Mitochondria as a Potential Target for the Development of Prophylactic and Therapeutic Drugs against Schistosoma mansoni Infection.

The emergence of parasites resistant to praziquantel, the only therapeutic agent, and its ineffectiveness as a prophylactic agent (inactive against the migratory/juvenile Schistosoma mansoni), make the development of new antischistosomal drugs urgent. The parasite's mitochondrion is an attractive target for drug development, because this organelle is essential for survival throughout the parasite's life cycle. We investigated the effects of 116 compounds against Schistosoma mansoni cercaria motility that have been reported to affect mitochondrion-related processes in other organisms. Next, eight compounds plus two controls (mefloquine and praziquantel) were selected and assayed against the motility of schistosomula (in vitro) and adults (ex vivo). Prophylactic and therapeutic assays were performed using infected mouse models. Inhibition of oxygen consumption rate (OCR) was assayed using Seahorse XFe24 analyzer. All selected compounds showed excellent prophylactic activity, reducing the worm burden in the lungs to less than 15% of that obtained in the vehicle control. Notably, ascofuranone showed the highest activity, with a 98% reduction of the worm burden, suggesting the potential for the development of ascofuranone as a prophylactic agent. The worm burden of infected mice with S. mansoni at the adult stage was reduced by more than 50% in mice treated with mefloquine, nitazoxanide, amiodarone, ascofuranone, pyrvinium pamoate, or plumbagin. Moreover, adult mitochondrial OCR was severely inhibited by ascofuranone, atovaquone, and nitazoxanide, while pyrvinium pamoate inhibited both mitochondrial and nonmitochondrial OCRs. These results demonstrate that the mitochondria of S. mansoni are a feasible target for drug development.

Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target.

Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc1 complex inhibitor.

Oral Administration of Surface-Deacetylated Chitin Nanofibers and Chitosan Inhibit 5-Fluorouracil-Induced Intestinal Mucositis in Mice.

This study investigated the prophylactic effects of orally administered surface-deacetylated chitin nanofibers (SDACNFs) and chitosan against 5-fluorouracil (5-FU)-induced intestinal mucositis, which is a common side effect of 5-FU chemotherapy. SDACNFs and chitosan abolished histological abnormalities associated with intestinal mucositis and suppressed hypoproliferation and apoptosis of intestinal crypt cells. These results indicate that SDACNF and chitosan are useful agents for preventing mucositis induced by anti-cancer drugs.

Biobased Wrinkled Surfaces Induced by Wood Mimetic Skins upon Drying: Effect of Mechanical Properties on Wrinkle Morphology.

We previously developed biobased wrinkled surfaces based on wood mimetic skins in which microscopic wrinkles were fabricated on a chitosan film by immersion in a phenolic acid solution, horseradish peroxidase-catalyzed surface reaction, and drying. Here, we prepared a diverse range of wrinkled films by immersion treatment at 30, 40, 50, and 60 °C in p-coumaric acid and then investigated the correlation between wrinkle morphology and mechanical properties. Wrinkle wavelengths gradually decreased as the immersion temperature increased as well as the previous report. In order to clarify the mechanisms responsible for the different wrinkle morphologies, the films were subjected to elastic moduli measurement and GPC analysis after immersion treatment. These experiments provided evidence that the chitosan around the film surface decomposed along with the immersion process. The decomposition was accelerated by higher immersion temperature, suggesting that higher temperatures led to the formation of softer skins, inducing smaller wrinkles. In fact, wrinkle morphologies with this system were predominately determined by the hardness of the wood mimetic skins. This phenomenon is consistent with the fundamentals of surface wrinkling in nature. This study is the first to demonstrate that artificial wrinkling triggered by water evaporation can be controlled by precise control of the surface hardness of soft material.

Structure of the trypanosome cyanide-insensitive alternative oxidase.

In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction.

Protein/CaCO3/Chitin Nanofiber Complex Prepared from Crab Shells by Simple Mechanical Treatment and Its Effect on Plant Growth.

