Low Dextran Research Articles

A200 CROHN’S-ASSOCIATED E. COLI WORSEN COLITIS SEVERITY IN MICE COLONIZED WITH DEFINED MICROBIOTA

Abstract Background Bacterial infections have been postulated to drive the dysregulated inflammation found in inflammatory bowel disease (IBD). In particular, adherent-invasive Escherichia coli (AIEC) isolated from patients with IBD have pathobiont characteristics and have been implicated in IBD pathogenesis. Aims Our aim was to characterize and compare the level of intestinal inflammation and potential microbiota shifts induced by E. coli clinical isolates using a gnotobiotic mouse model of colitis. Methods Adult germ-free C57BL/6 mice were transferred to ISO positive cages in a gnotobiotic facility and colonized with altered Schaedler flora-like (ASF) microbiota and one of three clinical E. coli isolates: E. coli C0004 (n = 5), E. coli LF82 (n = 9), E. coli NRG857c (n = 6), or ASF alone (n = 6). Three weeks later, mice were treated for 5 days with low dose dextran sodium sulphate in drinking water (2%; DSS), followed by 2 days of water. Mice were monitored daily for clinical symptoms (weight, stool consistency, and occult blood). At sacrifice, colon tissue was collected for histological analysis. Cecum contents were cultured to determine bacterial load. Fecal samples were collected for 16S rRNA gene sequencing analysis before and after DSS treatment. Results All mice colonized with an E. coli isolate displayed significantly greater clinical and microscopic scores of colitis compared to ASF alone, but the severity was dependent on the colonized E. coli strain. E. coli NRG857c-colonized mice exhibited more severe symptoms (p < 0.001) two days earlier than mice colonized with other E. coli isolates. Mice colonized with E. coli LF82 or E. coli NRG857c had higher histological scores of colitis compared to mice colonized with E. coli C0004, which were also significantly greater than ASF alone (p < 0.0001). 16S rRNA gene sequencing revealed that ASF-alone-colonized mice lacked Proteobacteria. All E. coli-colonized mice had comparable bacterial loads, which were verified by 16S rRNA analysis. Following DSS, E. coli LF82 and E. coli NRG857c relative abundance remained stable, whereas the relative abundance of Roseburia/ASF 492 declined significantly in all E. coli colonized mice. Conclusions The presence of E. coli pathobionts in mice drive the severity of chemically induced colitis, with AIEC NRG857c inducing the greatest severity. This gnotobiotic mouse model enables us to control the severity of colitis in a well-defined microbiota that is dependent on the colonized E. coli isolate. Using this model, we will be able to assess therapeutic candidates that aim to treat colitis at varying stages of its disease progression. Funding Agencies CCC, CIHRNSERC

Effect of dextran and enzymatically decomposed dextran on the sucrose crystal shape

The effect of dextran’s molecular mass distribution on the sucrose crystal shape was key to this study. Therefore, sucrose crystals were produced by evaporating crystallization experiments using synthetic thick juices in the form of pure sugar syrups containing high (T2000) and low (T40) molecular mass dextran fractions as well as enzymatically decomposed dextran. The combined analysis of molecular mass distributions by size exclusion chromatography and sucrose crystal shapes by static image analysis were used to identify the least harmful reaction products resulting from the enzymatic decomposition of dextran. The combined evaluation of two shape parameters, circularity and width-to-length ratio, has shown that three different shape modifications can be related to the presence of dextran, namely cube-shaped crystals, elongated needle-shaped crystals and agglomerates. In the main, the data indicated that high T2000 contents and generally all T40 dextran contents led to an increased occurrence of agglomerated and occasionally elongated crystals. The latter was especially found for high T2000 dextran contents. In contrast, low T2000 dextran contents predominantly increased the amount of cube-like crystals. The enzymatic decomposition of dextran resulted in a gradual reduction of the molecular mass. It was shown that an insufficient decomposition to broadly distributed low molecular mass dextran fragments, which are realistic to assume for technical cane and beet juices, still dramatically affected the sucrose crystal shape. Once dextran was decomposed to molecules with molecular masses of less than 5 kDa, no dextran-related effects on the sucrose crystal shape were found.

