Gram-scale chemical synthesis of galactosyllactoses and their impact on infant gut microbiota in vitro.

Galactooligosaccharides (GOS) are widely used as a supplement in infant nutrition to mimic the beneficial effects found in prebiotic human milk oligosaccharides (HMOs). However, the complexity of the GOS mixture makes it challenging to ascertain which of the GOS components contribute most to their health benefits. Galactosyllactoses (GLs) are lactose-based trisaccharides containing a ß-galactopyranosyl residue at the 3'-position (3'galactosyllactose, 3'-GL), 4'-position (4'-galactosyllactose, 4'-GL), or the 6'-position (6'-galactosyllactose, 6'-GL). These GLs are of particular interest as they are present in both GOS mixtures and human milk at early stages of lactation. However, research on the potential health benefits of these individual GLs has been limited. Gram quantities are needed to assess their health benefits but these GLs are not readily available at this scale. In this study, we report the gram-scale chemical synthesis of 3'-GL, 4'-GL, and 6'-GL. All three galactosyllactoses were obtained on a gram scale in good purity from cheap and commercially available lactose. Furthermore, in vitro incubation of GLs with infant faecal microbiota demonstrates that the GLs were able to increase the abundance of Bifidobacterium and stimulate short chain fatty acid production.

Ameliorating effect of 2'-fucosyllactose and 6'-sialyllactose on lipopolysaccharide-induced intestinal inflammation.

Human milk oligosaccharides (HMO) affect gut microbiota during neonatal development, particularly with respect to the immune system. Bovine milk-based infant formulas have low oligosaccharide contents. Thus, efforts to fortify infant formulas with HMO are being undertaken. Two major HMO, 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL), exert anti-inflammatory effects; however, the associations between anti-inflammatory effects induced by 2'-FL and 6'-SL cotreatment and gut microbiota composition and metabolite modulation remain unclear. Therefore, in this study, we evaluated the effects of a mixture of these HMO. To determine the optimal HMO ratio for anti-inflammatory effects and elucidate its mode of action, LPS-induced inflammatory HT-29 epithelial cells and intestinal-inflamed suckling mice were treated with various mixtures of 2'-FL and 6'-SL. A 2'-FL:6'-SL ratio of 5:1 was identified as the most effective pretreatment HMO mixture in vitro; thus, this ratio was selected and used for low-, middle-, and high-dose treatments for subsequent in vivo studies. In vivo, high-dose HMO treatment restored LPS-induced inflammation symptoms, such as BW loss, colon length reduction, histological structural damage, and intestinal gene expression related to inflammatory responses. High-dose HMO was the only treatment that modulated the major phyla Bacteroidetes and Firmicutes and the genera Ihubacter, Mageeibacillus, and Saccharofermentans. These changes in microbial composition were correlated with intestinal inflammation-related gene expression and short-chain fatty acid production. To our knowledge, our study is the first to report the effects of Ihubacter, Mageeibacillus, and Saccharofermentans on short-chain fatty acid levels, which can subsequently affect inflammatory cytokine and tight junction protein levels. Conclusively, the HMO mixture exerted anti-inflammatory effects through changes in microbiota and metabolite production. These findings suggest that supplementation of infant formula with HMO may benefit formula-fed infants by forming unique microbiota contributing to neonatal development.

Main active components of Si-Miao-Yong-An decoction (SMYAD) attenuate autophagy and apoptosis via the PDE5A-AKT and TLR4-NOX4 pathways in isoproterenol (ISO)-induced heart failure models.

