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Oligomeric species arising during aggregation of α-synuclein are proposed to be a major source of toxicity in Parkinson’s disease, and thus a major potential drug target. However, their mechanism of formation and role in aggregation are largely unresolved. Here we first show that, at physiological pH, α-synuclein aggregates by secondary nucleation, rather than fragmentation, 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 physiologically relevant conditions.

Parkinson’s disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerisation and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a unique binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.Abstract Figure

Effective public-health measures and vaccination campaigns against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against the ectodomain and the receptor-binding domain of the spike protein as well as the nucleocapsid protein of SARS-CoV-2. We used TRABI for continuous seromonitoring of hospital patients and healthy blood donors (n=72’222) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). Seroprevalence peaked in May 2020 and rose again in November 2020 in both cohorts. Validations of results included antibody diffusional sizing and Western Blotting. Using an extended Susceptible-Exposed-Infectious-Removed model, we found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020 in the population of the canton of Zurich. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19 and up to the timepoint of survey participation. Crucially, we found no evidence for a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2- infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

Sorting cells is an essential primary step in many biological and clinical applications such as high-throughput drug screening, cancer research and cell transplantation. Cell sorting based on their mechanical properties has long been considered as a promising label-free biomarker that could revolutionize the isolation of cells from heterogeneous populations. Recent advances in microfluidic image-based cell analysis combined with subsequent label-free sorting by on-chip actuators demonstrated the possibility of sorting cells based on their physical properties. However, the high purity of sorting is achieved at the expense of a sorting rate that lags behind the analysis throughput. Furthermore, stable and reliable system operation is an important feature in enabling the sorting of small cell fractions from a concentrated heterogeneous population. Here, we present a label-free cell sorting method, based on the use of focused travelling surface acoustic wave (FTSAW) in combination with real-time deformability cytometry (RT-DC). We demonstrate the flexibility and applicability of the method by sorting distinct blood cell types, cell lines and particles based on different physical parameters. Finally, we present a new strategy to sort cells based on their mechanical properties. Our system enables the sorting of up to 400 particles per s. Sorting is therefore possible at high cell concentrations (up to 36 million per ml) while retaining high purity (>92%) for cells with diverse sizes and mechanical properties moving in a highly viscous buffer. Sorting of small cell fraction from a heterogeneous population prepared by processing of small sample volume (10 μl) is also possible and here demonstrated by the 667-fold enrichment of white blood cells (WBCs) from raw diluted whole blood in a continuous 10-hour sorting experiment. The real-time analysis of multiple parameters together with the high sensitivity and high-throughput of our method thus enables new biological and therapeutic applications in the future.

Mutations in the SNCA gene, which encodes the protein α-synuclein, have been linked with early onset Parkinson's disease. The exact nature of this association, however, is still poorly understood. To investigate this problem, we started from the observation that α-synuclein is constitutively N-terminally acetylated, a post-translational modification that alters the charge and structure of α-synuclein molecules and affects their interaction with lipid membranes, as well as their aggregation process. We thus studied five N-terminal acetylated familial variants (A30P, E46K, H50Q, G51D and A53T) of α-synuclein through a wide range of biophysical assays to probe the microscopic steps in their aggregation process and the structures of the resulting aggregates. Our results reveal a great complexity in the combined effects of the disease-related mutations with N-terminal acetylation on the aggregation of α-synuclein, which underscores the great sensitivity to even relatively small perturbations of the behaviour of this protein.

Fibrillar protein aggregates are a hallmark of the pathology of a range of human disorders, from prion diseases to dementias. Yet, the same aggregated structures that are formed in disease are also encountered in several functional contexts. The fundamental properties that determine whether these protein assembly processes are functional or, by contrast, pathological, have remained elusive. Here, we address this question by analysing the aggregation kinetics of a large set of self-assembling proteins, from those associated with disease, over those whose aggregates fulfil functional roles in biology, to those that aggregate only under artificial conditions. Remarkably, we find that essentially all systems that assemble by a nucleated-growth mechanism are capable of significant self-replication on experimentally accessible timescales. However, comparing the intrinsic timescales of self-replication with the timescales over which the corresponding aggregates form in a biological context yields a clear distinction; for aggregates which have evolved to fulfil a structural role, the rate of self-replication is too low to be significant on the biologically relevant timescale. By contrast, all analysed proteins that aggregate in the context of disease are able to self-replicate quickly compared to the timescale of the associated disease. Our findings establish the ability to self-replicate as both a ubiquitous property of protein aggregates and one that has the potential to be a key process across aggregation-related disorders.

