Optimization of the Preparation Process of Glucuronomannan Oligosaccharides and Their Effects on the Gut Microbiota in MPTP-Induced PD Model Mice.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, and accumulating evidence suggests a link between dysbiosis of the gut microbiota and the onset and progression of PD. In our previous investigations, we discovered that intraperitoneal administration of glucuronomannan oligosaccharides (GMn) derived from Saccharina japonica exhibited neuroprotective effects in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. However, the complicated preparation process, difficulties in isolation, and remarkably low yield have constrained further exploration of GMn. In this study, we optimized the degradation conditions in the preparation process of GMn through orthogonal experiments. Subsequently, an MPTP-induced PD model was established, followed by oral administration of GMn. Through a stepwise optimization, we successfully increased the yield of GMn, separated from crude fucoidan, from 1~2/10,000 to 4~8/1000 and indicated the effects on the amelioration of MPTP-induced motor deficits, preservation of dopamine neurons, and elevation in striatal neurotransmitter levels. Importantly, GMn mitigated gut microbiota dysbiosis induced by MPTP in mice. In particular, GM2 significantly reduced the levels of Akkermansia, Verrucomicrobiota, and Lactobacillus, while promoting the abundance of Roseburia and Prevotella compared to the model group. These findings suggest that GM2 can potentially suppress PD by modulating the gut microbiota, providing a foundation for the development of a novel and effective anti-PD marine drug.

Glucuronomannan GM2 from Saccharina japonica Enhanced Mitochondrial Function and Autophagy in a Parkinson's Model.

Parkinson's disease (PD), one of the most common neurodegenerative disorders, is caused by dopamine depletion in the striatum and dopaminergic neuron degeneration in the substantia nigra. In our previous study, we hydrolyzed the fucoidan from Saccharina japonica, obtaining three glucuronomannan oligosaccharides (GMn; GM1, GM2, and GM3) and found that GMn ameliorated behavioral deficits in Parkinsonism mice and downregulated the apoptotic signaling pathway, especially with GM2 showing a more effective role in neuroprotection. However, the neuroprotective mechanism is unclear. Therefore, in this study, we aimed to assess the neuroprotective effects of GM2 in vivo and in vitro. We applied GM2 in 1-methyl-4-phenylpyridinium (MPP+)-treated PC12 cells, and the results showed that GM2 markedly improved the cell viability and mitochondrial membrane potential, inhibited MPP+-induced apoptosis, and enhanced autophagy. Furthermore, GM2 contributed to reducing the loss of dopaminergic neurons in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice through enhancing autophagy. These data indicate that a possible protection of mitochondria and upregulation of autophagy might underlie the observed neuroprotective effects, suggesting that GM2 has potential as a promising multifunctional lead disease-modifying therapy for PD. These findings might pave the way for additional treatment strategies utilizing carbohydrate drugs in PD.

Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies.

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed "starch-mannose-fermentation" biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.

Preparation and Neuroprotective Activity of Glucuronomannan Oligosaccharides in an MPTP-Induced Parkinson's Model.

Parkinson's disease (PD), characterized by dopaminergic neuron degeneration in the substantia nigra and dopamine depletion in the striatum, affects up to 1% of the global population over 50 years of age. Our previous study found that a heteropolysaccharide from Saccharina japonica exhibits neuroprotective effects through antioxidative stress. In view of its high molecular weight and complex structure, we degraded the polysaccharide and subsequently obtained four oligosaccharides. In this study, we aimed to further detect the neuroprotective mechanism of the oligosaccharides. We applied MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to induce PD, and glucuronomannan oligosaccharides (GMn) was subsequently administered. Results showed that GMn ameliorated behavioral deficits in Parkinsonism mice. Furthermore, we observed that glucuronomannan oligosaccharides contributed to down-regulating the apoptotic signaling pathway through enhancing the expression of tyrosine hydroxylase (TH) in dopaminergic neurons. These results suggest that glucuronomannan oligosaccharides protect dopaminergic neurons from apoptosis in PD mice.

Biopolymer monolith for protein purification.

Porous glycopolymers, "glycomonoliths", were prepared by radical polymerization based on polymerization-induced phase separation with an acrylamide derivative of α-mannose, acrylamide and cross-linker in order to investigate protein adsorption and separation. The porous structure was induced by a porogenic alcohol. The pore diameter and surface area were controlled by the type of alcohol. The protein adsorption was measured in both batch and continuous flow systems. The glycomonoliths showed specific interaction with the sugar recognition protein of concanavalin A, and non-specific interaction to other proteins was negligible. The amount of protein adsorption to the materials was determined by the sugar density and the composition of the glycomonoliths. Fundamental knowledge regarding the glycomonoliths for protein separation was obtained.

