Glycoconjugate nanoribbons from the self-assembly of carbohydrate-peptide block molecules for controllable bacterial cell cluster formation

We demonstrate here the rational design strategy to control the length of 1-dimensional beta-sheet peptide nanoassembly. We synthesized the beta-sheet peptides with attached coils and carbohydrates. We reasoned that the bulkiness of the coils affects the final length of the assembled beta-sheet peptide nanostructures because of the steric crowding effect. The nanostructure from the peptide with a small and linear coil was several micrometers long, whereas the one from the peptide with a high-volume-fraction dendritic coil was only about 150 nm long. For potential biological applications of the peptide nanoassemblies, we investigated the interactions of the carbohydrate-coated nanoassemblies with E. coli cells containing cognate binding proteins. The results showed that both of the nanoassemblies could immobilize and/or aggregate bacterial cells. The degrees of immobilization were similar for both nanoassemblies; however, only the long nanoribbon was shown to induce the formation of bacterial clusters.     

Mechanisms of B cell tolerance. I. Tolerance to dextran B1355 induced with the oxidized dextran.

The bacterial dextran B1355, which is normally a potent thymus-independent immunogen, was made tolerogenic by oxidation. The injection of the oxidized dextran into BALB/c mice before, at the same time, or up to 4 days after the injection of the immunogenic form of the dextran resulted in a marked immunologically specific suppression of the number of anti-dextran antibody-forming cells found in the spleen. This suppression resulted from a direct inactivation of antibody-forming cell precursors rather than from either inhibition of antibody secretion or the exhaustive utilization of precursor B cells that have been observed in other tolerance systems. A substantial degree of tolerance was achieved after only a 1-hr in vivo exposure of the spleen cells to the tolerogen. At a dose of 1 mg of oxidized dextran per mouse, tolerance persised for at least 3 weeks. A complete recovery was apparent by 10 weeks. The stability of the tolerance was demonstrated by transferring tolerant spleen cells to irradiated recipients. The response in the recipient animals to an immunogenic dextran challenge remained suppressed. It appears that the tolerogenicity of the oxidized dextran is due to its ability to couple covalently with free amino groups in or near the receptor site of the cell membrane via the reactive dialdehyde groups of the dextran.     

Extended shelf life of enzyme-based biosensors using a novel stabilization system

The storage stability of amperometric enzyme electrodes has been enhanced by a combination of a soluble, positively charged polymer, diethylaminoethyl (DEAE)-dextran, and a sugar alcohol, lactitol. Two different types of alcohol biosensor have been produced using the enzyme alcohol oxidase, isolated from the methylotrophic yeast Hansenula polymorpha. The first employs enzyme entrapment between two membranes with direct hydrogen peroxide amperometry at +0·65 V. The second was based on the mediated, coupled reaction with horseradish peroxidase and N-methyl phenazimiumtetracyanoquinonedimethane (NMP-TCNQ) on a graphite electrode. In both cases, addition of the stabilizers promoted a considerable increase in the storage stability of the enzyme component, as indicated by an increase in the shelf life of desiccated biosensors under conditions of thermal stress at 37°C. In addition, an L-glutamate biosensor constructed from NMP-TCNQ-modified graphite electrodes and L-glutamate oxidase also exhibited an increase in shelf life when stored, desiccated in the presence of stabilizers.     

Calculation of ionic flocculation concentrations for biological membranes bearing ionizable groups

The flocculation concentrations in the DLVO theory of colloid stability have previously been calculated under conditions of constant plate potential or constant plate charge. These boundary conditions are not appropriate in the case of biological membranes bearing ionizable surface groups. In this case the surfaces undergo charge regulation as they approach each other and both the surface charge and potential vary. In this paper a numerical method is used to calculate the ionic flocculation concentrations for the two membranes undergoing surface charge regulation. It is shown that the flocculation concentration lies between that for constant charge and that for constant potential. Flocculation concentrations are calculated as a function of the surface density of ionizable groups and are shown to be dependent on both the pKa of the surface groups and the bulk pH of the solution.     