Antibody profiling is a fundamental component of understanding the humoral response in a wide range of disease areas. Most currently used approaches operate by capturing antibodies onto functionalised surfaces. Such measurements of surface binding are governed by an overall antibody titre, while the two fundamental molecular parameters, antibody affinity and antibody concentration, are challenging to determine individually from such approaches. Here, by applying microfluidic diffusional sizing (MDS), we show how we can overcome this challenge and demonstrate reliable quantification of alloantibody binding affinity and concentration of alloantibodies binding to Human Leukocyte Antigens (HLA), an extensively used clinical biomarker in organ transplantation, both in buffer and in crude human serum. Capitalising on the ability to vary both serum and HLA concentrations during MDS, we show that both affinity and concentration of HLA-specific antibodies can be determined directly in serum when neither of these parameters is known. Finally, we provide proof of principle in clinical transplant patient sera that our assay enables differentiation of alloantibody reactivity against HLA proteins of highly similar structure, providing information not attainable through currently available techniques. These results outline a path towards detection and in-depth profiling of humoral immunity and may enable further insights into the clinical relevance of antibody reactivity in clinical transplantation and beyond.

The amyloid cascade hypothesis, according to which the self-assembly of amyloid-β peptide (Aβ) is a causative process in Alzheimer’s disease, has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating Aβ-targeted therapies have been interpreted as evidence against this hypothesis, irrespective of the characteristics and mechanisms of action of the therapeutic agents, which are highly challenging to assess. Here, we combine kinetic analyses with quantitative binding measurements to address the mechanism of action of four clinical stage anti-Aβ antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We quantify the influence of these antibodies on the aggregation kinetics and on the production of oligomeric aggregates and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of Aβ. Our results reveal that, uniquely among these four antibodies, aducanumab dramatically reduces the flux of Aβ oligomers. The effects of four antibodies on the aggregation pathway of Aβ are examined via an in-depth kinetics approach, revealing the specific molecular steps affected by each antibody.

Neurodegenerative diseases such as Alzheimer's and Parkinson's are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, the improved optical properties (extinction coefficient, quantum yield and brightness) of ThX can be used to monitor structural differences in oligomeric species, not observed via traditional ThT imaging., Introducing ThX, a next-generation ThT derivative that allows for the early detection of amyloid aggregates at the bulk and single-aggregate levels.

Parkinson's disease is associated with the aberrant aggregation of alpha-synuclein. Although the causes of this process are still unclear, post-translational modifications of alpha-synuclein are likely to play a modulatory role. Since alpha-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behavior of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and slows down both elongation and fibrilcatalyzed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and nonacetylated forms in both lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated alpha-synuclein exhibit a lower beta-sheet content. These findings indicate that N-terminal acetylation of alpha-synuclein alters its lipid-dependent aggregation behavior, reduces its rate of in vitro aggregation, and affects the structural properties of its fibrillar aggregates.

The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation.

Understanding the factors that contribute to antibody escape of SARS-CoV-2 and its variants is key for the development of drugs and vaccines that provide broad protection against a variety of virus variants. Using microfluidic diffusional sizing, we determined the dissociation constant (KD) for the interaction between receptor binding domains (RBDs) of SARS-CoV-2 in its original version (WT) as well as alpha and beta variants with the host-cell receptor angiotensin converting enzyme 2 (ACE2). For RBD-alpha, the ACE2-binding affinity was increased by a factor of ten when compared with RBD-WT, while ACE2-binding of RBD-beta was largely unaffected. However, when challenged with a neutralizing antibody that binds to both RBD-WT and RBD-alpha with low nanomolar KD values, RBD-beta displayed no binding, suggesting a substantial epitope change. In SARS-CoV-2 convalescent sera, RBD-binding antibodies showed low nanomolar affinities to both wild-type and variant RBD proteins—strikingly, the concentration of antibodies binding to RBD-beta was half that of RBD-WT and RBD-alpha, again indicating considerable epitope changes in the beta variant. Our data therefore suggests that one factor contributing to the higher transmissibility and antibody evasion of SARS-CoV-2 alpha and beta is a larger fraction of viruses that can form a complex with ACE2. However, the two variants employ different mechanisms to achieve this goal. While SARS-CoV-2 alpha RBD binds with greater affinity to ACE2 and is thus more difficult to displace from the receptor by neutralizing antibodies, RBD-beta is less accessible to antibodies due to epitope changes which increases the chances of ACE2-binding and infection.