A protein/CaCO3/chitin nanofiber complex was prepared from crab shells by a simple mechanical treatment with a high-pressure water-jet (HPWJ) system. The preparation process did not involve chemical treatments, such as removal of protein and calcium carbonate with sodium hydroxide and hydrochloric acid, respectively. Thus, it was economically and environmentally friendly. The nanofibers obtained had uniform width and dispersed homogeneously in water. Nanofibers were characterized in morphology, transparency, and viscosity. Results indicated that the shell was mostly disintegrated into nanofibers at above five cycles of the HPWJ system. The chemical structure of the nanofiber was maintained even after extensive mechanical treatments. Subsequently, the nanofiber complex was found to improve the growth of tomatoes in a hydroponics system, suggesting the mechanical treatments efficiently released minerals into the system. The homogeneous dispersion of the nanofiber complex enabled easier application as a fertilizer compared to the crab shell flakes.

Protective Effect of Chitin Urocanate Nanofibers against Ultraviolet Radiation.

Urocanic acid is a major ultraviolet (UV)-absorbing chromophore. Chitins are highly crystalline structures that are found predominantly in crustacean shells. Alpha-chitin consists of microfibers that contain nanofibrils embedded in a protein matrix. Acid hydrolysis is a common method used to prepare chitin nanofibrils (NFs). We typically obtain NFs by hydrolyzing chitin with acetic acid. However, in the present study, we used urocanic acid to prepare urocanic acid chitin NFs (UNFs) and examined its protective effect against UVB radiation. Hos: HR-1 mice coated with UNFs were UVB irradiated (302 nm, 150 mJ/cm²), and these mice showed markedly lower UVB radiation-induced cutaneous erythema than the control. Additionally, sunburn cells were rarely detected in the epidermis of UNFs-coated mice after UVB irradiation. Although the difference was not as significant as UNFs, the number of sunburn cells in mice treated with acetic acid chitin nanofibrils (ANFs) tended to be lower than in control mice. These results demonstrate that ANFs have a protective effect against UVB and suggest that the anti-inflammatory and antioxidant effects of NFs influence the protective effect of ANFs against UVB radiation. The combination of NFs with other substances that possess UV-protective effects, such as urocanic acid, may provide an enhanced protective effect against UVB radiation.

Characterization of Chitosan Nanofiber Sheets for Antifungal Application.

Chitosan produced by the deacetylation of chitin is a cationic polymer with antimicrobial properties. In this study, we demonstrate the improvement of chitosan properties by nanofibrillation. Nanofiber sheets were prepared from nanofibrillated chitosan under neutral conditions. The Young's modulus and tensile strength of the chitosan NF sheets were higher than those of the chitosan sheets prepared from dissolving chitosan in acetic acid. The chitosan NF sheets showed strong mycelial growth inhibition against dermatophytes Microsporum and Trichophyton. Moreover, the chitosan NF sheets exhibited resistance to degradation by the fungi, suggesting potentials long-lasting usage. In addition, surface-deacetylated chitin nanofiber (SDCNF) sheets were prepared. The SDCNF sheet had a high Young's modulus and tensile strength and showed antifungal activity to dermatophytes. These data indicate that nanofibrillation improved the properties of chitosan. Thus, chitosan NF and SDCNF sheets are useful candidates for antimicrobial materials.

Effects of Surface-Deacetylated Chitin Nanofibers in an Experimental Model of Hypercholesterolemia.

This study evaluated the effects of oral administration of surface-deacetylated chitin nanofibers (SDACNFs) on hypercholesterolemia using an experimental model. All rats were fed a high cholesterol diet with 1% w/w cholesterol and 0.5% w/w cholic acid for 28 days. Rats were divided equally into four groups: the control group was administered 0.05% acetic acid dissolved in tap water, and the SDACNF, chitosan (CS), and cellulose nanofiber (CLNF) groups were administered 0.1% CNF, CS, or CLNF dissolved in the tap water, respectively, during the experimental period. Changes in body weight, intake of food and water, and organ weight were measured. Serum blood chemistry and histopathological examination of the liver were performed. Administration of SDACNF did not affect body weight change, food and water intake, or organ weights. Administration of SDACNF and CS decreased the diet-induced increase in serum total cholesterol, chylomicron, very-low-density lipoprotein, and phospholipid levels on day 14. Moreover, oral administration of SDACNFs suppressed the increase of alanine transaminase levels on day 29 and suppressed vacuolar degeneration and accumulation of lipid droplets in liver tissue. These data indicate that SDACNF has potential as a functional food for patients with hypercholesterolemia.

