Small-Angle Neutron Scattering Study of High Fat Fish Oil-In-Water Emulsion Stabilized with Sodium Caseinate and Phosphatidylcholine.

We report on small-angle neutron scattering (SANS) investigations of separate phase domains in high fat (70%) oil-in-water emulsions emulsified with the combination of sodium caseinate (CAS) and phosphatidylcholine (PC). The emulsion as a whole was studied by contrast variation to identify scattering components dominated by individual emulsifiers. The emulsion was subsequently separated into the aqueous phase and the oil-rich droplet phase, which were characterized separately. Emulsions produced with 1.05% (w/w) CAS and PC fraction which varies between 1.75% (w/w) and 0.35% (w/w) provided droplets between 10 and 19 µm in surface weighted mean in 70% fish oil-in-water emulsions. At least two-third of the overall CAS is associated with the interface, while the rest remains with the aqueous phase. Six percent of PC formed a monolayer in the interface, while the rest of the PC remains in the droplet phase in the form of multilayers. When the separated components were resuspended, the resuspended emulsion showed similar characteristics compared to the original emulsion in terms of droplet size distribution and neutron scattering. Instead, CAS in the aqueous phase separated from the emulsion shows aggregation not present in the corresponding CAS-in-D2O system.

Interfacial structure of 70% fish oil-in-water emulsions stabilized with combinations of sodium caseinate and phosphatidylcholine.

We report on the structural evaluation of high fat fish oil-in-water emulsions emulsified with sodium caseinate (CAS) and phosphatidylcholine (PC). The microemulsions contained 70% (w/w) fish oil with 1.05-1.4% (w/w) CAS and 0.4-1.75% (w/w) PC and were studied by the combination of light scattering together with small-angle X-ray and neutron scattering (SAXS/SANS). Aqueous CAS forms aggregates having a denser core of about 100 kDa and less dense shell about 400 kDa with the hard sphere diameter of 20.4 nm. PC appears as multilayers whose coherence length spans from 40 to 100 nm. PC monolayer separates oil and water phases. Moreover, 80% CAS particles are loosely bound to the interface but are not forming continuous coverage. The distance between aggregated CAS particles in microemulsion is increased compared to CAS aggregates in pure CAS-in-water system. PC multilayers become larger in the presence of oil-water interface compared to the pure PC mixtures. Bilayers become larger with increasing PC concentration. This study forms a structural base for the combination of CAS and PC emulsifiers forming a well-defined thin and dense PC layer together with thick but less dense CAS layer, which is assumed to explain its better oxidative stability compared to single emulsifiers.

Correlating Charge Transport with Structure in Deconstructed Diketopyrrolopyrrole Oligomers: A Case Study of a Monomer in Field-Effect Transistors.

Copolymers based on diketopyrrolopyrrole (DPP) cores have attracted a lot of attention because of their high p-type as well as n-type carrier mobilities in organic field-effect transistors (FETs) and high power conversion efficiencies in solar cell structures. We report the structural and charge transport properties of n-dialkyl side-chain-substituted thiophene DPP end-capped with a phenyl group (Ph-TDPP-Ph) monomer in FETs which were fabricated by vacuum deposition and solvent coating. Grazing-incidence X-ray diffraction (GIXRD) from bottom-gate, bottom-contact FET architectures was measured with and without biasing. Ph-TDPP-Ph reveals a polymorphic structure with π-conjugated stacking direction oriented in-plane. The unit cell comprises either one monomer with a = 20.89 Å, b = 13.02 Å, c = 5.85 Å, α = 101.4°, ß = 90.6°, and γ = 94.7° for one phase (TR1) or two monomers with a = 24.92 Å, b = 25.59 Å, c = 5.42 Å, α = 80.3°, ß = 83.5°, and γ = 111.8° for the second phase (TR2). The TR2 phase thus signals a shift from a coplanar to herringbone orientation of the molecules. The device performance is sensitive to the ratio of the two triclinic phases found in the film. Some of the best FET performances with p-type carrier mobilities of 0.1 cm2/V s and an on/off ratio of 106 are for films that comprise mainly the TR1 phase. GIXRD from in operando FETs demonstrates the crystalline stability of Ph-TDPP-Ph.

Structural Evaluation of 5,5'-Bis(naphth-2-yl)-2,2'-bithiophene in Organic Field-Effect Transistors with n-Octadecyltrichlorosilane Coated SiO2 Gate Dielectric.