Heart failure (HF), the main cause of death in patients with many cardiovascular diseases, has been reported to be closely related to the complicated pathogenesis of autophagy, apoptosis, and inflammation. Notably, Si-Miao-Yong-An decoction (SMYAD) is a traditional Chinese medicine (TCM) used to treat cardiovascular disease; however, the main active components and their relevant mechanisms remain to be discovered. Based on our previous ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) results, we identified angoriside C (AC) and 3,5-dicaffeoylquinic acid (3,5-DiCQA) as the main active components of SMYAD. In vivo results showed that AC and 3,5-DiCQA effectively improved cardiac function, reduced the fibrotic area, and alleviated isoproterenol (ISO)-induced myocarditis in rats. Moreover, AC and 3,5-DiCQA inhibited ISO-induced autophagic cell death by inhibiting the PDE5A/AKT/mTOR/ULK1 pathway and inhibited ISO-induced apoptosis by inhibiting the TLR4/NOX4/BAX pathway. In addition, the autophagy inhibitor 3-MA was shown to reduce ISO-induced apoptosis, indicating that ISO-induced autophagic cell death leads to excess apoptosis. Taken together, the main active components AC and 3,5-DiCQA of SMYAD inhibit the excessive autophagic cell death and apoptosis induced by ISO by inhibiting the PDE5A-AKT and TLR4-NOX4 pathways, thereby reducing myocardial inflammation and improving heart function to alleviate and treat a rat ISO-induced heart failure model and cell heart failure models. More importantly, the main active components of SMYAD will provide new insights into a promising strategy that will promote the discovery of more main active components of SMYAD for therapeutic purposes in the future.

Recent advances on 2'-fucosyllactose: physiological properties, applications, and production approaches.

The trisaccharide, 2'-fucosyllactose (Fucα1-2Galß1-4Glc; 2'-FL), is the most abundant oligosaccharide in human milk. It has numerous significant biological properties including prebiotics, antibacterial, antiviral, and immunomodulating effects, and has been approved as "generally recognized as safe" (GRAS) by the Food and Drug Administration (FDA) and as a novel food (NF) by the European Food Safety Authority (EFSA). 2'-FL not only serves as a food ingredient added in infant formula, but also as a dietary supplement and medical food material in food bioprocesses. There is considerable commercial interest in 2'-FL for its irreplaceable nutritional applications. This review aims at systematically elaborating key functional properties of 2'-FL as well as its applications. In addition, several approaches for 2'-FL production are described in this review, including chemical, chemo-enzymatical, and cell factory approaches, and the pivotal research results also have been summarized. With the rapid development of metabolic engineering and synthetic biology strategies, using the engineered cell factory for 2'-FL large-scale production might be a promising approach. From an economic and safety point of view, microbial selection for cell factory engineering in 2'-FL bioprocess also should be taken into consideration.

Human Milk Oligosaccharide 2'-Fucosyllactose Inhibits Ligand Binding to C-Type Lectin DC-SIGN but Not to Langerin.

Human milk oligosaccharides (HMOs) and their most abundant component, 2'-Fucosyllactose (2'-FL), are known to be immunomodulatory. Previously, it was shown that HMOs and 2'-FL bind to the C-type lectin receptor DC-SIGN. Here we show, using a ligand-receptor competition assay, that a whole mixture of HMOs from pooled human milk (HMOS) and 2'-FL inhibit the binding of the carbohydrate-binding receptor DC-SIGN to its prototypical ligands, fucose and the oligosaccharide Lewis-B, (Leb) in a dose-dependent way. Interestingly, such inhibition by HMOS and 2'-FL was not detected for another C-type lectin, langerin, which is evolutionarily similar to DC-SIGN. The cell-ligand competition assay using DC-SIGN expressing cells confirmed that 2'-FL inhibits the binding of DC-SIGN to Leb. Molecular dynamic (MD) simulations show that 2'-FL exists in a preorganized bioactive conformation before binding to DC-SIGN and this conformation is retained after binding to DC-SIGN. Leb has more flexible conformations and utilizes two binding modes, which operate one at a time via its two fucoses to bind to DC-SIGN. Our hypothesis is that 2'-FL may have a reduced entropic penalty due to its preorganized state, compared to Leb, and it has a lower binding enthalpy, suggesting a better binding to DC-SIGN. Thus, due to the better binding to DC-SIGN, 2'-FL may replace Leb from its binding pocket in DC-SIGN. The MD simulations also showed that 2'-FL does not bind to langerin. Our studies confirm 2'-FL as a specific ligand for DC-SIGN and suggest that 2'-FL can replace other DC-SIGN ligands from its binding pocket during the ligand-receptor interactions in possible immunomodulatory processes.

