Discovery of potent inhibitors of α-synuclein aggregation using structure-based iterative learning.

Machine learning methods hold the promise to reduce the costs and the failure rates of conventional drug discovery pipelines. This issue is especially pressing for neurodegenerative diseases, where the development of disease-modifying drugs has been particularly challenging. To address this problem, we describe here a machine learning approach to identify small molecule inhibitors of α-synuclein aggregation, a process implicated in Parkinson's disease and other synucleinopathies. Because the proliferation of α-synuclein aggregates takes place through autocatalytic secondary nucleation, we aim to identify compounds that bind the catalytic sites on the surface of the aggregates. To achieve this goal, we use structure-based machine learning in an iterative manner to first identify and then progressively optimize secondary nucleation inhibitors. Our results demonstrate that this approach leads to the facile identification of compounds two orders of magnitude more potent than previously reported ones.

Unusual similarity of DNA solvation dynamics in high-salinity crowding with divalent cations of varying concentrations.

Double-stranded DNA bears the highest linear negative charge density (2e- per base-pair) among all biopolymers, leading to strong interactions with cations and dipolar water, resulting in the formation of a dense 'condensation layer' around DNA. Interactions involving proteins and ligands binding to DNA are primarily governed by strong electrostatic forces. Increased salt concentrations impede such electrostatic interactions - a situation that prevails in oceanic species due to their cytoplasm being enriched with salts. Nevertheless, how these interactions' dynamics are affected in crowded hypersaline environments remains largely unexplored. Here, we employ steady-state and time-resolved fluorescence Stokes shifts (TRFSS) of a DNA-bound ligand (DAPI) to investigate the static and dynamic solvation properties of DNA in the presence of two divalent cations, magnesium (Mg2+), and calcium (Ca2+) at varying high to very-high concentrations of 0.15 M, 1 M and 2 M. We compare the results to those obtained in physiological concentrations (0.15 M) of monovalent Na+ ions. Combining data from fluorescence femtosecond optical gating (FOG) and time-correlated single photon counting (TCSPC) techniques, dynamic fluorescence Stokes shifts in DNA are analysed over a broad range of time-scales, from 100 fs to 10 ns. We find that while divalent cation crowding strongly influences the DNA stability and ligand binding affinity to DNA, the dynamics of DNA solvation remain remarkably similar across a broad range of five decades in time, even in a high-salinity crowded environment with divalent cations, as compared to the physiological concentration of the Na+ ion. Steady-state and time-resolved data of the DNA-groove-bound ligand are seemingly unaffected by ion-crowding in hypersaline solution, possibly due to ions being mostly displaced by the DNA-bound ligand. Furthermore, the dynamic coupling of cations with nearby water may possibly contribute to a net-neutral effect on the overall collective solvation dynamics in DNA, owing to the strong anti-correlation of their electrostatic interaction energy fluctuations. Such dynamic scenarios may persist within the cellular environment of marine life and other biological cells that experience hypersaline conditions.

The influence of claw morphology on gripping efficiency.

This paper considers the effects of claw morphology on the gripping efficiency of arboreal (Varanus varius) and burrowing (Varanus gouldii and Varanus panoptes) lizards. To ensure a purely morphological comparison between the lizards, we circumvent the material effects of claws from different species, by modelling and testing claw replicates of the same material properties. We correlate climbing efficiency to critical morphological features including; claw height (hc), width (wc), length (lc), curvature () and tip angle (γ), which are expressed as ratios to normalise mechanically beneficial claw structures. We find that there is strong correlation between the static grip force Fsg and the claw aspect and the cross-sectional rigidity ratio , and milder correlation (i.e. higher scatter) with the profile rigidity ratio . These correlations are also true for the interlocking grip force Fint over different shaped and sized protuberances, though we note that certain protuberance size-shape couplings are of detriment to the repeatability of Fint. Of the three lizard species, the claws of the arboreal (V. varius) are found to be superior to those of the burrower lizards (V. gouldii and V. panoptes) as a result of the V. varius claws having a smaller aspect, a higher cross-sectional rigidity ratio and a small profile rigidity ratio, which are deemed noteworthy morphological parameters that influence a claw's ability to grip effectively.

Drug repurposing screens identify compounds that inhibit α-synuclein oligomers' membrane disruption and block antibody interactions.

