Higher Extrusion Temperature Induces Greater Formation of Less Digestible Type V and Retrograded Starch in Iron-Fortified Rice Grains But Does Not Affect Iron Bioavailability: Stable Isotope Studies in Young Women.

BACKGROUND: Hot extrusion is widely used to produce iron-fortified rice, but heating may increase resistant starch and thereby decrease iron bioavailability. Cold-extruded iron-fortified rice may have higher bioavailability but has higher iron losses during cooking. Thus, warm extrusion could have nutritional benefits, but this has not been tested. Whether the addition of citric acid (CA) and trisodium citrate (TSC) counteracts any detrimental effect of high-extrusion temperature on iron bioavailability is unclear. OBJECTIVES: Our aim was to assess the effects of varying processing temperatures on the starch microstructure of extruded iron-fortified rice and resulting iron solubility and iron bioavailability. METHODS: We produced extruded iron-fortified rice grains at cold, warm, and hot temperatures (40°C, 70°C, and 90°C), with and without CA/TSC at a molar ratio of iron to CA/TSC of 1:0.3:5.5. We characterized starch microstructure using small- and wide-angle X-ray scattering and differential scanning calorimetry, assessed color over 6 mo, and measured in vitro iron solubility. In standardized rice and vegetable test meals consumed by young women (n = 22; mean age: 23 y; geometric mean plasma ferritin: 29.3 µg/L), we measured iron absorption from the fortified rice grains intrinsically labeled with 57ferric pyrophosphate (57FePP), compared with ferrous sulfate (58FeSO4) solution added extrinsically to the meals. RESULTS: Warm and hot extrusion altered starch morphology from native type A to type V and increased retrograded starch. However, extrusion temperature did not significantly affect iron solubility or iron bioavailability. The geometric mean fractional iron absorption of iron from fortified rice extruded with CA/TSC (8.2%; 95% CI: 7.9%, 11.0%) was more than twice that from extruded rice without CA/TSC (3.0%; 95% CI: 2.7%, 3.4%; P < 0.001). CONCLUSIONS: Higher extrusion temperatures did not affect iron bioavailability from extruded rice in young women, but co-extrusion of CA/TSC with FePP sharply increased iron absorption independently from extrusion temperature. This trial is registered at www.clinicaltrials.gov as NCT03703726.

Understanding the Formation of Apoferritin Amyloid Fibrils.

We present the optimization of experimental conditions to yield long, rigid apoferritin protein amyloid fibrils, as well as the corresponding fibrillation pathway. Fibril growth kinetics was followed using atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS), circular dichroism (CD), fourier-transform infrared spectroscopy (FTIR), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among the morphologies identified, we show that the conditions result in small aggregates, as well as medium and long fibrils. Extended incubation times led to progressive unfolding and hydrolysis of the proteins into very short peptide fragments. AFM, SDS-PAGE, and CD support a universal common fibrillation mechanism in which hydrolyzed fragments play the central role. These collective results provide convincing evidence that protein unfolding and complete hydrolysis of the proteins into very short peptide sequences are essential for the formation of the final apoferritin amyloid-like fibrils.

Water-processable, biodegradable and coatable aquaplastic from engineered biofilms.

Petrochemical-based plastics have not only contaminated all parts of the globe, but are also causing potentially irreversible damage to our ecosystem because of their non-biodegradability. As bioplastics are limited in number, there is an urgent need to design and develop more biodegradable alternatives to mitigate the plastic menace. In this regard, we report aquaplastic, a new class of microbial biofilm-based biodegradable bioplastic that is water-processable, robust, templatable and coatable. Here, Escherichia coli was genetically engineered to produce protein-based hydrogels, which are cast and dried under ambient conditions to produce aquaplastic, which can withstand strong acid/base and organic solvents. In addition, aquaplastic can be healed and welded to form three-dimensional architectures using water. The combination of straightforward microbial fabrication, water processability and biodegradability makes aquaplastic a unique material worthy of further exploration for packaging and coating applications.

Covalent ß-lactoglobulin-maltodextrin amyloid fibril conjugate prepared by the Maillard reaction.

