Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX.

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

Time-resolved serial femtosecond crystallography at the European XFEL.

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

Formation kinetics and physicochemical properties of mesoscopic Alpha-Synuclein assemblies modulated by sodium chloride and a distinct pulsed electric field.

Alpha-Synuclein (ASN), a presynaptic protein, has been widely reported to form amyloid-rich hydrogel clusters through liquid-liquid phase separation (LLPS) and liquid-to-solid transition. However, in-depth investigations about the parameters that influence the assembling kinetics, structures, and physicochemical properties of intermediate ASN assemblies are still missing. Therefore, we monitored for the first time the assembling and ordering kinetics of ASN by polarized/depolarized light scattering (DLS/DDLS) under the effect of ionic strength and a pulsed electric field (EF), followed by characterizing the resultant ASN assemblies applying thermostability assays, fluorescence/autofluorescence assays, and TEM. The underlying molecular mechanism was discussed based on experimental evidence. Results showed that in the presence of 150-250 mM NaCl, monomeric ASN is highly soluble in a temperature range of 20-70 °C and could form dissoluble liquid dense clusters via LLPS in crowded environments, while the ionic strength of 50 mM NaCl could trigger conformational changes and attractive diffusion interactions of ASN monomers towards the formation of mesoscopic assemblies with ordered internal structures and high thermostabilities. We discovered that pulsed EFs and ionic strength can modulate effectively the thermostability and autofluorescence effect of mesoscopic ASN assemblies by tuning the molecular interaction and arrangement. Remarkably, a specie of thermostable ASN assemblies showing a maximum autofluorescence emission at approx. 700 nm was synthesized applying 250 mM NaCl and the distinct pulsed EF, which could be attributed to the increase of ß-sheet structures and hydrogen-bond networks within ASN assemblies. In summary, the presented data provide novel insights for modulating the growth kinetics, structures, and physicochemical properties of bio-macromolecular mesoscopic assemblies.

3D printed devices and infrastructure for liquid sample delivery at the European XFEL.

The Sample Environment and Characterization (SEC) group of the European X-ray Free-Electron Laser (EuXFEL) develops sample delivery systems for the various scientific instruments, including systems for the injection of liquid samples that enable serial femtosecond X-ray crystallography (SFX) and single-particle imaging (SPI) experiments, among others. For rapid prototyping of various device types and materials, sub-micrometre precision 3D printers are used to address the specific experimental conditions of SFX and SPI by providing a large number of devices with reliable performance. This work presents the current pool of 3D printed liquid sample delivery devices, based on the two-photon polymerization (2PP) technique. These devices encompass gas dynamic virtual nozzles (GDVNs), mixing-GDVNs, high-viscosity extruders (HVEs) and electrospray conical capillary tips (CCTs) with highly reproducible geometric features that are suitable for time-resolved SFX and SPI experiments at XFEL facilities. Liquid sample injection setups and infrastructure on the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument are described, this being the instrument which is designated for biological structure determination at the EuXFEL.

Biosynthesis of silver nanoparticles using extracts of Stevia rebaudiana and evaluation of antibacterial activity.

The present study reveals a simple, non-toxic and eco-friendly method for the "green" synthesis of Ag-NPs using hydroponic and soil medicinal plant Stevia rebaudiana extracts, the characterization of biosynthesized nanoparticles, as well as the evaluation of their antibacterial activity. Transmission electronic microscopy (TEM) and Dynamic Light Scattering (DLS) analysis confirmed that biosynthesized Ag-NPs are in the nano-size range (50-100 nm) and have irregular morphology. Biogenic NPs demonstrate antibacterial activity against Escherichia coli BW 25,113, Enterococcus hirae ATCC 9790, and Staphylococcus aureus MDC 5233. The results showed a more pronounced antibacterial effect on E. coli growth rate, in comparison with Gram-positive bacteria, which is linked to the differences in the structure of bacterial cell wall. Moreover, the Ag-NPs not only suppressed the growth of bacteria but also changed the energy-dependent H+-fluxes across the bacterial membrane. The change of H+-fluxes in presence of H+-translocating systems inhibitor, N,N'-dicyclohexylcarbodiimide (DCCD), proves the effect of Ag-NPs on the structure and permeability of the bacterial membrane. Overall, our findings indicate that the Ag-NPs synthesized by medicinal plant Stevia extracts may be an excellent candidate as an alternative to antibiotics against the tested bacteria.

Structure and dynamics of UBA5-UFM1 complex formation showing new insights in the UBA5 activation mechanism.