Small soluble oligomers of the protein α-synuclein (αSO) have been linked to disruptions in neuronal homeostasis, contributing to the development of Parkinson's Disease (PD). While this makes αSO an obvious drug target, the development of effective therapeutics against αSO is challenged by its low abundance and structural and morphological complexity. Here, we employ two different approaches to neutralize toxic interactions made by αSOs with different cellular components. First, we use available data to identify four neuronal proteins as likely candidates for αSO interactions, namely Cfl1, Uchl1, Sirt2 and SerRS. However, despite promising results when immobilized, all 4 proteins only bind weakly to αSO in solution in microfluidic assays, making them inappropriate for screening. In contrast, the formation of stable contacts formed between αSO and vesicles consisting of anionic lipids not only mimics a likely biological role of αSO but also provided a platform to screen two small molecule libraries for disruptors of these contacts. Of the 7 best leads obtained in this way, 2 significantly impaired αSO contacts with other proteins in a sandwich ELISA assay using αSO-binding monoclonal antibodies and nanobodies. In addition, 5 of these leads suppressed α-synuclein amyloid formation. Thus, a repurposing screening that directly targets a key culprit in PD pathogenesis shows therapeutic potential.

From trash to treasure: Sourcing high-value, sustainable cellulosic materials from living bioreactor waste streams.

The appreciation of how conventional and fossil-based materials could be harmful to our planet is growing, especially when considering single-use and non-biodegradable plastics manufactured from fossil fuels. Accordingly, tackling climate change and plastic waste pollution entails a more responsible approach to sourcing raw materials and the adoption of less destructive end-of-life pathways. Livestock animals, in particular ruminants, process plant matter using a suite of mechanical, chemical and biological mechanisms through the act of digestion. The manure from these "living bioreactors" is ubiquitous and offers a largely untapped source of lignocellulosic biomass for the development of bio-based and biodegradable materials. In this review, we assess recent studies made into manure-based cellulose materials in terms of their material characteristics and implications for sustainability. Despite the surprisingly diverse body of research, it is apparent that progress towards the commercialisation of manure-derived cellulose materials is hindered by a lack of truly sustainable options and robust data to assess the performance against conventional materials alternatives. Nanocellulose, a natural biopolymer, has been successfully produced by living bioreactors and is presented as a candidate for future developments. Life cycle assessments from non-wood sources are however minimal, but there are some initial indications that manure-derived nanocellulose would offer environmental benefits over traditional wood-derived sources.

Cryo-EM of prion strains from the same genotype of host identifies conformational determinants.

Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 Å resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors [anchorless (a) 22L]. Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular ß-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register ß-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype.

A penetratin-derived peptide reduces the membrane permeabilization and cell toxicity of α-synuclein oligomers.

Parkinson's disease is a neurodegenerative movement disorder associated with the intracellular aggregation of α-synuclein (α-syn). Cytotoxicity is mainly associated with the oligomeric species (αSOs) formed at early stages in α-syn aggregation. Consequently, there is an intense focus on the discovery of novel inhibitors such as peptides to inhibit oligomer formation and toxicity. Here, using peptide arrays, we identified nine peptides with high specificity and affinity for αSOs. Of these, peptides p194, p235, and p249 diverted α-syn aggregation from fibrils to amorphous aggregates with reduced ß-structures and increased random coil content. However, they did not reduce αSO cytotoxicity and permeabilization of large anionic unilamellar vesicles. In parallel, we identified a non-self-aggregating peptide (p216), derived from the cell-penetrating peptide penetratin, which showed 12-fold higher binding affinity to αSOs than to α-syn monomers (Kdapp 2.7 and 31.2 µM, respectively). p216 reduced αSOs-induced large anionic unilamellar vesicle membrane permeability at 10-1 to 10-3 mg/ml by almost 100%, was not toxic to SH-SY5Y cells, and reduced αSOs cytotoxicity by about 20%. We conclude that p216 is a promising starting point from which to develop peptides targeting toxic αSOs in Parkinson's disease.

Polarized α-synuclein trafficking and transcytosis across brain endothelial cells via Rab7-decorated carriers.

Parkinson's disease is mainly caused by aggregation of α-synuclein (α-syn) in the brain. Exchange of α-syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of α-syn across the blood brain-barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of α-syn monomers and oligomers across an in vitro BBB model system. Both α-syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric α-syn in brain endothelial cells, we investigated co-localization of α-syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric α-syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric α-syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric α-syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric α-syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric α-syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinson's disease and related synucleinopathies.

