Biology subjects

Xu, C. K.

Publications and source records attributed to Xu, C. K..

17 recordsLinked to original sources

bioRxiv2025

Neurodegeneration emerges at a cellular tipping point between protein accumulation and removal.

Protein aggregates are a pathological hallmark across neurodegenerative diseases. Yet, the disconnect between molecular-level aggregation and the emergence of disease severely limits mechanistic understanding of neurodegeneration. Here, we bridge this disconnect by showing that a cellular tipping point emerges as a universal feature across diseases from the competition between aggregate accumulation and removal. We map the resulting cellular phase transition with our high-throughput live-cell assay, measuring the tipping point that separates healthy cells from those with large aggregate loads. Using super-resolution imaging of brain tissue from Alzheimers and Parkinsons disease, we quantify how the balance of accumulation and removal is shifted in disease. We validate our framework by predicting how designed aggregation inhibitors shift the tipping point to restore cellular homeostasis. Our results provide a mechanistic framework connecting molecular-level aggregation to disease, paving the way for a quantitative, unified understanding of neurodegeneration and enabling predictions of therapeutic efficacy.

neuroscience↗

bioRxiv2025

Direct, high-throughput linking of single cell imaging and gene expression

Understanding sequence-phenotype relationships in biology requires large-scale datasets that provide one-to-one mapping of sequence to phenotype. Here, we address this challenge by introducing multimodal droplet barcoding, enabling individual microfluidic droplets to be tracked across both optical and sequencing measurements. We demonstrate the power of this approach through multimodal single-cell analysis. We pair imaging with gene expression profiling of individual cells, identifying genes that correlate with phenotypic features.

genomics↗

bioRxiv2023

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells

Colorectal cancer tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is unknown. Using CRC patient derived organoids (PDOs), we found that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing scRNA-seq expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

cancer biology↗

bioRxiv2023

Microfluidic antibody profiling after repeated SARS-CoV-2 vaccination links antibody affinity and concentration to impaired immunity and variant escape in patients on anti-CD-20 therapy

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 four to six 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 a novel microfluidic-based immunoassay was used to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. Comparative antibody profiling was performed at equivalent time points 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.

immunology↗

bioRxiv2023

α-Synuclein oligomers form by secondary nucleation

Oligomeric species arising during aggregation of -synuclein are proposed to be a major source of toxicity in Parkinsons 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, -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.

biophysics↗

bioRxiv2023

Alpha-synuclein oligomers displace monomeric alpha-synuclein from lipid membranes

Parkinsons disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of -synuclein (S) protein aggregates in the nervous system. While S binding to membranes in its monomeric state is correlated to its physiological role, S 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 S (OS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OS-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 OS-membrane interactions. We find that OS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OS and monomeric S (MS), we further demonstrate that OS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OS compete with and displace bound MS from the membrane surface, suggesting that disruption to the functional binding of MS 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/533646v2_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@4ab089org.highwire.dtl.DTLVardef@18c0709org.highwire.dtl.DTLVardef@220077org.highwire.dtl.DTLVardef@4c85b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Uncovering the universality of self-replication in protein aggregation and its link to disease.

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.

biophysics↗

bioRxiv2021

Single-molecule sizing through nano-cavity confinement

An approach relying on nano-cavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nano-cavity diffusional sizing (NDS), measures particle residence times within fluidic nano-cavities to determine their hydrodynamic radii. Using theoretical modeling and simulation, we show that the residence time of particles within nano-cavities above a critical timescale depends on the diffusion coefficient of the particle, which allows estimation of the particles size. We demonstrate this approach experimentally through measurement of particle residence times within nano-fluidic cavities using single-molecule confocal microscopy. Our data show that the residence times scale linearly with the sizes of nanoscale colloids, protein aggregates and single DNA oligonucleotides. NDS thus constitutes a new single molecule optofluidic approach that allows rapid and quantitative sizing of nanoscale particles for potential application in nanobiotechnology, biophysics, and clinical diagnostics. Table of content graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/472369v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@15abf7eorg.highwire.dtl.DTLVardef@d2180dorg.highwire.dtl.DTLVardef@1290f57org.highwire.dtl.DTLVardef@ae2087_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

2-photon-fabricated nano-fluidic traps for extended detection of single macromolecules and colloids in solution

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, -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.

biophysics↗

bioRxiv2021

Mutations in two SARS-CoV-2 variants of concern reflect two distinct strategies of antibody escape

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.

biochemistry↗

bioRxiv2021

Understanding the role of memory re-activation and cross-reactivity in the defense against SARS-CoV-2

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.

biophysics↗

bioRxiv2020

Amplification, not spreading limits rate of tau aggregate accumulation in Alzheimer's disease

Both the replication of protein aggregates and their spreading throughout the brain are implicated in the progression of Alzheimers 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 assays in vitro to 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.

biophysics↗

bioRxiv2020

Microfluidic Antibody Affinity Profiling for In-Solution Characterisation of Alloantibody - HLA Interactions in Human Serum

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.

biophysics↗

bioRxiv2020

Templating S100A9 amyloids on Aβ fibrillar surfaces revealed by charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses

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 A{beta}42 peptide in Alzheimers 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 A{beta}42 amyloids. Kinetic analysis further corroborates that the surfaces available for the A{beta}42 secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to A{beta}42 fibrils is validated by a microfuidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research.

biophysics↗

bioRxiv2020

Rapid Fractionation and Characterisation of Alpha-Synuclein Oligomers in Solution

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.

biophysics↗

bioRxiv2019

Kinetic fingerprint of antibody therapies predicts outcomes of Alzheimer clinical trials

The amyloid cascade hypothesis, according to which the self-assembly of amyloid-{beta} peptide (A{beta}) is a causative process in Alzheimers disease, has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating A{beta}-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. We bring together kinetic analysis with quantitative binding measurements to address the mechanisms of action of four clinical stage anti-A{beta} antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We reveal and quantify the striking differences 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 A{beta}. Our results uncover that, uniquely amongst these four antibodies, aducanumab dramatically reduces the flux of oligomeric forms of A{beta}.

biophysics↗

bioRxiv2019

ThX - A next-generation probe for the early detection of amyloid aggregates

Neurodegenerative diseases such as Alzheimers and Parkinsons 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.

biophysics↗