The first study on the impact of osmolytes in whole cells of high temperature-adapted microorganisms.

The hyperthermophilic piezophile, Thermococcus barophilus displays a strong stress response characterized by the accumulation of the organic osmolyte, mannosylglycerate during growth under sub-optimal pressure conditions (0.1 MPa). Taking advantage of this known effect, the impact of osmolytes in piezophiles in an otherwise identical cellular context was investigated, by comparing T. barophilus cells grown under low or optimal pressures (40 MPa). Using neutron scattering techniques, we studied the molecular dynamics of live cells of T. barophilus at different pressures and temperatures. We show that in the presence of osmolytes, cells present a higher diffusion coefficient of hydration water and an increase of bulk water motions at a high temperature. In the absence of osmolytes, the T. barophilus cellular dynamics is more responsive to high temperature and high hydrostatic pressure. These results therefore give clear evidence for a protecting effect of osmolytes on proteins.

Distortion of mannoimidazole supports a B2,5 boat transition state for the family GH125 α-1,6-mannosidase from Clostridium perfringens.

Enzyme transition-state mimics can act as powerful inhibitors and allow structural studies that report on the conformation of the transition-state. Here, mannoimidazole, a mimic of the transition state of mannosidase catalyzed hydrolysis of mannosides, is shown to bind in a B2,5 conformation on the Clostridium perfringens GH125 α-1,6-mannosidase, providing additional evidence of a OS2-B2,5-1S5 conformational itinerary for enzymes of this family.

A Unique Sugar l-Perosamine (4-Amino-4,6-dideoxy-l-mannose) Is a Compound Building Two O-Chain Polysaccharides in the Lipopolysaccharide of Aeromonas hydrophila Strain JCM 3968, Serogroup O6.

Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-d-GalpNAc and two α-l-Rhap4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1 →3)-α-l-Rhap4NAc-(1→4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc-(1→ O-PS2 →2)-α-l-Rhap4NAc-(1→ The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides.

Structural and Serological Studies of the O6-Related Antigen of Aeromonas veronii bv. sobria Strain K557 Isolated from Cyprinus carpio on a Polish Fish Farm, which Contains L-perosamine (4-amino-4,6-dideoxy-L-mannose), a Unique Sugar Characteristic for Aeromonas Serogroup O6.

Amongst Aeromonas spp. strains that are pathogenic to fish in Polish aquacultures, serogroup O6 was one of the five most commonly identified immunotypes especially among carp isolates. Here, we report immunochemical studies of the lipopolysaccharide (LPS) including the O-specific polysaccharide (O-antigen) of A. veronii bv. sobria strain K557, serogroup O6, isolated from a common carp during an outbreak of motile aeromonad septicemia (MAS) on a Polish fish farm. The O-polysaccharide was obtained by mild acid degradation of the LPS and studied by chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-antigen was composed of two O-polysaccharides, both containing a unique sugar 4-amino-4,6-dideoxy-L-mannose (N-acetyl-L-perosamine, L-Rhap4NAc). The following structures of the O-polysaccharides (O-PS 1 and O-PS 2) were established.

Leishmania-Derived Trimannose Modulates the Inflammatory Response To Significantly Reduce Leishmania major-Induced Lesions.

Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated with Leishmania major and trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated with L. major alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected with L. major alone, coinoculated with carrier beads and L. major, or coinoculated with trimannose-coated beads and L. major Trimannose treatment of L. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR-/-) mice lack the ability to detect trimannose. When MR-/- mice were infected with L. major and treated with trimannose beads, they did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.

Top-Down Chemoenzymatic Approach to Synthesizing Diverse High-Mannose N-Glycans and Related Neoglycoproteins for Carbohydrate Microarray Analysis.