RT-QuIC and Related Assays for Detecting and Quantifying Prion-like Pathological Seeds of α-Synuclein.

Various disease-associated forms or strains of α-synuclein (αSynD) can spread and accumulate in a prion-like fashion during synucleinopathies such as Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA). This capacity for self-propagation has enabled the development of seed amplification assays (SAAs) that can detect αSynD in clinical samples. Notably, α-synuclein real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays have evolved as ultrasensitive, specific, and relatively practical methods for detecting αSynD in a variety of biospecimens including brain tissue, CSF, skin, and olfactory mucosa from synucleinopathy patients. However, αSyn SAAs still lack concordance in detecting MSA and familial forms of PD/DLB, and the assay parameters show poor correlations with various clinical measures. End-point dilution analysis in αSyn RT-QuIC assays allows for the quantitation of relative amounts of αSynD seeding activity that may correlate moderately with clinical measures and levels of other biomarkers. Herein, we review recent advancements in α-synuclein SAAs for detecting αSynD and describe in detail the modified Spearman-Karber quantification algorithm used with end-point dilutions.

The C-terminal tail of α-synuclein protects against aggregate replication but is critical for oligomerization.

Aggregation of the 140-residue protein α-synuclein (αSN) is a key factor in the etiology of Parkinson's disease. Although the intensely anionic C-terminal domain (CTD) of αSN does not form part of the amyloid core region or affect membrane binding ability, truncation or reduction of charges in the CTD promotes fibrillation through as yet unknown mechanisms. Here, we study stepwise truncated CTDs and identify a threshold region around residue 121; constructs shorter than this dramatically increase their fibrillation tendency. Remarkably, these effects persist even when as little as 10% of the truncated variant is mixed with the full-length protein. Increased fibrillation can be explained by a substantial increase in self-replication, most likely via fragmentation. Paradoxically, truncation also suppresses toxic oligomer formation, and oligomers that can be formed by chemical modification show reduced membrane affinity and cytotoxicity. These remarkable changes correlate to the loss of negative electrostatic potential in the CTD and highlight a double-edged electrostatic safety guard.

Mixed-Mode Interlaminar Fracture Toughness of Glass and Carbon Fibre Powder Epoxy Composites-For Design of Wind and Tidal Turbine Blades.

Powder epoxy composites have several advantages for the processing of large composite structures, including low exotherm, viscosity and material cost, as well as the ability to carry out separate melting and curing operations. This work studies the mode I and mixed-mode toughness, as well as the in-plane mechanical properties of unidirectional stitched glass and carbon fibre reinforced powder epoxy composites. The interlaminar fracture toughness is studied in pure mode I by performing Double Cantilever Beam tests and at 25% mode II, 50% mode II and 75% mode II by performing Mixed Mode Bending testing according to the ASTM D5528-13 test standard. The tensile and compressive properties are comparable to that of standard epoxy composites but both the mode I and mixed-mode toughness are shown to be significantly higher than that of other epoxy composites, even when comparing to toughened epoxies. The mixed-mode critical strain energy release rate as a function of the delamination mode ratio is also provided. This paper highlights the potential for powder epoxy composites in the manufacturing of structures where there is a risk of delamination.

The Impact Behaviour of Crab Carapaces in Relation to Morphology.

Brachyuran crab carapaces are protective, impact-resistant exoskeletons with elaborate material microstructures. Though several research efforts have been made to characterise the physical, material and mechanical properties of the crab carapace, there are no studies detailing how crab morphologies might influence impact resistance. The purpose of this paper is to characterise and compare Brachyuran crab carapace morphologies in relation to their impact properties, using opto-digital, experimental and numerical methods. We find that crab carapaces with both extended carapace arc-lengths and deep carapace grooves lose stiffness rapidly under cyclic impact loading, and fail in a brittle manner. Contrarily, carapaces with smaller arc lengths and shallower, more broadly distributed carapace grooves are more effective in dissipating stresses caused by impact throughout the carapace structure. This allows them to retain stiffness for longer, and influences their failure mode, which is ductile (denting), rather than brittle fracture. The findings in this paper provide new bioinspired approaches for the geometrical designs by which means material failure under cyclic impact can be controlled and manipulated.