[Components and lipid-lowering effect of total saponins from underground part of Gynostemma pentaphyllum].

The saponins in different parts of Gynostemma pentaphyllum were analyzed via UPLC-Q-TOF-MS~E. A total of 46 saponins were identified, and the underground part had 26 saponins more than the aboveground part, most of which were trisaccharide saponins. The rat model of hyperlipidemia was established with high-fat diet. This study explored the lipid-lowering activity of total saponins in the underground part of G. pentaphyllum, so as to provide a theoretical basis for the comprehensive utilization of the underground part of G. pentaphyllum. A total of 99 healthy SD rats were randomly assigned into a blank group, a model group, a positive drug group, an aboveground total saponins group, and low-, medium-, and high-dose underground total saponins groups. Except the blank group, the other groups were fed with high-fat diet for 6 weeks. Then, the blood was collected from the orbital cavity to determine whether the modeling was successful according to the serum levels of total cholesterol(TC) and triglyceride(TG). After intragastric administration of the corresponding agents for 30 continuous days, the physical state of the rats were observed, and the body weight and liver specific gravity were measured. Furthermore, the levels of TC, TG, low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), alanine transaminase(ALT), aspartate transaminase(AST), bilirubin, and total bile acids in serum, as well as the levels of superoxide dismutase(SOD), malondialdehyde(MDA), peroxidase proliferator-activated receptor(PPAR-γ) in the liver tissue, were determined. The pathological changes of liver was observed via HE staining. The results showed that the aboveground total saponins and medium-and high-dose underground total saponins can treat hepatocyte steatosis, lower TC, TG, LDL-C, ALT, AST, total bilirubin, MDA, and PPAR-γ levels, and increase HDL-C and SOD levels in the model rats. The effect tended to be more obvious with the increase in dosage. Therefore, the total saponins in the underground part of G. pentaphyllum have good pharmacological effect of reducing blood lipid, which provides a theoretical basis for the comprehensive utilization of the underground part of G. pentaphyllum.

The human milk oligosaccharides 2'-fucosyllactose and 6'-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling.

BACKGROUND: Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) can reduce NEC through inhibition of TLR4 signaling. METHODS: NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2'-FL, 6'-SL, or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, and human tissue explants and via in silico modeling. RESULTS: Supplementation of infant formula with either 2'-FL and/or 6'-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2'-FL and 6'-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa light-chain enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2'-FL and 6'-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling. CONCLUSIONS: 2'-FL and 6'-SL, but not lactose, prevent NEC in mice and piglet models and attenuate NEC inflammation in the human ileum, in part through TLR4 inhibition. IMPACT: Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants that occurs in the setting of bacterial colonization of the gut and administration of formula feeds and activation by the innate immune receptor toll-like receptor 4 (TLR4). Breast milk prevents NEC through unclear mechanisms. We now show that breast milk-enriched human milk oligosaccharides (HMOs) that are derived from lactose prevent NEC through inhibition of TLR4. The human milk oligosaccharides 2'-FL and 6'-SL, but not the backbone sugar lactose, prevent NEC in mice and piglets. 2'-FL and 6'-SL but not lactose inhibited TLR4 signaling in cultured enterocytes, in enteroids derived from mouse intestine, and in human intestinal explants obtained at the time of surgical resection for patients with NEC. In seeking the mechanisms involved, 2'-FL and 6'-SL but not lactose were found to directly bind to TLR4, explaining the inhibition and protection against NEC. These findings may impact clinical practice by suggesting that administration of HMOs could serve as a preventive strategy for premature infants at risk for NEC development.

Mannotriose induced differentiation of mesenchymal stem cells into neuron-like cells.