High-mannose-type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope glycoprotein gp120. They also serve as signals for protein folding, trafficking, and degradation in protein quality control. A number of lectins and antibodies recognize high-mannose-type N-glycans, and glycan array technology has provided an avenue to probe these oligomannose-specific proteins. We describe in this paper a top-down chemoenzymatic approach to synthesize a library of high-mannose N-glycans and related neoglycoproteins for glycan microarray analysis. The method involves the sequential enzymatic trimming of two readily available natural N-glycans, the Man9GlcNAc2Asn prepared from soybean flour and the sialoglycopeptide (SGP) isolated from chicken egg yolks, coupled with chromatographic separation to obtain a collection of a full range of natural high-mannose N-glycans. The Asn-linked N-glycans were conjugated to bovine serum albumin (BSA) to provide neoglycoproteins containing the oligomannose moieties. The glycoepitopes displayed were characterized using an array of glycan-binding proteins, including the broadly virus-neutralizing agents, glycan-specific antibody 2G12, Galanthus nivalis lectin (GNA), and Narcissus pseudonarcissus lectin (NPA).

Split-and-Combine Approach Towards Branched Precision Glycomacromolecules and Their Lectin Binding Behavior.

Previously, monodisperse and sequence-controlled oligo(amidoamine) scaffolds were synthesized based on the step-wise assembly of tailor-made building blocks on a solid support that allow for the multivalent presentation of sugar ligands. Here, we extend on this concept using a split-and-combine approach to gain access to a small library of linear and branched glycomacromolecules. Azide side chains were introduced in the scaffold by the use of a novel building block allowing for copper-mediated azide-alkyne cycloaddition (CuAAC) of readily available propargyl-functionalized glycans. In the first stage, after assembly of the linear scaffold on solid support, the batch was divided into two. One part of the resin-bound oligomers was end-capped and further used as backbone and the other part was functionalized with propargylated α-d-mannopyranoside in the sidechain, end capped with an alkyne functionality and finally cleaved from solid support to give the branching arm. In the second stage, the linear, glycosylated and alkynylated arms were then coupled to the end capped backbone via CuAAC. In this way, branched glycomacromolecules with two and three branches, respectively, have been synthesized carrying from two to six sugar residues per molecule. Both, linear arms and branched glycomacromolecules were then subjected to a lectin binding assay using surface plasmon resonance (SPR) and model lectin Concanavalin A (Con A) showing the effect of branching as well as valency on the binding kinetics.

Single-Chain Glycopolymer Folding via Host-Guest Interactions and Its Unprecedented Effect on DC-SIGN Binding.

Reversible self-folding actions of natural biomacromolecules play crucial roles for specific and unique biological functions in Nature. Hence, controlled folding of single polymer chains has attracted significant attention in recent years. Herein, reversible single-chain folded glycopolymer structures in α-shape with different density of sugar moieties in the knot were created. The influence of folding as well as the sugar density in the knot was investigated on the binding capability with lectins, such as ConA, DC-SIGN, and DC-SIGNR. The synthesis of triblock glycocopolymers bearing ß-CD and adamantane for the host-guest interaction and also mannose residues for the lectin interaction was achieved using the reversible addition-fragmentation chain transfer (RAFT) polymerization technique. The reversible single-chain folding of glycopolymers was achieved under a high dilution of an aqueous solution and the self-assembled folding was monitored by 2D nuclear overhauser enhancement spectroscopy (NOESY) NMR and dynamic light scattering. The lectin binding profiles consistently provided an unprecedented effect of single chain folding as the single-chain folded structures enhanced greatly the binding ability in comparison to the unfolded linear structures.

Production of mannosylglycerate in Saccharomyces cerevisiae by metabolic engineering and bioprocess optimization.

BACKGROUND: Mannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward. However, the current prohibitive production costs impose severe constraints towards large-scale applications. All known microbial producers synthesize MG from GDP-mannose and 3-phosphoglycerate via a two-step pathway in which mannosyl-3-phosphoglycerate is the intermediate metabolite. In an early work, this pathway was expressed in Saccharomyces cerevisiae with the goal to confirm gene function (Empadinhas et al. in J Bacteriol 186:4075-4084, 2004), but the level of MG accumulation was low. Therefore, in view of the potential biotechnological value of this compound, we decided to invest further effort to convert S. cerevisiae into an efficient cell factory for MG production. RESULTS: To drive MG production, the pathway for the synthesis of GDP-mannose, one of the MG biosynthetic precursors, was overexpressed in S. cerevisiae along with the MG biosynthetic pathway. MG production was evaluated under different cultivation modes, i.e., flask bottle, batch, and continuous mode with different dilution rates. The genes encoding mannose-6-phosphate isomerase (PMI40) and GDP-mannose pyrophosphorylase (PSA1) were introduced into strain MG01, hosting a plasmid encoding the MG biosynthetic machinery. The resulting engineered strain (MG02) showed around a twofold increase in the activity of PMI40 and PSA1 in comparison to the wild-type. In batch mode, strain MG02 accumulated 15.86 mgMG g DCW -1 , representing a 2.2-fold increase relative to the reference strain (MG01). In continuous culture, at a dilution rate of 0.15 h-1, there was a 1.5-fold improvement in productivity. CONCLUSION: In the present study, the yield and productivity of MG were increased by overexpression of the GDP-mannose pathway and optimization of the mode of cultivation. A maximum of 15.86 mgMG g DCW -1 was achieved in batch cultivation and maximal productivity of 1.79 mgMG g DCW -1  h-1 in continuous mode. Additionally, a positive correlation between MG productivity and growth rate/dilution rate was established, although this correlation is not observed for MG yield.