Genetic analysis of SARS-CoV-2 isolates collected from Bangladesh: insights into the origin, mutation spectrum, and possible pathomechanism

As the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), rages across the world, killing hundreds of thousands and infecting millions, researchers are racing against time to elucidate the viral genome. Some Bangladeshi institutes are also in this race, sequenced a few isolates of the virus collected from Bangladesh. Here, we present a genomic analysis of 14 isolates. The analysis revealed that SARS-CoV-2 isolates sequenced from Dhaka and Chittagong were the lineage of Europe and the Middle East, respectively. Our analysis identified a total of 42 mutations, including three large deletions, half of which were synonymous. Most of the missense mutations in Bangladeshi isolates found to have weak effects on the pathogenesis. Some mutations may lead the virus to be less pathogenic than the other countries. Molecular docking analysis to evaluate the effect of the mutations on the interaction between the viral spike proteins and the human ACE2 receptor, though no significant interaction was observed. This study provides some preliminary insights into the origin of Bangladeshi SARS-CoV-2 isolates, mutation spectrum and its possible pathomechanism, which may give an essential clue for designing therapeutics and management of COVID-19 in Bangladesh.

The water-hopping kinematics of the tree-climbing fish, Periophthalmus variabilis.

In this communication, we describe the water-hopping kinematics of the dusky-gilled mudskipper (Periophthalmus variabilis), and by doing so elucidate an entirely new form of fish locomotion that has yet to be reported in the public domain. Water-hopping is defined herein as an ability to hop once, or in succession, on the surface of water without full submergence and without a fin-guided glide. We find that taxiing on the water surface is the predominating kinematic movement used for the execution of successful water-hops. We observe that an initial concentric ripple forms as the mudskipper impacts the water, and that subsequent taxiing on the water surface generates a sinusoid-like ripple pattern in the water prior to take off. Interestingly whilst airborne, the pectoral fins of P. variabilis appear to remain stationary, only to be deployed upon contact with the water. When landing back onto the surface of the water, P. variabilis makes the initial contact via its pelvic region, occasionally extending its pectoral fins during its descent. The reasons for pectoral and pelvic fin extension are unclear, however, there may be either aerodynamic or hydrodynamic benefits in its doing so. This motion furthermore prepares the mudskipper for either, a follow-on water-hop, or a discontinuation of movement altogether, as the body of the mudskipper becomes aligned in a way conducive to either. P. variabilis will launch and land using both, horizontal surfaces such as littorals, and inclined-to-vertical surfaces such as rocks and trees.

Both beta sheet breaker and alpha helix forming pentapeptide inhibits protein fibrillation: Implication for the treatment of amyloid disorders.

For the first time, the effect of two novel designed pentapeptides on amyloid growth of human insulin using combined biophysical, microscopic, cell viability and computational approaches. Collective experimental data from ThT, ANS, and TEM demonstrate that in spite of having contrasting features, both peptides can effectively inhibit amyloid formation by prolonging lag phase, slowing down aggregation rate, and reducing final fibril formation (up to 84.26% and 85.24% by P1 and P7 respectively). Although pure amyloid caused profound cellular toxicity in SH-SY5Y neuronal cells, amyloid formed in the presence of peptides showed much reduced cellular toxicity. Such an inhibitory effect can be attributed to interference with the structural transition of insulin toward ß-sheet structure by peptides. Furthermore, molecular dynamic simulations confirm that peptide preferentially binds to nearby region which is more prone to form aggregates that consequently disrupts self-assembly into amyloid fibrils (P1 and P7 possess inhibition constant value of 0.000183 and 0.000216 nm, respectively), supporting our experimental observations. This study underscores the information about the sequence based inhibition mechanism of peptides that might dictate their inhibition or modulation capacity, which might be helpful in designing anti-amyloid therapeutics.

Origin of Slow Solvation Dynamics in DNA: DAPI in Minor Groove of Dickerson-Drew DNA.