Directly crosslinked dextran gels for SWCNT separation

The gel column chromatography method allows high-throughput and high-purity single-wall carbon nanotube (SWCNT) separation. However, the role of the gel in this system has not been fully understood. In this study, five gels with different dextran concentrations were synthesized by directly crosslinking dextran with epichlorohydrin. The low dextran concentration gels work for metal/semiconductor separation and diameter sorting of semiconducting SWCNTs. This is evidence that the chemical structure in the gel that is responsible for the selective adsorption of semiconducting SWCNTs is dextran. However, high dextran concentration gels did not adsorb SWCNTs, although the adsorption site was dextran. Micro-Raman measurements on gel slices revealed that the high dextran concentration gel has a pore size too small to allow deep diffusion of SWCNTs. Not only the chemical structure but also the physical structure is important for designing high-performance gels for SWCNT separation in the near future.

Microbial Diversity Profiling of Polysaccharide (gum)-Producing Bacteria Isolated from a South African Sugarcane Processing Factory.

Polysaccharide (gum)-producing bacteria are responsible for severe economic losses in the sugarcane processing industry. Increased polysaccharide levels in raw sugar are normally an indication that biodeterioration occurred in the cane, soon after harvesting. Once in the sugar processing plant, the cell numbers of gum-producing bacteria escalate and may reach levels difficult to control. We have isolated 430 gum-producing bacteria from sugarcane and different sampling points in a South African sugarcane processing factory. As expected, high cell numbers of gum-producing bacteria were isolated from the factory during a time when sugar with a high dextran content was produced. What we did not expect was to find the same species in the factory at a time when sugar with a low dextran content was produced. Phylogenetic analyses of the 16S rRNA gene sequences differentiated the gum-producing bacteria into four genera and nine species. The majority of these isolates belonged to the genus Weissella (47%), followed by members of Bacillus (24%), Leuconostoc (19%) and Lactobacillus (10%). For the first time, we report on the isolation of Weissella confusa, Weissella cibaria and Bacillus amyloliquefaciens from a sugarcane processing factory.

Reduced early life mucosal integrity decreases thymic cell counts and increases local, but not thymic regulatory, T cell recruitment: Gut mucosal integrity breach and thymic T cells

Early life immune gut microbiota contact is critical for regulatory T cell–mediated oral tolerance induction. We induced a mucosal integrity breach with low dextran sulfate sodium dose right after weaning in BALB/c mice along with a standard high dose to study the impact of increased gut microbiota lymphatic tissue contact on the thymus. Both doses increased gut permeability, which caused a short-term generalized thymic involution and regulatory T cell induction in the mesenteric lymph nodes, even in the absence of clinically apparent inflammation in the low-dose group. The thymic regulatory T cells resisted thymic involution. In the low-dose group, we found acutely altered gut mobilization patterns characterized by changed gut-homing marker CD103 expression on mesenteric lymph node CD4+T cells as well as on mature CD8+T cells and developing CD4−/CD8−thymocytes. Furthermore, CD218a (IL-18-receptor-a) expression was acutely decreased on both mature CD8+T cells and regulatory T cells, while increased on the mesenteric lymph node CD8+T cells, indicating a direct link between the thymus and the mesenteric lymph nodes with CD218a in a functional role in thymic involution. Acute and non-persisting regulatory responses in the mesenteric lymph nodes were induced in the form of a relative regulatory T cell increase. We saw no changes in total thymic regulatory T cells and thus the thymus does not seem to play a major role of in the regulatory immunity induced by increased gut microbiota lymphatic tissue contact around weaning, which in our study primarily was located to the gut.

The characterization of the human nasal epithelial cell line RPMI 2650 under different culture conditions and their optimization for an appropriate in vitro nasal model.

