Preparation of Well-Defined Fluorescent Nanoplastic Particles by Confined Impinging Jet Mixing.

Research on the origin, distribution, detection, identification, and quantification of polymer nanoparticles (NPs) in the environment and their possible impact on animal and human health is surging. For different types of studies in this field, well-defined reference materials or mimics are needed. While isolated reports on the preparation of such materials are available, a simple and broadly applicable method that allows for the production of different NP types with well-defined, tailorable characteristics is still missing. Here, we demonstrate that a confined impinging jet mixing process can be used to prepare colloidally stable NPs based on polystyrene, polyethylene, polypropylene, and poly(ethylene terephthalate) with diameters below < 100 nm. Different fluorophores were incorporated into the NPs, to allow their detection in complex environments. To demonstrate their utility and detectability, fluorescent NPs were exposed to J774A.1 macrophages and visualized using laser scanning microscopy. Furthermore, we modified the NPs in a postfabrication process and changed their shape from spherical to heterogeneous geometries, in order to mimic environmentally relevant morphologies. The methodology used here should be readily applicable to other polymers and payloads and thus a broad range of NPs that enable studies of their behavior, uptake, translocation, and biological end points in different systems.

Nanoscale clustering by O-antigen-Secretory Immunoglobulin-A binding limits outer membrane diffusion by encaging individual Salmonella cells.

Secreted immunoglobulins, predominantly SIgA, influence the colonization and pathogenicity of mucosal bacteria. While part of this effect can be explained by SIgA-mediated bacterial aggregation, we have an incomplete picture of how SIgA binding influences cells independently of aggregation. Here we show that akin to microscale crosslinking of cells, SIgA targeting the Salmonella Typhimurium O-antigen extensively crosslinks the O-antigens on the surface of individual bacterial cells at the nanoscale. This crosslinking results in an essentially immobilized bacterial outer membrane. Membrane immobilization, combined with Bam-complex mediated outer membrane protein insertion results in biased inheritance of IgA-bound O-antigen, concentrating SIgA-bound O-antigen at the oldest poles during cell growth. By combining empirical measurements and simulations, we show that this SIgA-driven biased inheritance increases the rate at which phase-varied daughter cells become IgA-free: a process that can accelerate IgA escape via phase-variation of O-antigen structure. Our results show that O-antigen-crosslinking by SIgA impacts workings of the bacterial outer membrane, helping to mechanistically explain how SIgA may exert aggregation-independent effects on individual microbes colonizing the mucosae.

Bactericidal and Antiviral Bionic Metalized Nanocoatings.

In diverse living organisms, bionanocoatings provide multiple functionalities, to the surfaces they cover. We have, previously, identified the molecular mechanisms of Turing-based self-assembly of insect corneal nanocoatings and developed forward-engineering approaches to construct multifunctional soft bionic nanocoatings, encompassing the Drosophila protein Retinin. Here, we expand the versatility of the bionic nanocoatings, by identifying and using diverse Retinin-like proteins and different methods of their metallization, using nickel, silver, and copper ions. Comparative assessment, of the resulting bactericidal, antiviral, and cytotoxic properties, identifies the best protocols, to construct safe and anti-infective metalized bionic nanocoatings. Upscaled application of these protocols, to various public surfaces, may represent a safe and economic approach to limit hazardous infections.

Liquid Crystalline Properties of Symmetric and Asymmetric End-Grafted Cellulose Nanocrystals.

The hydrophilic polymer poly[2-(2-(2-methoxy ethoxy)ethoxy)ethylacrylate] (POEG3A) was grafted onto the reducing end-groups (REGs) of cellulose nanocrystal (CNC) allomorphs, and their liquid crystalline properties were investigated. The REGs on CNCs extracted from cellulose I (CNC-I) are exclusively located at one end of the crystallite, whereas CNCs extracted from cellulose II (CNC-II) feature REGs at both ends of the crystallite, so that grafting from the REGs affords asymmetrically and symmetrically decorated CNCs, respectively. To confirm the REG modification, several complementary analytical techniques were applied. The grafting of POEG3A onto the CNC REGs was evidenced by Fourier transform infrared spectroscopy, atomic force microscopy, and the coil-globule conformational transition of this polymer above 60 °C, i.e., its lower critical solution temperature. Furthermore, we investigated the self-assembly of end-tethered CNC-hybrids into chiral nematic liquid crystalline phases. Above a critical concentration, both end-grafted CNC allomorphs form chiral nematic tactoids. The introduction of POEG3A to CNC-I does not disturb the surface of the CNCs along the rods, allowing the modified CNCs to approach each other and form helicoidal textures. End-grafted CNC-II formed chiral nematic tactoids with a pitch observable by polarized optical microscopy. This is likely due to their increase in hydrodynamic radius or the introduced steric stabilization of the end-grafted polymer.

