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Abstract The deposition of the amyloid-β (Aβ) peptide into amyloid fibrils is a hallmark of Alzheimer’s disease. Recently, it has been reported that some proteins can aggregate and form amyloids through an intermediate pathway involving a liquid-like condensed phase. These observations prompted us to investigate the phase space of Aβ. We thus explored the ability of Aβ to undergo liquid–liquid phase separation, and the subsequent liquid-to-solid transition that takes place within the resulting condensates. Through the use of microfluidic approaches, we observed that the 40-residue form of Αβ (Αβ40) can undergo liquid–liquid phase separation, and that accessing a liquid-like intermediate state enables Αβ40 to self-assemble and aggregate into amyloid fibrils through this pathway. These results prompt further studies to investigate the possible role of Αβ liquid–liquid phase separation and its subsequent aggregation in the context of Alzheimer’s disease and more generally on neurodegenerative processes.

Abstract The physical characterization of proteins in terms of their sizes, interactions, and assembly states is key to understanding their biological function and dysfunction. However, this has remained a difficult task because proteins are often highly polydisperse and present as multicomponent mixtures. Here, we address this challenge by introducing single-molecule microfluidic diffusional sizing (smMDS). This approach measures the hydrodynamic radius of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. smMDS allows for ultrasensitive sizing of proteins down to femtomolar concentrations and enables affinity profiling of protein interactions at the single-molecule level. We show that smMDS is effective in resolving the assembly states of protein oligomers and in characterizing the size of protein species within complex mixtures, including fibrillar protein aggregates and nanoscale condensate clusters. Overall, smMDS is a highly sensitive method for the analysis of proteins in solution, with wide-ranging applications in drug discovery, diagnostics, and nanobiotechnology.

Abstract Oligomeric species arising during the aggregation of α-synuclein are implicated as a major source of toxicity in Parkinson’s disease, and thus a major potential drug target. However, both their mechanism of formation and role in aggregation are largely unresolved. Here we show that, at physiological pH and in the absence of lipid membranes, α-synuclein aggregates form by secondary nucleation, rather than simple primary nucleation, and that this process is enhanced by agitation. Moreover, using a combination of single molecule and bulk level techniques, we identify secondary nucleation on the surfaces of existing fibrils, rather than formation directly from monomers, as the dominant source of oligomers. Our results highlight secondary nucleation as not only the key source of oligomers, but also the main mechanism of aggregate formation, and show that these processes take place under conditions which recapitulate the neutral pH and ionic strength of the cytosol.

Abstract α-Synuclein (α-syn) accumulates as insoluble amyloid but also forms soluble α-syn oligomers (αSOs), thought to be even more cytotoxic than fibrils. To detect and block the unwanted activities of these αSOs, we have raised 30 monoclonal antibodies (mAbs) against different forms of αSOs, ranging from unmodified αSOs to species stabilized by lipid peroxidation products and polyphenols, αSOs formed by C-terminally truncated α-syn, and multivalent display of α-syn on capsid virus-like particles (cVLPs). While the mAbs generally show a preference for αSOs, they also bind fibrils, but to variable extents. Overall, we observe great diversity in the mAbs’ relative affinities for monomers and αSOs, varied requirements for the C-terminal extension of α-syn, and only a modest effect on α-syn fibrillation. Several mAbs show several orders of magnitude preference for αSOs over monomers in in-solution studies, while the commercial antibody MJF14 only bound 10-fold more strongly to αSOs than monomeric α-syn. Gratifyingly, seven mAbs almost completely block αSO permeabilization of membrane vesicles. Five selected mAbs identified α-syn-related pathologies like Lewy bodies (LBs) and Lewy Neurites, as well as Glial Cytoplasmic Inclusions in postmortem brains from people diagnosed for PD, dementia with LBs or multiple system atrophy, although to different extents. Three mAbs were particularly useful for pathological evaluation of postmortem brain human tissue, including early stages of PD. Although there was no straightforward connection between the mAbs’ biophysical and immunohistochemical properties, it is encouraging that this comprehensive collection of mAbs able to recognize different aggregated α-syn species in vitro also holds diagnostic potential.