This article demonstrates that mannotriose effectively induces the differentiation of mesenchymal stem cells into neuron-like cells in vitro. Rat-derived mesenchymal stem cells were investigated on their potential to differentiate into neuron-like cells induced by mannotriose purified from Radix Rehmanniae Preparata in vitro. The percentage of the neuron-specific enolase positive cells and the Nissl positive cells after mannotriose treatment was increased. The mRNA levels of neurofilament medium and neuron-specific enolase were upregulated in the mannotriose group compared to the control. These findings demonstrate that mannotriose purified from Radix Rehmanniae Preparata can effectively induce differentiation of rat-derived mesenchymal stem cells into neuron-like cells.

Human Milk Oligosaccharide 2'-Fucosyllactose Induces Neuroprotection from Intracerebral Hemorrhage Stroke.

Intracerebral hemorrhage (ICH) occurs when brain blood vessels rupture, causing inflammation and cell death. 2-Fucosyllactose (2FL), a human milk oligosaccharide, has potent antiapoptotic and anti-inflammatory effects. The purpose of this study was to examine the protective effect of 2FL in cellular and rodent models of ICH. Hemin was added to a primary rat cortical neuronal and BV2 microglia coculture to simulate ICH in vitro. IBA1 and MAP2 immunoreactivities were used to determine inflammation and neuronal survival. Hemin significantly increased IBA1, while it reduced MAP2 immunoreactivity. 2FL significantly antagonized both responses. The protective effect of 2FL was next examined in a rat ICH model. Intracerebral administration of type VII collagenase reduced open-field locomotor activity. Early post-treatment with 2FL significantly improved locomotor activity. Brain tissues were collected for immunohistochemistry and qRT-PCR analysis. 2FL reduced IBA1 and CD4 immunoreactivity in the lesioned striatum. 2FL downregulated the expression of ER stress markers (PERK and CHOP), while it upregulated M2 macrophage markers (CD206 and TGFß) in the lesioned brain. Taken together, our data support that 2FL has a neuroprotective effect against ICH through the inhibition of neuroinflammation and ER stress. 2FL may have clinical implications for the treatment of ICH.

Biosynthesis of the anti-diabetic metabolite montbretin A: glucosylation of the central intermediate mini-MbA.

Type 2 diabetes (T2D) affects over 320 million people worldwide. Healthy lifestyles, improved drugs and effective nutraceuticals are different components of a response against the growing T2D epidemic. The specialized metabolite montbretin A (MbA) is being developed for treatment of T2D and obesity due to its unique pharmacological activity as a highly effective and selective inhibitor of the human pancreatic α-amylase. MbA is an acylated flavonol glycoside found in small amounts in montbretia (Crocosmia × crocosmiiflora) corms. MbA cannot be obtained in sufficient quantities for drug development from its natural source or by chemical synthesis. To overcome these limitations through metabolic engineering, we are investigating the genes and enzymes of MbA biosynthesis. We previously reported the first three steps of MbA biosynthesis from myricetin to myricetin 3-O-(6'-O-caffeoyl)-glucosyl rhamnoside (mini-MbA). Here, we describe the sequence of reactions from mini-MbA to MbA, and the discovery and characterization of the gene and enzyme responsible for the glucosylation of mini-MbA. The UDP-dependent glucosyltransferase CcUGT3 (UGT703E1) catalyzes the 1,2-glucosylation of mini-MbA to produce myricetin 3-O-(glucosyl-6'-O-caffeoyl)-glucosyl rhamnoside. Co-expression of CcUGT3 with genes for myricetin and mini-MbA biosynthesis in Nicotiana benthamiana validated its biological function and expanded the set of genes available for metabolic engineering of MbA.

Influence of 2'-fucosyllactose and galacto-oligosaccharides on the growth and adhesion of Streptococcus mutans.

