Cation Triggered Self-Assembly of α-Lactalbumin Nanotubes.

Metal ions play a dual role in biological systems. Although they actively participate in vital life processes, they may contribute to protein aggregation and misfolding and thus contribute to development of diseases and other pathologies. In nanofabrication, metal ions mediate the formation of nanostructures with diverse properties. Here, we investigated the self-assembly of α-lactalbumin into nanotubes induced by coordination with metal ions, screened among the series Mn2+, Co2+, Ni2+, Zn2+, Cd2+, and Au3+. Our results revealed that the affinity of metal ions toward hydrolyzed α-lactalbumin peptides not only impacts the kinetics of nanotube formation but also influences their length and rigidity. These findings expand our understanding of supramolecular assembly processes in protein-based materials and pave the way for designing novel materials such as metallogels in biochip and biosensor applications.

Surface Cross-Linking by Macromolecular Tethers Enhances Virus-like Particles' Resilience to Mucosal Stress Factors.

Virus-like particles (VLPs) are emerging as nanoscaffolds in a variety of biomedical applications including delivery of vaccine antigens and cargo such as mRNA to mucosal surfaces. These soft, colloidal, and proteinaceous structures (capsids) are nevertheless susceptible to mucosal environmental stress factors. We cross-linked multiple capsid surface amino acid residues using homobifunctional polyethylene glycol tethers to improve the persistence and survival of the capsid to model mucosal stressors. Surface cross-linking enhanced the stability of VLPs assembled from Acinetobacter phage AP205 coat proteins in low pH (down to pH 4.0) and high protease concentration conditions (namely, in pig and mouse gastric fluids). Additionally, it increased the stiffness of VLPs under local mechanical indentation applied using an atomic force microscopy cantilever tip. Small angle X-ray scattering revealed an increase in capsid diameter after cross-linking and an increase in capsid shell thickness with the length of the PEG cross-linkers. Moreover, surface cross-linking had no effect on the VLPs' mucus translocation and accumulation on the epithelium of in vitro 3D human nasal epithelial tissues with mucociliary clearance. Finally, it did not compromise VLPs' function as vaccines in mouse subcutaneous vaccination models. Compared to PEGylation without cross-linking, the stiffness of surface cross-linked VLPs were higher for the same length of the PEG molecule, and also the lifetimes of surface cross-linked VLPs were longer in the gastric fluids. Surface cross-linking using macromolecular tethers, but not simple conjugation of these molecules, thus offers a viable means to enhance the resilience and survival of VLPs for mucosal applications.

Food amyloid fibrils are safe nutrition ingredients based on in-vitro and in-vivo assessment.

Food protein amyloid fibrils have superior technological, nutritional, sensorial, and physical properties compared to native monomers, but there is as yet insufficient understanding of their digestive fate and safety for wide consumption. By combining SDS-PAGE, ELISA, fluorescence, AFM, MALDI-MS, CD, microfluidics, and SAXS techniques for the characterization of ß-lactoglobulin and lysozyme amyloid fibrils subjected to in-vitro gastrointestinal digestion, here we show that either no noticeable conformational differences exist between amyloid aggregates and their monomer counterparts after the gastrointestinal digestion process (as in ß-lactoglobulin), or that amyloid fibrils are digested significantly better than monomers (as in lysozyme). Moreover, in-vitro exposure of human cell lines and in-vivo studies with C. elegans and mouse models, indicate that the digested fibrils present no observable cytotoxicity, physiological abnormalities in health-span, nor accumulation of fibril-induced plaques in brain nor other organs. These extensive in-vitro and in-vivo studies together suggest that the digested food amyloids are at least equally as safe as those obtained from the digestion of corresponding native monomers, pointing to food amyloid fibrils as potential ingredients for human nutrition.

Ion-Induced Reassembly between Protein Nanotubes and Nanospheres.