We report on the structure and morphology of 5,5'-bis(naphth-2-yl)-2,2'-bithiophene (NaT2) films in bottom-contact organic field-effect transistors (OFETs) with octadecyltrichlorosilane (OTS) coated SiO2 gate dielectric, characterized by atomic force microscopy (AFM), grazing-incidence X-ray diffraction (GIXRD), and electrical transport measurements. Three types of devices were investigated with the NaT2 thin-film deposited either on (1) pristine SiO2 (corresponding to higher surface energy, 47 mJ/m2) or on OTS deposited on SiO2 under (2) anhydrous or (3) humid conditions (corresponding to lower surface energies, 20-25 mJ/m2). NaT2 films grown on pristine SiO2 form nearly featureless three-dimensional islands. NaT2 films grown on OTS/SiO2 deposited under anhydrous conditions form staggered pyramid islands where the interlayer spacing corresponds to the size of the NaT2 unit cell. At the same time, the grain size measured by AFM increases from hundreds of nanometers to micrometers and the crystal size measured by GIXRD from 30 nm to more than 100 nm. NaT2 on OTS/SiO2 deposited under humid conditions also promotes staggered pyramids but with smaller crystals 30-80 nm. The NaT2 unit cell parameters in OFETs differ 1-2% from those in bulk. Carrier mobilities tend to be higher for NaT2 layers on SiO2 (2-3 × 10-4 cm2/(V s)) compared to NaT2 on OTS (2 × 10-5-1 × 10-4 cm2/(V s)). An applied voltage does not influence the unit cell parameters when probed by GIXRD in operando.

Hydrophobin film structure for HFBI and HFBII and mechanism for accelerated film formation.

Hydrophobins represent an important group of proteins from both a biological and nanotechnological standpoint. They are the means through which filamentous fungi affect their environment to promote growth, and their properties at interfaces have resulted in numerous applications. In our study we have combined protein docking, molecular dynamics simulation, and electron cryo-microscopy to gain atomistic level insight into the surface structure of films composed of two class II hydrophobins: HFBI and HFBII produced by Trichoderma reesei. Together our results suggest a unit cell composed of six proteins; however, our computational results suggest P6 symmetry, while our experimental results show P3 symmetry with a unit cell size of 56 Å. Our computational results indicate the possibility of an alternate ordering with a three protein unit cell with P3 symmetry and a smaller unit cell size, and we have used a Monte Carlo simulation of a spin model representing the hydrophobin film to show how this alternate metastable structure may play a role in increasing the rate of surface coverage by hydrophobin films, possibly indicating a mechanism of more general significance to both biology and nanotechnology.

Drug release from nanoparticles embedded in four different nanofibrillar cellulose aerogels.

Highly porous nanocellulose aerogels prepared by freeze-drying from various nanofibrillar cellulose (NFC) hydrogels are introduced as nanoparticle reservoirs for oral drug delivery systems. Here we show that beclomethasone dipropionate (BDP) nanoparticles coated with amphiphilic hydrophobin proteins can be well integrated into the NFC aerogels. NFCs from four different origins are introduced and compared to microcrystalline cellulose (MCC). The nanocellulose aerogel scaffolds made from red pepper (RC) and MCC release the drug immediately, while bacterial cellulose (BC), quince seed (QC) and TEMPO-oxidized birch cellulose-based (TC) aerogels show sustained drug release. Since the release of the drug is controlled by the structure and interactions between the nanoparticles and the cellulose matrix, modulation of the matrix formers enable a control of the drug release rate. These nanocomposite structures can be very useful in many pharmaceutical nanoparticle applications and open up new possibilities as carriers for controlled drug delivery.

Structural and functional roles of carotenoids in chlorosomes.

Chlorosomes are large light-harvesting complexes found in three phyla of anoxygenic photosynthetic bacteria. Chlorosomes are primarily composed of self-assembling pigment aggregates. In addition to the main pigment, bacteriochlorophyll c, d, or e, chlorosomes also contain variable amounts of carotenoids. Here, we use X-ray scattering and electron cryomicroscopy, complemented with absorption spectroscopy and pigment analysis, to compare the morphologies, structures, and pigment compositions of chlorosomes from Chloroflexus aurantiacus grown under two different light conditions and Chlorobaculum tepidum. High-purity chlorosomes from C. aurantiacus contain about 20% more carotenoid per bacteriochlorophyll c molecule when grown under low light than when grown under high light. This accentuates the light-harvesting function of carotenoids, in addition to their photoprotective role. The low-light chlorosomes are thicker due to the overall greater content of pigments and contain domains of lamellar aggregates. Experiments where carotenoids were selectively extracted from intact chlorosomes using hexane proved that they are located in the interlamellar space, as observed previously for species belonging to the phylum Chlorobi. A fraction of the carotenoids are localized in the baseplate, where they are bound differently and cannot be removed by hexane. In C. tepidum, carotenoids cannot be extracted by hexane even from the chlorosome interior. The chemical structure of the pigments in C. tepidum may lead to π-π interactions between carotenoids and bacteriochlorophylls, preventing carotenoid extraction. The results provide information about the nature of interactions between bacteriochlorophylls and carotenoids in the protein-free environment of the chlorosome interior.

