Particle-stabilized emulsions offer a strategy for forming mechanically robust microcapsules based on coassembly of silica nanoparticles, polyelectrolytes, and surfactants at oil-water interfaces. Such systems have complicated distributions of inorganic colloidal solids, surfactants, solvents, and ions that influence their compositions and structures over multiple length scales, which have been challenging to characterize and establish. To do so, silica-polyelectrolyte microcapsules were prepared with nearly monodisperse dimensions in the submicron range from water-in-oil (W/O) emulsions that were stabilized by a combination of nonionic surfactants, anionic silica nanoparticles, and cationic polyelectrolyte chains. Nonionic surfactants were used to establish oil as the continuous phase, while silica-polyelectrolyte complexes, self-assembled at the oil-water interfaces, prevented coalescence between droplets and provided mechanical elasticity. Particle charge measurements show that the surface charge density of the silica nanoparticles can be controlled by adjusting the pH conditions or by substituting aluminate ions at their surfaces. These promoted strong electrostatic and hydrogen-bonding interactions with the cationic polyelectrolyte and nonionic surfactant species, direct atomic-scale evidence of which is provided by solid-state two-dimensional (2D) 29Si{1H} NMR. For nanoparticles with higher surface charge densities, strong electrostatic interactions are the basis for particle coassembly with cationic polyelectrolyte species, and the resulting silica-polyelectrolyte complexes adsorb at the oil-water interface, as revealed by cryogenic electron microscopy. Relative to larger spherical nanoparticles, the elongated nanoparticles exhibit more extensive hydrogen bonding with polar organic moieties, which contributes to polyelectrolyte bridging between particles with lower densities of surface negative charges, consistent with interfacial rheology analyses. In addition to interfacial compositions and conditions, noncovalent polyelectrolyte-silica interactions, governed by nanoparticle surface compositions, charge density, and surface area, can be adjusted to control the macroscopic mechanical properties of the microcapsule interfaces.

The reversible folding and assembly of the human brain protein tau are regulated by charge neutralization through limited and reversible phosphorylation, enabling tau to bind tubulin and maintain the structural integrity of neuronal microtubules. However, in neurodegenerative diseases like Alzheimer's and related tauopathies, tau becomes hyperphosphorylated, detaches from tubulin, and irreversibly assembles into β-structured amyloid filaments responsible for neuronal death. In previous work, we showed that charge neutralization via Faradaic electroreduction of cationic residues in tau and other intrinsically disordered proteins can mimic phosphorylation to trigger protein condensation, folding, and assembly. Here, we demonstrate that even non-Faradaic effects─including large electric fields and concentration gradients in the electric double layer, together with spatial ordering of ions at the solution-electrode interface─can induce folding and assembly of tau, its microtubule-binding region K18, and a 19-residue tau peptide (jR2R3 P301L) containing a mutation known to induce early aggregation in vitro and in vivo. Assembly occurs on different electrode materials at identical effective electric fields, demonstrating independence from the electrode hydrophobicity and electronic structure. Surface-enhanced infrared absorption and plasmon resonance spectroscopies show that near-surface electric fields of ∼1 MV/cm trigger K18 folding and assembly. Ion ordering and charge screening near electrodes at higher salt concentrations (50 vs 1 mM) also reduce Coulombic repulsion between protein monomers and their cationic residues, promoting folding and assembly. Overall, these results show that interfacial electric fields and other non-Faradaic processes can reveal and drive protein misfolding and aggregation, hallmarks of tauopathies and prion-related neurodegenerative diseases.