Effects of Oral Administration of Chitin Nanofiber on Plasma Metabolites and Gut Microorganisms.

The aim of this study was to examine the effects of oral administration of chitin nanofibers (CNFs) and surface-deacetylated (SDA) CNFs on plasma metabolites using metabolome analysis. Furthermore, we determined the changes in gut microbiota and fecal organic acid concentrations following oral administrations of CNFs and SDACNFs. Healthy female mice (six-week-old) were fed a normal diet and administered tap water with 0.1% (v/v) CNFs or SDACNFs for 28 days. Oral administration of CNFs increased plasma levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and serotonin (5-hydroxytryptamine, 5-HT). Oral administration of SDACNFs affected the metabolisms of acyl-carnitines and fatty acids. The fecal organic level analysis indicated that oral administration of CNFs stimulated and activated the functions of microbiota. These results indicate that oral administration of CNFs increases plasma levels of ATP and 5-HT via activation of gut microbiota.

Effect of fucoidan extracted from mozuku on experimental cartilaginous tissue injury.

We investigated the effect of fucoidan, a sulfated polysaccharide, on acceleration of healing of experimental cartilage injury in a rabbit model. An injured cartilage model was surgically created by introduction of three holes, one in the articular cartilage of the medial trochlea and two in the trochlear sulcus of the distal femur. Rabbits in three experimental groups (F groups) were orally administered fucoidan of seven different molecular weights (8, 50, 146, 239, 330, 400, or 1000 kD) for 3 weeks by screening. Control (C group) rabbits were provided water ad libitum. After the experimental period, macroscopic examination showed that the degree of filling in the fucoidan group was higher than that in the C group. Histologically, the holes were filled by collagen fiber and fibroblasts in the C group, and by chondroblasts and fibroblasts in the F groups. Image analysis of Alcian blue- and safranin O-stained F-group specimens showed increased production of glycosaminoglycans (GAGs) and proteoglycans (PGs), respectively. Some injured holes were well repaired both macroscopically and microscopically and were filled with cartilage tissues; cartilage matrices such as PGs and GAGs were produced in groups F 50, F 146, and F 239. Thus, fucoidan administration enhanced morphologically healing of cartilage injury.

Effects of oral administration of fucoidan extracted from Cladosiphon okamuranus on tumor growth and survival time in a tumor-bearing mouse model.

We evaluated the anti-tumor activities of the oral administration of fucoidan extracted from Cladosiphon okamuranus using a tumor (colon 26)-bearing mouse model. The materials used included low-molecular-weight fucoidan (LMWF: 6.5-40 kDa), intermediate-molecular-weight fucoidan (IMWF: 110-138 kDa) and high-molecular-weight fucoidan (HMWF: 300-330 kDa). The IMWF group showed significantly suppressed tumor growth. The LMWF and HMWF groups showed significantly increased survival times compared with that observed in the control group (mice fed a fucoidan-free diet). The median survival times in the control, LMWF, IMWF and HMWF groups were 23, 46, 40 and 43 days, respectively. It was also found that oral administration of fucoidan increased the population of natural killer cells in the spleen. Furthermore, from the results of the experiment using Myd-88 knockout mice, it was found that these effects are related to gut immunity. These results suggest that fucoidan is a candidate anti-tumor functional food.

Hierarchical surface wrinkles and bumps generated on chitosan films having double-skin layers comprising topmost carrageenan layers and polyion complex layers.