Proteins used as building blocks to template nanostructures with manifold morphologies have been widely reported. Understanding their self-assembly and reassembly mechanism is important for designing functional biomaterials. Herein, we show that enzyme-hydrolyzed α-lactalbumin (α-lac) can self-assemble into either nanotubes in the presence of Ca2+ ions or nanospheres in the absence of Ca2+ in solution. Remarkably, such assembled α-lac nanotubes can be elongated by adding preassembled α-lac nanospheres and Ca2+ solution, which suggests that the self-assembled α-lac nanospheres undergo disassembly and reassembly processes into existing nanotube nuclei. By performing atomic force microscopy (AFM), transmission electron microscopy (TEM), and confocal laser scanning microscopy (CLSM), it indicates that there is an equilibrium among nanotubes, nanospheres, hydrolyzed α-lac, and Ca2+ in solution. The structural transition between nanotubes and nanospheres is driven from a less stable structure into a more stable structure determined by the conditions. During the transition from nanospheres into nanotubes, the hydrolyzed α-lac in nanospheres transfers into helical ribbon form at both nanotube extremities. Then helical ribbons close into mature nanotubes, extending the length of the initial nuclei. Besides, by dilution or adding ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), the decreased Ca2+ concentration in solution drives the Ca2+ dissociating from nanotubes into solution, leading to the transitions from nanotubes into nanospheres. The reversible transformation between nanotubes and nanospheres is achieved by adjusting the pH value from 7.5 to 5.0 and back to 7.5. This is because the stability of nanotubes decreases from pH 7.5 to 5 but increases from 5 to 7.5. Significantly, this approach can be used for the fabrication of various responsive nanomaterials from the same starting material.

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.

Mechanical tuning of virus-like particles.

HYPOTHESIS: Virus-like particles (VLPs) are promising scaffolds for developing mucosal vaccines. For their optimal performance, in addition to design parameters from an immunological perspective, biophysical properties may need to be considered. EXPERIMENTS: We investigated the mechanical properties of VLPs scaffolded on the coat protein of Acinetobacter phage AP205 using atomic force microscopy and small angle X-ray scattering. FINDINGS: Investigations showed that AP205 VLP is a tough nanoshell of stiffness 93 ± 23 pN/nm and elastic modulus 0.11 GPa. However, its mechanical properties are modulated by attaching muco-inert polyethylene glycol to 46 ± 10 pN/nm and 0.05 GPa. Addition of antigenic peptides derived from SARS-CoV2 spike protein by genetic fusion increased the stiffness to 146 ± 54 pN/nm although the elastic modulus remained unchanged. These results, which are interpreted in terms of shell thickness and coat protein net charge variations, demonstrate that surface conjugation can induce appreciable changes in the biophysical properties of VLP-scaffolded vaccines.

Probing DNA-Amyloid Interaction and Gel Formation by Active Magnetic Wire Microrheology.

Recent studies have shown that bacterial nucleoid-associated proteins (NAPs) can bind to DNA and result in altered structural organization and bridging interactions. Under spontaneous self-assembly, NAPs may also form anisotropic amyloid fibers, whose effects are still more significant on DNA dynamics. To test this hypothesis, microrheology experiments on dispersions of DNA associated with the amyloid terminal domain (CTR) of the bacterial protein Hfq were performed using magnetic rotational spectroscopy (MRS). In this chapter, we survey this microrheology technique based on the remote actuation of magnetic wires embedded in a sample. MRS is interesting as it is easy to implement and does not require complex procedures regarding data treatment. Pertaining to the interaction between DNA and amyloid fibers, it is found that DNA and Hfq-CTR protein dispersions behave like a gel, an outcome that suggests the formation of a network of amyloid fibers cross-linked with the DNA strands. In contrast, the pristine DNA and Hfq-CTR dispersions behave as purely viscous liquids. To broaden the scope of the MRS technique, we include theoretical predictions for the rotation of magnetic wires regarding the generic behaviors of basic rheological models from continuum mechanics, and we list the complex fluids studied by this technique over the past 10 years.

Magnetic wire active microrheology of human respiratory mucus.

