the development of new clinical approaches for treatment of patients with C. krusei infection.

Fusigen Reduces Intracellular Reactive Oxygen Species and Nitric Oxide Levels.

BACKGROUND/AIM: Oxidative stress caused by the production of excessive cellular reactive oxygen species (ROS) and high levels of nitric oxide contribute to several human pathologies. This study aimed to examine the anti-oxidant effects of fusigen, a compound produced from Aureobasidium melanogenum. MATERIALS AND METHODS: Extracts of A. melanogenum were selected as a source for the isolation of fusigen. The anti-oxidant, nitric oxide suppression, as well as the free radical scavenging activities of fusigen were tested in BEAS-2B human bronchial epithelial cell line (BEAS-2B cells) and human dermal papilla cells (DP cells) using specific fluorescence dyes and flow cytometry analysis. Cell viability was determined by the MTT assay. RESULTS: Fusigen did not exert cytotoxicity in the human normal BEAS-2B and DP cells at concentrations up to 100 µM. Fusigen decreased basal levels of cellular ROS, as well as the levels of ROS induced by hydrogen peroxide and ferrous ion enrichment. ROS decreasing effect was confirmed in DP cells. In addition, fusigen treatment suppressed intracellular NO levels in both BEAS-2B and DP cells. CONCLUSION: The optimal process of production of purified fusigen from A. melanogenum was determined. Fusigen exhibited a low cytotoxic effect and the potential to suppress ROS and NO. These results demonstrated that fusigen may be used for the treatment or prevention of human diseases.

Enzymatic hydrolysis of tropical weed xylans using xylanase from Aureobasidium melanogenum PBUAP46 for xylooligosaccharide production.

The maximum yield of xylanase from Aureobasidium melanogenum PBUAP46 was 5.19 ± 0.08 U ml-1 when cultured in a production medium containing 3.89% (w/v) rice straw and 0.75% (w/v) NaNO3 as carbon and nitrogen sources, respectively, for 72 h. This enzyme catalyzed well and was relatively stable at pH 7.0 and room temperature (28 ± 2 °C). The produced xylanase was used to hydrolyze xylans from four tropical weeds, whereupon it was found that the highest amounts of reducing sugars in the xylan hydrolysates of cogon grass (Imperata cylindrical), Napier grass (Pennisetum purpureum), and vetiver grass (Vetiveria zizanioides) were at 20.44 ± 0.84, 17.50 ± 0.29, and 19.44 ± 0.40 mg 100 mg xylan-1, respectively, but it was not detectable in water hyacinth (Eichhornia crassipes) hydrolysate. The highest combined amount of xylobiose and xylotriose was obtained from vetiver grass; thus, it was selected for further optimization. After optimization, xylanase digestion of vetiver grass xylan at 27.94 U g xylan-1 for 92 h 19 min gave the highest amount of reducing sugars (23.65 ± 1.34 mg 100 mg xylan-1), which were principally xylobiose and xylotriose. The enriched XOs exhibited a prebiotic property, significantly stimulating the growth of Lactobacillus brevis and L. casei by a factor of up to 3.5- and 6.5-fold, respectively, compared to glucose.

Cell wall mannan of Candida krusei mediates dendritic cell apoptosis and orchestrates Th17 polarization via TLR-2/MyD88-dependent pathway.