Molecular photoswitches capable of controlled pH changes are often limited in a critical application, biological systems, by poor aqueous solubility, which hinders their ability to generate large and sustained pH shifts. Herein, we report the synthesis and photophysical characterization of a highly water-soluble, photoswitchable diarylethene base that modulates pH through light-controlled changes in the pKa of an N-heterocyclic imine (NHI) moiety. Using experimentally determined pKa values, we developed and validated a model showing that this NHI-diarylethene exhibits photoacidic behavior over a wide range of pH 6-11 and photobasic behavior over the range pH 3-6, making it the first photoswitch reported to demonstrate dual functionality and to operate in the basic pH regime. By alternating UV and visible light irradiation, this NHI-diarylethene switch achieved a change of >1.7 pH units and was reversibly cycled in both buffered and unbuffered aqueous solutions. We applied this photoswitch to reversibly control the assembly of the pH-responsive, cationic reflectin protein, which shows promise for biophotonic applications. This photoswitch is a powerful tool for bioinspired systems that operate in aqueous environments, where precise, sustained, and reversible pH control is essential for mimicking dynamic biological functions such as assembly, signaling, and responsiveness.

The intrinsically disordered reflectin proteins drive tunable reflectivity for dynamic camouflage and communication in the recently evolved Loliginidae family of squid. Previous work revealed that reflectin A1 forms discrete assemblies whose size is precisely predicted by protein net charge density and charge screening by the local anion concentration. Using dynamic light scattering, FRET, and confocal microscopy, we show that these assemblies, of which 95 to 99% of bulk protein in solution is partitioned into, are dynamic intermediates to liquid protein-dense condensates formed by liquid-liquid phase separation (LLPS). Increasing salt concentration drives this progression by anionic screening of the cationic protein's Coulombic repulsion, and by increasing the contribution of the hydrophobic effect which tips the balance between short-range attraction and long-range repulsion to drive protein assembly and ultimately LLPS. Measuring fluorescence recovery after photobleaching and droplet fusion dynamics, we demonstrate that reflectin diffusivity in condensates is tuned by protein net charge density. These results illuminate the physical processes governing reflectin A1 assembly and LLPS and demonstrate the potential for reflectin A1 condensate-based tunable biomaterials. They also compliment previous observations of liquid phase separation in the Bragg lamellae of activated iridocytes and suggest that LLPS behavior may serve a critical role in governing the tunable and reversible dehydration of the membrane-bounded Bragg lamellae and vesicles containing reflectin in biophotonically active cells.

As the molecular driver for tunable iridescence in cephalopods, the tunable phase behavior of reflectin A1 protein continues to be a focus of biomaterial engineering. Modulating salt concentration and protein net charge density of reflectin A1 drives the protein to form dynamic assemblies as intermediates to liquid-liquid phase separation. Reflectin assemblies, while limited in size by the extent of charge neutralization of the protein's cationic, Coulombic repulsion, are initially in dynamic exchange with monomers or oligomers from the surrounding solution. A novel fluorescence resonance energy transfer (FRET) dilution assay was used, in conjunction with dynamic light scattering (DLS) and protein concentration assays, to characterize the two-way flux of protein between reflectin A1 assemblies and a dilute phase as a function of assembly age. This FRET dilution assay distinguishes between one-way and two-way flux of protein into and out of protein assemblies and, therefore, can be applied during assembly formation. Differentiating between dynamic and kinetically arrested protein assemblies is crucial to understanding their biophysical origins, and this novel FRET dilution assay can be adapted to supplement biophysical investigations of other protein assemblies.

Neuronally triggered phosphorylation drives the dynamic condensation of reflectin proteins, enabling squid to fine tune the colors reflected from specialized skin cells (iridocytes) for camouflage and communication. Reflectin, the primary component of iridocyte lamellae, forms alternating layers of protein and low refractive index extracellular space within membrane-encapsulated structures, acting as a biologically tunable distributed Bragg reflector. In vivo, reflectin condensation induces osmotic dehydration of these lamellae, reducing their thickness and shifting the wavelength of reflected light. Inspired by this natural mechanism, we demonstrate that electrochemical reduction of imidazolium moieties within the protein provides a reversible and tunable method to control the water volume fraction in reflectin thin films, allowing precise, dynamic modulation of the film's refractive index and thickness - mimicking the squid's dynamic color adaptation. To unravel the underlying mechanisms, we developed electrochemical correlative ellipsometry and surface plasmon resonance spectroscopy, enabling real-time analysis of optical property changes of reflectin films. This electrochemically driven approach offers unprecedented control over reflectin condensation dynamics. Our findings not only deepen the understanding of biophysical processes governing cephalopod coloration but also pave the way for bio-inspired materials and devices that seamlessly integrate biological principles with synthetic systems to bridge the biotic-abiotic gap.