Loosening of globular structure under alkaline pH affects accessibility of ß-lactoglobulin to tyrosinase-induced oxidation and subsequent cross-linking.

Globular proteins such as ß-lactoglobulin (BLG) are poorly accessible to enzymes. We have studied susceptibility of BLG to oxidation by Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) tyrosinases and subsequent intermolecular cross-linking with respect to pH-induced structural changes. We evaluated pH-induced structural changes in BLG using circular dichroism, tryptophan fluorescence and small angle X-ray scattering (SAXS) measurements, where after these results were correlated with the analysis of cross-linking by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Oxygen consumption measurement and changes in radii of gyration determined by SAXS during the enzyme-induced oxidation at the respective reaction conditions were also followed. Intermolecular cross-linking of BLG by TrTyr was found at pH 9 but not at pH 7.5. AbTyr was unable to catalyze cross-linking at pH 7.5 or pH 9. Increased accessibility and cross-linking by TrTyr was addressed to loosening of the three dimensional structure of the protein, increased flexibility of the backbone as well as partial hydrolysis. In addition to basic research of the effect of protein folding on enzymatic cross-linking the research results have significance on the exploitation of TrTyr at alkaline conditions.

Amorphous characteristics of an ultrathin cellulose film.

Swelling behavior and rearrangements of an amorphous ultrathin cellulose film (20 nm thickness) exposed to water and subsequently dried were investigated with grazing incidence X-ray diffraction, neutron reflectivity, atomic force microscopy, and surface energy calculations obtained from contact angle measurements. The film swelled excessively in water, doubling its thickness, but shrunk back to the original thickness upon water removal. Crystallinity (or amorphousness) and morphology remained relatively unchanged after the wetting/drying cycle, but surface free energy increased considerably (ca. 15%) due to an increase in its polar component, that is, the hydrophilicity of the film, indicating that rearrangements occurred during the film's exposure to water. Furthermore, stability of the films in aqueous NaOH solution was investigated with quartz crystal microbalance with dissipation monitoring. The films were stable at 0.0001 M NaOH but already 0.001 M NaOH partially dissolved the film. The surprising susceptibility to dissolve in dilute NaOH was hypothetically attributed to the lack of hierarchical morphology in the amorphous film.

The lamellar spacing in self-assembling bacteriochlorophyll aggregates is proportional to the length of the esterifying alcohol.

Chlorosomes from green photosynthetic bacteria are large photosynthetic antennae containing self-assembling aggregates of bacteriochlorophyll c, d, or e. The pigments within chlorosomes are organized in curved lamellar structures. Aggregates with similar optical properties can be prepared in vitro, both in polar as well as non-polar solvents. In order to gain insight into their structure we examined hexane-induced aggregates of purified bacteriochlorophyll c by X-ray scattering. The bacteriochlorophyll c aggregates exhibit scattering features that are virtually identical to those of native chlorosomes demonstrating that the self-assembly of these pigments is fully encoded in their chemical structure. Thus, the hexane-induced aggregates constitute an excellent model to study the effects of chemical structure on assembly. Using bacteriochlorophyllides transesterified with different alcohols we have established a linear relationship between the esterifying alcohol length and the lamellar spacing. The results provide a structural basis for lamellar spacing variability observed for native chlorosomes from different species. A plausible physiological role of this variability is discussed. The X-ray scattering also confirmed the assignments of peaks, which arise from the crystalline baseplate in the native chlorosomes.

Structure of chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus.

The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols. C. aurantiacus chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.

Surface location of individual residues of SlpA provides insight into the Lactobacillus brevis S-layer.