The further characterization of the cell line RPMI 2650 and the evaluation of different culture conditions for an in vitro model for nasal mucosa. Cells were cultured in media MEM or A-MEM at air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 weeks. Different cryopreservation methods and cell culture techniques were evaluated with immunolabelling of junctional proteins, ultrastructural analysis using electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran and jacalin, and gene expression profiling of 84 drug transporters. Cell proliferation and differentiation depended on the used medium. The established epithelia expressed occludin, claudin-1, and E-cadherin under all conditions. Cells grown at the A-L interface formed more layers and exhibited a higher TEER and lower dextran and jacalin permeability than at the L-L interface, where cells morphologically exhibited a more differentiated phenotype. The expression of ABC and SLC transporters depended on culture duration and interface. The RPMI 2650 cells form a polarized epithelium resembling nasal mucosa. However, different culture conditions have a significant effect on cell ultrastructure, barrier integrity, and gene expression, and should be considered when using this cell line as an in vitro model for drug permeability studies and screening of nasal drug candidates.

Production and spouted bed drying of acerola juice containing oligosaccharides

In the present study, acerola (Malpighiae marginata) juice was used as substrate for oligosaccharides synthesis by the dextransucrase acceptor reaction. Due to the low sugar content of acerola, external sugars (sucrose, glucose and fructose) were added to the juice and the oligosaccharides synthesis was carried out using a central composite rotated design. High oligosaccharide and low dextran yields were obtained indicating that the acceptor reaction had occurred. The sugar conversion into oligosaccharides was also high (>60%) and oligosaccharides with a degree of polymerization up to 12 were obtained. The prebiotic juice was dried in a spouted bed using maltodextrin as carrier. The powder dried at 60°C showed low hygroscopicity (7.90%), low moisture (1.91%) and low water activity (0.18).

Reversible Self-Assembling of Comb-Like Functionalized Dextran Surfactant Polymers as an SPR-Sensing Surfaces

Novel surface plasmon resonance (SPR) sensing surfaces were developed from the perspective of reversible self-assembling of the mixed dextran surfactant polymers onto SPR gold chips. Two comb-like dextran surfactant polymers, which are different in their dextran molecular weight (MW) distribution and the presence of amine functional groups, and their characterization were synthesized. These structurally well-defined surfactant polymers include a bimodal amine dextran surfactant polymer and a monomodal dextran surfactant polymer. The bimodal one consists of poly (vinyl amine) (PVAm) backbone with amine functionalized high MW dextran, non-functionalized low MW dextran and hydrophobic hexyl pendant groups. Allyl high MW dextran aldonic acid and low MW dextran aldonic acid chains with various ratios were sequentially attached to PVAm. The unreacted amine groups on the backbone were further grafted with hexanoic acid and the allyl groups on the high MW dextran were finally converted to amine. The monomodal surfactant polymer, PVAm with non-functionalized low MW dextran and hexyl branches, was synthesized and characterized using similar procedures, except with the absence of allyl high MW dextran aldonic acid and the amine functionalization. The molecular compositions of the surfactant polymers were determined by FTIR and 1H-NMR. Surface active properties at the air-water interface were determined using tensiometer. Reduction in water contact angles was observed on the surfactant treated surfaces. Reversible self-assembling of the synthesized surfactant polymers on SPR gold chip surface was monitored using SPR analysis. Reproducibility of the mixed surfactant assembling process was preliminarily confirmed by a consistent increase in reflectivity change, while no detectable changes in the reflectivity under a 1-h buffer flow indicated stability of the absorbed surfactant layer. Moreover, a simple, quick flow of a basis regeneration solution completely removed the adsorbed surfactant from the surface. Our preliminary investigation also exhibited that the regenerated SPR gold chip was effectively reusable.