Surface wrinkling to fabricate hierarchical surface topographies has attracted much attention because of the potential and multifunctional applications of hierarchical surface wrinkles beyond uniform wrinkles. Although many reports have described the preparation of hierarchical wrinkles induced by mechanical stress and heat, fabrication through drying-induced shrinkage has hardly been reported. Here we introduce hierarchical surface wrinkles and bumps generated on a chitosan film via the preparation of double-skin layers with κ- and ι-carrageenans, respectively, and subsequent drying. Double-skin layers are fabricated on a swollen chitosan film, called a chitosan hydrogel film, that is soaked first in κ- or ι-carrageenan solution and then in water to remove excess adsorbed κ- or ι-carrageenan. After the film is dried, hierarchical microscopic surface architectures are observed. In the case of the κ-carrageenan system, the wrinkles are hierarchical, consisting of wrinkles (6.2 ± 2.8 µm) that have smaller buckles (0.23 ± 0.09 µm). We reveal that the wrinkles or the smaller buckles are caused by plane inhomogeneous shrinkage between the κ-carrageenan layer and the chitosan film or by the aggregation of the κ-carrageenan layer upon drying, respectively. Interestingly, the ι-carrageenan system showed hierarchical bumps consisting of semispherical bumps (5.6 ± 2.1 µm) that have smaller bumps (0.78 ± 0.27 µm). We reveal that the larger bumps are generated during the immersion of the chitosan hydrogel film into ι-carrageenan solution. The smaller bumps are generated by the aggregation of the ι-carrageenan layer that occurs during drying; this process requires the plane compression strain caused by the shrinkage of the chitosan hydrogel film.

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei.

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K(m) of 338microM and V(max) of 601micromol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.

Unique Photophysical Properties of 1,8-Naphthalimide Derivatives: Generation of Semi-stable Radical Anion Species by Photo-Induced Electron Transfer from a Carboxy Group.

The development of anion sensors for selective detection of a specific anion is a crucial research topic. We previously reported a selective photo-induced colorimetric reaction of 1-methyl-3-(N-(1,8-naphthalimidyl)ethyl)imidazolium (MNEI) having a cationic receptor in the presence of molecules having multiple carboxy groups, such as succinate, citrate, and polyacrylate. However, the mechanism underlying this reaction was not clarified. Here, we investigate the photo-induced colorimetric reaction of N-[2-(trimethylammonium)ethyl]-1,8-naphthalimide (TENI), which has a different cationic receptor from MNEI and undergoes the photo-induced colorimetric reaction, and its analogues to clarify the reaction mechanism. The TENI analogues having substituents on the naphthalene ring provide important evidence, suggesting that the colorimetric chemical species were radical anions generated via photo-induced electron transfer from carboxylate to the naphthalimide derivative. The generation of the naphthalimide-based radical anion is verified by 1H NMR and cyclic voltammetry analyses, and photo-reduction of methylene blue is mediated by TENI. In addition, the role of the cationic receptor for the photo-induced colorimetric reaction is investigated with TENI analogues having different hydrophilic groups instead of the trimethylammonium group. Interestingly, the photo-induced colorimetric reaction is observed in a nonionic analogue having a polyethylene glycol group, indicating that the colorimetric reaction does not require a cationic receptor. On the other hand, we reveal that the trimethylammonium group stabilizes the radical anion species. These generation and stabilization phenomena of naphthalimide-based radical anion species will contribute to the development of sophisticated detection systems specific for carboxylate.

Acetylation of chitin nanofibers and their transparent nanocomposite films.

Chitin nanofibers were acetylated to modify the fiber surface and were characterized in detail. The acetyl DS could be controlled from 0.99 to 2.96 by changing the reaction time. FT-IR spectra indicate that chitin nanofibers were acetylated completely after 50 min reaction time. X-ray diffraction profiles and TGA curves show that the chitin nanofibers were acetylated heterogeneously from the surface to the core. SEM images show that fiber shape was maintained even in the high-DS sample and that the thickness of the nanofibers increased with the introduction of bulky acetyl groups. Acetylated chitin nanofiber composites were fabricated with acrylic resin with the fiber content of approximately 25 wt %. Due to the size effect, all nanocomposites had high transparency, despite the variety of acetyl DS, and the transparency of the chitin nanofiber composite was less sensitive to acetylation. By only 1 min acetylation, the moisture absorption of the nanocomposite drastically decreased from 4.0 to 2.2%. Although the coefficient of thermal expansion (CTE) of the tricyclodecane dimethanol dimethacrylate (TCDDMA) resin was 6.4 x 10(-5) degrees C(-1), the CTE of the chitin nanofiber/TCDDMA composite decreased to 2.3 x 10(-5) degrees C(-1) by the reinforcement effect of the chitin nanofibers with low thermal expansion.