Human milk oligosaccharides, such as 2'-fucosyllactose (2'-FL), and galacto-oligosaccharides (GOS), a prebiotic carbohydrate mixture, are being increasingly added to infant formulas, necessitating the understanding of their impact on the oral microbiota. Here, for the first time, the effects of 2'-FL and GOS on the planktonic growth and adhesion characteristics of the caries-associated oral pathogen Streptococcus mutans were assessed, and the results were compared against the effects of xylitol, lactose and glucose. There were differences in S. mutans growth between 2'-FL and GOS. None of the three S. mutans strains grew with 2'-FL, while they all grew with GOS as well as lactose and glucose. Xylitol inhibited S. mutans growth. The adhesion of S. mutans CI 2366 to saliva-coated hydroxyapatite was reduced by 2'-FL and GOS. Exopolysaccharide-mediated adhesion of S. mutans DSM 20523 to a glass surface was decreased with 2'-FL, GOS and lactose, and the adhesion of strain CI 2366 strain was reduced only by GOS. Unlike GOS, 2'-FL did not support the growth of any S. mutans strain. Neither 2'-FL nor GOS enhanced the adhesive properties of the S. mutans strains, but they inhibited some of the tested strains. Thus, the cariogenic tendency may vary between infant formulas containing different types of oligosaccharides.

A trisaccharide phenylethanoid glycoside from Scrophularia flava Grau with potential anti-type 2 diabetic properties by inhibiting α-glucosidase enzyme and decreasing oxidative stress.

The Scrophularia genus is a rich source of phenylethanoid glycosides, with diverse biological activities including anti-diabetic properties. This study investigated anti-type 2 diabetic potential and active component of Scrophularia flava Grau. A new phenylethanoid glycoside was isolated from aerial parts of the plant and identified as 2-(4-hydroxy-3-methoxyphenyl) ethyl 6-deoxy-3-O-[(2E)-3-(3 hydroxy-4-methoxyphenyl) prop-2-enoyl]-α-rhamnopyranosyl-(1 â†’ 3)-[α-rhamnopyranosyl-(1 â†’ 6)]-4-O-[(2E)-3-(4-hydroxy-3-methoxyphenyl) prop-2-enoyl]-ß-glucopyranoside. It was named flavaioside. The structure of flavaioside was identified based on 1H NMR, 13C NMR, DEPT-HSQC, COSY, HMBC, NOESY and LC-ESI-MS-MS. Total methanol extract, fractions (A-F) and specific main phenylethanoid glycoside (flavaioside), were assessed for inhibitory effects against the α-glucosidase enzyme (in vitro anti-type 2 diabetic assay). The antioxidant activities of methanol extracts, all fractions and isolated flavaioside were identified based on 2, 2'-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity, 2, 2'-azino-bis (3-ethylbenzothiazoline)-6-sulphonic acid radical cation (ABTS+) scavenging activity, phosphomolybdenum method, and metal chelating activity. In comparison to the other fractions, the best antioxidant result was observed in fraction E and its main compound, flavaioside, in DPPH (IC50 = 4.26, 2.57 µg/mL) and ABTS+ (EC50 = 55.45, 6.34 µg/mL) scavenging activities. Flavaioside showed significantly stronger activities than α-tocopherol and ascorbic acid in DPPH and ABTS+ assays. Furthermore, flavaioside showed a potent inhibitory activity on the α-glucosidase enzyme which was comparable with the known anti-type 2 diabetic drug, acarbose (91.85%, and 92.87%, respectively). Fraction E and flavaioside showed α-glucosidase inhibitory activities with IC50 values, 65.05 and 6.50 µg/mL. The plant and its isolated flavaioside can possess acceptable anti-type 2 diabetic potential and anti-oxidant activity.

2'-Fucosyllactose promotes Bifidobacterium bifidum DNG6 adhesion to Caco-2 cells.