Dendritic cells (DCs) abundantly express diverse receptors to recognize mannans in the outer surface of Candida cell wall, and these interactions dictate the host immune responses that determine disease outcomes. C. krusei prevalence in candidiasis worldwide has increased since this pathogen has developed multidrug resistance. However, little is known how the immune system responds to C. krusei. Particularly, the molecular mechanisms of the interplay between C. krusei mannan and DCs remain to be elucidated. We investigated how C. krusei mannan affected DC responses in comparison to C. albicans, C. tropicalis and C. glabrata mannan. Our results showed that only C. krusei mannan induced massive cytokine responses in DCs, and led to apoptosis. Although C. krusei mannan-activated DCs underwent apoptosis, they were still capable of initiating Th17 response. C. krusei mannan-mediated DC apoptosis was obligated to the TLR2 and MyD88 pathway. These pathways also controlled Th1/Th17 switching possibly by virtue of the production of the polarizing cytokines IL-12 and IL-6 by the C. krusei mannan activated-DCs. Our study suggests that TLR2 and MyD88 pathway in DCs are dominant for C. krusei mannan recognition, which differs from the previous reports showing a crucial role of C-type lectin receptors in Candida mannan sensing.

An α-1,6-and α-1,3-linked glucan produced by Leuconostoc citreum ABK-1 alternansucrase with nanoparticle and film-forming properties.

Alternansucrase catalyses the sequential transfer of glucose residues from sucrose onto another sucrose molecule to form a long chain polymer, known as "alternan". The alternansucrase-encoding gene from Leuconostoc citreum ABK-1 (Lcalt) was successfully cloned and expressed in Escherichia coli. Lcalt encoded LcALT of 2,057 amino acid residues; the enzyme possessed an optimum temperature and pH of 40 °C and 5.0, respectively, and its' activity was stimulated up to 2.4-fold by the presence of Mn2+. Kinetic studies of LcALT showed a high transglycosylation activity, with Km 32.2 ± 3.2 mM and kcat 290 ± 12 s-1. Alternan generated by LcALT (Lc-alternan) harbours partially alternating α-1,6 and α- 1,3 glycosidic linkages confirmed by NMR spectroscopy, methylation analysis, and partial hydrolysis of Lc-alternan products. In contrast to previously reported alternans, Lc-alternan can undergo self-assembly, forming nanoparticles with an average size of 90 nm in solution. At concentrations above 15% (w/v), Lc-alternan nanoparticles disassemble and form a high viscosity solution, while this polymer forms a transparent film once dried.

Synthesis and DNA/RNA Binding Properties of Conformationally Constrained Pyrrolidinyl PNA with a Tetrahydrofuran Backbone Deriving from Deoxyribose.

Sugar-derived cyclic ß-amino acids are important building blocks for designing of foldamers and other biomimetic structures. We report herein the first synthesis of a C-activated N-Fmoc-protected trans-(2S,3S)-3-aminotetrahydrofuran-2-carboxylic acid as a building block for Fmoc solid phase peptide synthesis. Starting from 2-deoxy-d-ribose, the product is obtained in a 6.7% overall yield following an 11-step reaction sequence. The tetrahydrofuran amino acid is used as a building block for a new peptide nucleic acid (PNA), which exhibits excellent DNA binding affinity with high specificity. It also shows preference for binding to DNA over RNA and specifically in the antiparallel orientation. In addition, the presence of the hydrophilic tetrahydrofuran ring in the PNA structure reduces nonspecific interactions and self-aggregation, which is a common problem in PNA due to its hydrophobic nature.

Synthesis and reactivity of 4'-deoxypentenosyl disaccharides.

4-Deoxypentenosides (4-DPs) are versatile synthons for rare or higher-order pyranosides, and they provide an entry for structural diversification at the C5 position. Previous studies have shown that 4-DPs undergo stereocontrolled DMDO oxidation; subsequent epoxide ring-openings with various nucleophiles can proceed with both anti or syn selectivity. Here, we report the synthesis of α- and ß-linked 4'-deoxypentenosyl (4'-DP) disaccharides, and we investigate their post-glycosylational C5' additions using the DMDO oxidation/ring-opening sequence. The α-linked 4'-DP disaccharides were synthesized by coupling thiophenyl 4-DP donors with glycosyl acceptors using BSP/Tf2O activation, whereas ß-linked 4'-DP disaccharides were generated by the decarboxylative elimination of glucuronyl disaccharides under microwave conditions. Both α- and ß-linked 4'-DP disaccharides could be epoxidized with high stereoselectivity using DMDO. In some cases, the α-epoxypentenosides could be successfully converted into terminal l-iduronic acids via the syn addition of 2-furylzinc bromide. These studies support a novel approach to oligosaccharide synthesis, in which the stereochemical configuration of the terminal 4'-DP unit is established at a post-glycosylative stage.