The measurement and understanding of collective solvation dynamics in DNA have vital biological implications, as protein and ligand binding to DNA can be directly controlled by complex electrostatic interactions of anionic DNA and surrounding dipolar water, and ions. Time-resolved fluorescence Stokes shift (TRFSS) experiments revealed anomalously slow solvation dynamics in DNA much beyond 100 ps that follow either power-law or slow multiexponential decay over several nanoseconds. The origin of such dispersed dynamics remains difficult to understand. Here we compare results of TRFSS experiments to molecular dynamics (MD) simulations of well-known 4',6-diamidino-2-phenylindole (DAPI)/Dickerson-Drew DNA complex over five decades of time from 100 fs to 10 ns to understand the origin of such dispersed dynamics. We show that the solvation time-correlation function (TCF) calculated from 200 ns simulation trajectory (total 800 ns) captures most features of slow dynamics as measured in TRFSS experiments. Decomposition of TCF into individual components unravels that slow dynamics originating from dynamically coupled DNA-water motion, although contribution from coupled water-Na+ motion is non-negligible. The analysis of residence time of water molecules around the probe (DAPI) reveals broad distribution from ∼6 ps to ∼3.5 ns: Several (49 nos.) water molecules show residences time greater than 500 ps, of which at least 14 water molecules show residence times of more than 1 ns in the first solvation shell of DAPI. Most of these slow water molecules are found to occupy two hydration sites in the minor groove near DAPI binding site. The residence time of Na+, however, is found to vary within ∼17-120 ps. Remarkably, we find that freezing the DNA fluctuations in simulation eliminates slower dynamics beyond ∼100 ps, where water and Na+ dynamics become faster, although strong anticorrelation exists between them. These results indicate that primary origin of slow dynamics lies within the slow fluctuations of DNA parts that couple with nearby slow water and ions to control the dispersed collective solvation dynamics in DNA minor groove.

α-synuclein oligomers and fibrils: a spectrum of species, a spectrum of toxicities.

This review article provides an overview of the different species that α-synuclein aggregates can populate. It also attempts to reconcile conflicting views regarding the cytotoxic roles of oligomers versus fibrils. α-synuclein, while highly dynamic in the monomeric state, can access a large number of different assembly states. Depending on assembly conditions, these states can interconvert over different timescales. The fibrillar state is the most thermodynamically favored due to the many stabilizing interactions formed between each monomeric unit, but different fibrillar types form at different rates. The end distribution is likely to reflect kinetic partitioning as much as thermodynamic equilibra. In addition, metastable oligomeric species, some of which are on-pathway and others off-pathway, can be populated for remarkably long periods of time. Chemical modifications (phosphorylation, oxidation, covalent links to ligands, etc.) perturb these physical interconversions and invariably destabilize the fibrillar state, leading to small prefibrillar assemblies which can coalesce into amorphous states. Both oligomeric and fibrillar species have been shown to be cytotoxic although firm conclusions require very careful evaluation of particle concentrations and is complicated by the great variety and heterogeneity of different experimentally observed states. The mechanistic relationship between oligomers and fibrils remains to be clarified, both in terms of assembly of oligomers into fibrils and potential dissolution of fibrils into oligomers. While oligomers are possibly implicated in the collapse of neuronal homeostasis, the fibrillar state(s) appears to be the most efficient at propagating itself both in vitro and in vivo, pointing to critical roles for multiple different aggregate species in the progression of Parkinson's disease (https://onlinelibrary.wiley.com/page/journal/14714159/homepage/virtual_issues.htm). This article is part of the Special Issue "Synuclein".

Elucidating the inhibitory potential of Vitamin A against fibrillation and amyloid associated cytotoxicity.

Protein aggregation and amyloid fibrillation are associated with many serious human pathophysiologies like Alzheimer's, Parkinson's diseases, type II diabetes etc. A powerful strategy for controlling and understanding amyloid protein aggregation is the modulation of protein self-assembly. In this study, anti-fibrillation activity of vitamin A (VA) and its effect on the kinetics of amyloid formation of Aß-42 peptide was investigated by employing various spectroscopic, imaging and computational approaches. The present data of Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), dynamic light scattering assay, transmission electron microscopy and cell cytotoxicity assay demonstrated that vitamin A significantly inhibits fibril formation. Our experimental studies inferred that Vitamin A protects human neuroblastoma cell line (SH-SY5Y) and the neuroprotective effect against amyloid induced cytotoxicity is through modification of the amyloid formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin A interacts with Aß-42 through hydrophobic interactions as well as hydrogen bonding. Therefore, the study signifies the role of vitamin A as a potential molecule in preventing Aß-42 aggregation and associated pathophysiology. Hence, Vitamin A and related compounds can thus act as effective inhibitors in the therapeutic development to combat systemic amyloidosis.