Synthesis of silver nanoparticles templated by TEMPO-mediated oxidized bacterial cellulose nanofibers.

We have prepared silver nanoparticles on the surface of bacterial cellulose (BC) nanofibers. The synthesis of silver nanoparticles incorporates 2,2,6,6-tetramethylpiperidine-1-oxyradical (TEMPO)-mediated oxidation to introduce carboxylate groups on the surface of BC nanofibers. An ion exchange of the sodium to the silver salt was performed in AgNO(3) solution, followed by thermal reduction. By using oxidized BC nanofibers as a reaction template, we have prepared stable silver nanoparticles with a narrow size distribution and high density through strong ion interactions between host carboxylate groups and guest silver cations, which have been investigated by scanning electron microscopy, UV-visible spectroscopy, and a small-angle X-ray scattering method.

Preparation of chitin nanofibers with a uniform width as alpha-chitin from crab shells.

Chitin nanofibers were prepared from dried crab shells by a simple grinding treatment in a never-dried state under an acidic condition after the removal of proteins and minerals. The obtained nanofibers were observed by FE-SEM and found to have a uniform width of approximately 10-20 nm and high aspect ratio; both these findings were similar to those for nanofibers from prawns. Furthermore, it was confirmed that the nanofibers were extracted from the natural chitin/protein/mineral composites of crab shell in their original state. That is, the N-acetyl group was not removed and the alpha-chitin crystal structure was maintained, as confirmed by elemental analysis data, FT-IR spectra, and X-ray diffraction profiles.

Preparation of stable chitosan-carboxymethyl dextran nanoparticles.

Chitosan-carboxymethyl dextran nanoparticles (CHI-CMD NPs) were prepared by the formation of polyelectrolyte complex via the interaction of the positive charged amino group of chitosan and the negative charged carboxyl group of carboxymethyl dextran. Biocompatible organic reagents such as lactic acid, phosphate-buffered saline, and carbonate-bicarbonate buffer were used to make CHI-CMD NPs. The pH value of narrowly distributive CHI-CMD NP suspension was close to the physiological value of 6.8, which made it possible for it to be directly applied in an in vitro test without further additional operations. In the presence of sugars (30 mM), the colloidal stability of CHI-CMD NPs could be extended to 5 to 7 days when the particle size could be maintained within a nanometer scale, while the addition of xylose or lactose was found to most effectively extend the colloidal stability of CHI-CMD NPs to 10 days. Scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction analysis revealed that the structural integrity of CHI-CMD NPs was not destroyed after high steam pressure sterilization (121 degrees C, 30 min), which confirmed the autoclavable property of CHI-CMD NPs.

Guanidinylation of Chitooligosaccharides Involving Internal Cyclization of the Guanidino Group on the Reducing End and Effect of Guanidinylation on Protein Binding Ability.

In order to synthesize a promising material for developing a novel peptide/protein delivery system, guanidinylation of chitooligosaccharides with 1-amidinopyrazole hydrochloride was investigated herein. The production of guanidinylated chitooligosaccharides was demonstrated by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and elemental analyses. Interestingly, we found that the reducing end in the guanidinylated chitooligosaccharides was converted to a cyclic guanidine structure (2-[(aminoiminomethyl)amino]-2-deoxy-d-glucose structure). This reaction was carefully proven by the guanidinylation of d-glucosamine. Although this is not the first report on the synthesis of the 2-[(aminoiminomethyl)amino]-2-deoxy-d-glucose, it has provided a rational synthetic route using the high reactivity of the reducing end. Furthermore, we found that the interaction between chitooligosaccharides and bovine serum albumin is weak when in a neutral pH environment; however, it is significantly improved by guanidinylation. The guanidinylated chitooligosaccharides are useful not only for the development of a novel drug delivery system but also as a chitinase/chitosanase inhibitor and an antibacterial agent.