A Relationship between the Structures and Neurotoxic Effects of Aß Oligomers Stabilized by Different Metal Ions.

Oligomeric assemblies of the amyloid ß peptide (Aß) have been investigated for over two decades as possible neurotoxic agents in Alzheimer's disease. However, due to their heterogeneous and transient nature, it is not yet fully established which of the structural features of these oligomers may generate cellular damage. Here, we study distinct oligomer species formed by Aß40 (the 40-residue form of Aß) in the presence of four different metal ions (Al3+, Cu2+, Fe2+, and Zn2+) and show that they differ in their structure and toxicity in human neuroblastoma cells. We then describe a correlation between the size of the oligomers and their neurotoxic activity, which provides a type of structure-toxicity relationship for these Aß40 oligomer species. These results provide insight into the possible role of metal ions in Alzheimer's disease by the stabilization of Aß oligomers.

Maturation-dependent changes in the size, structure and seeding capacity of Aß42 amyloid fibrils.

Many proteins self-assemble to form amyloid fibrils, which are highly organized structures stabilized by a characteristic cross-ß 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-ß 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.

Interconnectable 3D-printed sample processing modules for portable mycotoxin screening of intact wheat.

BACKGROUND: The increasing demand for food and feed products is stretching the capacity of the food value chain to its limits. A key step for ensuring food safety is checking for mycotoxin contamination of wheat. However, this analysis is typically performed by rather complex and expensive chromatographic methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). These costly methods require extensive sample preparation that is not easily carried out at different points along the food supply chain. To overcome such challenges in sample processing, an inexpensive and portable sample preparation device was needed, that required low skill, for rapid sample-to-result mycotoxin screening. RESULTS: We describe 3D-printed and interconnectable modules for simple, integrated and on-site sample preparation, including grinding of wheat kernels, and solvent-based extraction. We characterized these 3D-printed modules for mycotoxin screening and benchmarked them against a laboratory mill using commercial lateral flow device(s) (LFD) and in-house validated LC-MS/MS analysis. Different integrated sieve configurations were compared based on grinding efficiency, and we selected a sieve size of 2 mm allowing grinding of 10 g of wheat within 5 min. Moreover, 10 first time-users were able to operate the grinder module with minimal instructions. Screening for deoxynivalenol (DON) in naturally contaminated samples at the regulatory/legal limit (1.25 mg kg-1) was demonstrated using the developed 3D-printed prototype. The whole process only takes 15 min, from sample preparation to screening result. The results showed a clear correlation (R2 = 0.96) between the LFD and LC-MS/MS. SIGNIFICANCE: Our findings demonstrate the potential of 3D-printed sample handling equipment as a valuable extension of existing analytical procedures, facilitating the on-site implementation of rapid methods for the determination of mycotoxins in grains. The presented prototype is inexpensive with material costs of 2.5€, relies on biodegradable 3D printing filament and can be produced with consumer-grade printers, making the prototype readily available. As a future perspective, the modular character of our developed tool kit will allow for adaptation to other hard food commodities beyond the determination of DON in wheat.

The liquid-to-solid transition of FUS is promoted by the condensate surface.

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.

Formation of amyloid loops in brain tissues is controlled by the flexibility of protofibril chains.

Neurodegenerative diseases, such as Alzheimer's disease (AD), are associated with protein misfolding and aggregation into amyloid fibrils. Increasing evidence suggests that soluble, low-molecular-weight aggregates play a key role in disease-associated toxicity. Within this population of aggregates, closed-loop pore-like structures have been observed for a variety of amyloid systems, and their presence in brain tissues is associated with high levels of neuropathology. However, their mechanism of formation and relationship with mature fibrils have largely remained challenging to elucidate. Here, we use atomic force microscopy and statistical theory of biopolymers to characterize amyloid ring structures derived from the brains of AD patients. We analyze the bending fluctuations of protofibrils and show that the process of loop formation is governed by the mechanical properties of their chains. We conclude that ex vivo protofibril chains possess greater flexibility than that imparted by hydrogen-bonded networks characteristic of mature amyloid fibrils, such that they are able to form end-to-end connections. These results explain the diversity in the structures formed from protein aggregation and shed light on the links between early forms of flexible ring-forming aggregates and their role in disease.