Recent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potential beneficial as well as detrimental effects of cross-reactive antibodies derived from memory immunity. Specifically, due to a significant degree of sequence similarity between SARS-CoV-2 and other members of the coronavirus family, memory B-cells that emerged from previous infections with endemic human coronaviruses (HCoVs) could be re-activated upon encountering the newly emerged SARS-CoV-2, thus prompting the production of cross-reactive antibodies. Understanding the affinity and concentration of these potentially cross-reactive antibodies to the new SARS-CoV-2 antigens is therefore particularly important when assessing both existing immunity against common HCoVs and adverse effects like antibody-dependent enhancement (ADE) in COVID-19. However, these two fundamental parameters cannot easily be deconvoluted by surface-based assays like enzyme-linked immunosorbent assays (ELISAs) which are routinely used to assess cross-reactivity.Here, we have used microfluidic antibody-affinity profiling (MAAP) to quantitatively evaluate the humoral immune response in COVID-19 convalescent patients by determining both antibody affinity and concentration against spike antigens of SARS-CoV-2 directly in nine convalescent COVID-19 patient and three pre-pandemic sera that were seropositive for common HCoVs. All 12 sera contained low concentrations of high affinity antibodies against spike antigens of HCoV-NL63 and HCoV-HKU1, indicative of past exposure to these pathogens, while the affinity against the SARS-CoV-2 spike protein was lower. These results suggest that cross-reactivity as a consequence of memory re-activation upon an acute SARS-CoV-2 infection may not be a significant factor in generating immunity against SARS-CoV-2.

Aberrant soluble oligomers formed by the amyloid-β peptide (Aβ) are major pathogenic agents in the onset and progression of Alzheimer's disease. A variety of biomolecules can influence the formation of these oligomers in the brain, although their mechanisms of action are still largely unknown. Here, we studied the effects on Aβ aggregation of DOPAL, a reactive catecholaldehyde intermediate of dopamine metabolism. We found that DOPAL is able to stabilize Aβ oligomeric species, including dimers and trimers, that exert toxic effects on human neuroblastoma cells, in particular increasing cytosolic calcium levels and promoting the generation of reactive oxygen species. These results reveal an interplay between Aβ aggregation and key biochemical processes regulating cellular homeostasis in the brain.

Aberrant soluble oligomers formed by the amyloid-β peptide (Aβ) are major pathogenic agents in the onset and progression of Alzheimer’s disease. A variety of biomolecules can influence the formation of these oligomers in the brain, although their mechanisms of action are still largely unknown. Here, we studied the effects on Aβ aggregation of DOPAL, a reactive catecholaldehyde intermediate of dopamine metabolism. We found that DOPAL is able to stabilize Aβ oligomeric species, including dimers and trimers, that exert toxic effects on human neuroblastoma cells, in particular increasing cytosolic calcium levels and promoting the generation of reactive oxygen species. These results reveal an interplay between Aβ aggregation and key biochemical processes regulating cellular homeostasis in the brain., 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.

Both the replication of protein aggregates and their spreading throughout the brain are implicated in the progression of Alzheimer’s disease (AD). However, the rates of these processes are unknown and the identity of the rate-determining process in humans has therefore remained elusive. By bringing together chemical kinetics with measurements of tau seeds and aggregates across brain regions, we are able to quantify their replication rate in human brains. Remarkably, we obtain comparable rates in several different datasets, with 5 different methods of tau quantification, from seed amplification assaysin vitroto tau PET studies in living patients. Our results suggest that the overall rate of accumulation of tau in neocortical regions is limited not by spreading between brain regions but by local replication, which doubles the number of seeds every ~5 years. Thus, we propose that limiting local replication constitutes the most promising strategy to control tau accumulation during AD.

The clinical outcome of SARS-CoV-2 infections can range from asymptomatic to lethal, and is thought to be crucially shaped by the quality of the immune response which includes antibody titres and affinity for their targets. Using Microfluidic Antibody Affinity Profiling (MAAP), we determined the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 23 of whom were confirmed to be SARS-CoV-2-positive by PCR testing. We found that dissociation constants (Kd) of anti-RBD antibodies spanned more than two orders of magnitude from 80 pM to 25 nM, despite having similar antibody concentrations. The tested individuals showed progressively higher antibody concentrations but constant Kd values, suggesting that affinities did not mature over time. 33 sera showed affinities higher than that of the CoV2 spike for its ACE2 receptor. Accordingly, addition of seropositive plasma to pre-formed spike-ACE2 receptor complexes led to their dissociation. Finally, we observed that the RBD of HKU1, OC43, and SARS-CoV coronaviruses, but not unrelated control proteins, were able to compete substantially with the RBD of SARS-CoV-2 in solution. Therefore, the affinity of total plasma immunoglobulins to SARS-CoV-2 is an indicator of the quality of the immune response to SARS-CoV-2, and may help select the most efficacious samples for therapeutic plasmapheresis.