Experimental evaluation of mechanical and electrical properties of RBC suspensions in Dextran and PEG under flow II. Role of RBC deformability and morphology

Mechanical and electrical properties of the normal RBCs suspensions and of hardened after treatment with glutaraldehyde (0.01-2.5%) RBCs in isotonic physiological solution and Dextran 70,000 (Dextran 70) and Polyethylene glycol 35,000 (PEG) and adjusted to hematocrit of 40%, were evaluated. Apparent viscosity and conductivity were measured under steady and transient flow regimes at low shear rates and at different local structure of the flow at 37 °C. A time course of conductivity was recorded in parallel with the rheological properties of the RBC suspensions and conductivity and apparent viscosity dependences on shear rates were studied and compared at different concentrations of Dextran 70, PEG and glutaraldehyde. Low shear viscosity decreased after RBCs treatment with glutaraldehyde and at 0.5-2.5% it is constant. Echinocytes are observed at low Dextran 70 and PEG concentrations while spherocytes are found mainly in smears treated with higher concentrations. The results show that the apparent viscosity and conductivity of RBCs suspensions in Dextran 70 and PEG are strongly influenced by flow, shear rates, concentration, cell deformability and morphology and the method is sensitive to study the mechanical and electrical properties of RBC suspension and to provide experimental description of RBCs and other cell-to-cell interactions.

Block Copolymer Photo-Grafted Poly(Ethylene Terephthalate) Capillary Pore Membranes Distinctly Switchable by Two Different Stimuli

Track-etched poly(ethylene terephthalate) capillary pore membranes with a pore diameter of 690 nm were functionalized via photo-initiated “living” radical graft polymerization with block copolymers of acrylic acid and N-isopropylacrylamide. Preadsorbed xanthone was more efficient than benzophenone in order to achieve higher grafting efficiency and “living” character, including the option to reinitiate a grafted homopolymer to obtain grafted diblock copolymers. Characterizations were mainly done with water flux and dextran diffusion experiments at temperatures below and above the lower critical solution temperature (32°C) of poly(N-isopropylacrylamide) and pH values below and above the pKa value (4.5) of poly(acrylic acid). Block sequences and relative block lengths were identified to obtain stimuli-responsive membranes which have no measurable water flux and allow only low dextran diffusion rates at 25°C and pH 7 (“closed state”), and which reversibly open their pores by either increase of temperature or decrease of pH, or by the combination of both stimuli.

The Design by Molecular Dynamics Modeling and Simulations of Porous Polymer Adsorbent Media Immobilized on the Throughpore Surfaces of Polymeric Monoliths

Ion-exchange porous adsorbent media having intermediate and low surface densities of dextran polymer grafted on the surface of the throughpores of polymeric monoliths are constructed and characterized by a molecular dynamics modeling and simulation approach that has also been shown to be effective in the construction and characterization of porous ion-exchange adsorbent media whose number of immobilized dextran polymer chains per unit surface area is high. The activation step that prepares the surface of the pores of the dextran polymer layer for the immobilization of the charged ligands insignificantly affected the pore structure of the dextran polymer layer, while this was found to not be the case for previously studied systems that involved high dextran polymer surface densities. Compared to the high dextran polymer density system studied previously, the intermediate dextran polymer density system can generate significantly larger pores but still possesses relatively high interconnection and mutual steric support between dextran chains to exhibit similar structural characteristics and responses to charged ligand immobilization, including dextran layer thickness, stability, monomer distribution, ligand-induced compact chain structures, dextran layer shrinkage, distributions of ligands and counterions, and local nonelectroneutrality. The low dextran polymer density system having relatively isolated dextran chains and insufficient mutual steric support can result in even larger pores than those obtained in the intermediate dextran polymer density system, but a significantly thinner porous dextran polymer layer and different dextran monomer distributions are obtained in the low dextran polymer density system. More importantly, the gradient of the local nonelectroneutrality after the immobilization of the charged ligands is significantly smaller in magnitude in the low dextran polymer density system than that obtained in the system having intermediate dextran polymer density, and, despite a lack of porous layer depth to accommodate adsorbate biomolecules in large amounts, it could still be useful for the effective transport and adsorption of very large biomolecules. Compared with the polymeric monoliths without a porous dextran polymer layer grafted on the surface of their throughpores, the intermediate and low dextran polymer density systems explored and studied in this work provide pore structures with desirable characteristics for the effective transport of adsorbate biomolecules and substantially larger effective surface areas and throughput capacities for the adsorption of the adsorbate biomolecules.