Abstract Biomolecular condensates help cells organise their content in space and time. Cells harbour a variety of condensate types with diverse composition and many are likely yet to be discovered. Here, we develop a methodology to predict the composition of biomolecular condensates. We first analyse available proteomics data of cellular condensates and find that the biophysical features that determine protein localisation into condensates differ from known drivers of homotypic phase separation processes, with charge mediated protein-RNA and hydrophobicity mediated protein-protein interactions playing a key role in the former process. We then develop a machine learning model that links protein sequence to its propensity to localise into heteromolecular condensates. We apply the model across the proteome and find many of the top-ranked targets outside the original training data to localise into condensates as confirmed by orthogonal immunohistochemical staining imaging. Finally, we segment the condensation-prone proteome into condensate types based on an overlap with biomolecular interaction profiles to generate a Protein Condensate Atlas. Several condensate clusters within the Atlas closely match the composition of experimentally characterised condensates or regions within them, suggesting that the Atlas can be valuable for identifying additional components within known condensate systems and discovering previously uncharacterised condensates.

Abstract Phase separation and percolation contribute to phase transitions of multivalent macromolecules. Contributions of percolation are evident through the viscoelasticity of condensates and through the formation of heterogeneous distributions of nano- and mesoscale pre-percolation clusters in sub-saturated solutions. Here, we show that clusters formed in sub-saturated solutions of FET (FUS-EWSR1-TAF15) proteins are affected differently by glutamate versus chloride. These differences on the nanoscale, gleaned using a suite of methods deployed across a wide range of protein concentrations, are prevalent and can be unmasked even though the driving forces for phase separation remain unchanged in glutamate versus chloride. Strikingly, differences in anion-mediated interactions that drive clustering saturate on the micron-scale. Beyond this length scale the system separates into coexisting phases. Overall, we find that sequence-encoded interactions, mediated by solution components, make synergistic and distinct contributions to the formation of pre-percolation clusters in sub-saturated solutions, and to the driving forces for phase separation.

Abstract Aggregated forms of α-synuclein constitute the major component of Lewy bodies, the proteinaceous aggregates characteristic of Parkinson’s disease. Emerging evidence suggests that α-synuclein aggregation may occur within liquid condensates formed through phase separation. This mechanism of aggregation creates new challenges and opportunities for drug discovery for Parkinson’s disease, which is otherwise still incurable. Here we show that the condensation-driven aggregation pathway of α-synuclein can be inhibited using small molecules. We report that the aminosterol claramine stabilizes α-synuclein condensates and inhibits α-synuclein aggregation within the condensates both in vitro and in a Caenorhabditis elegans model of Parkinson’s disease. By using a chemical kinetics approach, we show that the mechanism of action of claramine is to inhibit primary nucleation within the condensates. These results illustrate a possible therapeutic route based on the inhibition of protein aggregation within condensates, a phenomenon likely to be relevant in other neurodegenerative disorders.

Abstract Amyloid polymorphism is a hallmark of almost all amyloid species, yet the mechanisms underlying the formation of amyloid polymorphs and their complex architectures remain elusive. Commonly, two main mesoscopic topologies are found in amyloid polymorphs characterized by non‐zero Gaussian and mean curvatures: twisted ribbons and helical fibrils, respectively. Here, a rich heterogeneity of configurations is demonstrated on insulin amyloid fibrils, where protofilament packing can occur, besides the common polymorphs, also in a combined mode forming mixed‐curvature polymorphs. Through AFM statistical analysis, an extended array of heterogeneous architectures that are rationalized by mesoscopic theoretical arguments are identified. Notably, an unusual fibrillization pathway is also unraveled toward mixed‐curvature polymorphs via the widespread recruitment and intertwining of protofilaments and protofibrils. The results present an original view of amyloid polymorphism and advance the fundamental understanding of the fibrillization mechanism from single protofilaments into mature amyloid fibrils.