Enhanced water-solubility and mucoadhesion of N,N,N-trimethyl-N-gluconate-N-homocysteine thiolactone chitosan.

A water-soluble chitosan with improved mucoadhesion was prepared by modifying 19.4% of the amine groups of chitosan to trimethylammonium and conjugation of gluconolactone (GLU) and homocysteine thiolactone (HT) onto the remaining amine groups of the chitosan backbone. The derived trimethyl-gluconate-HT-chitosan (TM-GN-HT-chitosan) was confirmed by Fourier Transform Infrared spectroscopy, NMR and thermogravimetric analysis. The total thiol and disulfide group level on the TM-GN-HT-chitosan were 17.96 ± 0.03 and 7.36 ± 0.03 µmol/g, respectively. The water solubility of the TM-GN-HT-chitosan conjugate was 79.0 ± 0.15%, more than that of TM-chitosan and chitosan, with an enhanced solubility over a broad pH range ranging from 85.6 ± 10.4% to 58.5±1.1% maximal solubility at pH 2 to 11. Finally, TM-GN-HT-chitosan showed a nearly ∼9.5-, 5.0- and 5.6-fold higher mucoadhesiveness than chitosan at pH 1.2, 4.0 and 6.4, respectively, and was optimal at pH 4.0.

Glycosyl dithiocarbamates: ß-selective couplings without auxiliary groups.

In this article, we evaluate glycosyl dithiocarbamates (DTCs) with unprotected C2 hydroxyls as donors in ß-linked oligosaccharide synthesis. We report a mild, one-pot conversion of glycals into ß-glycosyl DTCs via DMDO oxidation with subsequent ring opening by DTC salts, which can be generated in situ from secondary amines and CS2. Glycosyl DTCs are readily activated with Cu(I) or Cu(II) triflate at low temperatures and are amenable to reiterative synthesis strategies, as demonstrated by the efficient construction of a tri-ß-1,6-linked tetrasaccharide. Glycosyl DTC couplings are highly ß-selective despite the absence of a preexisting C2 auxiliary group. We provide evidence that the directing effect is mediated by the C2 hydroxyl itself via the putative formation of a cis-fused bicyclic intermediate.

PRACTICAL SYNTHESIS OF AROMATIC DITHIOCARBAMATES.

Oxidation-sensitive N,N-diaryl dithiocarbamates (DTCs) are synthesized in good yields by the generation of metal amide salts from N-benzoyl precursors, followed by addition of CS2. para-Substituted diphenylamines are prepared by electrophilic aromatic substitution of diphenylbenzamide and saponification. Deacylation of electron-rich species such as bis(p-dimethylaminophenyl)benzamide is challenging because of the oxidative sensitivity of the anionic intermediate but could be achieved in good yield by using n-BuLi to generate a hemiaminal adduct, prior to acidification. The N,N-diaryl DTCs are stable as alkali salts and can be used to produce densely packed monolayers on gold surfaces.

Glycal assembly by the in situ generation of glycosyl dithiocarbamates.

Glycal assembly offers an expedient entry into ß-linked oligosaccharides, but epoxyglycal donors can be capricious in their reactivities. Treatment with Et(2)NH and CS(2) enables their in situ conversion into glycosyl dithiocarbamates, which can be activated by copper triflate for coupling with complex or sterically congested acceptors. The coupling efficiency can be further enhanced by in situ benzoylation, as illustrated in an 11-step synthesis of a branched hexasaccharide from glucals in 28% isolated yield and just four chromatographic purifications.