Different Folding States from the Same Protein Sequence Determine Reversible vs Irreversible Amyloid Fate.

The propensity to self-assemble into amyloid fibrils with a shared cross-ß architecture is a generic feature of proteins. Amyloid-related diseases affect millions of people worldwide, yet they are incurable and cannot be effectively prevented, largely due to the irreversible assembly and extraordinary stability of amyloid fibrils. Recent studies suggest that labile amyloids may be possible in certain proteins containing low-complexity domains often involved in the formation of subcellular membraneless organelles. Although the fundamental understanding of this reversible amyloid folding process is completely missing, the current view is that a given protein sequence will result in either irreversible, as in most of the cases, or reversible amyloid fibrils, as in few exceptions. Here we show that two common globular proteins, human lysozyme and its homologue from hen egg white, can self-assemble into both reversible and irreversible amyloid fibrils depending on the folding path followed by the protein. In both folding states, the amyloid nature of the fibrils is demonstrated at the molecular level by its cross-ß structure, yet with substantial differences on the mesoscopic polymorphism and the labile nature of the amyloid state. Structural analysis shows that reversible and irreversible amyloid fibrils possess the same full-length protein sequence but different fibril core structures and ß-sheet arrangements. These results illuminate a mechanistic link between the reversible and irreversible nature of amyloids and highlight the central role of protein folding states in regulating the lability and reversibility of amyloids.

A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants.

The ability of gut bacterial pathogens to escape immunity by antigenic variation-particularly via changes to surface-exposed antigens-is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

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.

Engineering of biofilms with a glycosylation circuit for biomaterial applications.

Glycosylation is a crucial post-translational modification for a wide range of functionalities. Adhesive protein-based biomaterials in nature rely on heavily glycosylated proteins such as spider silk and mussel adhesive proteins. Engineering protein-based biomaterials genetically enables desired functions and characteristics. Additionally, utilization of glycosylation for biomaterial engineering can expand possibilities by including saccharides to the inventory of building blocks. Here, de novo glycosylation of Bacillus subtilis amyloid-like biofilm protein TasA using a Campylobacter jejuni glycosylation circuit is proposed to be a novel biomaterial engineering method for increasing adhesiveness of TasA fibrils. A C. jejuni glycosylation motif is genetically incorporated to tasA gene and expressed in Escherichia coli containing the C. jejuni pgl protein glycosylation pathway. Glycosylated TasA fibrils indicate enhanced adsorption on the gold surface without disruption of fibril formation. Our findings suggest that N-linked glycosylation can be a promising tool for engineering protein-based biomaterials specifically regarding adhesion.

Evolution of Conformation, Nanomechanics, and Infrared Nanospectroscopy of Single Amyloid Fibrils Converting into Microcrystals.

Nanomechanical properties of amyloid fibrils and nanocrystals depend on their secondary and quaternary structure, and the geometry of intermolecular hydrogen bonds. Advanced imaging methods based on atomic force microscopy (AFM) have unravelled the morphological and mechanical heterogeneity of amyloids, however a full understanding has been hampered by the limited resolution of conventional spectroscopic methods. Here, it is shown that single molecule nanomechanical mapping and infrared nanospectroscopy (AFM-IR) in combination with atomistic modelling enable unravelling at the single aggregate scale of the morphological, nanomechanical, chemical, and structural transition from amyloid fibrils to amyloid microcrystals in the hexapeptides, ILQINS, IFQINS, and TFQINS. Different morphologies have different Young's moduli, within 2-6 GPa, with amyloid fibrils exhibiting lower Young's moduli compared to amyloid microcrystals. The origins of this stiffening are unravelled and related to the increased content of intermolecular ß-sheet and the increased lengthscale of cooperativity following the transition from twisted fibril to flat nanocrystal. Increased stiffness in Young's moduli is correlated with increased density of intermolecular hydrogen bonding and parallel ß-sheet structure, which energetically stabilize crystals over the other polymorphs. These results offer additional evidence for the position of amyloid crystals in the minimum of the protein folding and aggregation landscape.