Abstract Many proteins self-assemble to form amyloid fibrils, which are highly organized structures stabilized by a characteristic cross-β network of hydrogen bonds. This process underlies a variety of human diseases and can be exploited to develop versatile functional biomaterials. Thus, protein self-assembly has been widely studied to shed light on the properties of fibrils and their intermediates. A still open question in the field concerns the microscopic processes that underlie the long-time behaviour and properties of amyloid fibrillar assemblies. Here, we use atomic force microscopy with angstrom-sensitivity to observe that amyloid fibrils undergo a maturation process, associated with an increase in both fibril length and thickness, leading to a decrease of their density, and to a change in their cross-β sheet content. These changes affect the ability of the fibrils to catalyse the formation of new aggregates. The identification of these changes helps us understand the fibril maturation processes, facilitate the targeting of amyloid fibrils in drug discovery, and offer insight into the development of biocompatible and sustainable protein-based materials.

Abstract Antimicrobial peptides (AMPs), which combat bacterial infections by disrupting the bacterial cell membrane or interacting with intracellular targets, are naturally produced by a number of different organisms, and are increasingly also explored as therapeutics. However, the mechanisms by which AMPs act on intracellular targets are not well understood. Using machine learning-based sequence analysis, we identified a significant number of AMPs that have a strong tendency to form liquid-like condensates in the presence of nucleic acids through phase separation. We demonstrate that this phase separation propensity is linked to the effectiveness of the AMPs in inhibiting transcription and translation in vitro, as well as their ability to compact nucleic acids and form clusters with bacterial nucleic acids in bacterial cells. These results suggest that the AMP-driven compaction of nucleic acids and modulation of their phase transitions constitute a previously unrecognised mechanism by which AMPs exert their antibacterial effects. The development of antimicrobials that target nucleic acid phase transitions may become an attractive route to finding effective and long-lasting antibiotics.

BackgroundPatients with autoimmune/inflammatory conditions on anti-CD20 therapies, such as rituximab, have suboptimal humoral responses to vaccination and are vulnerable to poorer clinical outcomes following SARS-CoV-2 infection. We aimed to examine how the fundamental parameters of antibody responses, namely, affinity and concentration, shape the quality of humoral immunity after vaccination in these patients.MethodsWe performed in-depth antibody characterisation in sera collected 4 to 6 weeks after each of three vaccine doses to wild-type (WT) SARS-CoV-2 in rituximab-treated primary vasculitis patients (n = 14) using Luminex and pseudovirus neutralisation assays, whereas we used a novel microfluidic-based immunoassay to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. We performed comparative antibody profiling at equivalent timepoints in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one infection and two vaccinations; n = 15) and in convalescent patients after WT SARS-CoV-2 infection (n = 30).ResultsRituximab-treated patients had lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in rituximab-treated patients and in healthy individuals. In the rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT [median (range) KD: 21.6 (9.7–38.8) nM vs. 4.6 (2.3–44.8) nM, p = 0.0004]. By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in rituximab-treated patients [median (range) KD: 1.05 (0.45–1.84) nM vs. 20.25 (13.2–38.8) nM, p = 0.0002], underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients had similar anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV-2 infection, which was not observed in rituximab-treated patients, despite repeated vaccination.DiscussionOur results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response.

There are limitations with current protein sensing methods. Here the authors report DigitISA, a digital immunosensor assay based on microchip electrophoretic separation and single-molecule detection that enables quantitation of protein biomarkers in a single, solution-phase step.

Biomolecular condensates with internal structure allow cells to further organise their processes. In this work the authors investigate how condensates can obtain an internal structure with droplets of dilute phase inside via kinetic, rather than purely thermodynamic driving forces.