Mucus is a viscoelastic gel secreted by the pulmonary epithelium in the tracheobronchial region of the lungs. The coordinated beating of cilia moves mucus upwards towards the pharynx, removing inhaled pathogens and particles from the airways. The efficacy of this clearance mechanism depends primarily on the rheological properties of mucus. Here we use magnetic wire based microrheology to study the viscoelastic properties of human mucus collected from human bronchus tubes. The response of wires between 5 and 80 µm in length to a rotating magnetic field is monitored by optical time-lapse microscopy and analyzed using constitutive equations of rheology, including those of Maxwell and Kelvin-Voigt. The static shear viscosity and elastic modulus can be inferred from low frequency (3 × 10-3-30 rad s-1) measurements, leading to the evaluation of the mucin network relaxation time. This relaxation time is found to be widely distributed, from one to several hundred seconds. Mucus is identified as a viscoelastic liquid with an elastic modulus of 2.5 ± 0.5 Pa and a static viscosity of 100 ± 40 Pa s. Our work shows that beyond the established spatial variations in rheological properties due to microcavities, mucus exhibits secondary inhomogeneities associated with the relaxation time of the mucin network that may be important for its flow properties.

Effect of water on the electroresponsive structuring and friction in dilute and concentrated ionic liquid lubricant mixtures.

The effect of water on the electroactive structuring of a tribologically relevant ionic liquid (IL) when dispersed in a polar solvent has been investigated at a gold electrode interface using neutron reflectivity (NR). For all solutions studied, the addition of small amounts of water led to clear changes in electroactive structuring of the IL at the electrode interface, which was largely determined by the bulk IL concentration. At a dilute IL concentration, the presence of water gave rise to a swollen interfacial structuring, which exhibited a greater degree of electroresponsivity with applied potential compared to an equivalent dry solution. Conversely, for a concentrated IL solution, the presence of water led to an overall thinning of the interfacial region and a crowding-like structuring, within which the composition of the inner layer IL layers varied systematically with applied potential. Complementary nanotribotronic atomic force microscopy (AFM) measurements performed for the same IL concentration, in dry and ambient conditions, show that the presence of water reduces the lubricity of the IL boundary layers. However, consistent with the observed changes in the IL layers observed by NR, reversible and systematic control of the friction coefficient with applied potential was still achievable. Combined, these measurements provide valuable insight into the implications of water on the interfacial properties of ILs at electrified interfaces, which inevitably will determine their applicability in tribotronic and electrochemical contexts.

Electroresponsive structuring and friction of a non-halogenated ionic liquid in a polar solvent: effect of concentration.

Neutron reflectivity (NR) measurements have been employed to study the interfacial structuring and composition of electroresponsive boundary layers formed by an ionic liquid (IL) lubricant at an electrified gold interface when dispersed in a polar solvent. The results reveal that both the composition and extent of the IL boundary layers intricately depend on the bulk IL concentration and the applied surface potential. At the lowest concentration (5% w/w), a preferential adsorption of the IL cation at the gold electrode is observed, which hinders the ability to electro-induce changes in the boundary layers. In contrast, at higher IL bulk concentrations (10 and 20% w/w), the NR results reveal a significantly larger concentration of the IL ions at the gold interface that exhibit significantly greater electroresponsivity, with clear changes in the layer composition and layer thickness observed for different potentials. In complementary atomic force microscopy (AFM) measurements on an electrified gold surface, such IL boundary layers are demonstrated to provide excellent friction reduction and electroactive friction (known as tribotronics). In agreement with the NR results obtained, clear concentration effects are also observed. Together such results provide valuable molecular insight into the electroactive structuring of ILs in solvent mixtures, as well as provide mechanistic understanding of their tribotronic behaviours.

Interactions between DNA and the Hfq Amyloid-like Region Trigger a Viscoelastic Response.