Enhanced surface nanoanalytics of transient biomolecular processes.

Fundamental knowledge of the physical and chemical properties of biomolecules is key to understanding molecular processes in health and disease. Bulk and single-molecule analytical methods provide rich information about biomolecules but often require high concentrations and sample preparation away from physiologically relevant conditions. Here, we present the development and application of a lab-on-a-chip spray approach that combines rapid sample preparation, mixing, and deposition to integrate with a range of nanoanalytical methods in chemistry and biology, providing enhanced spectroscopic sensitivity and single-molecule spatial resolution. We demonstrate that this method enables multidimensional study of heterogeneous biomolecular systems over multiple length scales by nanoscopy and vibrational spectroscopy. We then illustrate the capabilities of this platform by capturing and analyzing the structural conformations of transient oligomeric species formed at the early stages of the self-assembly of α-synuclein, which are associated with the onset of Parkinson's disease.

Rapid Single Particle Atmospheric Solids Analysis Probe-Mass Spectrometry for Multimodal Analysis of Microplastics.

Despite mass spectrometry (MS) being proven powerful for the characterization of synthetic polymers, its potential for the analysis of single particle microplastics (MPs) is yet to be fully disclosed. To date, MPs are regarded as ubiquitous contaminants, but the limited availability of techniques that enable full characterizations of MPs results in a lack of systematic data regarding their occurrence. In this study, an atmospheric solid analysis probe (ASAP) coupled to a compact quadrupole MS is proposed for the chemical analysis of single particle microplastics, while maintaining full compatibility with complementary staining and image analysis approaches. A two-stage ASAP probe temperature program was optimized for the removal of additives and surface contaminants followed by the actual polymer characterization. The method showed specific mass spectra for a wide range of single particle MPs, including polyolefins, polyaromatics, polyacrylates, (bio)polyesters, polyamides, polycarbonates, and polyacrylonitriles. The single particle size detection limits for polystyrene MPs were found to be 30 and 5 µm in full scan and selected ion recording mode, respectively. Moreover, results are presented of a multimodal microplastic analysis approach in which filtered particles are first characterized by staining and fluorescence microscopy, followed by simple probe picking of individual particles for subsequent analysis by ASAP-MS. The method provides a full characterization of MP contamination, including particle number, particle size, particle shape, and chemical identity. The applicability of the developed multimodal method was successfully demonstrated by the analysis of MPs in bioplastic bottled water.

Characterization of full-length p53 aggregates and their kinetics of formation.

Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis.

Synthesis of well-defined linear-bottlebrush-linear triblock copolymer towards architecturally-tunable soft materials.

Linear-bottlebrush-linear (LBBL) triblock copolymers are emerging systems for topologically-tunable elastic materials. In this paper, a new synthetic methodology is presented to synthesize LBBL polystyrene-block-bottlebrushpolydimethylsiloxane-block-polystyrene (PS-b-bbPDMS-b-PS) triblock copolymer via the "grafting onto" approach where the precursors are individually synthesized through living anionic polymerization and selective coupling reaction. In this two-step approach, polystyrene-block-polymethylvinylsiloxane (PS-b-PMVS) diblock copolymer with a low dispersity couples with another living PS block to form PS-b-PMVS-b-PS triblock copolymer. Secondly, this is followed by grafting of separately prepared monohydride-terminated PDMS chains with controllable grafting density through a hydrosilylation reaction. In addition to fully tunable architectural parameters, this approach permits a quantitative determination of the ratio of diblock and triblock bottlebrush copolymers and consistency between batches, highlighting the feasibility for scaled-up production. These LBBL triblock copolymers self-assemble into soft, low-modulus thermoplastic elastomers, and the precise knowledge of the composition is crucial for correlating microstructure to mechanical properties.

Stabilized tilted-octahedra halide perovskites inhibit local formation of performance-limiting phases.