Abstract Astrocytes are diverse brain cells that form large networks communicating via gap junctions and chemical transmitters. Despite recent advances, the functions of astrocytic networks in information processing in the brain are not fully understood. In culture, brain slices, and in vivo, astrocytes, and neurons grow in tight association, making it challenging to establish whether signals that spread within astrocytic networks communicate with neuronal groups at distant sites, or whether astrocytes solely respond to their local environments. A multi‐electrode array (MEA)‐based device called AstroMEA is designed to separate neuronal and astrocytic networks, thus allowing to study the transfer of chemical and/or electrical signals transmitted via astrocytic networks capable of changing neuronal electrical behavior. AstroMEA demonstrates that cortical astrocytic networks can induce a significant upregulation in the firing frequency of neurons in response to a theta‐burst charge‐balanced biphasic current stimulation (5 pulses of 100 Hz × 10 with 200 ms intervals, 2 s total duration) of a separate neuronal‐astrocytic group in the absence of direct neuronal contact. This result corroborates the view of astrocytic networks as a parallel mechanism of signal transmission in the brain that is separate from the neuronal connectome. Translationally, it highlights the importance of astrocytic network protection as a treatment target.

Extrusion bioprinting can be used to produce living materials but controlling cell microenvironments is challenging. Here, the authors use a type of core-shell microgel ink that decouples cell culture from material processing to produce functional materials with a range of potential applications.

A central concept for characterising phase-separating systems is the phase diagram but generation of such diagrams for biomolecular systems is typically slow and low-throughput. Here the authors describe PhaseScan, a combinatorial droplet microfluidic platform for high-resolution acquisition of multidimensional biomolecular phase diagrams.

Abstract The effectiveness of therapeutic monoclonal antibodies (mAbs) against variants of the SARS-CoV-2 virus is highly variable. As target recognition of mAbs relies on tight binding affinity, we assessed the affinities of five therapeutic mAbs to the receptor binding domain (RBD) of wild type (A), Delta (B.1.617.2), and Omicron BA.1 SARS-CoV-2 (B.1.1.529.1) spike using microfluidic diffusional sizing (MDS). Four therapeutic mAbs showed strongly reduced affinity to Omicron BA.1 RBD, whereas one (sotrovimab) was less impacted. These affinity reductions correlate with reduced antiviral activities suggesting that affinity could serve as a rapid indicator for activity before time-consuming virus neutralization assays are performed. We also compared the same mAbs to serological fingerprints (affinity and concentration) obtained by MDS of antibodies in sera of 65 convalescent individuals. The affinities of the therapeutic mAbs to wild type and Delta RBD were similar to the serum antibody response, indicating high antiviral activities. For Omicron BA.1 RBD, only sotrovimab retained affinities within the range of the serum antibody response, in agreement with high antiviral activity. These results suggest that serological fingerprints provide a route to evaluating affinity and antiviral activity of mAb drugs and could guide the development of new therapeutics.

α-synuclein aggregates cause neuronal damage, but their heterogeneity complicates studying their toxic properties. Here, the authors analyze α-synuclein aggregates in vitro and study post-mortem brain samples, providing evidence that small aggregates are the main culprit for neuronal death in Parkinson’s disease.

Farzadfard et al. present a comprehensive analysis of a range of C-terminal truncations of aSN, linking the importance of high C-terminus charge for decreased fibrillation rates. The ability to formation oligomers, to disrupt synthetic vesicles and cell toxicity was reduced with truncated aSN, aiding in understanding of the intramolecular interactions of aSN which promote/inhibit aggregation.

Molecular chaperones from the Hsp70 family can break up protein aggregates, including amyloids. Here, the authors utilize microfluidic diffusional sizing to assess the mechanism of α-synuclein (αS) disaggregation by the Hsc70–DnaJB1–Apg2 system, and show that single αS molecules are removed directly from the fibril ends.