The detection and characterisation of antibodies in human blood is a key for clinical diagnostics and risk assessment for autoimmunity, infectious diseases and transplanta-tion. Antibody titre derived from immunoassays is a commonly used measure for anti-body response, but this metric does not resolve readily the two fundamental properties of antibodies in solution, namely their affinity and concentration. This difficulty originates from the fact that the fundamental parameters describing the binding interaction, affinity and ligand concentration, are convoluted into the titre measurement; moreover, the difficulty of controlling the surface concentration and activity of the immobilised ligand can make it challenging to distinguish between avidity and affinity. To address these challenges, we developed microfluidic antibody affinity profiling, an assay which allows the simultaneous determination of both affinity and antibody concentration, directly in solution, without surface immobilisation or antibody purification. We demonstrate these measurements in the context of alloantibody characterisation in organ transplantation, using complex patient sera, and quantify the concentration and affinity of alloantibodies against donor Human Leukocyte Antigens (HLA), an extensively used clinical biomarker to access the risk of allograft rejection. These results outline a path towards detection and in depth profiling of antibody response in patient sera.

10 pags., 5 figs., The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer’s disease and α-synuclein (αS) in Parkinson’s disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases., This work was supported by the Cambridge Centre for Misfolding Diseases (R.L., B.M., F.S.R., C.K.X., M.P., S.C., S.W.C., J.H., T.K., J.R.K., T.P.J.K., M.V., and C.M.D.), the UK Biotechnology and Biochemical Sciences Research Council (M.V. and C.M.D.), the Wellcome Trust (203249/Z/16/Z to T.P.J.K and M.V.), the Frances and Augustus Newman Foundation (T.P.J.K.), the Regione Toscana – FAS Salute, project SUPREMAL (R.C., A.B., C.C., and F.C.), the Gates Cambridge Trust and St. John’s College Cambridge (R.L.), Darwin College Cambridge (F.S.R.), the Herchel Smith Fund (C.K.X.), a Faculty Development Research Fund grant from the United States Military Academy, West Point (R.L.) and a DTRA Service Academy Research Initiative grant (HDTRA1033862 to R.L.).

Oligomeric intermediates are implicated as neurotoxins in the pathogenesis of protein mis-folding diseases. Structural, biophysical and biochemical characterisation of these species is challenging due to their heterogeneous and transient nature, and their typically low abun-dance. Here, we show that microfluidic free-flow electrophoresis is capable of separating heterogeneous oligomer mixtures on a timescale of seconds, at least two orders of magnitude faster than conventional techniques. This enables analysis of oligomer structural heterogeneity, zeta-potential and immunochemistry with minimal sample perturbation under physiologically-relevant conditions.

The amyloid cascade hypothesis, according to which the self-assembly of amyloid-β peptide (Aβ) is a causative process in Alzheimer's disease, has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating Aβ-targeted therapies have been interpreted as evidence against this hypothesis, irrespective of the characteristics and mechanisms of action of the therapeutic agents, which are highly challenging to assess. Here, we combine kinetic analyses with quantitative binding measurements to address the mechanism of action of four clinical stage anti-Aβ antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We quantify the influence of these antibodies on the aggregation kinetics and on the production of oligomeric aggregates and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of Aβ. Our results reveal that, uniquely among these four antibodies, aducanumab dramatically reduces the flux of Aβ oligomers.

16 pags., 6 figs., The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson’s disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species., This research was supported by the University of Florence (Fondi Ateneo to F.C. and C.C.), the Ministry of Education, Universities and Research of Italy (Progetto Dipartimento di Eccellenza “Gender Medicine” to C.C.), Parkinson’s UK (G-1508 to S.W.C. and C.M.D.), the Center for Misfolding Diseases of the University of Cambridge (S.W.C. and C.M.D.), the UK Medical Research Council (MR/N000676/1 to C.M.D.), the Agency of Science, Technology and Research of Singapore (to S.W.C.), and the Ministry of Economy and Competitiveness of Spain (MINECO RYC-2012-12068 and MINECO/FEDER EU BFU2015-64119-P to N.C.).