Efforts to stabilize photoactive formamidinium (FA)­based halide perovskites for perovskite photovoltaics have focused on the growth of cubic formamidinium lead iodide (α-FAPbI3) phases by empirically alloying with cesium, methylammonium (MA) cations, or both. We show that such stabilized FA-rich perovskites are noncubic and exhibit ~2° octahedral tilting at room temperature. This tilting, resolvable only with the use of local nanostructure characterization techniques, imparts phase stability by frustrating transitions from photoactive to hexagonal phases. Although the bulk phase appears stable when examined macroscopically, heterogeneous cation distributions allow microscopically unstable regions to form; we found that these transitioned to hexagonal polytypes, leading to local trap-assisted performance losses and photoinstabilities. Using surface-bound ethylenediaminetetraacetic acid, we engineered an octahedral tilt into pure α-FAPbI3 thin films without any cation alloying. The templated photoactive FAPbI3 film was extremely stable against thermal, environmental, and light stressors.

In situ Sub-Cellular Identification of Functional Amyloids in Bacteria and Archaea by Infrared Nanospectroscopy.

Formation of amyloid structures is originally linked to human disease. However, amyloid materials are found extensively in the animal and bacterial world where they stabilize intra- and extra-cellular environments like biofilms or cell envelopes. To date, functional amyloids have largely been studied using optical microscopy techniques in vivo, or after removal from their biological context for higher-resolution studies in vitro. Furthermore, conventional microscopies only indirectly identify amyloids based on morphology or unspecific amyloid dyes. Here, the high chemical and spatial (≈20 nm) resolution of Infrared Nanospectroscopy (AFM-IR) to investigate functional amyloid from Escherichia coli (curli), Pseudomonas (Fap), and the Archaea Methanosaeta (MspA) in situ is exploited. It is demonstrated that AFM-IR identifies amyloid protein within single intact cells through their cross ß-sheet secondary structure, which has a unique spectroscopic signature in the amide I band of protein. Using this approach, nanoscale-resolved chemical images and spectra of purified curli and Methanosaeta cell wall sheaths are provided. The results highlight significant differences in secondary structure between E. coli cells with and without curli. Taken together, these results suggest that AFM-IR is a new and powerful label-free tool for in situ investigations of the biophysical state of functional amyloid and biomolecules in general.

The Hsc70 disaggregation machinery removes monomer units directly from α-synuclein fibril ends.

Molecular chaperones contribute to the maintenance of cellular protein homoeostasis through assisting de novo protein folding and preventing amyloid formation. Chaperones of the Hsp70 family can further disaggregate otherwise irreversible aggregate species such as α-synuclein fibrils, which accumulate in Parkinson's disease. However, the mechanisms and kinetics of this key functionality are only partially understood. Here, we combine microfluidic measurements with chemical kinetics to study α-synuclein disaggregation. We show that Hsc70 together with its co-chaperones DnaJB1 and Apg2 can completely reverse α-synuclein aggregation back to its soluble monomeric state. This reaction proceeds through first-order kinetics where monomer units are removed directly from the fibril ends with little contribution from intermediate fibril fragmentation steps. These findings extend our mechanistic understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and inform on possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies.

Alpha Synuclein only Forms Fibrils In Vitro when Larger than its Critical Size of 70 Monomers.

The aggregation of α-synuclein into small soluble aggregates and then fibrils is important in the development and spreading of aggregates through the brain in Parkinson's disease. Fibrillar aggregates can grow by monomer addition and then break into fragments that could spread into neighboring cells. The rate constants for fibril elongation and fragmentation have been measured but it is not known how large an aggregate needs to be before fibril formation is thermodynamically favorable. This critical size is an important parameter controlling at what stage in an aggregation reaction fibrils can form and replicate. We determined this value to be approximately 70 monomers using super-resolution and atomic force microscopy imaging of individual α-synuclein aggregates formed in solution over long time periods. This represents the minimum size for a stable α-synuclein fibril and we hypothesis the formation of aggregates of this size in a cell represents a tipping point at which rapid replication occurs.

Controlled self-assembly of plant proteins into high-performance multifunctional nanostructured films.

The abundance of plant-derived proteins, as well as their biodegradability and low environmental impact make them attractive polymeric feedstocks for next-generation functional materials to replace current petroleum-based systems. However, efforts to generate functional materials from plant-based proteins in a scalable manner have been hampered by the lack of efficient methods to induce and control their micro and nanoscale structure, key requirements for achieving advantageous material properties and tailoring their functionality. Here, we demonstrate a scalable approach for generating mechanically robust plant-based films on a metre-scale through controlled nanometre-scale self-assembly of water-insoluble plant proteins. The films produced using this method exhibit high optical transmittance, as well as robust mechanical properties comparable to engineering plastics. Furthermore, we demonstrate the ability to impart nano- and microscale patterning into such films through templating, leading to the formation of hydrophobic surfaces as well as structural colour by controlling the size of the patterned features.

