Optimized Adeno-Associated Viral-Mediated Human Factor VIII Gene Therapy in Cynomolgus Macaques.

Hemophilia A is a common hereditary bleeding disorder that is characterized by a deficiency of human blood coagulation factor VIII (hFVIII). Previous studies with adeno-associated viral (AAV) vectors identified two liver-specific promoter and enhancer combinations (E03.TTR and E12.A1AT) that drove high level expression of a codon-optimized, B-domain-deleted hFVIII transgene in a mouse model of the disease. This study further evaluated these enhancer/promoter combinations in cynomolgus macaques using two different AAV capsids (AAVrh10 and AAVhu37). Each of the four vector combinations was administered intravenously at a dose of 1.2 × 1013 genome copy/kg into five macaques per group. Delivery of the hFVIII transgene via the AAVhu37 capsid resulted in a substantial increase in hFVIII expression compared to animals administered with AAVrh10 vectors. Two weeks after administration of E03.TTR packaged within the AAVhu37 capsid, average hFVIII expression was 20.2 ± 5.0% of normal, with one animal exhibiting peak expression of 37.1% of normal hFVIII levels. The majority of animals generated an anti-hFVIII antibody response by week 8-10 post vector delivery. However, two of the five macaques administered with AAVhu37.E03.TTR were free of a detectable antibody response for 30 weeks post vector administration. Overall, the study supports the continued development of AAV-based gene therapeutics for hemophilia A using the AAVhu37 capsid.

Characterisation of minimalist co-assembled fluorenylmethyloxycarbonyl self-assembling peptide systems for presentation of multiple bioactive peptides.

UNLABELLED: The nanofibrillar structures that underpin self-assembling peptide (SAP) hydrogels offer great potential for the development of finely tuned cellular microenvironments suitable for tissue engineering. However, biofunctionalisation without disruption of the assembly remains a key issue. SAPS present the peptide sequence within their structure, and studies to date have typically focused on including a single biological motif, resulting in chemically and biologically homogenous scaffolds. This limits the utility of these systems, as they cannot effectively mimic the complexity of the multicomponent extracellular matrix (ECM). In this work, we demonstrate the first successful co-assembly of two biologically active SAPs to form a coassembled scaffold of distinct two-component nanofibrils, and demonstrate that this approach is more bioactive than either of the individual systems alone. Here, we use two bioinspired SAPs from two key ECM proteins: Fmoc-FRGDF containing the RGD sequence from fibronectin and Fmoc-DIKVAV containing the IKVAV sequence from laminin. Our results demonstrate that these SAPs are able to co-assemble to form stable hybrid nanofibres containing dual epitopes. Comparison of the co-assembled SAP system to the individual SAP hydrogels and to a mixed system (composed of the two hydrogels mixed together post-assembly) demonstrates its superior stable, transparent, shear-thinning hydrogels at biological pH, ideal characteristics for tissue engineering applications. Importantly, we show that only the coassembled hydrogel is able to induce in vitro multinucleate myotube formation with C2C12 cells. This work illustrates the importance of tissue engineering scaffold functionalisation and the need to develop increasingly advanced multicomponent systems for effective ECM mimicry. STATEMENT OF SIGNIFICANCE: Successful control of stem cell fate in tissue engineering applications requires the use of sophisticated scaffolds that deliver biological signals to guide growth and differentiation. The complexity of such processes necessitates the presentation of multiple signals in order to effectively mimic the native extracellular matrix (ECM). Here, we establish the use of two biofunctional, minimalist self-assembling peptides (SAPs) to construct the first co-assembled SAP scaffold. Our work characterises this construct, demonstrating that the physical, chemical, and biological properties of the peptides are maintained during the co-assembly process. Importantly, the coassembled system demonstrates superior biological performance relative to the individual SAPs, highlighting the importance of complex ECM mimicry. This work has important implications for future tissue engineering studies.

Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability.