Defeating pathogen drug resistance: guidance from evolutionary theory

Many of the greatest challenges in medicine and public health involve the evolution of drug resistance by pathogens. Recent advances in the theory of natural selection suggest that there are two broad classes of pathogen traits that can be targeted by drugs or vaccines. The first class, consisting of traits that benefit the individual organisms bearing them, causes a strong evolutionary response and the rapid emergence of drug resistance. The second class, consisting of traits that benefit groups of pathogen organisms including the individual provider, causes a weaker evolutionary response and less drug resistance. Although most previous drug development has targeted the first class, it would be advantageous to focus on the second class as targets for drug and vaccine development. Specific examples and test cases are discussed.     

Growling for food: acoustic emissions during competitive feeding of the streaked gurnard

The streaked gurnard Trigloporus lastoviza produced only one sound type, a growl, lasting up to 3 s and consisting of repeated groups of typically one to three pulses. The foraging fish followed two different strategies. In the first, the fish circled the feeding area, grasped a food item and fled, sometimes displaying aggressively to competitors. With this foraging strategy, fish usually made sounds as they circled, grasped and fled. Fish that growled while circling were more likely to grasp a food item subsequently than were silent fish. The second feeding strategy occurred when a fish had already ingested food or failed to get any. In this case, typically fish searched for food on the substratum or approached and touched other individuals that were feeding, sometimes grabbing food that was spat out during food handling by the other fish. Although payback experiments would be needed to draw firm conclusions on the communicative function of growling during competitive feeding in the streaked gurnard, the results suggest that sound production confers advantages to individuals competing for limited food resources.     

Reactor optimization for alpha-1,2 glucooligosaccharide synthesis by immobilized dextransucrase

The immobilization of dextransucrase in Ca-alginate beads relies on the close association between dextran polymer and dextransucrase. However, high amounts of dextran in the enzyme preparation drastically limit the specific activity of the immobilized enzyme (4 U/mL of alginate beads). Moreover, even in the absence of diffusion limitation at the batch conditions used, the enzyme behavior is modified by entrapment so that the dextran yield increases and the alpha-1,2 glucooligosaccharides (GOS) are produced with a lower yield (46.6% instead of 56.7%) and have a lower mean degree of polymerization than with the free dextransucrase. When the immobilized catalyst is used in a continuous reaction, the reactor flow rate necessary to obtain high conversion of the substrates is very low, leading to external diffusion resistance. As a result, dextran synthesis is even higher than in the batch reaction, and its accumulation within the alginate beads limits the operational stability of the catalyst and decreases glucooligosaccharide yield and productivity. This effect can be limited by using reactor columns with length to diameter ratio > or =20, and by optimizing the substrate concentrations in the feed solution: the best productivity obtained was 3.74 g. U(-1). h(-1), with an alpha-1,2 GOS yield of 36%.     

Effect of dextran and dextran modifications on the thermal and proteolytic stability of conjugated bovine testis beta-galactosidase and human serum albumin

In order to study carbohydrate-induced protein stabilization bovine testis beta-galactosidase and human serum albumin were conjugated with dextran, partially acetylated dextran and partially methylated dextran. The conjugates and the free proteins were compared with respect to thermal stability at 50 degrees C and resistance to proteolytic digestion by subtilopeptidase A. Both beta-galactosidase and serum albumin were stabilized by conjugation with polysaccharide. However, higher stability was achieved by conjugating the proteins with the hydrophilic polysaccharides, dextran and acetylated dextran, than by conjugation with the hydrophobic polysaccharide, methylated dextran. The results are discussed in relation to possible explanations of carbohydrate-induced protein stabilization.     