The surface modification of ß-lactoglobulin amyloid fibrils (AFs) was investigated by performing the Maillard reaction with the free anomeric carbon of the maltodextrin in water at pH 9.0 and 90 °C. The bonding of maltodextrin to fibrils was confirmed by determining the free amino group content and the presence of final products from the Maillard reaction. The secondary structure of AFs was preserved as observed by circular dichroism analysis. Atomic force microscopy evidenced that prolonged heat treatment caused hydrolysis of the attached polysaccharide and consequently lowered the height of the fibrils from 8.0 nm (after 1 h) to 6.0 nm (after 24 h), which led to the reduction of hydrophilicity of resulting conjugate. Increasing the reaction time, however, resulted in the improvement of colloidal stability and decrease in turbidity ascribed to the increment of glycation degree, as well as, a decrease in the isoelectric point of the protein-based supramolecular object.

The hierarchical bulk molecular structure of poly(acrylamide) hydrogels: beyond the fishing net.

The polymeric structure of hydrogels is commonly presented in the literature as resembling a fishing net. However, this simple view cannot fully capture all the unique properties of these materials. Crucial for a detailed description of the bulk structure in free-radical polymerized vinylic hydrogels is a thorough understanding of the cross-linker distribution. This work focuses on the precise role of the tetra-functional cross-linker in the hydrogel system: acrylamide-N,N'-methylenebis(acrylamide). Clusters of crosslinker smaller than 4 nm and their agglomerates, as well as polymer domains with sizes from the 100 nm to the µm-range, have been identified by means of both X-ray and visible-light scattering. Placed in the context of the extensive literature on this system, these observations demonstrate the heterogeneous organisation of the polymer within the hydrogel network structure, and can be accounted for by the different polymerization behavior of the monomer and crosslinker. Together with polymer-network chain-length approximations based on swelling experiments and structural observations with scanning electron microscopy, these results indicate a hierarchical structure of the polymer network surrounding pockets of water.

Structure-property relationships of cellulose nanofibril hydro- and aerogels and their building blocks.

As abundant and renewable materials with excellent mechanical and functional properties, cellulose nanomaterials are utilized in advanced structural, optical and electronic applications. However, in order to further improve and develop new cellulose nanomaterials, a better understanding of the interplay between the self-assembled materials and their building blocks is crucial. This paper describes the structure-property relationships between cellulose nanofibrils (CNFs) and their resulting self-assembled structures in the form of hydrogels and aerogels. Rheological experiments revealed that the transition from viscous to elastic state with the corresponding evolution of the properties of the CNF dispersion depends on the aspect ratio and can be described in terms of the dynamic overlap concentration. The elastic shear modulus was dependent on the aspect ratio at very low CNF concentrations, reaching a plateau, where only the concentration of CNFs was relevant. This transition point in shear modulus was exploited to determine the mesh size of the fibril network, which was found to be in excellent agreement with predictions from scaling arguments. These findings highlight the possibility to tune the self-assembled materials response directly from the bottom-up by the CNF particle structure and thus, suggest new assembly routes starting directly from the CNF design.

Metal ions confinement defines the architecture of G-quartet, G-quadruplex fibrils and their assembly into nematic tactoids.

G-quadruplex, assembled from a square array of guanine (G) molecules, is an important structure with crucial biological roles in vivo but also a versatile template for ordered functional materials. Although the understanding of G-quadruplex structures is the focus of numerous studies, little is known regarding the control of G-quartet stacking modes and the spontaneous orientation of G-quadruplex fibrils. Here, the effects of different metal ions and their concentrations on stacking modes of G-quartets are elucidated. Monovalent cations (typically K+) facilitate the formation of G-quadruplex hydrogels with both heteropolar and homopolar stacking modes, showing weak mechanical strength. In contrast, divalent metal ions (Ca2+, Sr2+, and Ba2+) at given concentrations can control G-quartet stacking modes and increase the mechanical rigidity of the resulting hydrogels through ionic bridge effects between divalent ions and borate. We show that for Ca2+ and Ba2+ at suitable concentrations, the assembly of G-quadruplexes results in the establishment of a mesoscopic chirality of the fibrils with a regular left-handed twist. Finally, we report the discovery of nematic tactoids self-assembled from G-quadruplex fibrils characterized by homeotropic fibril alignment with respect to the interface. We use the Frank-Oseen elastic energy and the Rapini-Papoular anisotropic surface energy to rationalize two different configurations of the tactoids. These results deepen our understanding of G-quadruplex structures and G-quadruplex fibrils, paving the way for their use in self-assembly and biomaterials.