Disordered proteins often undergo a stimuli-responsive, disorder-to-order transition which facilitates dynamic processes that modulate the physiological activities and material properties of cells, such as strength, chemical composition, and reflectance. It remains challenging to gain rapid and spatiotemporal control over such disorder-to-order transitions, which limits the incorporation of these proteins into novel materials. The reflectin protein is a cationic, disordered protein whose assembly is responsible for dynamic color camouflage in cephalopods. Stimuli-responsive control of reflectin's assembly would enable the design of biophotonic materials with tunable color. Herein, a novel, multivalent azobenzene photoswitch is shown to be an effective and non-invasive strategy for co-assembling with reflectin molecules and reversibly controlling assembly size. Photoisomerization between the trans and cis (E and Z) photoisomers promotes or reduces Coulombic interactions, respectively, with reflectin proteins to repeatedly cycle the sizes of the photoswitch-reflectin assemblies between 70 nm and 40 nm. The protein assemblies formed with the trans and cis isomers show differences in interaction stoichiometry and secondary structure, which indicate that photoisomerization modulates the photoswitch-protein interactions to change assembly size. Our results highlight the utility of photoswitchable interactions to control reflectin assembly and provide a tunable synthetic platform that can be adapted to the structure, assembly, and function of other disordered proteins.

The pathological aggregation of the microtubule-associated protein tau is a hallmark of various neurodegenerative disorders, including Alzheimer’s disease (AD), Pick’s disease and frontotemporal dementia. Tau protein’s reversible assembly and binding to microtubules in brain neurons are regulated by charge-neutralizing phosphorylation. Hyperphosphorylation, on the other hand, leads to irreversible formation of cytotoxic filaments. Under physiological conditions, tau remains remarkably stable, exhibiting intrinsically disordered behavior and reversible assembly. Irreversible aggregation requires specific mutations, preformed seeds or nucleating polyanionic cofactors. However, heparin-induced fibrils differ structurally from brain-derived filaments, and the presence of such cofactors can impede therapeutic agent development. We have developed a quick and easy method to generate cofactor-free filaments using heterogeneous electroreduction as a surrogate for charge-neutralization by hyperphosphorylation. We combine electrochemistry with in situ UV absorbance, circular dichroism and dynamic light scattering spectroscopies to dynamically follow tau’s conformational changes and assembly. Electroreduction of positively charged residues of the protein leads to rapid formation of b-rich structures, even at times as short as 15 minutes. The extent of assembly can be controlled by fine-tuning electroreductive potentials and electrolyte conditions. Relevance of this new method was confirmed by examining the impact of an early-onset AD-causing tau mutant and known inhibitors on aggregation kinetics. Our results demonstrate comparable effects to heparin-induced fibrils, as assessed by thioflavin-T fluorescence, dynamic light scattering, circular dichroism and electron microscopy. Low-voltage electroreduction provides a platform to study the effects of tau mutations and effectors, as well as to develop rapid assays to test how additional effectors may inhibit, disassemble or direct the trajectory of tau assembly. The versatility of our approach also suggests its potential applicability to a broader spectrum of neurodegenerative amyloid diseases.

Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, the neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing their refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that the reduction in repulsive net charge of the purified, recombinant reflectin—either (for the first time) by generalized anionic screening with salt or by pH titration—drives a finely tuned, precisely calibrated increase in the size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. The precise proportionality between the assembly size and charge neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth by a continual, equilibrium tuning of the balance between the protein’s Coulombic repulsion and short-range interactive forces. The resulting stability of reflectin assemblies with time ensures a reciprocally precise control of the particle number concentration, encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The charge regulation of reflectin assembly precisely fine-tunes a colligative property-based nanostructured biological machine. A physical mechanism is proposed.