Prevention of interfacial inactivation of enzymes by coating the enzyme surface with dextran-aldehyde

Interactions between soluble enzymes and interfaces of organic solvent drops or gas bubbles have a very negative effect on the operational stability of the soluble enzymes. In this study, the formation of a hydrophilic shell around the enzyme has been attempted using dextran-aldehyde which would prevent the interaction between enzyme and hydrophobic interfaces with minimal modification of the enzyme surface. After optimizing the size of the dextran (that was found to play a critical role), three different enzymes (glucose oxidase, d-amino acid oxidase, and trypsin) have been conjugated with dextran-aldehyde and their stability towards organic-aqueous and air-liquid interfaces has been evaluated. The treatment itself proved to be very low-cost in terms of activity and was highly stabilizing for the three enzymes assayed. The conjugated preparation of the three assayed enzymes remained fully active in the presence of air-liquid interfaces for at least 10h. However, the unmodified enzymes lost more than 50% of activity within the first hour of the experiments except for trypsin which kept 38% activity after 12h while the trypsin dextran-aldehyde conjugate maintained 100% enzyme activity. Similar results were achieved in the presence of stirred organic solvent-aqueous buffer biphasic system, although in this case some activity was lost by the action of the soluble portion of the organic solvent. In fact, this treatment seems to be also effective to improve the resistance to the action of organic solvent.     

Depletion flocculation and depletion stabilization of erythrocytes.

At dextran (Mw approximately 500,000) concentrations from 2 to approximately 10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations greater than 10%, the erythrocytes resegregate in a stable monodisperse suspension. At all these dextran concentrations, the erythrocytes are coated with considerable amounts of dextran. It can be argued that at dextran concentrations from 2 to 10%, as well as at dextran concentrations greater than 10%, there is a thin layer, which is depleted of dextran, between the dextran layer adsorbed onto the erythrocytes and the bulk dextran solution. It can also be shown that there is a repulsive interaction between the two layers of dextran: one adsorbed and one free. When the adsorbed dextran layer is the most concentrated, stability must ensue, and when the dextran in free solution is the most concentrated, flocculation should occur. Below 7% dextran, the concentration of free dextran is higher than the adsorbed concentration; above 10% dextran that situation is reversed. These data correlate well with the depletion flocculation predicted for the lower concentration and the depletion stabilization predicted for the higher dextran concentration.     

High stabilization of immobilized Rhizomucor miehei lipase by additional coating with hydrophilic crosslinked polymers: Poly-allylamine/Aldehyde–dextran

Immobilized enzymes have a very large surface region which is not in contact with the support surface and, thus, have potential as a target for novel stabilization strategies. In this paper, coating the surfaces of such enzymes with a highly hydrophilic and compact cross-linked poly-aminated polymer as a strategy to increase the thermal stability of the immobilized enzymes is proposed. In particular, Rhizomucor miehei lipase (RML) was immobilized by interfacial adsorption onto octyl-agarose and further coated with poly-allylamine (PAA), a polymer that is very rich in primary amino groups. Cross-linking of the PAA layer to coat the immobilized enzyme was carried out, in situ, by reaction with freshly oxidized dextran (aldehyde–dextran). The PAA layer only exerted moderate stabilizing effects (around 4-fold), but further cross-linking with aldehyde–dextran highly increased the stabilizing effects; the new derivative was 440-fold more stable than uncoated derivative at 55 °C and pH 7 and exhibited 6-fold more catalytic activity compared to the soluble enzyme used for immobilization. We hypothesize that the hydrophilicity of PAA reduces the exposure of internal hydrophobic pockets to the enzyme surface at high temperatures. Besides, the compactness of the polymer may reduce distortion of the enzyme surface during inactivation.     

Stroma-free hemoglobin: its presence in plasma does not improve oxygen supply to the resting hindlimb vascular bed of hemodiluted dogs.