Abstract Droplet microfluidic methods have opened up the possibility of studying a plethora of phenomena ranging from biological to physical or chemical processes at ultra low volumes and high throughput. A key component of such approaches is the ability to trap droplets for observation, and many device architectures for achieving this objective have been developed. A challenge with such approaches is, however, recovering the droplets following their confinement for applications involving further analysis. Here, we present a device capable of generating, confining and releasing microdroplets in a sequential manner. Through a combination of experimental and computational simulations, we shed light on the key features required for successful droplet storage and retrieval. Moreover, we explore the effect of the flow rate of the continuous phase on droplet release, determining that a critical rate is needed to ensure complete droplet deformation through constrictions holding the droplets in place prior to release. Finally, we find that once released, droplets can be retrieved and collected off chip. The ability to generate, store and sequentially release droplets renders such a device particularly promising for future applications where reactions may not only be monitored on-chip, but droplets can also be retrieved for further analysis, facilitating new exploratory avenues in the fields of analytical chemistry and biology.

Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aβ, the peptide associated with Alzheimer’s disease, the mechanism and rate of aggregation have been established for a range of variants and conditions in vitro and in bodily fluids. A key outstanding question is how the relative stabilities of monomers, fibrils and intermediates affect each step in the fibril formation process. By monitoring the kinetics of aggregation of Aβ42, in the presence of urea or guanidinium hydrochloride (GuHCl), we here determine the rates of the underlying microscopic steps and establish the importance of changes in relative stability induced by the presence of denaturant for each individual step. Denaturants shift the equilibrium towards the unfolded state of each species. We find that a non-ionic denaturant, urea, reduces the overall aggregation rate, and that the effect on nucleation is stronger than the effect on elongation. Urea reduces the rate of secondary nucleation by decreasing the coverage of fibril surfaces and the rate of nucleus formation. It also reduces the rate of primary nucleation, increasing its reaction order. The ionic denaturant, GuHCl, accelerates the aggregation at low denaturant concentrations and decelerates the aggregation at high denaturant concentrations. Below approximately 0.25 M GuHCl, the screening of repulsive electrostatic interactions between peptides by the charged denaturant dominates, leading to an increased aggregation rate. At higher GuHCl concentrations, the electrostatic repulsion is completely screened, and the denaturing effect dominates. The results illustrate how the differential effects of denaturants on stability of monomer, oligomer and fibril translate to differential effects on microscopic steps, with the rate of nucleation being most strongly reduced.

Green use of plant derived proteins in functional materials has been limited by inefficient methods to control micro and nanoscale structure. Here, the authors use nanoscale assembly of water-insoluble plant proteins to make meter scale films with comparable properties to conventional plastics.

Through advances in the past decades, the central role of aberrant protein aggregation has been established in many neurodegenerative diseases. Crucially, however, the molecular mechanisms that underlie aggregate proliferation in the brains of affected individuals are still only poorly understood. Under controlled in vitro conditions, significant progress has been made in elucidating the molecular mechanisms that take place during the assembly of purified protein molecules, through advances in both experimental methods and the theories used to analyse the resulting data. The determination of the aggregation mechanism for a variety of proteins revealed the importance of intermediate oligomeric species and of the interactions with promotors and inhibitors. Such mechanistic insights, if they can be achieved in a disease-relevant system, provide invaluable information to guide the design of potential cures to these devastating disorders. However, as experimental systems approach the situation present in real disease, their complexity increases substantially. Timescales increase from hours an aggregation reaction takes in vitro, to decades over which the process takes place in disease, and length-scales increase to the dimension of a human brain. Thus, molecular level mechanistic studies, like those that successfully determined mechanisms in vitro, have only been applied in a handful of living systems to date. If their application can be extended to further systems, including patient data, they promise powerful new insights. Here we present a review of the existing strategies to gain mechanistic insights into the molecular steps driving protein aggregation and discuss the obstacles and potential paths to achieving their application in disease. First, we review the experimental approaches and analysis techniques that are used to establish the aggregation mechanisms in vitro and the insights that have been gained from them. We then discuss how these approaches must be modified and adapted to be applicable in vivo and review the existing works that have successfully applied mechanistic analysis of protein aggregation in living systems. Finally, we present a broad mechanistic classification of in vivo systems and discuss what will be required to further our understanding of aggregate formation in living systems.