A Short Peptide Hydrogel with High Stiffness Induced by 310-Helices to ß-Sheet Transition in Water.

Biological gels generally require polymeric chains that produce long-lived physical entanglements. Low molecular weight colloids offer an alternative to macromolecular gels, but often require ad-hoc synthetic procedures. Here, a short biomimetic peptide composed of eight amino acid residues derived from squid sucker ring teeth proteins is demonstrated to form hydrogel in water without any cross-linking agent or chemical modification and exhibits a stiffness on par with the stiffest peptide hydrogels. Combining solution and solid-state NMR, circular dichroism, infrared spectroscopy, and X-ray scattering, the peptide is shown to form a supramolecular, semiflexible gel assembled from unusual right-handed 310-helices stabilized in solution by π-π stacking. During gelation, the 310-helices undergo conformational transition into antiparallel ß-sheets with formation of new interpeptide hydrophobic interactions, and molecular dynamic simulations corroborate stabilization by cross ß-sheet oligomerization. The current study broadens the range of secondary structures available to create supramolecular hydrogels, and introduces 310-helices as transient building blocks for gelation via a 310-to-ß-sheet conformational transition.

Structural Transformation in Vesicles upon Hydrolysis of Phosphatidylethanolamine and Phosphatidylcholine with Phospholipase C.

This study provides insights into dynamic nanostructural changes in phospholipid systems during hydrolysis with phospholipase C, the fate of the hydrolysis products, and the kinetics of lipolysis. The effect of lipid restructuring of the vesicle was investigated using small-angle X-ray scattering and cryogenic scanning electron microscopy. The rate and extent of phospholipid hydrolysis were quantified using nuclear magnetic resonance. Hydrolysis of two phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), results in the cleavage of the molecular headgroup, causing two strikingly different changes in lipid self-assembly. The diacylglycerol product of PC escapes the lipid bilayer, whereas the diacylglycerol product adopts a different configuration within the lipid bilayer of the PE vesicles. These results are then discussed concerning the change of the lipid configuration upon the lipid membrane and its potential implications in vivo, which is of significant importance for the detailed understanding of the fate of lipidic particles and the rational design of enzyme-responsive lipid-based drug delivery systems.

Can one determine the density of an individual synthetic macromolecule?

Dendronized polymers (DPs) are large and compact main-chain linear polymers with a cylindrical shape and cross-sectional diameters of up to ∼15 nm. They are therefore considered molecular objects, and it was of interest whether given their experimentally accessible, well-defined dimensions, the density of individual DPs could be determined. We present measurements on individual, deposited DP chains, providing molecular dimensions from scanning and transmission electron microscopy and mass-per-length values from quantitative scanning transmission electron microscopy. These results are compared with density values obtained from small-angle X-ray scattering on annealed bulk specimen and with classical envelope density measurements, obtained using hydrostatic weighing or a density gradient column. The samples investigated comprise a series of DPs with side groups of dendritic generations g = 1-8. The key findings are a very large spread of the density values over all samples and methods, and a consistent increase of densities with g over all methods. While this work highlights the advantages and limitations of the applied methods, it does not provide a conclusive answer to the question of which method(s) to use for the determination of densities of individual molecular objects. We are nevertheless confident that these first attempts to answer this challenging question will stimulate more research into this important aspect of polymer and soft matter science.

Primary, Secondary, Tertiary and Quaternary Structure Levels in Linear Polysaccharides: From Random Coil, to Single Helix to Supramolecular Assembly.