In hemodilution, red cell spacing in the microcirculation is increased, flow distribution may become more heterogeneous, and, as a result, oxygen supply to tissues may suffer. We tested the hypothesis that oxygen extraction from diluted blood may be enhanced by the presence of hemoglobin in the plasma phase in relatively low concentrations. In anesthetized dogs, the hindlimb vascular bed was isolated and perfused with the animal's own blood by a roller pump. One group of dogs (n = 6) was hemodiluted (hematocrit = 15.0 +/- 1.0%) with a 6% solution of dextran. A second group of dogs (n = 6) was similarly hemodiluted (hematocrit = 16.0 +/- 0.4%) with dextran containing stroma-free hemoglobin solution whereby plasma-phase hemoglobin concentration was raised to 1.1 +/- 0.1 g.dL-1. Systemic hemodynamic observations were made repeatedly over the subsequent 2.5 h, while blood flow to the hindlimb was progressively reduced in stepwise decrements. The hemoglobin-hemodiluted group showed increased systemic arterial blood pressure and total peripheral resistance when compared with the control (dextran diluted) group. The isolated hindlimb also showed evidence of increased vascular resistance in the hemoglobin-treated group. In each individual animal, critical oxygen delivery and extraction were determined by finding the intercept of the supply-independent and supply-dependent portions of the oxygen uptake/oxygen delivery relationship. Neither the critical oxygen delivery rates (5.75 +/- 0.83 vs. 6.41 +/- 0.53 mL.kg-1.min-1) nor critical oxygen extraction ratios (0.75 +/- 0.03 vs. 0.76 +/- 0.04) were found to be significantly different in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cost-Effectiveness of HIV Drug Resistance Testing to Inform Switching to Second Line Antiretroviral Therapy in Low Income Settings

Background

To guide future need for cheap resistance tests for use in low income settings, we assessed cost-effectiveness of drug resistance testing as part of monitoring of people on first line ART - with switching from first to second line ART being conditional on NNRTI drug resistance mutations being identified.

Methods

An individual level simulation model of HIV transmission, progression and the effect of ART which accounts for adherence and resistance development was used to compare outcomes of various potential monitoring strategies in a typical low income setting in sub-Saharan Africa. Underlying monitoring strategies considered were based on clinical disease, CD4 count or viral load. Within each we considered a strategy in which no further measures are performed, one with a viral load measure to confirm failure, and one with both a viral load measure and a resistance test. Predicted outcomes were assessed over 2015–2025 in terms of viral suppression, first line failure, switching to second line regimen, death, HIV incidence, disability-adjusted-life-years averted and costs. Potential future low costs of resistance tests ($30) were used.

Results

The most effective strategy, in terms of DALYs averted, was one using viral load monitoring without confirmation. The incremental cost-effectiveness ratio for this strategy was $2113 (the same as that for viral load monitoring with confirmation). ART monitoring strategies which involved resistance testing did not emerge as being more effective or cost effective than strategies not using it. The slightly reduced ART costs resulting from use of resistance testing, due to less use of second line regimens, was of similar magnitude to the costs of resistance tests.

Conclusion

Use of resistance testing at the time of first line failure as part of the decision whether to switch to second line therapy was not cost-effective, even though the test was assumed to be very inexpensive.     

Depletion flocculation and depletion stabilization of erythrocytes

At dextran (Mw ≈ 500,000) concentrations from 2 to ≈10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations > 10%, the erythrocytes resegregate in a stable monodisperse suspension. At all these dextran concentrations, the erythrocytes are coated with considerable amounts of dextran. It can be argued that at dextran concentrations from 2 to 10%, as well as at dextran concentrations > 10%, there is a thin layer, which is depleted of dextran, between the dextran layer adsorbed onto the erythrocytes and the bulk dextran solution. It can also be shown that there is a repulsive interaction between the two layers of dextran: one adsorbed and one free. When the adsorbed dextran layer is the most concentrated, stability must ensue, and when the dextran in free solution is the most concentrated, flocculation should occur. Below 7% dextran, the concentration of free dextran is higher than the adsorbed concentration; above 10% dextran that situation is reversed. These data correlate well with the depletion flocculation predicted for the lower concentration and the depletion stabilization predicted for the higher dextran concentration.