Reflectin is an intrinsically disordered protein known for its ability to modulate the biophotonic camouflage of cephalopods based on its assembly-induced osmotic properties. Its reversible self-assembly into discrete, size-controlled clusters and condensed droplets are known to depend sensitively on the net protein charge, making reflectin stimuli-responsive to pH, phosphorylation, and electric fields. Despite considerable efforts to characterize this behavior, the detailed physical mechanisms of reflectin's assembly are not yet fully understood. Here, we pursue a coarse-grained molecular understanding of reflectin assembly using a combination of experiments and simulations. We hypothesize that reflectin assembly and phase behavior can be explained from a remarkably simple colloidal model whereby individual protein monomers effectively interact via a short-range attractive and long-range repulsive (SA-LR) pair potential. We parameterize a coarse-grained SA-LR interaction potential for reflectin A1 from small-angle x-ray scattering measurements, and then extend it to a range of pH values using Gouy-Chapman theory to model monomer-monomer electrostatic interactions. The pH-dependent SA-LR interaction is then used in molecular dynamics simulations of reflectin assembly, which successfully capture a number of qualitative features of reflectin, including pH-dependent formation of discrete-sized nanoclusters and liquid-liquid phase separation at high pH, resulting in a putative phase diagram for reflectin. Importantly, we find that at low pH size-controlled reflectin clusters are equilibrium assemblies, which dynamically exchange protein monomers to maintain an equilibrium size distribution. These findings provide a mechanistic understanding of the equilibrium assembly of reflectin, and suggest that colloidal-scale models capture key driving forces and interactions to explain thermodynamic aspects of native reflectin behavior. Furthermore, the success of SA-LR interactions presented in this study demonstrates the potential of a colloidal interpretation of interactions and phenomena in a range of intrinsically disordered proteins.

Introduction: Chimeric antigen receptor-modified T cell (CAR-T) therapy is emerging as a useful therapy for B-cell acute lymphoblastic leukemia (B-ALL) with remaining uncertainty regarding the timing of response. We present a patient with extramedullary cutaneous involvement, initially stimulated by CAR-T infusion, with a rapid resolution of the lesions and serial biopsies providing information on the timing of reactivity. Case presentation: An 18-year-old male with relapsed and refractory B-ALL developed extramedullary involvement, leukemia cutis. After receiving chemotherapy, the lesion resolved, and he then proceeded to CAR-T therapy. Starting 9 days post-CAR-T infusion, he experienced progression of leukemia cutis. Skin biopsy confirmed leukemia cutis with less than 1% of T-cells. Due to worsening, he underwent another biopsy on day 13 of CAR-T therapy, which showed CAR-T cells comprising ~50% of all cells while peripheral blood CAR-T counts were negligible. The leukemia cutis lesions then cleared over the next week, and he achieved remission, lasting 5 months. Discussion: These findings suggest an initial stimulation of residual leukemia cutis cells, likely in response to chemokine and cytokine release related to CAR-T cell infusion. This was followed by an expansion of CAR-T cells at the site of involvement but not in the peripheral blood, and subsequent eradication of the leukemia. The time course of events and the biopsies 5 days apart provide insight into the timing of potential responses. The latter highlight the need to give CAR-T cells time to respond and that the correlation between peripheral blood CAR-T levels and activity can be disparate.

Neuronally triggered phosphorylation drives the calibrated and cyclable assembly of the reflectin signal transducing proteins, resulting in their fine tuning of colours reflected from specialized skin cells in squid for camouflage and communication. In close parallel to this physiological behaviour, we demonstrate for the first time that electrochemical reduction of reflectin A1, used as a surrogate for charge neutralization by phosphorylation, triggers voltage-calibrated, proportional and cyclable control of the size of the protein's assembly. Electrochemically triggered condensation, folding and assembly were simultaneously analysed using in situ dynamic light scattering, circular dichroism and UV absorbance spectroscopies. The correlation of assembly size with applied potential is probably linked to reflectin's mechanism of dynamic arrest, which is controlled by the extent of neuronally triggered charge neutralization and the corresponding fine tuning of colour in the biological system. This work opens a new perspective on electrically controlling and simultaneously observing reflectin assembly and, more broadly, provides access to manipulate, observe and electrokinetically control the formation of intermediates and conformational dynamics of macromolecular systems.