The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and Aβ42 peptide in Alzheimer's disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of Aβ42 amyloids. Kinetic analysis further corroborates that the surfaces available for the Aβ42 secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to Aβ42 fibrils is validated by a microfluidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research., Templating mechanism of S100A9 amyloids on Aβ fibrillar surfaces during amyloid co-aggregation process was revealed by synergy of biophysical methods including charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses.

Phase transitions of protein molecules are central to biological function and malfunction. One such transition commonly encountered in nature is the conversion of soluble monomeric states into solid phases, which include crystals and amyloid fibrils, the latter of which are associated with the onset and development of neurodegenerative diseases. Monitoring aggregate formation and protein phase behavior is essential in gaining mechanistic insights into these fundamental processes. Fluorescence techniques have proven invaluable in observing biological molecules; yet, most such approaches rely on the use of an extrinsic fluorophore that binds to the molecule of interest, the installation of which can perturb the molecular systems under study. However, most proteins also possess aromatic amino acids within their peptide sequence and therefore exhibit intrinsic fluorescence. Here, we show that by measuring in space and time tryptophan autofluorescence for three proteins, reconstituted silk fibroin, β-lactoglobulin, and lysozyme, fibrillar self-assembly can be monitored accurately and without the need for extrinsic dyes. When fibrillar protein self-assembly takes place, hydrophobic burial occurs, resulting in the minimization of exposed tryptophan residues to the solvent and consequently leading to an increase in protein autofluorescence. Moreover, by employing a droplet-microfluidic approach to confine protein self-assembly in space, we demonstrate that intrinsic fluorescence can be used to image protein nanofibrils in a label-free manner and that the microstructural analysis obtained from intrinsic fluorescence microscopy correlates well with that from samples treated with extrinsic dyes. Finally, our results show that protein autofluorescence is not limited to the observation of β-sheet-rich structures, but can also be used to distinguish between different types of solid phases including spherulites and crystals, making this approach suitable for overall characterization of protein phase transition phenomena.

Funder: Herchel Smith Fund, Funder: St John’s College Cambridge, Funder: Centre for Misfolding Diseases, Cambridge, Funder: Swiss FCS and the Forschungskredit of the University of Zurich, Funder: Frances and Augustus Newman Foundation, Funder: BBRSC, Funder: NOMIS Foundation, The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.

Alzheimer9s disease affects nearly 50 million people worldwide with an overall cost corresponding to more than 1% of the global economy. The amyloid cascade hypothesis, according to which the misfolding and self-assembly of the amyloid-β peptide (Aβ) into oligomeric and fibrillar aggregates is a causative pathogenic process in this disease (1,2), has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating Aβ-targeted therapies (3-6), however, have been interpreted as evidence against this hypothesis, irrespective of the characteristics and mechanisms of action of the therapeutic agents, which have proved highly challenging to assess. Here, we bring together kinetic analysis with quantitative binding measurements to address the mechanisms of action of four clinical stage anti-Aβ antibodies, aducanumab (7), gantenerumab, bapineuzumab and solanezumab. We reveal and quantify the striking differences that these antibodies have on the aggregation kinetics and on the production of oligomeric aggregates, and link these effects to the affinity and stoichiometry of each antibody for the monomeric and fibrillar forms of Aβ. Our results uncover that, uniquely amongst these four antibodies, aducanumab (3) dramatically reduces the flux of oligomeric forms of Aβ.

Neurodegenerative diseases such as Alzheimer’s and Parkinson’s are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, we demonstrate that ThX can be used to detect a distinct oligomeric species, not observed via traditional ThT imaging.

Parkinson's disease is an increasingly prevalent and currently incurable neurodegenerative disorder. At the molecular level, this disease is characterized by the formation of aberrant intracellular protein deposits known as Lewy bodies. Oligomeric forms of the protein α-synuclein (αS), which are believed to be both intermediates and by-products of Lewy body formation, are considered to be the main pathogenic species. Interactions of such oligomers with lipid membranes are increasingly emerging as a major molecular pathway underpinning their toxicity. Here we review recent progress in our understanding of the interactions of αS oligomers with lipid membranes. We highlight key structural and biophysical features of αS oligomers, the effects of these features on αS oligomer membrane binding properties, and resultant implications for understanding the etiology of Parkinson's disease. We discuss mechanistic modes of αS oligomer–lipid membrane interactions and the effects of environmental factors to such modes. Finally, we provide an overview of the current understanding of the main molecular determinants of αS oligomer toxicity in vivo.

RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the −10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD. DOI: http://dx.doi.org/10.7554/eLife.22520.001