Structural Features of Sulfated Glucuronomannan Oligosaccharides and Their Antioxidant Activity.

Glucuronomannan oligosaccharides (Gs) were derived from fucoidan, which was extracted from the brown alga Sargassum thunbergii. Sulfated glucuronomannan oligosaccharides (SGs) were obtained by the sulfation of Gs. NMR techniques were used to reveal that the order of sulfation was Man-C6 > Man-C4 > Man-C1R > GlcA-C3 > Man-C3 > GlcA-C2. Finally, the antioxidant activities (hydroxyl radical scavenging activity, superoxide radical scavenging activity, reducing power and DPPH radical scavenging activity) of Gs and SGs were determined. The findings showed that the higher the degree of polymerization, the better the activity, except for the hydroxyl radical scavenging activity. In addition, the higher the sulfate content, the lower the activities for the reducing power and DPPH radical scavenging activity. Opposite results were found for the superoxide radical scavenging activity. Finally, compared with fucoidan, most Gs and SGs had higher antioxidant activity, suggesting that they might be good candidates for antioxidants.

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di-N-acetyllegionaminic Acid-Containing Glycosides.

A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9-di-N-acetyllegionaminic acid (Leg5,7Ac2 ), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2-3/6-linked Leg5,7Ac2 -glycosides was built by using chemically synthesized 2,4-diazido-2,4,6-trideoxymannose as a chemoenzymatic synthon for highly efficient one-pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d-fucose and had an overall yield of 34-52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac2 monosaccharide was also synthesized by a sialic acid aldolase-catalyzed reaction.

Computational Design of Experiment Unveils the Conformational Reaction Coordinate of GH125 α-Mannosidases.

Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B2,5 or 3H4 transition-state (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-α-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a nonhydrolyzable 1,6-α-thiomannoside substrate mimic bound across the active site revealed an undistorted 4C1 conformation and provided no insight into the catalytic pathway of this enzyme. We show through a purely computational approach (QM/MM metadynamics) that sulfur-for-oxygen substitution in the glycosidic linkage fundamentally alters the energetically accessible conformational space of a thiomannoside when bound within the GH125 active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative 4C1 conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an OS2 conformation (consistent with catalysis through a B2,5 TS). This prediction was tested experimentally by determination of the 3D X-ray structure of the pseudo-Michaelis complex of an inactive (D220N) variant of C. perfringens GH125 enzyme in complex with 1,6-α-mannobiose. This complex revealed unambiguous distortion of the -1 subsite mannoside to an OS2 conformation, matching that predicted by theory and supporting an OS2 → B2,5 → 1S5 conformational itinerary for GH125 α-mannosidases. This work highlights the power of the QM/MM approach and identified shortcomings in the use of nonhydrolyzable substrate analogues for conformational analysis of enzyme-bound species.

Mannose-Functionalized Hyperbranched Polyglycerol Loaded with Zinc Porphyrin: Investigation of the Multivalency Effect in Antibacterial Photodynamic Therapy.