     

Complexes of glucose oxidase with chitosan and dextran possessing enhanced stability

Abstract

In this study, the different mole ratios of glucose oxidase/chitosan/dextran–aldehyde and glucose oxidase/chitosan/dextran–sulfate complexes were synthesized. The modification of glucose oxidase by non-covalent complexation with dextran and chitosan in different molar ratios was studied in order to increase the enzyme activity. The enzyme/polymer complexes obtained were investigated by UV spectrophotometer and dynamic light scattering. Activity determination of synthesized complexes and free enzyme were performed at a temperature range. The best results were obtained by C_chitosan/C_{dextran–aldehyde} = 10/1 ratio and C_chitosan/C_{dextran–sulfate} = 1/5 ratio that were used in thermal stability, shelf life, salt stress, and ethanol effect experiments. The results demonstrated that both complexes were thermally stable at 60?°C and had superior storage stability compared to the free glucose oxidase. Complexes showed higher enzymatic activity than free enzyme in the organic solvent environment using 10% ethanol. The complexes were resistant to salt stress containing 0.1?M NaCl or CaCl₂. The particle size distribution results of the triple complex evaluated the complexation of the chitosan, dextran derivative, and glucose oxidase. The average size of the triple complex in diameter was found to be 325.8?±?9.3?nm. Overall findings suggest that the complexes of glucose oxidase, chitosan, and dextran showed significant enhancement in the enzyme activity.     

Description of the thermodynamic incompatibility of the guar–dextran aqueous two-phase system by light scattering

The phase diagram of the guar–dextran aqueous two-phase system has been described on the basis of static light scattering measurements in the dilute regime. By determining the molar weight and second virial coefficient from the two single polymers and the second virial cross coefficient from mixtures at constant guar/dextran ratio (either 17/83 or 28/72), the thermodynamic models based on the virial expansion or the Flory–Huggins theory were successfully applied. The second virial coefficient of guar was difficult to estimate with enough accuracy by light scattering and therefore was obtained by adjustment using a simple criterion stating that the calculated spinodal passes through the experimental critical point. The obtained value was within the confidence interval given by light scattering. Virial expansion and Flory–Huggins approaches yielded quite similar theoretical phase diagrams that satisfactorily fitted the experimental one. The slight discrepancies observed on the position of binodals and critical points has been attributed to the polydispersity of guar or the difficulty in extrapolating from the dilute regime to the semidilute one. The slope of the tie-lines was predicted with a good accuracy, especially with the virial expansion model. The fact that both approaches gave such similar results is probably related to the fact that the expressions of chemical potentials are equivalent if the polymer concentrations are low enough. In this particular case, both models are based on excluded volume interactions and equally describe the phase behavior of the guar–dextran aqueous system.     

Cross-linked nanoassemblies from poly(ethylene glycol)-poly(aspartate) block copolymers as stable supramolecular templates for particulate drug delivery

Block copolymer cross-linked nanoassemblies (CNAs) were developed as stable supramolecular templates for particulate drug delivery. Poly(ethylene glycol)-poly(aspartate) [PEG-p(Asp)] block copolymers, consisting of PEG (5 or 12 kDa) and Asp (5, 14, 25, 33, and 37 repeating units), were used as scaffolds and grafts in combination to prepare a nanoassembly library of grafted nanoassemblies (GNAs) and CNAs. Four synthesis routes were tested to maximize the number of drug-binding Asp units per nanoassembly. Grafting-onto-scaffold and grafting-from-scaffold methods were used for GNA synthesis. Either partially or completely deprotected PEG-p(Asp) was cross-linked with diamine compounds to prepare CNAs. (1)H NMR and GPC measurements showed that GNAs and CNAs contained the maximum 183 and 253 Asp units, respectively. Initial screening of the nanoassemblies revealed that GNAs would be impractical for further development as drug carriers due to variable grafting efficiency and low product yields. CNAs were obtained in high yields and identified as a promising supramolecular template that can entrap and release ionizable drugs (doxorubicin), enhancing the particle stability of nanoassemblies in the pharmaceutically relevant pH ranges between 4 and 9. Light scattering measurements demonstrated that the particle size of CNAs remained uniform before and after drug entrapment, causing neither aggregation nor dissociation (<5 mg/mL).     