Effects of different means of liquid recucitation on mirrocircultation in rats with hemorrhagic shock

Objective To investigate the effects of different means of liquid resuscitation on microcirculation in rats with hemorrhagic shock. Method A model of rats with hemorrhagic shock was established in 40 Wistar rats. The rats were randomly divided into 4 groups according on different means of liquid resuscitation, and killed 24 hours after the examination, group A: without liquid resuscitation; group B: low-molecular-weight-40; group C: 7.5% hypertonic saline,group D:low-molecular-weight-40 + hyperonic saline. As well as the microcirculation in kidney was observed using electron microscope. Results In group without resuscitation, the microcirculation in kidney damaged severely; While in group with hypertonic or low molecule dextran alone, there was harm with dif-ferent degrees, and the microcirculation in kidney was nearly perfect in group with both hypertonic and low molecule dextran alone. Conclusions Different means of liquid resuscitation may infect the microcirculation in kidney, which with both hypertonic and low molecule dextran can protect the microcirculation in kidney. Key words: Shock; Hemorrhagic; Liquid resuscitation; Renal cortex; Microcirculation kidney

CE analysis of the acidic organelles of a single cell

The properties of organelles within a cell have been shown to be highly heterogeneous. Until now, it has been unclear just how much of this heterogeneity is endemic to the organelle subpopulations themselves and how much is actually due to stochastic cellular noise. An attractive approach for investigating the origins of heterogeneity among the organelles of a single cell is CE with LIF detection (CE-LIF). As a proof of principle, in this report we optimize and use a single cell CE-LIF method to investigate the properties of endocytic (acidic) organelles. Our results show that the properties of individual acidic organelles containing Alexa Fluor 488 Dextran suggest that there are two groups of CCRF-CEM cells: a group with a high dextran content per cell, and a group with a low dextran content per cell. Furthermore, the individual organelle measurements of the single cells allow us to compare in each group the distributions of doxorubicin content per acidic organelle and electrophoretic mobilities of these organelles.

Tuning of hollow fiber membrane properties using different bore fluids

Polymer solutions of a modified poly(ether ether ketone) and poly(vinyl pyrrolidone) in dimethylformamide were spun to prepare hollow fiber membranes by dry–wet spinning method. The influence of R OH bore fluids (BF), with R H; CH 3; C 2H 5; n-C 3H 7 or n-C 4H 9, on morphology and transport and mechanical properties was studied by electron scanning microscopy (SEM) and by measurements of water permeability (WP), dextrane rejection, tensile strength and elastic modulus. The results were discussed on the basis of ternary phase diagrams and Hildebrand solubility parameters, δ. The use of water as the bore fluid resulted in typical UF membranes with the lowest water permeability (158 l h −1 m −2 bar −1) and the highest dextrane rejection (99%). On the other hand, n-alkyl alcohols as the bore fluid produced MF membranes with a water permeability, increasing from about 600 to 1200 l h −1 m −2 bar −1 with increasing alkyl chain length, and with a low dextrane rejection.

Enzymatic Synthesis of Prebiotic Oligosaccharides

Prebiotic oligosaccharides are nondigestible carbohydrates that can be obtained by enzymatic synthesis. Glucosyltransferases can be used to produce these carbohydrates through an acceptor reaction synthesis. When maltose is the acceptor a trisaccharide composed of one maltose unit and one glucose unit linked by an alpha-1,6-glycosidic bond (panose) is obtained as the primer product of the dextransucrase acceptor reaction. In this work, panose enzymatic synthesis was evaluated by a central composite experimental design in which maltose and sucrose concentration were varied in a wide range of maltose/sucrose ratios in a batch reactor system. A partially purified enzyme was used in order to reduce the process costs, because enzyme purification is one of the most expensive steps in enzymatic synthesis. Even using high maltose/sucrose ratios, dextran and higher-oligosaccharide formation were not avoided. The results showed that intermediate concentrations of sucrose and high maltose concentration resulted in high panose productivity with low dextran and higher-oligosaccharide productivity.