The antibacterial photodynamic activity of hyperbranched polyglycerol (hPG) loaded with zinc porphyrin photosensitizers and mannose units was investigated. hPG, with a MW of 19.5 kDa, was functionalized with about 15 molecules of the photosensitizer {5,10,15-tris(3-hydroxyphenyl)-20-[4-(prop-2-yn-1-ylamino)tetrafluorophenyl]porphyrinato}-zinc(II) by using copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC). These nanoparticle conjugates were functionalized systematically with increasing loadings of mannose in the range of approximately 20 to 110 groups. With higher mannose loadings (ca. 58-110 groups) the water-insoluble zinc porphyrin photosensitizer could thus be transferred into a water-soluble form. Targeting of the conjugates was proven in binding studies to the mannose-specific lectin concanavalin A (Con A) by using surface plasmon resonance (SPR). The antibacterial phototoxicity of the conjugates on Staphylococcus aureus (as a typical Gram-positive germ) was investigated in phosphate-buffered saline (PBS). It was shown that conjugates with approximately 70-110 mannose units exhibit significant antibacterial activity, whereas conjugates with approximately 20-60 units did not induce bacterial killing at all. These results give an insight into the multivalency effect in combination with photodynamic therapy (PDT). On addition of serum to the bacterial cultures, a quenching of this antibacterial phototoxicity was observed. In fluorescence studies with the conjugates in the presence of increasing bovine serum albumin (BSA) concentrations, protein-conjugate associations could be identified as a plausible cause for this quenching.

Isolation of a methylated mannose-binding protein from terrestrial worm Enchytraeus japonensis.

To elucidate a biological role of the methylated mannose residues found in N-glycans of terrestrial worm Enchytraeus japonensis, we first synthesized 3-O-methyl mannose- and 4-O-methyl mannose-derivatives and immobilized them to Sepharose 4B beads in order to isolate the sugar-binding protein. When whole protein extracts from the worms was applied to a series of the columns immobilized with the modified and unmodified mannose-derivatives, respectively, a protein with a molecular weight of 25,000 was isolated by 4-O-methyl mannose-immobilized column chromatography, and termed as a methylated mannose-binding protein (mMBP). mMBP bound weakly to a mannose-immobilized column and moderately to a 3-O-methyl mannose-immobilized column. The N-terminal amino acid sequences of mMBP and its endoprotease-digested peptides were determined. Using the degenerate first primers synthesized based on the primary sequence, a genomic DNA fragment was isolated. Then, the second primers were synthesized based on the genomic DNA fragment, and with use of them two cDNA fragments were obtained by the 3'- and 5'-RACE methods. Finally, the third primers were synthesized based on the sequences of the two cDNA fragments and one genomic DNA fragment, and with use of them a full-length cDNA of mMBP was isolated and shown to comprise a putative 633 bp open reading frame encoding 210 amino acid residues. BLAST analysis revealed that mMBP has identities by 26 ~ 55% to several proteins including the regeneration-upregulated protein 3 from the same species. Whether mMBP is involved in the regeneration of the worm is under investigation.

A non-covalent "click chemistry" strategy to efficiently coat highly porous MOF nanoparticles with a stable polymeric shell.

BACKGROUND: Metal-organic framework nanoparticles (nanoMOFs) are biodegradable highly porous materials with a remarkable ability to load therapeutic agents with a wide range of physico-chemical properties. Engineering the nanoMOFs surface may provide nanoparticles with higher stability, controlled release, and targeting abilities. Designing postsynthetic, non-covalent self-assembling shells for nanoMOFs is especially appealing due to their simplicity, versatility, absence of toxic byproducts and minimum impact on the original host-guest ability. METHODS: In this study, several ß-cyclodextrin-based monomers and polymers appended with mannose or rhodamine were randomly phosphorylated, and tested as self-assembling coating building blocks for iron trimesate MIL-100(Fe) nanoMOFs. The shell formation and stability were studied by isothermal titration calorimetry (ITC), spectrofluorometry and confocal imaging. The effect of the coating on tritium-labeled AZT-PT drug release was estimated by scintillation counting. RESULTS: Shell formation was conveniently achieved by soaking the nanoparticles in self-assembling agent aqueous solutions. The grafted phosphate moieties enabled a firm anchorage of the coating to the nanoMOFs. Coating stability was directly related to the density of grafted phosphate groups, and did not alter nanoMOFs morphology or drug release kinetics. CONCLUSION: An easy, fast and reproducible non-covalent functionalization of MIL-100(Fe) nanoMOFs surface based on the interaction between phosphate groups appended to ß-cyclodextrin derivatives and iron(III) atoms is presented. GENERAL SIGNIFICANCE: This study proved that discrete and polymeric phosphate ß-cyclodextrin derivatives can conform non-covalent shells on iron(III)-based nanoMOFs. The flexibility of the ß-cyclodextrin to be decorated with different motifs open the way towards nanoMOFs modifications for drug delivery, catalysis, separation, imaging and sensing. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.