Synthesis of ultrasmall superparamagnetic iron oxides using reduced polysaccharides

Investigation into the effect of the reducing sugar of dextran on formation and stability of dextran-coated ultrasmall superparamagnetic iron oxides (USPIO) has demonstrated that reduction of the terminal reducing sugar can have a significant effect on particle size, coating stability, and magnetic properties. Four aspects of polysaccharide-coated USPIO particle synthesis were investigated: (i) the effect reduction of the terminal polysaccharide sugar has upon polysaccharide usage, particle size, stability, and magnetic susceptibility; (ii) the effect an exogenous reducing sugar can have upon particle synthesis; (iii) the effect the molecular weight of the reduced polysaccharide has on particle synthesis; and (iv) the effectiveness of reduced and native dextrans in stabilizing a preformed magnetic sol. For low molecular weight dextrans (MW 20,000 x 10(-6) cgs). Similar results were obtained with a 12 kDa pullulan. The effect of polysaccharide molecular weight on particle size was studied, wherein higher molecular weight reduced dextrans produced larger particles. The effectiveness of the reduced and native dextrans in stabilizing a preformed magnetic sol was compared. Reduced dextrans were found to be superior for stabilizing the magnetic sol. The observed effects of reduction of the terminal sugar in dextran compared with the native dextran were modeled using the Langmuir adsorption isotherm. A good fit of experimental data with this model was found.     

Insulin resistance and impaired glucose tolerance in obese children and adolescents

In developed countries, obesity prevalence has strongly increased in the last decades. This has also been observed in children and adolescents. Until recently, type 2 diabetes mellitus was considered very rare among children and adolescents; however, in the last decades, some cases have been observed mainly in obese adolescents of some minority populations. The aim of our study was to assess the prevalence of type 2 diabetes, impaired glucose tolerance (IGT) and insulin resistance, and the metabolic features, in obese children and adolescents. We have studied 95 obese children and adolescents, 53 males and 42 females, aged 4-16 years. The prevalence of IGT in obese children and adolescents studied was 7.4%; there was not any child with type 2 diabetes. Fasting glucose and insulin serum concentrations did not show significant differences between obese children with or without IGT; however, 120 minutes after an oral glucose tolerance test, glucose and insulin serum concentrations showed statistically significant differences between both groups. Insulin resistance is defined as a HOMA index higher than 4. The prevalence of insulin resistance in obese children studied was 35.8%. Trygliceride serum concentrations were higher and HDL-C serum concentrations were lower in obese children with IGT than in those without IGT, but the differences were not statistically significant. IGT and insulin resistance are frequent in obese children and adolescents; early treatment in obese children and adolescents with IGT constitutes a strategy of reversing progression to beta-cell failure and in preventing type 2 diabetes.     

Selective effects of anti-Ia serum and complement treatment upon polyclonal B lymphocyte responses to dextran sulfate and lipopolysaccharide.

Subpopulations of B lymphocytes have been shown to vary in their expression of Ia alloantigens and polyclonal responsiveness to thymic independent antigens. We have demonstrated that the polyclonal B cell antibody response to dextran sulfate is less sensitive to removal of Ia-positive cells than is the response to LPS. This is a consistent finding whether alloantibody and complement (C) pretreatment is directed toward cells bearing Ia antigens coded for by the entire I region or by the I-A or I-E subregions. Heterogeneity appears to exist within the dextran sulfate-sensitive population in that using high antibody; cell ratios during antibody and C-mediated cell selection results in an inhibition of the proliferative but not the antibody response. This result may indicate a differential expression of Ia antigens on dextran sulfate-sensitive B cells that respond by proliferation versus those cells that produce antibody. Alternatively, proliferative responses to dextran sulfate may be more dependent upon Ia-positive accessory cells than is the polyclonal antibody response.