It has been known for almost one hundred years that a lower surface tension can be achieved at the air-water interface by spreading protein from a concentrated solution than by adsorption from an equivalent total bulk concentration. Nevertheless, the factors that control this nonequilibrium process have not been fully understood. In the present work, we apply ellipsometry, neutron reflectometry, X-ray reflectometry, and Brewster angle microscopy to elaborate the surface loading of human serum albumin in terms of both the macroscopic film morphology and the spreading dynamics. We show that the dominant contribution to the surface loading mechanism is the Marangoni spreading of protein from the bulk of the droplets rather than the direct transfer of their surface films. The films can be spread on a dilute subphase if the concentration of the spreading solution is sufficient; if not, dissolution of the protein occurs, and only a textured adsorbed layer slowly forms. The morphology of the spread protein films comprises an extended network with regions of less textured material or gaps. Further, mechanical cycling of the surface area of the spread films anneals the network into a membrane that approach constant compressibility and has increased durability. Our work provides a new perspective on an old problem in colloid and interface science. The scope for optimization of the surface loading mechanism in a range of systems leading to its exploitation in deposition-based technologies in the future is discussed.

Chemical delithiation and exfoliation of [Formula: see text].

Progressive chemical .delithiation of commercially available lithium cobalt oxide ([Formula: see text]) showed consecutive changes in the crystal properties. Rietveld refinement of high resolution X-ray and neutron diffraction revealed an increased lattice parameter c and a reduced lattice parameter a for chemically delithiated samples. Using electron microscopy we have also followed the changes in the texture of the samples towards what we have found is a critical layer stoichiometry of about [Formula: see text] with x=1/3 that causes the grains to exfoliate. The pattern of etches by delithiation suggests that unrelieved strain fields may produce chemical activity.

Human serum albumin binding to silica nanoparticles--effect of protein fatty acid ligand.

Neutron reflectivity shows that fatted (F-HSA) and defatted (DF-HSA) versions of human serum albumin behave differently in their interaction with silica nanoparticles premixed in buffer solutions although these proteins have close to the same surface excess when the silica is absent. In both cases a silica containing film is quickly established at the air-water interface. This film is stable for F-HSA at all relative protein-silica concentrations measured. This behaviour has been verified for two small silica nanoparticle radii (42 Å and 48 Å). Contrast variation and co-refinement have been used to find the film composition for the F-HSA-silica system. The film structure changes with protein concentration only for the DF-HSA-silica system. The different behaviour of the two proteins is interpreted as a combination of three factors: increased structural stability of F-HSA induced by the fatty acid ligand, differences in the electrostatic interactions, and the higher propensity of defatted albumin to self-aggregate. The interfacial structures of the proteins alone in buffer are also reported and discussed.

The intercalation of N,N,N',N'-tetramethyl-ethane-1,2-diamine (tmeda) into C6Li and C12Li.

Liquid N,N,N',N'-tetramethyl-ethane-1,2-diamine (tmeda) was intercalated into preprepared C(6)Li or C(12)Li. X-ray diffraction from the ternary compound indicates an identity period of 11.5 Å. The (13)C NMR line shifts show that tmeda molecules form a screen between the graphene planes and the lithium ions. Small-angle X-ray scattering showed that no higher structural ordering was present but revealed a progressive roughening of the surfaces with successive intercalation of lithium and amine into the graphite galleries.

Complex layering observed in high internal phase emulsions at a silicon surface by neutron reflectometry.

The neutron reflectivity profiles from the interface between silicon and aqueous phase-in-oil high internal phase emulsions of steadily increasing surfactant hydrophilicity, are reported for two isotopic contrasts for each surfactant. Layered models are required to fit the structured reflectivity profiles that demonstrate that the oxidised top layer of the silicon is always covered by a surfactant monolayer. Interposed between the surfactant monolayer and the bulk emulsion is a layer of oil--a geometric effect caused by reorganisation of the aqueous droplets. As the surfactant hydrophilicity increases, alternating aqueous and oil+surfactant layers are inserted between this topmost oil layer and the oxide attached surfactant monolayer. The resulting structures have compositions and layer spacings suggestive of sections from lamellar phases. This increase in layer ordering with increasing surfactant hydrophilicity is expected. The bulk emulsions are observed to exhibit lamellar or sponge phases increasingly as surfactant hydrophilicity increases.