Polysaccharides are ubiquitous in nature and represent an essential class of biopolymers with multiple levels of conformation and structural hierarchy. However, a standardized structural nomenclature, as in the case of proteins, is still lacking due to uncertainty on their hierarchical organization. In this work we use carrageenans as model polysaccharides to demonstrate that several structural levels exist and can be unambiguously resolved by statistical analysis on high resolution Atomic Force Microscopy images, supported by spectroscopic, X-ray scattering and rheological techniques. In direct analogy with proteins, we identify primary, secondary, tertiary and quaternary structures. The structure-property relationship induced by monovalent ions for κ-, ι- and the non-gelling control λ-carrageenan is established from the single chain regime to the occurrence of hydrogels at higher concentrations. For κ-carrageenan in the presence of potassium, a disorder-order transition from random coil to single helix is first observed (secondary structure), followed by intrachain supercoiling events (tertiary structure) and macroscopic anisotropic domains which are parts of a network (quaternary structure) with tunable elasticity up to ∼103 Pa. In contrast, κ-carrageenan in the presence of sodium only produces changes in secondary structure without supercoiling events, prior to formation of gels, highlighting the ion-specificity of the process. Loosely intertwined single helices are observed for ι-carrageenan in the presence of sodium and potassium chloride, providing an elastic mesh with many junction zones, while λ-carrageenan does not undergo any structural change. A generality of the observed behavior may be inferred by extending these observations to a distinct class of polysaccharides, the weak carboxylic polyelectrolyte Gellan gum. These results advance our understanding of ion-specific structural changes of polysaccharides and the physical mechanisms responsible for their gelation.

Ion-Induced Formation of Nanocrystalline Cellulose Colloidal Glasses Containing Nematic Domains.

Controlling the assembly of colloids in dispersion is a fundamental approach toward the production of functional materials. Nanocrystalline cellulose (NCC) is a charged nanoparticle whose colloidal interactions can be modulated from repulsive to attractive by increasing ionic strength. Here, we combine polarized optical microscopy, rheology, and small-angle scattering techniques to investigate (i) the concentration-driven transition from isotropic dispersion to cholesteric liquid crystals and (ii) salt-induced NCC phase transitions. In particular, we report on the formation of NCC attractive glasses containing nematic domains. At increasing NCC concentration, a structure peak was observed in small-angle X-ray scattering (SAXS) patterns. The evolution of the structure peak demonstrates the decrease in NCC interparticle distance, favoring orientational order during the isotropic-cholesteric phase transition. Small amounts of salt reduce the cholesteric volume fraction and pitch by a decrease in excluded volume. Beyond a critical salt concentration, NCC forms attractive glasses due to particle caging and reduced motility. This results in a sharp increase in viscosity and formation of viscoelastic glasses. The presence of nematic domains is suggested by the appearance of interference colors and the Cox-Merz rule failure and was confirmed by an anisotropic SAXS scattering pattern at q ranges associated with the presence of nematic domains. Thus, salt addition allows the formation of NCC attractive glasses with mechanical properties similar to those of gels while remaining optically active owed to entrapped nematic domains.

Nanostructural Properties and Twist Periodicity of Cellulose Nanofibrils with Variable Charge Density.

Cellulose nanofibrils (CNFs) are a renewable and facile to produce nanomaterial that recently gained a lot of attention in soft material research. The nanostructural properties of the fibrils largely determine their self-organizing functionalities, and the ability to tune the CNF nanostructure through control of the processing parameters is therefore crucial for developing new applications. In this study, we systematically altered the CNF production parameters (i.e., variation in cellulose source, chemical, and mechanical treatment) to observe their impact on the nanostructural properties of the resulting fibrils. Atomic force microscopy (AFM) allowed detailed topological examination of individual CNFs to elucidate fibril properties such as contour length, kink distribution and the right-handed twist periodicity of individual fibrils. Statistical analysis revealed a large dependency of the fibril properties on the industrial treatment of the cellulose source material. Our results furthermore confirm that the average charge density of the fibrils regulates both contour length and twist periodicity and, thus, has a very strong impact on the final morphology of CNFs. These results provide a route to tune the detailed nanostructure of CNFs with potential impact on the self-organization of these biological colloids and their optimal use in new nanomaterials.

Apoferritin Protein Amyloid Fibrils with Tunable Chirality and Polymorphism.