Molecular transport of biomolecules plays a pivotal role in the machinery of life. Yet, this role is poorly understood due the lack of quantitative information. Here, the role and properties of the C-terminal region of Escherichia coli Hfq is reported, involved in controlling the flow of a DNA solution. A combination of experimental methodologies has been used to probe the interaction of Hfq with DNA and to measure the rheological properties of the complex. A physical gel with a temperature reversible elasticity modulus is formed due to the formation of noncovalent cross-links. The mechanical response of the complexes shows that they are inhomogeneous soft solids. Our experiments indicate that the Hfq C-terminal region could contribute to the genome's mechanical response. The reported viscoelasticity of the DNA-protein complex might have implications for cellular processes involving molecular transport of DNA or segments thereof.

Common trends in the epidemic of Covid-19 disease.

ABSTRACT: The discovery of SARS-CoV-2, the responsible virus for the Covid-19 epidemic, has sparked a global health concern with many countries affected. Developing models that can interpret the epidemic and give common trend parameters are useful for prediction purposes by other countries that are at an earlier phase of the epidemic; it is also useful for future planning against viral respiratory diseases. One model is developed to interpret the fast-growth phase of the epidemic and another model for an interpretation of the entire data set. Both models agree reasonably with the data. It is shown by the first model that during the fast phase, the number of new infected cases depends on the total number of cases by a power-law relation with a scaling exponent equal to 0.82. The second model gives a duplication time in the range 1-3 days early in the start of the epidemic, and another parameter (α = 0.1-0.5) that deviates the progress of the epidemic from an exponential growth. Our models may be used for data interpretation and for guiding predictions regarding this disease, e.g., the onset of the maximum in the number of new cases.

Alveolar mimics with periodic strain and its effect on the cell layer formation.

We report on the development of a new model of alveolar air-tissue interface on a chip. The model consists of an array of suspended hexagonal monolayers of gelatin nanofibers supported by microframes and a microfluidic device for the patch integration. The suspended monolayers are deformed to a central displacement of 40-80 µm at the air-liquid interface by application of air pressure in the range of 200-1,000 Pa. With respect to the diameter of the monolayers, that is, 500 µm, this displacement corresponds to a linear strain of 2-10% in agreement with the physiological strain range in the lung alveoli. The culture of A549 cells on the monolayers for an incubation time of 1-3 days showed viability in the model. We exerted a periodic strain of 5% at a frequency of 0.2 Hz for 1 hr to the cells. We found that the cells were strongly coupled to the nanofibers, but the strain reduced the coupling and induced remodeling of the actin cytoskeleton, which led to a better tissue formation. Our model can serve as a versatile tool in lung investigations such as in inhalation toxicology and therapy.

Interfacial structuring of non-halogenated imidazolium ionic liquids at charged surfaces: effect of alkyl chain length.

Control of the interfacial structures of ionic liquids (ILs) at charged interfaces is important to many of their applications, including in energy storage solutions, sensors and advanced lubrication technologies utilising electric fields. In the case of the latter, there is an increasing demand for the study of non-halogenated ILs, as many fluorinated anions have been found to produce corrosive and toxic halides under tribological conditions. Here, the interfacial structuring of a series of four imidazolium ILs ([CnC1Im]) of varying alkyl chain lengths (n = 5, 6, 7, 10), with a non-halogenated borate-based anion ([BOB]), have been studied at charged interfaces using sum frequency generation (SFG) spectroscopy and neutron reflectivity (NR). For all alkyl chain lengths, the SFG spectra show that the cation imidazolium ring responds to the surface charge by modifying its orientation with respect to the surface normal. In addition, the combination of SFG spectra with electrochemical NR measurements reveals that the longest alkyl chain length (n = 10) forms a bilayer structure at all charged interfaces, independent of the ring orientation. These results demonstrate the tunability of IL interfacial layers through the use of surface charge, as well as effect of the cation alkyl chain length, and provide valuable insight into the charge compensation mechanisms of ILs.

Effect of Nanoparticles on the Bulk Shear Viscosity of a Lung Surfactant Fluid.