Candida albicans is the most predominant fungal species isolated from medical devices, including catheters, heart valves, and dental prostheses. In recent years, it has been demonstrated to be resistant to many antifungals; therefore, silver nanoparticles (AgNPs) have been proposed as an alternative. But only a handful of research is contributed to omic-based studies to study the various impacts of AgNPs on Candida species and other microorganisms. Thus, the study aims to biosynthesize AgNPs using Pelargonium-hortorum leaf and test its antifungal, cytotoxicity, and global gene expression on Candida through transcriptomic profiling. The leaf-assisted AgNPs resulted in spherical shapes with a particle size of 38 nm. The anticandidal effect demonstrated that the Minimum inhibitory concentration was 25 μg·mL −1 . Later, the cytotoxicity assay reported a moderate impact on the human gingival fibroblast cells. Finally, the transcriptomic analysis demonstrated the differential gene expression of 3,871 upregulated and 3,902 downregulated genes. Thus, proving the anticandidal effect of AgNPs on Candida through RNA-seq experiments and the regulated genes is highly important to cell wall integrity, adherence, and virulence.

Tau protein's reversible assembly and binding of microtubules in brain neurons are regulated by charge-neutralizing phosphorylation, while its hyperphosphorylation drives the irreversible formation of cytotoxic filaments associated with neurodegenerative diseases. However, the structural changes that facilitate these diverse functions are unclear. Here, we analyzed K18, a core peptide of tau, using newly developed spectroelectrochemical instrumentation that enables electroreduction as a surrogate for charge neutralization by phosphorylation, with simultaneous, real-time quantitative analyses of the resulting conformational transitions and assembly. We observed a tipping point between behaviors that paralleled the transition between tau's physiologically required, reversible folding and assembly and the irreversibility of assemblies. The resulting rapidly electroassembled structures represent the first fibrillar tangles of K18 that have been formed in vitro at room temperature without using heparin or other chargecomplementary anionic partners. These methods make it possible to (i) trigger and analyze in real time the early stages of conformational transitions and assembly without the need for preformed seeds, heterogenous coacervation, or crowding; (ii) kinetically resolve and potentially isolate never-before-seen early intermediates in these processes; and (iii) develop assays for additional factors and mechanisms that can direct the trajectory of assembly from physiologically benign and reversible to potentially pathological and irreversible structures. We anticipate wide applicability of these methods to other amyloidogenic systems and beyond.

Biofilms are naturally occurring communities of micro-organisms, attached to a surface and often embedded in a matrix of self-produced polymeric substances. Biofilms are widely implicated in human infections, particularly on prostheses and medical implants. Such biofilms are difficult to eradicate, often leading to replacement of the prosthesis and resulting in a significant burden to healthcare. Here we present a fun and engaging interactive activity targeted toward primary school/early secondary school children, introducing the concept of natural and healthcare-associated biofilms, using dental plaque as an archetypal example. Dental plaque forms as a result of poor oral/dental hygiene, and develops according to a typical series of defined stages: attachment and adherence to the surface, followed by colonization and maturation of the biofilm structure, and eventually, dispersal. This activity uses dental disclosing tablets to visualize real biofilms (plaque) on the participants teeth, and uses interlocking building-blocks to represent microorganisms, where children build three-dimensional 'biofilms' of varying shapes and structural integrities. Each of the stages of development are discussed in detail, and after building the biofilms, balls of different shapes, sizes and weights can be used as 'antimicrobials' to disrupt the biofilm structure. The outcomes of the activity are to enhance knowledge and general understanding of biofilms; their ubiquitous presence in the natural environment, development, implications in healthcare, and challenges of treatment. The various 'antimicrobial' balls also provide a basis to introduce and discuss drug selection for infections, and the importance of using the correct antimicrobial for different infections to avoid development of resistance.