Adhesion to the intestinal mucosa is the prerequisite for bifidobacteria to colonize and exert biological functions, whereas the choice of carbon source affects the ability of bifidobacteria to adhere to and interact with intestinal epithelial cells. However, knowledge about the relationship between human milk oligosaccharide consumption by bifidobacteria and its adhesion is still limited. In this study, we aim to investigate the effect of 2'-fucosyllactose (2'-FL) as the carbon source on the growth and adhesion properties of Bifidobacterium bifidum DNG6, and make comparisons with galactooligosaccharides and glucose. We found that the growth and adhesion properties of B. bifidum DNG6 grown in different carbon sources were varied. The 2'-FL as a carbon source improves the adhesion ability of B. bifidum DNG6. The expression of adhesion-associated genes was significantly higher in B. bifidum DNG6 grown in 2'-FL after incubation with Caco-2 cells compared with that in galactooligosaccharides and glucose. Our results indicated that 2'-FL may promote B. bifidum DNG6 adhesion to Caco-2 cells through high expression of genes encoding adhesion proteins. The findings of this study contribute to a better understanding of the involvement of human milk oligosaccharides in the adhesion of bifidobacteria and further support the potential application of 2'-FL as a prebiotic in infant nutritional supplements.

The Combination of 2'-Fucosyllactose with Short-Chain Galacto-Oligosaccharides and Long-Chain Fructo-Oligosaccharides that Enhance Influenza Vaccine Responses Is Associated with Mucosal Immune Regulation in Mice.

BACKGROUND: A critical role for host-microbe interactions and establishment of vaccine responses has been postulated. Human milk oligosaccharides, of which 2'-fucosyllactose (2'FL) is the most prevalent, are known to alter host-associated microbial communities and play a critical role in the immunologic development of breastfed infants. OBJECTIVES: Dietary supplementation with a combination of 2'FL and prebiotic short-chain (sc) galacto-oligosaccharides (GOS) and long-chain (lc) fructo-oligosaccharides (FOS) was employed to examine human milk oligosaccharide effects on immune responsiveness, within a murine influenza vaccination model. METHODS: Female mice (6 wk old, C57Bl/6JOlaHsd) were fed either control diet (CON) or scGOS/lcFOS/2'FL-containing diet (GF2F) for 45 d. After starting dietary intervention (day 14), mice received a primary influenza vaccination (day 0) followed by a booster vaccination (day 21), after which ear challenges were conducted to measure vaccine-specific delayed type hypersensitivity (DTH). Serum immunoglobulin (Ig) levels, fecal and cecal microbial community structure, short-chain fatty acids, host intestinal gene expression and cellular responses in the mesenteric lymph nodes (MLNs) were also measured. RESULTS: Relative to CON, mice fed the GF2F diet had increased influenza vaccine-specific DTH responses (79.3%; P < 0.01), higher levels of both IgG1 (3.2-fold; P < 0.05) and IgG2a (1.2-fold; P < 0.05) in serum, and greater percentages of activated B cells (0.3%; P < 0.05), regulatory T cells (1.64%; P < 0.05), and T-helper 1 cells (2.2%; P < 0.05) in their MLNs. GF2F-fed mice had elevated cecal butyric (P < 0.05) and propionic (P < 0.05) acid levels relative to CON, which correlated to DTH responses (R2 = 0.22; P = 0.05 and R2 = 0.39; P < 0.01, respectively). Specific fecal microbial taxa altered in GF2F diet fed mice relative to CON were significantly correlated with the DTH response and IgG2a level increases. CONCLUSIONS: Dietary GF2F improved influenza vaccine-specific T-helper 1 responses and B cell activation in MLNs and enhanced systemic IgG1 and IgG2a concentrations in mice. These immunologic changes are correlated with microbial community structure and metabolites.

The Sweet Taste of Acarbose and Maltotriose: Relative Detection and Underlying Mechanism.