Inhaled nanoparticles (<100 nm) reaching the deep lung region first interact with the pulmonary surfactant, a thin lipid film lining the alveolar epithelium. To date, most biophysical studies have focused on particle-induced modifications of the film interfacial properties. In comparison, there is less work on the surfactant bulk properties and on their changes upon particle exposure. Here we study the viscoelastic properties of a biomimetic pulmonary surfactant in the presence of various engineered nanoparticles. The microrheology technique used is based on the remote actuation of micron-sized wires via the application of a rotating magnetic field and on time-lapse optical microscopy. It is found that particles strongly interacting with lipid vesicles, such as cationic silica (SiO2, 42 nm) and alumina (Al2O3, 40 nm) induce profound modifications of the surfactant flow properties, even at low concentrations. In particular, we find that silica causes fluidification, while alumina induces a liquid-to-soft solid transition. Both phenomena are described quantitatively and accounted for in the context of colloidal physics models. It is finally suggested that the structure and viscosity changes could impair the fluid reorganization and recirculation occurring during breathing.

Size extensivity of elastic properties of alkane fragments.

Using MP2, CCSD, and B3LYP methods of computational chemistry, we show length dependence in the intrinsic elastic properties of short alkane fragments. For isolated alkane fragments of finite length in the gas phase and zero temperature, the intrinsic elasticity constants are found to vary with the number of carbon atoms and its parity. From extrapolation of the elasticity constants calculations to infinite chain length, and by comparing with in-situ elasticity constant of single poly(ethylene) molecule obtained with atomic force microscopy, we estimate the softening effect of environment on the extension response of the polymer.

Quantitative Nano-characterization of Polymers Using Atomic Force Microscopy.

The present article offers an overview on the use of atomic force microscopy (AFM) to characterize the nanomechanical properties of polymers. AFM imaging reveals the conformations of polymer molecules at solid- liquid interfaces. In particular, for polyelectrolytes, the effect of ionic strength on the conformations of molecules can be studied. Examination of force versus extension profiles obtained using AFM-based single molecule force spectroscopy gives information on the entropic and enthalpic elasticities in pN to nN force range. In addition, single molecule force spectroscopy can be used to trigger chemical reactions and transitions at the molecular level when force-sensitive chemical units are embedded in a polymer backbone.

Influence of Solvent Quality on the Force Response of Individual Poly(styrene) Polymer Chains.

Single molecule mechanics of poly(styrene) polymer chains is investigated in different organic solvents with atomic force microscopy (AFM). The acquired force-extension profiles can be well fitted with a modified freely jointed chain (FJC) model. The model describes the force-extension profiles in terms of an apparent Kuhn length and an elasticity constant. The elasticity constant is found to be the same for all different solvents investigated. Best fit of the force-extension profiles with the FJC model reveals that the Kuhn length varies systematically with solvent quality. In fact, one can establish a good correlation between the Kuhn length and the Flory-Huggins interaction parameter. The increase in the Kuhn length with increasing solvent quality reflects the larger extent of swelling of the polymer in good solvents.

Influence of ligand-receptor interactions on force-extension behavior within the freely jointed chain model.

We study the influence of receptor-ligand interactions on the force response of single polymer chains theoretically. The extension of the chain is modeled in terms of freely jointed chain or elastic freely jointed chain (EFJC) models. The situation involving noninteracting bonds is solved exactly, while effects of interactions are treated within a mean-field approximation. The form with shorter bonds governs the low force situation, while the form with longer bonds is relevant in the high force regime. We further discuss the accuracy of approximate relations, which were used to describe the response of the EFJC model.

Mechanically induced cis-to-trans isomerization of carbon-carbon double bonds using atomic force microscopy.

Cis-to-trans isomerization of carbon-carbon double bonds can be induced by the application of mechanical force. Using single molecule force spectroscopy by means of atomic force microscopy (AFM) we pulled polymer molecules which contained cis double bonds in the backbone. In the force versus extension profiles of these polymers, a sudden extension increase is observed which is due to the conversion of shorter cis isomers into longer trans isomers. The added length to the polymer results in relaxation in probed force. We find that the isomerization occurs at forces of 800 ± 60 pN, independent of AFM tip and solid substrate chemistries. Investigation of similar polymers which exclusively contained single bonds in the backbone showed no evidence of a similar transition.