Ferritin, a soluble and highly robust protein with subunits packed into well-defined helices, is a key component of the iron regulatory system in the brain and thus is widely recognized as a crucial protein for iron metabolism, but may also bear possible implications in some neurodegenerative disorders. Here, we present evidence of how human recombinant apoferritin can convert into an unusual structure from its folded native state; that is, amyloid fibrils analogue to those found in pathological disorders such as Alzheimer's and Parkinson's diseases. An extensive combination of advanced microscopy, spectroscopy and scattering techniques concur to reveal that apoferritin fibrils possess a common double stranded twisted ribbon structure which can result in a mesoscopic right-handed chirality. We highlight a direct connection between the chirality and morphology of the resulting amyloid fibrils, and the initial protein subunits composition, advancing our understanding on the possible role of misfolding in some ferritin-related pathologies and posing new bases for the design of chiral 1D functional nanostructures.

Cooperative Induction of Ordered Peptide and Fatty Acid Aggregates.

Interactions between biological membranes and disease-associated amyloids are well documented, and their prevalence suggests that an inherent affinity exists between these molecular assemblies. Our interest in the molecular origins of life have led us to investigate the nature of such interactions in the context of their molecular predecessors (i.e., vesicle-forming amphiphiles and small peptides). Under certain conditions, amyloidogenic peptides or fatty acids are each able to form ordered structures on their own; however, we report here on their cooperative assembly into novel, to our knowledge, highly ordered structures. We first examined an amyloidogenic eight-residue peptide, which forms amyloids at pH 11, yet because of its positive electrostatic character remains soluble at a neutral pH. In mixtures with simple fatty acids, this peptide is also able to form novel, to our knowledge, coaggregates at a neutral pH whose structures are sensitive to both the fatty acid concentration and identity. Below the critical vesicle concentration, the mixtures of fatty acid and peptide yield a flocculent precipitate with an underlying ß-structure. Above the critical vesicle concentration, the mixtures yield a translucent precipitate that consists of tube-like structures. Small-angle x-ray scattering and fiber diffraction data were used to model their structures as hollow-core two-shell cylinders in which the inner shell is a bilayer of fatty acid and the outer shell alternates between amyloid and bilayers of fatty acid. The further analysis of decanoic acid with a panel of 13 other basic amyloidogenic peptides confirmed the general nature of the observed interactions. The cooperativity within this heterogeneous system is attributed to the structurally repetitive natures of the fatty acid bilayer and the cross-ß-sheet motif, providing compatible scaffolds for attractive electrostatic interactions. We show these interactions to be mutually beneficial, expanding the phase space of both peptides and fatty acids while providing a simple yet robust physical connection between two distinct entities relevant for life.

Interplay between structure and relaxation in polyurea networks: the point of view from a novel method of cooperativity analysis of dielectric response.

The influence of structural constraints on the relaxation dynamics of three polyurea networks with a varying degree of crosslinking, has been studied by means of a thorough analysis of broadband dielectric spectroscopy measurements. Two different relaxation processes are observed, namely, a fast process involving the soft poly(propylene oxide) chains, and a slower and much broader process associated with the immediate surroundings of the hard crosslinkers. Microphase separation in soft and hard domains characterizes the systems in the presence of hydrogen bonding. In this case, different confinement conditions are explored by varying the soft chain length; overall, so called "adsorption" effects dominate. With respect to both cooperativity and the rearrangement energy threshold in fast relaxation, it is found that the enhancement of configurational constraints is similar to cooling, but only on qualitative grounds. An upper bound of the hard domains' interface thickness, in which the slow relaxation is believed to take place, is estimated from the analysis of the fast relaxation in the system characterized by the highest degree of confinement, taking into account the results of the structural analysis. Dropping the hydrogen bonding mechanism, phase separation does not occur anymore and the configurational constraints at the ends of the soft chains are reduced, leaving just those imposed by the rigid crosslinkers. This leads to a significant increase in cooperativity on approaching the glass transition, and to a complex behavior that is thoroughly discussed in comparison with those observed in the micro-segregated systems.

Controlling Supramolecular Chiral Nanostructures by Self-Assembly of a Biomimetic ß-Sheet-Rich Amyloidogenic Peptide.