Ubiquitin fold modifier 1 (UFM1) is an ubiquitin-like protein (Ubl) involved especially in endoplasmic stress response. Activation occurs via a three-step mechanism like other Ubls. Data obtained reveal that UFM1 regulates the oligomeric state of ubiquitin activating enzyme 5 (UBA5) to initiate the activation step. Mixtures of homodimers and heterotrimers are observed in solution at the equilibrium state, demonstrating that the UBA5-UFM1 complex undergoes several concentration dependent oligomeric translational states to form a final functional complex. The oligomerization state of unbound UBA5 is also concentration dependent and shifts from the monomeric to the dimeric state. Data describing different oligomeric states are complemented with binding studies that reveal a negative cooperativity for the complex formation and thereby provide more detailed insights into the complex formation mechanism.

Pulsed electric fields induce modulation of protein liquid-liquid phase separation.

The time-resolved dynamic assembly and the structures of protein liquid dense clusters (LDCs) were analyzed under pulsed electric fields (EFs) applying complementary polarized and depolarized dynamic light scattering (DLS/DDLS), optical microscopy, and transmission electron microscopy (TEM). We discovered that pulsed EFs substantially affected overall morphologies and spatial distributions of protein LDCs and microcrystals, and affected the phase diagrams of LDC formation, including enabling protein solutions to overcome the diffusive flux energy barrier to phase separate. Data obtained from DLS/DDLS and TEM showed that LDCs appeared as precursors of protein crystal nuclei, followed by the formation of ordered structures within LDCs applying a pulsed EF. Experimental results of circular dichroism spectroscopy provided evidence that the protein secondary structure content is changing under EFs, which may consequently modulate protein-protein interactions, and the morphology, dimensions, and internal structure of LDCs. Data and results obtained unveil options to modulate the phase diagram of crystallization, and physical morphologies of protein LDCs and microcrystals by irradiating sample suspensions with pulsed EFs.

Application of Quantitative Motor Assessments in Friedreich Ataxia and Evaluation of Their Relation to Clinical Measures.

Friedreich's ataxia (FRDA) is a rare autosomal-recessive slowly progressive neurodegenerative disorder. As common clinical measures for this devastating disease lack sensitivity, we explored whether (a) the quantitative motor assessments of the Q-Motor battery can enhance clinical characterisation of FRDA; (b) clinical measures can predict Q-Motor outcomes and (c) Q-Motor is sensitive to longitudinal change. At baseline 29 patients and 23 controls and in a 1-year follow-up 14 patients and 6 controls were included. The Q-Motor included lift (manumotography), finger tapping (digitomotography) and pronate/supinate (dysdiadochomotography) tasks. To model responses, a search of generalised linear models was conducted, selecting best fitting models, using demographic and clinical data as predictors. Predictors from selected models were used in linear mixed models to investigate longitudinal changes. Patients with FRDA performed worse than controls on most measures. Modelling of the pronate/supinate task was dominated by SCAFI (SCA functional index) subtasks, while tapping task and lift task models suggested a complex relationship with clinical measures. Longitudinal modelling implied minor changes from baseline to follow-up, while clinical scales mainly showed no change in this sample. Overall Q-Motor likely has favourable properties for assessing distinct motor aspects in severe FRDA as it can be administered in wheelchair-bound patients. Further longitudinal research is warranted to fully characterise its relation to routinely used measures and scales for FRDA.

Quantitative Measurements of Motor Function in Alzheimer's Disease, Frontotemporal Dementia, and Dementia with Lewy Bodies: A Proof-of-Concept Study.

BACKGROUND: This study examines the efficacy of using quantitative measurements of motor dysfunction, compared to clinical ratings, in Alzheimer's disease (AD), frontotemporal dementia (FTD), and dementia with Lewy bodies (DLB). METHODS: In this cross-sectional study, 49 patients with a diagnosis of AD (n = 17), FTD (n = 19), or DLB (n = 13) were included and underwent cognitive testing, clinical motor evaluation, and quantitative motor tests: pronation/supination hand tapping, grasping and lifting, and finger and foot tapping. RESULTS: Our results revealed significantly higher Q-Motor values in pronation/supination and in grip lift force assessment in AD, FTD, and DLB compared to healthy controls (HC). Q-Motor values detected significant differences between AD and HC, while clinical ratings did not. CONCLUSION: Our results suggest that quantitative measurements provide more objective and sensitive measurements of motor dysfunction in dementia.

Exosomal cellular prion protein drives fibrillization of amyloid beta and counteracts amyloid beta-mediated neurotoxicity.