High internal phase emulsions under shear. Co-surfactancy and shear stability.

Large changes in the rheology of high-internal phase aqueous-in-oil emulsions (HIPEs) using an oil-soluble polyisobutylene-based primary surfactant (PIBSA) are provoked by very small quantities of water-soluble polyamide-based cosurfactants (PAM with C(12), C(14), and C(16) tails). The structural origin of this was studied using small-angle neutron scattering (SANS) from sheared emulsions, with simultaneous in situ rheology measurements. The PAM drastically lowers the droplet-oil interfacial tension by displacing PIBSA, causing large droplet deformation under shear and much lowered emulsion yield stress. With PAM, the surfactant monolayer at the droplet surface becomes more responsive to droplet shape change and redistributes in response to shear which the PIBSA-only system does not. Although it is oil-insoluble, PAM also reaches the nanoscale PIBSA micelles in the oil phase, changing micelle size and content in ways predictable from the hydrophilicity of the different PAMs. PAM does not, however, strongly affect the viscosities at high shear rates; shear thinning and thickening are unaffected. Droplet size, droplet-droplet flattening, and linkage determine the viscosities observed, more so than droplet-oil interfacial tension. We infer from this that the droplet motion under shear does not involve much transient droplet deformation as the droplets move by each other.

The stability of high internal phase emulsions at low surfactant concentration studied by small angle neutron scattering.

The changes in structure of high internal phase emulsions at low concentrations and at elevated temperature are reported for comparison with the same emulsions under conditions well away from instability. Small angle neutron scattering measurements on aqueous ammonium nitrate droplets dispersed in hexadecane and stabilized by very small quantities of a polyisobutylene-based surfactant (PIBSA) as well as related inverse micellar solutions in hexadecane, have been made as a function of temperature and surfactant concentration. Experimental conditions here favour larger and more deformable droplets than in previous studies. Besides the expected micelles and adsorbed surfactant, planar bilayers of micron lateral extent between touching droplets cover 20% of the droplet surface. Another difference from previous experiments is that the oil phase in the emulsions, and corresponding inverse micellar solutions are different in micellar radii and composition. The differences, and changes with surfactant concentration and temperature, are attributed to fractionation of the polydisperse PIBSA in the emulsions, but not the inverse micellar solutions. At low PIBSA concentration and high temperature the SANS shows emulsion decomposing into separate oil and aqueous phases. This occurs when the micelle concentration reaches a very small but measurable value. The inverse micelles may suppress by steric action long wavelength unstable capillary waves in the bilayers. Depletion repulsion forces here have a minor role in the emulsion stabilization.

Nano- and microstructure of high-internal phase emulsions under shear.

High-internal phase aqueous-in-oil emulsions of two surfactant concentrations were studied using small-angle neutron scattering (SANS) and simultaneous in situ rheology measurements. They contained a continuous oil phase with differing amounts of hexadecane and d-hexadecane (for contrast matching experiments), a deuteroaqueous phase almost saturated with ammonium nitrate, and an oil-soluble stabilizing polyisobutylene-based surfactant. The emulsions' macroscopic rheological behavior has been related to quantify changes in microscale and nanoscale structures observed in the SANS measurements. The emulsions are rheologically unexceptional and show, inter alia, refinement to higher viscosity after high shear, and shear thinning. These are explained by changes observed in the SANS model parameters. Shear thinning is explained by SANS-observed shear disruption of interdroplet bilayer links, causing deflocculation to more spherical, less linked, aqueous droplets. Refinement to higher viscosity is accompanied by droplet size reduction and loss of surfactant from the oil continuous phase. Refinement occurs because of shear-induced droplet anisotropy, which we have also observed in the SANS experiment. This observed anisotropy and the emulsion refinement cannot be reproduced by either isolated molecule or mean-field models but require a more detailed consideration of interdroplet forces in the sheared fluid.