Abstract Microcapsules and microgels consisting of macromolecular networks have received increasing attention due to their biomedical and pharmaceutical applications. Protein microgels and in particular silk-based microcapsules have desirable properties due to their biocompatibility and lack of toxicity. Typically such structures formed through emulsion templating are spherical in geometry due to interfacial tension. However, approaches to synthesis particles with more complex and non-spherical geometries are sought due to their packing properties and cargo release characteristics. Here, we describe a droplet-microfluidic strategy for generating asymmetric tubular-like microgels from reconstituted silk fibroin; a major component of native silk. It was determined using fluorescence microscopy, that the shear stress within the microchannel promotes surface protein aggregation, resulting in the asymmetric morphology of the microgels. Moreover, the structural transition that the protein undergoes was confirmed using FTIR. Crucially, the core of the microgels remains liquid, while the surface has fully aggregated into a fibrillar network. Additionally, we show that microgel morphology could be controlled by varying the dispersed to continuous phase flow rates, while it was determined that the radius of curvature of the asymmetric microgels is correlated to the wall shear stress. By comparing the surface fluorescence intensity of the microgels as a function of radius of curvature, the effect of the shear stress on the amount of aggregation could be quantified. Finally, the potential use of these asymmetric microgels as carriers of cargo molecules is showcased. As the core of the microgel remains liquid but the shell has gelled, this approach is highly suitable for the storage of bio-active cargo molecules such as antibodies, making such a delivery system attractive in the context of biomedical and pharmaceutical applications.

Elucidating the molecular driving forces underlying liquid–liquid phase separation is a key objective for understanding biological function and malfunction. Here the authors show that a wide range of cellular proteins, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, which form condensates at low salt concentrations, can reenter a phase-separated regime at high salt concentrations.

Cataldi et al. investigates the impact of the dopamine derivative DOPAL on the Aβ peptide oligomer formation. They report that DOPAL promotes the formation of stable Aβ oligomers that exert toxicity on neuroblastoma cells by increasing cytosolic calcium levels and generating reactive oxygen species. This study connects Aβ aggregation with processes regulating cellular homeostasis in the brain.

Our understanding of the molecular mechanisms underlying pathological protein aggregation remains incomplete. Here, single molecule infrared nanospectroscopy (AFM-IR) offers insight into the structure of Aβ42 oligomeric and fibrillar species and their interaction with an aggregation inhibitor, paving the way for single molecule drug discovery studies.

Promising treatments for neurogenerative disorders may involve targeting kinetic intermediates, including α-synuclein oligomers. Here a kinetic method for quantifying oligomer populations is used to screen small molecule inhibitors of oligomerisation and gain mechanistic insight into their modes of action.

While infrared nanospectroscopy methods based on thermomechanical detection (AFM-IR) enables the acquisition of absorption spectra at the nanoscale, single molecule detection has not been possible so far. Here, the authors present off-resonance, low power and short pulse infrared nanospectroscopy (ORS-nanoIR), which allows measuring infrared absorption spectra at the single molecule level in a time scale of seconds with high throughput and demonstrate that the secondary structure of single protein molecules can be determined with this method.