Bacterial surface layer (S-layer) proteins are excellent candidates for in vivo and in vitro nanobiotechnological applications because of their ability to self-assemble into two-dimensional lattices that form the outermost layer of many Eubacteria and most Archaea species. Despite this potential, knowledge about their molecular architecture is limited. In this study, we investigated SlpA, the S-layer protein of the potentially probiotic bacterium Lactobacillus brevis ATCC 8287 by cysteine-scanning mutagenesis and chemical modification. We generated a series of 46 mutant proteins by replacing single amino acids with cysteine, which is not present in the wild-type protein. Most of the replaced amino acids were located in the self-assembly domain (residues 179 to 435) that likely faces the outer surface of the lattice. As revealed by electron microscopy, all the mutant proteins were able to form self-assembly products identical to that of the wild type, proving that this replacement does not dramatically alter the protein conformation. The surface accessibility of the sulfhydryl groups introduced was studied with two maleimide-containing marker molecules, TMM(PEG)(12) (molecular weight [MW], 2,360) and AlexaFluor488-maleimide (MW = 720), using both monomeric proteins in solution and proteins allowed to self-assemble on cell wall fragments. Using the acquired data and available domain information, we assigned the mutated residues into four groups according to their location in the protein monomer and lattice structure: outer surface of the lattice (9 residues), inner surface of the lattice (9), protein interior (12), and protein-protein interface/pore regions (16). This information is essential, e.g., in the development of therapeutic and other health-related applications of Lactobacillus S-layers.

Self-assembled films of hydrophobin proteins HFBI and HFBII studied in situ at the air/water interface.

Hydrophobins are a group of surface-active fungal proteins known to adsorb to the air/water interface and self-assemble into highly crystalline films. We characterized the self-assembled protein films of two hydrophobins, HFBI and HFBII from Trichoderma reesei, directly at the air/water interface using Brewster angle microscopy, grazing-incidence X-ray diffraction, and reflectivity. Already in zero surface pressure, HFBI and HFBII self-assembled into micrometer-sized rafts containing hexagonally ordered two-dimensional crystallites with lattice constants of 55 A and 56 A, respectively. Increasing the pressure did not change the ordering of the proteins in the crystallites. According to the reflectivity measurements, the thicknesses of the hydrophobin films were 28 A (HFBI) and 24 A (HFBII) at 20 mN/m. The stable films could also be transferred to a silicon substrate. Modeling of the diffraction data indicated that both hydrophobin films contained six molecules in the unit cell, but the ordering of the molecules was somewhat different for HFBI and HFBII, suggesting specific protein-protein interactions.

Hexanol-induced order-disorder transitions in lamellar self-assembling aggregates of bacteriochlorophyll c in Chlorobium tepidum chlorosomes.

Chlorosomes are light-harvesting complexes of green photosynthetic bacteria. Chlorosomes contain bacteriochlorophyll (BChl) c, d, or e aggregates that exhibit strong excitonic coupling. The short-range order, which is responsible for the coupling, has been proposed to be augmented by pigment arrangement into undulated lamellar structures with spacing between 2 and 3 nm. Treatment of chlorosomes with hexanol reversibly converts the aggregated chlorosome chlorophylls into a form with spectral properties very similar to that of the monomer. Although this transition has been extensively studied, the structural basis remains unclear due to variability in the obtained morphologies. Here we investigated hexanol-induced structural changes in the lamellar organization of BChl c in chlorosomes from Chlorobium tepidum by a combination of X-ray scattering, electron cryomicroscopy, and optical spectroscopy. At a low hexanol/pigment ratio, the lamellae persisted in the presence of hexanol while the short-range order and exciton interactions between chlorin rings were effectively eliminated, producing a monomer-like absorption. The result suggested that hexanol hydroxyls solvated the chlorin rings while the aliphatic tail partitioned into the hydrophobic part of the lamellar structure. This partitioning extended the chlorosome along its long axis. Further increase of the hexanol/pigment ratio produced round pigment-hexanol droplets, which lost all lamellar order. After hexanol removal the spectral properties were restored. In the samples treated under the high hexanol/pigment ratio, lamellae reassembled in small domains after hexanol removal while the shape and long-range order were irreversibly lost. Thus, all the interactions required for establishing the short-range order by self-assembly are provided by BChl c molecules alone. However, the long-range order and overall shape are imposed by an external structure, e.g., the proteinaceous chlorosome baseplate.

Human breast cancer in vitro: matching histo-pathology with small-angle x-ray scattering and diffraction enhanced x-ray imaging.