Squid sucker ring teeth (SRT) have emerged as a promising protein-only, thermoplastic biopolymer with an increasing number of biomedical and engineering applications demonstrated in recent years. SRT is a supra-molecular network whereby a flexible, amorphous matrix is mechanically reinforced by nanoconfined ß-sheets. The building blocks for the SRT network are a family of suckerin proteins that share a common block copolymer architecture consisting of amorphous domains intervened by smaller, ß-sheet forming modules. Recent studies have identified the peptide A1H1 (peptide sequence AATAVSHTTHHA) as one of the most abundant ß-sheet forming domains within the suckerin protein family. However, we still have little understanding of the assembly mechanisms by which the A1H1 peptide may assemble into its functional load-bearing domains. In this study, we conduct a detailed self-assembly study of A1H1 and show that the peptide undergoes ß-strands-driven elongation into amyloid-like fibrils with a rich polymorphism. The nanostructure of the fibrils was elucidated by small and wide-angle X-ray scattering (SAXS and WAXS) and atomic force microscopy (AFM). The presence of His-rich and Ala-rich segments results in an amphiphilic behavior and drives its assembly into fibrillar supramolecular chiral aggregates with helical ribbon configuration in solution, with the His-rich region exposed to the solvent molecules. Upon increase in concentration, the fibrils undergo gel formation, while preserving the same mesoscopic features. This complex phase behavior suggests that the repeat peptide modules of suckerins may be manipulated beyond their native biological environment to produce a wider variety of self-assembled amyloid-like nanostructures.

In Vivo Mitigation of Amyloidogenesis through Functional-Pathogenic Double-Protein Coronae.

Amyloid diseases are global epidemics with no cure available. Herein, we report a first demonstration of in vivo mitigation of amyloidogenesis using biomimetic nanotechnology. Specifically, the amyloid fragments (ba) of ß-lactoglobulin, a whey protein, were deposited onto the surfaces of carbon nanotubes (baCNT), which subsequently sequestered human islet amyloid polypeptide (IAPP) through functional-pathogenic double-protein coronae. Conformational changes at the ba-IAPP interface were studied by Fourier transform infrared, circular dichroism, and X-ray scattering spectroscopies. baCNT eliminated the toxic IAPP species from zebrafish embryos, as evidenced by the assays of embryonic development, cell morphology, hatching, and survival as well as suppression of oxidative stress. In addition to IAPP, baCNT also displayed high potency against the toxicity of amyloid-ß, thereby demonstrating the broad applicability of this biomimetic nanotechnology and the use of an embryonic zebrafish model for the high-throughput screening of a range of amyloidogenesis and their inhibitors in vivo.

Motorizing fibres with geometric zero-energy modes.

Responsive materials1-3 have been used to generate structures with built-in complex geometries4-6, linear actuators7-9 and microswimmers10-12. These results suggest that complex, fully functional machines composed solely from shape-changing materials might be possible 13 . Nonetheless, to accomplish rotary motion in these materials still relies on the classical wheel and axle motifs. Here we explore geometric zero-energy modes to elicit rotary motion in elastic materials in the absence of a rigid wheel travelling around an axle. We show that prestrained polymer fibres closed into rings exhibit self-actuation and continuous motion when placed between two heat baths due to elastic deformations that arise from rotational-symmetry breaking around the rod's axis. Our findings illustrate a simple but robust model to create active motion in mechanically prestrained objects.

Squid Suckerin Biomimetic Peptides Form Amyloid-like Crystals with Robust Mechanical Properties.

We present the self-assembly of fibers formed from a peptide sequence (A1H1) derived from suckerin proteins of squid sucker ring teeth (SRT). SRT are protein-only biopolymers with an unconventional set of physicochemical and mechanical properties including high elastic modulus coupled with thermoplastic behavior. We have identified a conserved peptide building block from suckerins that possess the ability to assemble into materials with similar mechanical properties as the native SRT. A1H1 displays amphiphilic characteristics and self-assembles from the bottom-up into mm-scale fibers initiated by the addition of a polar aprotic solvent. A1H1 fibers are thermally resistant up to 239 °C, coupled with an elastic modulus of ∼7.7 GPa, which can be explained by the tight packing of ß-sheet-enriched crystalline building blocks as identified by wide-angle X-ray scattering (WAXS), with intersheet and interstrand distances of 5.37 and 4.38 Å, respectively. A compact packing of the peptides at their Ala-rich terminals within the fibers was confirmed from molecular dynamics simulations, and we propose a hierarchical model of fiber assembly of the mature peptide fiber.