Although sweet-tasting saccharides possess similar molecular structures, their relative sweetness often varies to a considerable degree. Current understanding of saccharide structure/sweetness interrelationships is limited. Understanding how certain structural features of saccharides and/or saccharide analogs correlate to their relative sweetness can provide insight on the mechanisms underlying sweetness potency. Maltotriose is a short-chain glucose-based oligosaccharide, which we recently reported to elicit sweet taste. Acarbose, an α-glucosidase inhibitor, is a pseudo-saccharide that has an overall resemblance to a glucose-based oligosaccharide and thus may be viewed as a structural analog. During other studies, we recognized that acarbose can also elicit sweet taste. Here, we formally investigated the underlying taste detection mechanism of acarbose, while confirming our previous findings for maltotriose. We found that subjects could detect the sweet taste of acarbose and maltotriose in aqueous solutions but were not able to detect them in the presence of a sweet taste inhibitor lactisole. These findings support that both are ligands of the human sweet taste receptor, hT1R2/hT1R3. In a separate experiment, we measured the relative sweetness detection of acarbose, maltotriose, and other sweet-tasting mono- and disaccharides (glucose, fructose, maltose, and sucrose). Whereas maltotriose was found to have a similar discriminability profile to glucose and maltose, the discriminability of acarbose matched that of fructose at the concentrations tested (18, 32, and 56 mM). These findings are discussed in terms of how specific molecular features (e.g., degree of polymerization and monomer composition) may contribute to the relative sweetness of saccharides.

In vitro activation and maturation of human mononuclear phagocytes by stimulation with liposomes coated with a neoglycolipid containing α1-3, α1-6-mannotriose.

In this study, we assessed the potential of liposomes coated with a neoglycolipid containing α1-3,α1-6-mannotriose residues (Man3-DPPE; Manα1-6(Manα1-3)Manitol-DPPE) for in vitro activation and maturation of human mononuclear phagocytes. In response to treatment with Man3-DPPE-coated liposomes (Man3-OMLs), PMA-stimulated human THP-1 cells showed enhanced expression of CD40, CD80 and HLA-DR and secreted significant levels of IL-12p40. Among various linkages of Man2-DPPE-coated liposomes, only liposomes coated with Manα1-6Manitol-DPPE (α1-6Man2-DPPE) induced these cellular responses similarly to Man3-OML treatment. Liposomes coated with Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manitol-DPPE (Man5-DPPE) failed to activate the cells. These results suggest that an unsubstituted α1-6Man branch bound to a mannitol unit at the reducing end in Man3-DPPE is required for in vitro activation of human mononuclear phagocytes. Man3-OML-induced IL-12p40 production was not inhibited by BAY11-7082, an inhibitor of the MyD88-dependent signaling network, suggesting that TLRs are not involved in activation of human mononuclear phagocytes by Man3-OMLs. Stimulation of inflammatory monocytes or monocyte-derived dendritic cells (moDCs) with Man3-OMLs also induced enhanced expression of co-stimulatory molecules, HLA-DR, and CCR7, and IL-12p40 production from both types of cells. In response to Man3-OML treatment, moDCs but not inflammatory monocytes produced bioactive IL-12p70, which was enhanced by CD40 ligation. Thus, Man3-OMLs can activate naïve human mononuclear phagocytes and lead human moDCs to a fully matured status in vitro to elicit CTLs and a Th1 response without addition of inflammatory cytokines or TLR agonists.

2'-Fucosyllactose Attenuates Particulate Matter-Induced Inflammation via Inhibition of Hypoxia-Inducible Factor in Keratinocytes.

2'-Fucosyllactose (2FL) is the most abundant component of the oligosaccharide content in human milk. It has been reported that 2FL has the ability to protect against infectious disease caused by bacterial pathogens. In this study, we investigated the protective effects of 2FL on particulate matter (PM)10-induced pro-inflammatory cytokines in HaCaT keratinocytes. 2FL reduced PM10-induced excess expression of interleukin (IL)-6, IL-8, IL-1α and IL-1ß in HaCaT keratinocytes. In addition, PM10 also increased hypoxia-inducible factor (HIF)-1α protein levels; however, 2FL inhibited the accumulation of HIF-1α protein and the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt stimulated by PM10. Furthermore, 2FL improved PM10-induced the decrease in epidermal thickness and integrity of the cornified layer in the reconstructed human epidermal skin model (RHE). In our results, 2FL inhibited PM10-induced pro-inflammatory mediators by regulating the HIF-1α/PI3K/Akt pathway and protected the skin epidermis against PM10 irritation. Taken together, these results suggest that 2FL can be used as a primary ingredient in cosmeceutical products to alleviate skin irritation and inflammation caused by urban air pollution.