Infrared nanospectroscopy reveals the molecular interaction fingerprint of an aggregation inhibitor with single Aß42 oligomers.

Significant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer's and Parkinson's diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the structure and interaction between single Aß42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent Aß42 aggregation in vitro and reverses its neurotoxicity in cell and animal models of Alzheimer's disease. Our results demonstrate that the carboxyl group of this compound interacts with Aß42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases.

Evolution of Conformation, Nanomechanics, and Infrared Nanospectroscopy of Single Amyloid Fibrils Converting into Microcrystals.

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 ß-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 ß-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.

Small-molecule sequestration of amyloid-ß as a drug discovery strategy for Alzheimer's disease.

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid-ß (Aß) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aß and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric Aß while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aß-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes.

Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition.

Membrane-less organelles resulting from liquid-liquid phase separation of biopolymers into intracellular condensates control essential biological functions, including messenger RNA processing, cell signalling and embryogenesis1-4. It has recently been discovered that several such protein condensates can undergo a further irreversible phase transition, forming solid nanoscale aggregates associated with neurodegenerative disease5-7. While the irreversible gelation of protein condensates is generally related to malfunction and disease, one case where the liquid-to-solid transition of protein condensates is functional, however, is that of silk spinning8,9. The formation of silk fibrils is largely driven by shear, yet it is not known what factors control the pathological gelation of functional condensates. Here we demonstrate that four proteins and one peptide system, with no function associated with fibre formation, have a strong propensity to undergo a liquid-to-solid transition when exposed to even low levels of mechanical shear once present in their liquid-liquid phase separated form. Using microfluidics to control the application of shear, we generated fibres from single-protein condensates and characterized their structural and material properties as a function of shear stress. Our results reveal generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. These observations suggest that shear can play an important role in the irreversible liquid-to-solid transition of protein condensates, shed light on the role of physical factors in driving this transition in protein aggregation-related diseases and open a new route towards artificial shear responsive biomaterials.

Single molecule secondary structure determination of proteins through infrared absorption nanospectroscopy.

The chemical and structural properties of biomolecules determine their interactions, and thus their functions, in a wide variety of biochemical processes. Innovative imaging methods have been developed to characterise biomolecular structures down to the angstrom level. However, acquiring vibrational absorption spectra at the single molecule level, a benchmark for bulk sample characterization, has remained elusive. Here, we introduce off-resonance, low power and short pulse infrared nanospectroscopy (ORS-nanoIR) to allow the acquisition of infrared absorption spectra and chemical maps at the single molecule level, at high throughput on a second timescale and with a high signal-to-noise ratio (~10-20). This high sensitivity enables the accurate determination of the secondary structure of single protein molecules with over a million-fold lower mass than conventional bulk vibrational spectroscopy. These results pave the way to probe directly the chemical and structural properties of individual biomolecules, as well as their interactions, in a broad range of chemical and biological systems.

The Influence of Pathogenic Mutations in α-Synuclein on Biophysical and Structural Characteristics of Amyloid Fibrils.

Proteinaceous deposits of α-synuclein amyloid fibrils are a hallmark of human disorders including Parkinson's disease. The onset of this disease is also associated with five familial mutations of the gene encoding the protein. However, the mechanistic link between single point mutations and the kinetics of aggregation, biophysical properties of the resulting amyloid fibrils, and an increased risk of disease is still elusive. Here, we demonstrate that the disease-associated mutations of α-synuclein generate different amyloid fibril polymorphs compared to the wild type protein. Remarkably, the α-synuclein variants forming amyloid fibrils of a comparable structure, morphology, and heterogeneity show similar microscopic steps defining the aggregation kinetics. These results demonstrate that a single point mutation can significantly alter the distribution of fibrillar polymorphs in α-synuclein, suggesting that differences in the clinical phenotypes of familial Parkinson's disease could be associated with differences in the mechanism of formation and the structural characteristics of the aggregates.