Twenty-eight human breast tumour specimens were studied with small-angle x-ray scattering (SAXS), and 10 of those were imaged by the diffraction enhanced x-ray imaging (DEI) technique. The sample diameter was 20 mm and the thickness 1 mm. Two examples of ductal carcinoma are illustrated by histology images, DEI, and maps of the collagen d-spacing and scattered intensity in the Porod regime, which characterize the SAXS patterns from collagen-rich regions of the samples. Histo-pathology reveals the cancer-invaded regions, and the maps of the SAXS parameters show that in these regions the scattering signal differs significantly from scattering by the surrounding tissue, indicating a degradation of the collagen structure in the invaded regions. The DEI images show the borders between collagen and adipose tissue and provide a co-ordinate system for tissue mapping by SAXS. In addition, degradation of the collagen structure in an invaded region is revealed by fading contrast of the DEI refraction image. The 28 samples include fresh, defrosted tissue and formalin-fixed tissue. The d-values with their standard deviations are given. In the fresh samples there is a systematic 0.76% increase of the d-value in the invaded regions, averaged over 11 samples. Only intra-sample comparisons are made for the formalin-fixed samples, and with a long fixation time, the difference in the d-value stabilizes at about 0.7%. The correspondence between the DEI images, the SAXS maps and the histo-pathology suggests that definitive information on tumour growth and malignancy is obtained by combining these x-ray methods.

Self-assembled polymeric solid films with temperature-induced large and reversible photonic-bandgap switching.

In aqueous solutions the response of polymers and biological matter to external conditions, such as temperature and pH, is typically based on the hydrophobic/hydrophilic balance and its effects on the polymer conformation. In the solid state, related concepts using competing interactions could allow novel functions. In this work we demonstrate that polymeric self-assembly, reversibility of hydrogen bonding, and polymer-additive phase behaviour allow temperature response in the solid state with large and reversible switching of an optical bandgap. A complex of polystyrene-block-poly(4-vinylpyridinium methanesulphonate) and 3-n-pentadecylphenol leads to the supramolecular comb-shaped architecture with a particularly long lamellar period. The sample is green at room temperature, as an incomplete photonic bandgap due to a dielectric reflector is formed. On heating, hydrogen bonds are broken and 3-n-pentadecylphenol additionally becomes soluble in polystyrene, leading to a sharp and reversible transition at approximately 125 degrees C to uncoloured material due to collapse of the long period. This encourages further developments, for example, for functional coatings or sensors in the solid state.

Structural hierarchy in molecular films of two class II hydrophobins.

Hydrophobins are highly surface-active proteins that are specific to filamentous fungi. They function as coatings on various fungal structures, enable aerial growth of hyphae, and facilitate attachment to surfaces. Little is known about their structures and structure-function relationships. In this work we show highly organized surface layers of hydrophobins, representing the most detailed structural study of hydrophobin films so far. Langmuir-Blodgett films of class II hydrophobins HFBI and HFBII from Trichoderma reesei were prepared and analyzed by atomic force microscopy. The films showed highly ordered two-dimensional crystalline structures. By combining our recent results on small-angle X-ray scattering of hydrophobin solutions, we found that the unit cells in the films have dimensions similar to those of tetrameric aggregates found in solutions. Further analysis leads to a model in which the building blocks of the two-dimensional crystals are shape-persistent supramolecules consisting of four hydrophobin molecules. The results also indicate functional and structural differences between HFBI and HFBII that help to explain differences in their properties. The possibility that the highly organized surface assemblies of hydrophobins could allow a route for manufacturing functional surfaces is suggested.

Aggregation and self-assembly of hydrophobins from Trichoderma reesei: low-resolution structural models.

Hydrophobins are secreted fungal proteins, which have diverse roles in fungal growth and development. They lower the surface tension of water, work as adhesive agents and coatings, and function through self-assembly. One of the characteristic properties of hydrophobins is their tendency to form fibrillar or rod-like aggregates at interfaces. Their structure is still poorly known. In a step to elucidate the structure/function relation of hydrophobin self-assembly, we present the low-resolution structure of self-assembled fibrils of the class II hydrophobin HFBII from Trichoderma reesei based on small and wide-angle x-ray scattering. We first studied the solution state (10 mg/mL) of both HFBI and HFBII and showed that they formed assemblages in aqueous solution, which have a radius of gyration of ~24 A and maximum dimension of ~65 A, corresponding to the size of a tetramer. This result was supported by size-exclusion chromatography. Undried samples of HFBII fibrils had a monoclinic crystalline structure, which changed to hexagonal when the material was dried. A low-resolution structure for the HFBII fibrils is suggested. There are data in the literature based on staining properties suggesting that hydrophobins of class I form assemblies with an amyloid structure. Comparison of the HFBII data (x-ray results, staining with thioflavin T) to published data showed that the HFBII assemblages are not amyloid.