Phosphorus runoff from waste water treatment biosolids and poultry litter applied to agricultural soils.

Differences in the properties of organic phosphorus (P) sources, particularly those that undergo treatment to reduce soluble P, can affect soil P solubility and P transport in surface runoff. This 2-yr field study investigated soil P solubility and runoff P losses from two agricultural soils in the Mid-Atlantic region after land application of biosolids derived from different waste water treatment processes and poultry litter. Phosphorus speciation in the biosolids and poultry litter differed due to treatment processes and significantly altered soil P solubility and dissolved reactive P (DRP) and bioavailable P (FeO-P) concentrations in surface runoff. Runoff total P (TP) concentrations were closely related to sediment transport. Initial runoff DRP and FeO-P concentrations varied among the different biosolids and poultry litter applied. Over time, as sediment transport declined and DRP concentrations became an increasingly important component of runoff FeO-P and TP, total runoff P was more strongly influenced by the type of biosolids applied. Throughout the study, application of lime-stabilized biosolids and poultry litter increased concentrations of soil-soluble P, readily desorbable P, and soil P saturation, resulting in increased DRP and FeO-P concentrations in runoff. Land application of biosolids generated from waste water treatment processes that used amendments to reduce P solubility (e.g., FeCl(3)) did not increase soil P saturation and reduced the potential for DRP and FeO-P transport in surface runoff. These results illustrate the importance of waste water treatment plant process and determination of specific P source coefficients to account for differential P availability among organic P sources.

Denaturation resistance of beta-lactoglobulin in monomolecular films at the air-water interface.

Using X-ray reflectometry we report strong differences in the denaturation response of beta-lactoglobulin adsorbed as a monomolecular film at the air-water interface from that observed in mixed denaturant/beta-lactoglobulin bulk solutions. Using the "flow trough" technique an isolated monomolecular film of the protein showed little change in structure when subjected to a 4.0 M guanidinium hydrochloride substrate. Unlike the bulk solution where a new protein layer structure appears, small changes in the protein packing and the roughness of the film are the only evidence of change. These parameters have been studied as a function of denaturant concentration and film quality. The strength of the response depends on the degree of perfection of the originally formed film; quickly formed films are more easily denatured. As the response is so subtle, possible interfering effects such as denaturant release of protein adsorbed on the trough have been quantified.

Structure of high internal phase aqueous-in-oil emulsions and related inverse micelle solutions. 3. Variation of surfactant.

The small angle neutron scattering from high internal phase water-in-hexadecane and saturated ammonium nitrate-in-hexadecane emulsions is compared with that from related hexadecane-based inverse micellar solutions. Three molecular weights of the monodisperse polyisobutylene acid amide (PIBSA) surfactant 750, 1200, and 1700 were studied over a range of surfactant concentrations. As an additional comparison, emulsions based on sorbitan monooleate and isostearate surfactants were investigated. The scattering from molecular weight 1200 water-based PIBSA emulsions can be fitted at all concentrations to a model with a surfactant coated aqueous droplet-oil interface together with the majority of the surfactant in the oil phase of the emulsion in the form of inverse micelles. Variation of the molecular weight shows a variety of phases of increasing curvature: lamellar, sponge, and, most commonly, the emulsion structure described above. In addition, the molecular weight affects the oil component in the emulsions, which can contain either cylindrical micelles or spherical micelles of varying water but constant hexadecane content. Increased phase curvature is favored by both increased PIBSA molecular weight and ammonium nitrate dissolved in the water. These observations are consistent with "Wedge theory". The structures observed in the emulsions are close to those observed in related inverse micellar solutions made from hexadecane, the surfactant, and water. Lower concentrations of surfactant in the micellar solutions decrease micelle curvature, except where the inverse micelles are spherical and small; here, there is little effect of dilution. Substitution of sorbitan surfactants for PIBSAs produces slightly less organized but similar structures, with smaller spherical micelles containing proportionally more water. The aqueous-oil droplet interface has a relatively invariant monolayer of adsorbed surfactant. For all emulsions, we can infer from the mass balance that micelle concentrations are depressed in the inverse micellar solutions because up to half the added surfactant is present as individually dissolved molecules.