Synthesis, antiasthmatic, and insecticidal/antifungal activities of allosamidins.

Allosamidins come from the secondary metabolites of Streptomyces species, and they have the pseudotrisaccharide structures. Allosamidins are chitinase inhibitors that can be used to study the physiological effects of chitinases in a variety of organisms. They have the novel antiasthmatic activity and insecticidal/antifungal activities. Herein, the synthesis and activities of allosamidins were summarized and analyzed.

MytiLec-1 Shows Glycan-Dependent Toxicity against Brine Shrimp Artemia and Induces Apoptotic Death of Ehrlich Ascites Carcinoma Cells In Vivo.

MytiLec-1, a 17 kDa lectin with ß-trefoil folding that was isolated from the Mediterranean mussel (Mytilus galloprovincialis) bound to the disaccharide melibiose, Galα(1,6) Glc, and the trisaccharide globotriose, Galα(1,4) Galß(1,4) Glc. Toxicity of the lectin was found to be low with an LC50 value of 384.53 µg/mL, determined using the Artemia nauplii lethality assay. A fluorescence assay was carried out to evaluate the glycan-dependent binding of MytiLec-1 to Artemia nauplii. The lectin strongly agglutinated Ehrlich ascites carcinoma (EAC) cells cultured in vivo in Swiss albino mice. When injected intraperitoneally to the mice at doses of 1.0 mg/kg/day and 2.0 mg/kg/day for five consecutive days, MytiLec-1 inhibited 27.62% and 48.57% of cancer cell growth, respectively. Antiproliferative activity of the lectin against U937 and HeLa cells was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro in RPMI-1640 medium. MytiLec-1 internalized into U937 cells and 50 µg/mL of the lectin inhibited their growth of to 62.70% whereas 53.59% cell growth inhibition was observed against EAC cells when incubated for 24 h. Cell morphological study and expression of apoptosis-related genes (p53, Bax, Bcl-X, and NF-κB) showed that the lectin possibly triggered apoptosis in these cells.

Synthesis of Fucosylated Chondroitin Sulfate Glycoclusters: A Robust Route to New Anticoagulant Agents.

Fucosylated chondroitin sulfate (FuCS) is a structurally distinct glycosaminoglycan with excellent anticoagulant activity. Studies show that FuCS and its depolymerized fragments exhibit a different anticoagulant mechanism from that of heparin derivatives, with decreased risks of adverse effects and bleeding. However, further exploitation has been hindered by the scarcity of structurally defined oligosaccharides. Herein, facile method is reported for the synthesis of the repeating trisaccharide unit of FuCS based on the degradation of chondroitin sulfate polymers. A series of simplified FuCS glycomimetics that have highly tunable structures, controllable branches, and defined sulfation motifs were generated by copper-catalyzed alkyne-azide cycloaddition. Remarkable improvement in activated partial thromboplastin time (APTT) assay activities was observed as the branches increased, but no significant influences were observed for prothrombin time (PT) and thrombin time (TT) assay activities. Further FXase inhibition tests suggested that glycoclusters 33 b-40 b selectively inhibited intrinsic anticoagulant activities, but had little effect on the extrinsic and common coagulation pathways. Notably, glycoclusters with the 2,4-di-O-sulfated fucosyl residue displayed the most potency, which was in consistent with that of natural polysaccharides. These FuCS clusters demonstrated potency to mimic linear glycosaminoglycans and offer a new framework for the development of novel anticoagulant agents.