Design of immobilised dextransucrase for fluidised bed application

Immobilisation of dextransucrase from Leuconostoc mesenteroides NRRL B-512F in alginate is optimised for applications in a fluidised bed reactor with high concentrated sugar solutions, in order to allow a continuous formation of defined oligosaccharides as prebiotic isomalto-oligosaccharides. Efficient design of fluidised bed immobilised biocatalyst in high density solutions requires particles with elevated density, high effectiveness and both thermal and mechanical stability. Inert silica flour/sand (Mikrosil 300) as supplement turned out to be best suited for increasing the density up to 1400 kg m −3 of the alginate beads and generating a stable expanded bed without diffusional restrictions. Kinetic investigations demonstrate that low effectiveness of immobilised enzyme due to close association to dextranpolymers (dextran content of enzyme preparation >90%) is compensated by reducing the particle size and/or by decreasing the dextran content. A low dextran content (5%) is sufficient to immobilise and stabilise the enzyme, thus diffusional limitation is reduced essentially while operational stability is maintained. Fluidisation behaviour and bed expansion proved to be appropriate for the intended application. Both calculated and measured expansion coefficients showed good agreement for different conditions.

Glomerular Processing of Dextran Sulfate during Transcapillary Transport

The fractional clearance of dextran sulfate across the glomerular capillary wall in the isolated perfused kidney is shown to be dependent on the concentration of dextran sulfate in the perfusate and the degree of sulfate substitution on dextran sulfate. While normal charge selectivity is apparent at low dextran sulfate perfusate concentrations, the nature of the concentration dependence of the fractional clearance clearly eliminates charge-electrostatic interactions as being responsible. A strong correlation has been experimentally established between fractional clearance and the degree of desulfation of dextran sulfate found in the urine. The correlation was further established through the demonstration that the lysosomotropic agent NH4Cl partially inhibits charge selectivity as well as desulfation. The glomerular cellular processing of the dextran sulfate was further studied through dextran sulfate-mediated release of metabolically labeled glomerular heparan sulfate. A model of glomerular capillary wall transport, invoking endothelial cell processing of dextran sulfate, is proposed to explain the differences between the fractional clearance of dextran and dextran sulfate.

Determination of protein foam stability from time-dependent pressure monitoring

An apparatus is described for the determination of protein foam stability from time-dependent measurements of the small pressure changes above the collapsing foam in a closed chamber. Experimental data are reported for foams made with a fixed concentration (0.1% wt) of bovine serum albumin (BSA) and various concentrations of the nonionic polysaccharide dextran at neutral pH. The results show a loss of foam stability at low dextran contents, an increase in stability at high contents, and a minimum level of stability at a dextran:bovine serum albumin (BSA) molar ratio of ~2. The trends in the data obtained with the new pressure monitoring apparatus are consistent with those obtained by visual observations of the rate of foam volume decay.

Topo-optical investigations of human erythrocyte glycocalyx conformational changes induced by dextran

Cell surface properties are involved in the aggregation process of red blood cells. Using the topo-optical toluidine blue reaction, conformational changes of the glycocalyx (main component glycophorin A) were found when red blood cells were incubated and fixed in the presence of dextran. Relative differences in optical path as a measure of red blood cell membrane anisotropy decreased in relation to dextran concentration during fixation. These conformational changes could not be detected by electrophoretic measurements. When incubating, fixing and staining red blood cells in the presence of dextran, anisotropy decreased only at low dextran concentrations and increased at rising dextran concentrations. This biphasic course of differences in optical path seems to be due to different effects of dextran superimposing upon each other: a disturbing influence on the spatial order of sialic acid carrying oligosaccharide side chains due to H-bond interaction, and an increase in the size of dye aggregates and suppression of the thermal motion of macromolecules at higher dextran concentrations.

The incompatibility of concentrated aqueous solutions of dextran and amylose and its effect on amylose gelation

At 75°C concentrated aqueous solutions of dextran and amylose exhibit immiscibility. The presence of dextran has a major effect on the microstructure and mechanical behaviour of amylose gels formed on cooling. At low dextran concentrations the modulus of the gel increases with increasing dextran concentration, whereas at higher dextran concentrations, polymer incompatibility and segregation of dextran-rich droplets reduces the firmness of the gel.