Structure of high internal phase aqueous-in-oil emulsions and related inverse micelle solutions. 4. Surfactant mixtures.

The effects of combinations of surfactants on the structure and stability of high internal phase water-in-hexadecane and saturated ammonium nitrate-in-hexadecane oil-based emulsions and oil-based inverse micellar solutions are reported. The combinations were 750, 1,200, and 1,700 molecular weight monodisperse and 450 and 1,000 molecular weight polydisperse polyisobutylene acid amides, and sorbitan monooleate. The samples made from mixtures have qualitatively similar nanostructures to emulsions made from single surfactants. Again, for the emulsions, micrometer-scale aqueous droplets are dispersed in a continuous oil phase, which contains inverse spherical micelles composed of surfactant, hexadecane, and water. In quantitative terms, lower average surfactant molecular weight, lower ammonium nitrate content, and lower surfactant content increased the swelling of micelles, their water content, and the tendency of the emulsion to be unstable and form a sponge phase. This instability also allows micelle plasticity such that their geometry and content in mixed surfactant systems are not simply predictable by interpolation from single surfactant systems. An example was found of a mixed micelle 3 times larger than either single component micelle. The observed behavior suggests that mixing surfactant molecules of very different molecular weights destabilizes the emulsions, while mixing surfactants close in molecular weight has the opposite effect. The synergistic effects of surfactant molecular weight polydispersity and binary mixing are most marked for 1:1 molecular mixtures of surfactant.

Ultra-small-angle neutron scattering: a tool to study packing of relatively monodisperse polymer spheres and their binary mixtures.

We measured ultra-small-angle neutron scattering (USANS) from polymethylmethacrylate spheres tamped down in air. Two slightly polydisperse pure sphere sizes (1.5 and 7.5 microm diameters) and five mixtures of these were used. All were loose packed (packing fractions of 0.3-0.6) with nongravitational forces (e.g., friction) important, preventing close packing. The USANS data are rich in information on powder packing. A modified Percus-Yevick fluid model was used to parametrize the data-adequately but not well. The modifications required the introduction of small voids, less than the sphere size, and a parameter reflecting substantial deviation from the Percus-Yevick prediction of the sphere-sphere correlation function. The mixed samples fitted less well, and two further modifying factors were necessary. These were local inhomogeneities, where the concentration of same-size spheres, both large and small, deviated from the mean packing, and a factor accounting for the presence within these "clusters" of self-avoidance of the large spheres (that is, large spheres coated with more small spheres than Percus-Yevick would predict). The overall deviations from the hard-sphere Percus-Yevick model that we find here suggest that fluid models of loose packed powders are unlikely to be successful but lay the ground work for future theoretical and computational works.

Nano- and microstructure of air/oil/water interfaces.

We report the creation of air/oil/water interfaces with variable-thickness oil films using polyisobutylene-based (PIB) surfactants cospread with long-chain paraffinic alkanes on clean water surfaces. The resultant stable oil layers are readily measurable with simple surface techniques, exhibit physical densities the same as expected for bulk oils, and are up to approximately 100 A thick above the water surface as determined using X-ray reflectometry. This provides a ready system for studying the competition of surfactants at the oil/water interface. Results from the competition of a nonionic polyamide surfactant or an anionic sodium dodecyl sulfate with the PIB surfactant are reported. However, this smooth oil layer does not account for the total volume of spread oil nor is the increase in thickness proportional to the film compression. Brewster angle microscopy (BAM) reveals surfactant and oil structures on the scale of 1 to 10 microm at the interface. At low surface pressure (pi < 24 mN m(-1)) large, approximately 10 microm inhomogeneities are observed. Beyond a phase transition observed at pi approximately = 24 mN m(-1), a structure with a spongy appearance and a microscale texture develops. These structures have implications for understanding the microstructure at the oil/water interface in emulsions.