Reversible electrochemical triggering of the random coil to α-helix conformational transition of polylysine (Lys10, Lys20, Lys50) was accomplished at a Pt electrode at potentials < |1| V vs. Ag/AgCl. Direct electroreduction of the N-terminus vs ε-amino groups in Lys sidechains, as well as hydronium reduction and electrolysis, could be easily distinguished and deconvolved using differential pulse voltammetry. Electrochemistry was coupled with in situ UV absorbance and circular dichroism spectroscopies to dynamically follow the evolution of α-helix formation at different potentials. Isotope experiments in H2O vs. D2O unequivocally confirm that direct electroreduction of ε-NH3+/ND3+ groups in Lys sidechains, rather than electrochemically generated pH gradient-induced deprotonation, leads to subsequent α-helix formation. The site-selective electrochemistry and optical methodologies presented herein can be generalized and extended to interrogate other protonation-sensitive biomolecular systems, and potentially provide access to early intermediates and control over the dynamic structural evolution of peptides and proteins.

Platinum-catalyzed electrochemical reduction of dissociable protons at low potentials was used to investigate proton dissociation equilibria of freely diffusing and peptide-incorporated charged amino acids. We first demonstrate with five charged essential amino acids and their analogs that the electrochemically induced deprotonation of each amino acid occurs at distinct formal reduction potential. Moreover, the observed direct reduction for all the charged species, excluding arginine, occurs at low potentials suitable for investigation under aqueous conditions (-0.4 to -0.9 V vs Ag/AgCl). The direct proton reduction was resolved via deconvolution of the observed differential pulse voltammogram (DPV) from background hydronium reduction and water electrolysis. A linear correlation was found between the formal reduction potentials and the pKa values of the dissociable protons hosted by various molecular moieties in the amino acids and their analogs and further verified with tripeptides. DPV of poly(l-lysine) decamer (Lys10) distinctively resolved the pKa values of the amino groups in the side chains and N-terminus, at a resolution not possible by conventional acid-base titration. This work demonstrates selective electrochemical titration of dissociable protons in charged amino acids in the free state and as residues in biomolecules, as well as the utility of DPV to indirectly interrogate local electrostatic environments that are essential to the stability and function of biomolecules.

Reversible electrochemical triggering of the random coil to α-helix conformational transition of polylysine, and secondary folding of reflectin A1, was accomplished at a Pt electrode at potentials < |1| V vs. Ag/AgCl. Direct electroreduction of the N-terminus vs. ε-amino groups in lysine (Lys) sidechains, imidazolium groups of histidine-containing reflectin A1, as well as hydronium reduction and electrolysis, could be easily distinguished and deconvolved using differential pulse voltammetry. Electrochemistry was coupled with in situ UV absorbance, circular dichroism, and dynamic light scattering to dynamically follow the evolution of secondary folding and assembly of polylysine and reflectin at different potentials. Isotope experiments in H2O vs. D2O unequivocally confirm that direct electroreduction of ε-NH3 +/ND3 + groups in Lys sidechains, rather than electrochemically generated pH gradient-induced deprotonation, leads to subsequent α-helix formation in polylysine. The site-selective electrochemistry and optical methodologies to be presented herein can be generalized and extended to interrogate other protonation-sensitive biomolecular systems, and potentially provide access to early intermediates and control over the dynamic structural evolution of peptides and proteins. [1] E. Masquelier, S.P. Liang, L. Sepunaru, D. E. Morse, M. J. Gordon, "Reversible Electrochemical Triggering and Optical Interrogation of Polylysine α-helix formation " Bioelectrochemistry vol. 144, p. 108007, 04/2020 2022, doi: 10.1016/j.bioelechem.2021.108007 [2] S. P. Liang, R. Levenson, B. Malady, M. J. Gordon, D. E. Morse, and L. Sepunaru, "Electrochemistry as a surrogate for protein phosphorylation: voltage-controlled assembly of reflectin A1," J R Soc Interface, vol. 17, no. 173, Dec 23 2020, doi: 10.1098/rsif.2020.0774.