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.

Human neuropeptide substance P self-assembles into semi-flexible nanotubes that can be manipulated for nanotechnology.

Substance P neuropeptide is here reported to self-assemble into well-defined semi-flexible nanotubes. Using a blend of synchrotron small angle X-ray scattering, atomic force microscopy and other biophysical techniques, the natural peptide is shown to self-assemble into monodisperse 6 nm wide nanotubes, which can closely associate into nano-arrays with nematic properties. Using simple protocols, the nanotubes could be precipitated or mineralised while conserving their dimensions and core-shell morphology. Our discovery expands the small number of available monodisperse peptide nanotube systems for nanotechnology, beyond direct relevance to biologically functional peptide nanostructures since the substance P nanotubes are fundamentally different from typical amyloid fibrils.

Accelerated Amyloid Beta Pathogenesis by Bacterial Amyloid FapC.

The gut-brain axis has attracted increasing attention in recent years, fueled by accumulating symptomatic, physiological, and pathological findings. In this study, the aggregation and toxicity of amyloid beta (Aß), the pathogenic peptide associated with Alzheimer's disease (AD), seeded by FapC amyloid fragments (FapCS) of Pseudomonas aeruginosa that colonizes the gut microbiome through infections are examined. FapCS display favorable binding with Aß and a catalytic capacity in seeding the peptide amyloidosis. Upon seeding, twisted Aß fibrils assume a much-shortened periodicity approximating that of FapC fibrils, accompanied by a 37% sharp rise in the fibrillar diameter, compared with the control. The robust seeding capacity for Aß by FapCS and the biofilm fragments derived from P. aeruginosa entail abnormal behavior pathology and immunohistology, as well as impaired cognitive function of zebrafish. Together, the data offer the first concrete evidence of structural integration and inheritance in peptide cross-seeding, a crucial knowledge gap in understanding the pathological correlations between different amyloid diseases. The catalytic role of infectious bacteria in promoting Aß amyloidosis may be exploited as a potential therapeutic target, while the altered mesoscopic signatures of Aß fibrils may serve as a prototype for molecular assembly and a biomarker for screening bacterial infections in AD.

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.

Time-temperature-resolved functional and structural changes of phycocyanin extracted from Arthrospira platensis/Spirulina.

Arthrospira platensis, commonly known as Spirulina, gains increasing importance as alternative protein source for food production and biotechnological systems. A promising area is functional high-value algae extracts, rich in phycocyanin, a protein-pigment complex derived from A. platensis. This complex has proven functionality as the only natural blue colorant, fluorescent marker and therapeutic agent. The structure-function relationship is heat sensitive, making thermal processing in its production and its subsequent application a crucial aspect. In continuous high-temperature short-time treatments, it was shown how a purified phycocyanin (mixture of allophycocyanin and c-phycocyanin) disassembled and denatured between 50 and 70 °C. Three characteristic transition temperatures were allocated to specific quaternary aggregates. In contrast to sequential chemical denaturation, phycocyanin's chromophore and protein structure were simultaneously affected by thermal processing. Through a functionality assessment, the findings help optimize the efficiency of raw material usage by defining a processing window, enabling targeted process control resulting in desired product properties.

Formation of Higher Structural Levels in λ-Carrageenan Induced by the Antimalarial Drug Chloroquine.

The linear polysaccharide λ-carrageenan is the only one among the carrageenans not forming secondary, tertiary, and quaternary structures in the presence of inorganic ions. Chloroquine (CQ) is a well-established antimalaria drug also recently discussed in therapeutics against the COVID-19 pandemic. The interaction of this polysaccharide-ionic drug pair was investigated by combining UV-vis spectrophotometry and atomic force microscopy (AFM) imaging. A decrease of the UV peak assigned to free CQ and the occurrence of isosbestic points indicate the formation of complexes. High-resolution AFM height images revealed an increasing height of the single polysaccharide chains in the random coil state upon addition of CQ, indicating the formation of a secondary structure, followed by higher hierarchical aggregates. The disappearance of higher-ordered structures and the recovery of polysaccharide chains with primary structure were observed by introducing inorganic cations (Na+, K+, Ca2+), replacing the condensed CQ and paving the way to reversible ion-induced drug release.