Evaluation of phosphorus source coefficients as predictors of runoff phosphorus concentrations.

Many states have adopted a P site index (PSI) as a risk assessment tool to determine when P-based nutrient management is required for a given agricultural field. Some PSIs use a weighting factor, the phosphorus source coefficient (PSC), to account for differences in P solubility between organic P sources. Information relating to appropriate values of PSC for various organic P sources is limited. The objectives of this study were to determine PSCs for organic P sources and to examine the relationship between PSCs and P concentrations measured in simulated rainfall runoff. An incubation study was used to calculate PSCs based on the extractability of P from organic P sources (separated and unseparated liquid dairy manure, digested dairy manure, dairy manure solids, poultry litter, and compost) relative to P from triple superphosphate fertilizer. The PSCs from the 14-d incubations were the best predictors of runoff P after 14 d soil equilibration in the runoff boxes. The values for iron-oxide strip phosphorus (FeO-P) PSC ranged from 78% for compost to 28% for poultry litter and were significantly related to runoff DR-P (r(2) = 0.80***) and FeO-P (r(2) = 0.76***) during the 14-d runoff event. Mehlich 3 PSCs ranged from 59% for compost to 30% for unseparated dairy manure and were better predictors of DR-P and FeO-P during the 56-d event (r(2) = 0.73*** and 0.65***, respectively). The results of this study indicate that PSCs based on soil incubations may improve the ability of PSCs to predict the risk of runoff transport, particularly after manure incorporation.

Effect of the air-water interface on the structure of lysozyme in the presence of guanidinium chloride.

We report observations of the changes in the surface structure of lysozyme adsorbed at the air-water interface produced by the chemical denaturant guanidinium chloride. A primary result is the durability of the adsorbed surface layer to denaturation, as compared to the molecule in the bulk solution. Data on the surface film were obtained from X-ray and neutron reflectivity measurements and modeled simultaneously. The behavior of lysozyme in G.HCl solutions was determined by small-angle X-ray scattering. For the air-water interface, determination of the adsorbed protein layer dimensions shows that at low to moderate denaturant concentrations (up to 2 mol L(-1)), there is no significant distortion of the protein's tertiary structure at the interface, as changes in the orientation of the protein are sufficient to model data. At higher denaturant concentrations, time-dependent multilayer formation occurred, indicating molecular aggregation at the surface. Methodologies to predict the protein orientation at the interface, based on amino acid residues' surface affinities and charge, were critiqued and validated against our experimental data.

Surface movement in water of splendipherin, the aquatic male sex pheromone of the tree frog Litoria splendida.

The aquatic sex pheromone splendipherin (GLVSSIGKALGGLLADVVKSKGQPA-OH) of the male green tree frog Litoria splendida moves across the surface of water to reach the female. Surface pressure and X-ray reflectometry measurements confirm that splendipherin is a surface-active molecule, and are consistent with it having an ordered structure, whereby the hydrophilic portion of the peptide interacts with the underlying water and the hydrophobic region is adjacent to the vapour phase. The movement of splendipherin over the surface of water is caused by a surface pressure gradient. In order to better define the structure of splendipherin at the water/air interface we used 2D NMR studies of the pheromone with the solvent system trifluoroethanol/water (1 : 1 v/v). In this solvent system, splendipherin adopts a bent alpha helix from residues V3 to K21. The bending of the helix occurs in the centre of the peptide in the vicinity of G11 and G12. The region of splendipherin from V3 to G11 has well-defined amphipathicity, whereas the amphipathicity from G12 to A25 is reduced by K19 and P24 intruding into the hydrophobic and hydrophilic regions respectively. A helical structure is consistent with X-ray reflectometry data.