Phase separation of biomolecules gives rise to membraneless organelles that contribute to the spatiotemporal organization of the cell. In most cases, such biomolecular condensates contain multiple components, but the manner in which interactions between components control the stability of condensates have remained challenging to elucidate. Here, we develop an approach to determine tie-line gradients in ternary biomolecular phase-separation systems based on measurements of the dilute phase concentration of only one component. We show that the sign of the tie-line gradient is related to the cross-interaction energy between the polymers in the system and discriminates between associative and segregative phase separation. Using this approach, we study the interaction between protein fused in sarcoma (FUS) and polyethylene glycol (PEG) polymer chains and measure positive tie-line gradients. Our results show that PEG drives phase separation through an associative interaction with FUS, other than through acting as an inert crowder. We further study the interaction between poly(A) ribonucleic acid (RNA) (700–3500 kDa [kilodalton]) and the protein G3BP1, and using the tie-line gradient as a reporter for the stoichiometry of polymers in the condensate, we determine a G3BP1-to-poly(A) RNA molar ratio of 1∶0.003–0.015 in the dense phase. Our framework for measuring tie-line gradients opens up a route for the characterization of interaction types and compositions in ternary phase-separation systems.

Droplet sorting is one of the fundamental manipulations of droplet-based microfluidics. Although many sorting methods have already been proposed, there is still a demand to develop new sorting methods for various applications of droplet-based microfluidics. This work presents numerical investigations on droplet sorting with asymmetric acute junctions. It is found that the asymmetric acute junctions could achieve volume-based sorting and velocity-based sorting. The pressure distributions in the asymmetric junctions are discussed to reveal the physical mechanism behind the droplet sorting. The dependence of the droplet sorting on the droplet volume, velocity, and junction angle is explored. The possibility of the employment of the proposed sorting method in most real experiments is also discussed. This work provides a new, simple, and cost-effective passive strategy to separate droplets in microfluidic channels. Moreover, the proposed acute junctions could be used in combination with other sorting methods, which may boost more opportunities to sort droplets.

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-β peptide (Aβ) and of α-synuclein (αS), which are associated with Alzheimer’s and Parkinson’s diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aβ and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aβ and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer’s and Parkinson’s diseases.

Abstract Soluble aggregates of amyloid-β (Aβ) have been associated with neuronal and synaptic loss in Alzheimer’s disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we characterised soluble aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls using a range of sensitive biophysical methods. We used super-resolution imaging and atomic force microscopy to characterise the size and structure of the aggregates present in CSF and correlate this with their ability to permeabilise lipid membranes and induce an inflammatory response. We found that these aggregates are extremely heterogeneous and exist in a range of sizes, varying both structurally and in their mechanisms of toxicity during the disease progression. A higher proportion of small aggregates of Aβ that can cause membrane permeabilization are found in MCI CSF; in established AD, a higher proportion of the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells, while there was no detectable change in aggregate concentration. These results show that large aggregates, some longer than 100 nm, are present in the CSF of AD patients and suggest that different neurotoxic mechanisms are prevalent at different stages of AD.

Amyloid beta (Aβ42) peptides form heterogeneous mixtures of aggregates, which are closely linked to Alzheimer’s disease. This study shows how different types of Aβ42 aggregates are associated with distinct mechanisms of toxicity

Transient oligomeric species of the amyloid-β peptide (Aβ42) have been identified as key pathogenic agents in Alzheimer’s disease. Here the authors find that the aminosterol trodusquemine enhances Aβ42 aggregation and suppresses Aβ42-induced toxicity by displacing oligomers from cell membranes.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.

Chaperones protect other proteins against misfolding and aggregation, a key requirement for maintaining biological function. Experimental observations of changes in solubility of amyloid proteins in the presence of certain chaperones are discussed here in terms of thermodynamic driving forces. We outline how chaperones can enhance amyloid solubility through the formation of heteromolecular aggregates (co-aggregates) based on the second law of thermodynamics and the flux towards equal chemical potential of each compound in all phases of the system. Higher effective solubility of an amyloid peptide in the presence of chaperone implies that the chemical potential of the peptide is higher in the aggregates formed under these conditions compared to peptide-only aggregates. This must be compensated by a larger reduction in chemical potential of the chaperone in the presence of peptide compared to chaperone alone. The driving force thus relies on the chaperone being very unhappy on its own (high chemical potential), thus gaining more free energy than the amyloid peptide loses upon forming the co-aggregate. The formation of heteromolecular aggregates also involves the kinetic suppression of the formation of homomolecular aggregates. The unhappiness of the chaperone can explain the ability of chaperones to favour an increased population of monomeric client protein even in the absence of external energy input, and with broad client specificity. This perspective opens for a new direction of chaperone research and outlines a set of outstanding questions that aim to provide additional cues for therapeutic development in this area.