Rigid, Fibrillar Quaternary Structures Induced by Divalent Ions in a Carboxylated Linear Polysaccharide.

Polysaccharides are ubiquitous in nature; they serve fundamental roles in vivo and are used for a multitude of food, pharmaceutical, cosmetic biomaterials, and biomedical applications. Here, the structure-property function for low acetylated Gellan gum hydrogels induced by divalent ions was established by means of optical, rheological, and microscopic techniques. The hydrogels interacted with visible light as revealed by birefringence and multiple scattering, as a consequence of quaternary, supramolecular fibrillar structures. The molecular assembly and structure were elucidated by statistical analysis and polymer physics concepts applied to high-resolution AFM height images and further supported by FTIR. This revealed intramolecular coil-to-single helix transitions, followed by lateral aggregation of single helices into rigid, fibrillar quaternary structures, ultimately responsible for gelation of the system. Calcium and magnesium chloride were shown to lead to fibrils up to heights of 6.0 nm and persistence lengths of several micrometers. The change in molecular structure affected the macroscopic gel stiffness, with the plateau shear modulus reaching ∼105 Pa. These results shed light on the two-step gelation mechanism of linear polysaccharides, their conformational molecular changes at the single polymer level and ultimately the macroscale properties of the ensued gels.

Structure and dynamics of hagfish mucin in different saline environments.

The defense mechanism of hagfish against predators is based on its ability to form slime within a few milliseconds. Hagfish slime consists of two main components, namely mucin-like glycoproteins and long protein threads, which together entrap vast amounts of water and thus form a highly dilute hydrogel. Here, we investigate the mucin part of this hydrogel, in particular the role of the saline marine environment on the viscoelasticity and structure. By means of dynamic light scattering (DLS), shear and extensional rheology we probe the diffusion dynamics, the flow behavior, and the longest filament breaking time of hagfish mucin solutions. Using DLS we find a concentration-independent diffusion coefficient - characteristic for polyelectrolytes - up to the entanglement regime of 0.2 mg ml-1, which is about ten times higher than the natural concentration of hagfish mucin in hagfish slime. We also observe a slow relaxation process associated with clustering, probably due to electrostatic interactions. Shear rheology further revealed that hagfish mucin possesses pronounced viscoelastic properties at high concentrations (3 mg ml-1), showing that mucin alone achieves mechanical properties similar to those of natural hagfish slime (mucins and protein threads). The main effects of added seawater salts, and predominantly CaCl2 is to reduce the intensity of the slow relaxation process, which suggests that calcium ions lead to an ionotropic gelation of hagfish mucins.

Stable Immobilization of Enzymes in a Macro- and Mesoporous Silica Monolith.

Horseradish peroxidase isoenzyme C (HRP) and Engyodontium album proteinase K (proK) were immobilized inside macro- and mesoporous silica monoliths. Stable immobilization was achieved through simple noncovalent adsorption of conjugates, which were prepared from a polycationic, water-soluble second generation dendronized polymer (denpol) and the enzymes. Conjugates prepared from three denpols with the same type of repeating unit (r.u.), but different average lengths were compared. It was shown that there is no obvious advantage of using denpols with very long chains. Excellent results were achieved with denpols having on average 750 or 1000 r.u. The enzyme-loaded monoliths were tested as flow reactors. Comparison was made with microscopy glass coverslips onto which the conjugates were immobilized and with glass micropipettes containing adsorbed conjugates. High enzyme loading was achieved using the monoliths. Monoliths containing immobilized denpol-HRP conjugates exhibited good operational stability at 25 °C (for at least several hours), and good storage stability at 4 °C (at least for weeks) was demonstrated. Such HRP-containing monoliths were applied as continuous flow reactors for the quantitative determination of hydrogen peroxide in aqueous solution between 1 µM (34 ng/mL) and 50 µM (1.7 µg/mL). Although many methods for immobilizing enzymes on silica surfaces exist, there are only a few approaches with porous silica materials for the development of flow reactors. The work presented is a promising contribution to this field of research toward bioanalytical and biosynthetic applications.