Abstract 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 β‐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 β‐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.

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of β-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure–function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins.

Manual sample deposition on a substrate can introduce artifacts in quantitative AFM measurements. Here the authors present a microfluidic spray device for reliable deposition of subpicoliter droplets which dry out in milliseconds after landing on the surface, thereby avoiding protein self-assembly.

The cytoskeleton, a network of fibrils, controls how cells divide. Here, the authors show that synthetic protein fibrils added to an emulsion can control the division of droplets and that this method can be used to control the morphology of microparticles during biomaterial preparation.

Abstract Background Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, assembly of peptides already conjugated to proteins is limited to cargo species that do not interfere sterically with the assembly process or misfold under the harsh conditions often used for assembly. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled. Results Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen cycloaddition, one of the class of “click” reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology. Discussion Although previous studies have produced peptides that can both form amyloid fibrils and undergo “click”-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation.

Silk fibres currently used in biotechnology are chemically reconstituted silk fibroins (RSF), which are more stable than native silk fibroin (NSF) but possess different biophysical properties. Here, the authors use microfluidic droplets to encapsulate and store NSF, preserving their native structure.

Abstract Background The aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson’s disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS. Results We show that both nanobodies inhibit the formation of ɑS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ɑS, leading to a dramatic reduction in oligomer-induced cellular toxicity. Conclusions The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.

ABSTRACT A wide range of bacterial pathogens have been shown to form biofilms, which significantly increase their resistance to environmental stresses, such as antibiotics, and are thus of central importance in the context of bacterial diseases. One of the major structural components of these bacterial biofilms are amyloid fibrils, yet the mechanism of fibril assembly and its importance for biofilm formation are currently not fully understood. By studying fibril formation in vitro, in a model system of two common but unrelated biofilm-forming proteins, FapC from Pseudomonas fluorescens and CsgA from Escherichia coli, we found that the two proteins have a common aggregation mechanism. In both systems, fibril formation proceeds via nucleated growth of linear fibrils exhibiting similar measured rates of elongation, with negligible fibril self-replication. These similarities between two unrelated systems suggest that convergent evolution plays a key role in tuning the assembly kinetics of functional amyloid fibrils and indicates that only a narrow window of mechanisms and assembly rates allows for successful biofilm formation. Thus, the amyloid assembly reaction is likely to represent a means for controlling biofilm formation, both by the organism and by possible inhibitory drugs. IMPORTANCE Biofilms are generated by bacteria, embedded in the formed extracellular matrix. The biofilm's function is to improve the survival of a bacterial colony through, for example, increased resistance to antibiotics or other environmental stresses. Proteins secreted by the bacteria act as a major structural component of this extracellular matrix, as they self-assemble into highly stable amyloid fibrils, making the biofilm very difficult to degrade by physical and chemical means once formed. By studying the self-assembly mechanism of the fibrils from their monomeric precursors in two unrelated bacteria, our experimental and theoretical approaches shed light on the mechanism of functional amyloid assembly in the context of biofilm formation. Our results suggest that fibril formation may be a rate-limiting step in biofilm formation, which in turn has implications on the protein self-assembly reaction as a target for potential antibiotic drugs.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-021-01680-7

The organization of supramolecular peptide polymers determines their properties; however, controlling their dimensions still remains a problem. Here, Gazitet al. show the spontaneous elongation and shortening of these polymers at an individual nano-assembly level by using a microfluidic platform.