Alzheimer's disease is a common neurodegenerative, progressive, and fatal disorder. Generation and deposition of amyloid beta (Aß) peptides associate with its pathogenesis and small soluble Aß oligomers show the most pronounced neurotoxic effects and correlate with disease initiation and progression. Recent findings showed that Aß oligomers bind to the cellular prion protein (PrP(C) ) eliciting neurotoxic effects. The role of exosomes, small extracellular vesicles of endosomal origin, in Alzheimer's disease is only poorly understood. Besides serving as disease biomarkers they may promote Aß plaque formation, decrease Aß-mediated synaptotoxicity, and enhance Aß clearance. Here, we explore how exosomal PrP(C) connects to protective functions attributed to exosomes in Alzheimer's disease. To achieve this, we generated a mouse neuroblastoma PrP(C) knockout cell line using transcription activator-like effector nucleases. Using these, as well as SH-SY5Y human neuroblastoma cells, we show that PrP(C) is highly enriched on exosomes and that exosomes bind amyloid beta via PrP(C) . Exosomes showed highest binding affinity for dimeric, pentameric, and oligomeric Aß species. Thioflavin T assays revealed that exosomal PrP(C) accelerates fibrillization of amyloid beta, thereby reducing neurotoxic effects imparted by oligomeric Aß. Our study provides further evidence for a protective role of exosomes in Aß-mediated neurodegeneration and highlights the importance of exosomal PrP(C) in molecular mechanisms of Alzheimer's disease. We show that the prion protein (PrP(C) ) on exosomes captures neurotoxic species of amyloid beta (Aß) promoting its fibrillization. Our study provides evidence for a protective role of exosomes in Alzheimer`s disease and suggests that, depending on its membrane topology, PrP(C) holds a dual function: when expressed at the neuronal surface it acts as receptor for Aß leading to neurotoxic signaling, whereas it detoxifies Aß when present on exosomes. This provides further support for key roles of PrP(C) in Alzheimer's disease.

Comparative Study of Physicochemical Properties and Antibacterial Potential of Cyanobacteria Spirulina platensis-Derived and Chemically Synthesized Silver Nanoparticles.

The "green synthesis" of nanoparticles (NPs) offers cost-effective and environmentally friendly advantages over chemical synthesis by utilizing biological sources such as bacteria, algae, fungi, or plants. In this context, cyanobacteria and their components are valuable sources to produce various NPs. The present study describes the comparative analysis of physicochemical and antibacterial properties of chemically synthesized (Chem-AgNPs) and cyanobacteria Spirulina platensis-derived silver NPs (Splat-AgNPs). The physicochemical characterization applying complementary dynamic light scattering and transmission electron microscopy revealed that Splat-AgNPs have an average hydrodynamic radius of ∼ 28.70 nm and spherical morphology, whereas Chem-AgNPs are irregular-shaped with an average radius size of ∼ 53.88 nm. The X-ray diffraction pattern of Splat-AgNPs confirms the formation of face-centered cubic crystalline AgNPs by "green synthesis". Energy-dispersive spectroscopy analysis demonstrated the purity of the Splat-AgNPs. Fourier transform infrared spectroscopy analysis of Splat-AgNPs demonstrated the involvement of some functional groups in the formation of NPs. Additionally, Splat-AgNPs demonstrated high colloidal stability with a zeta-potential value of (-50.0 ± 8.30) mV and a pronounced bactericidal activity against selected Gram-positive (Enterococcus hirae and Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa and Salmonella typhimurium) bacteria compared with Chem-AgNPs. Furthermore, our studies toward understanding the action mechanism of NPs showed that Splat-AgNPs alter the permeability of bacterial membranes and the energy-dependent H+-fluxes via FoF1-ATPase, thus playing a crucial role in bacterial energetics. The insights gained from this study show that Spirulina-derived synthesis is a low-cost, simple approach to producing stable AgNPs for their energy-metabolism-targeted antibacterial applications in biotechnology and biomedicine.

Multifeature quantitative motor assessment of upper limb ataxia including drawing and reaching.

OBJECTIVE: Voluntary upper limb movements are an ecologically important yet insufficiently explored digital-motor outcome domain for trials in degenerative ataxia. We extended and validated the trial-ready quantitative motor assessment battery "Q-Motor" for upper limb movements with clinician-reported, patient-focused, and performance outcomes of ataxia. METHODS: Exploratory single-center cross-sectional assessment in 94 subjects (46 cross-genotype ataxia patients; 48 matched controls), comprising five tasks measured by force transducer and/or position field: Finger Tapping, diadochokinesia, grip-lift, and-as novel implementations-Spiral Drawing, and Target Reaching. Digital-motor measures were selected if they discriminated from controls (AUC >0.7) and correlated-with at least one strong correlation (rho ≥0.6)-to the Scale for the Assessment and Rating of Ataxia (SARA), activities of daily living (FARS-ADL), and the Nine-Hole Peg Test (9HPT). RESULTS: Six movement features with 69 measures met selection criteria, including speed and variability in all tasks, stability in grip-lift, and efficiency in Target Reaching. The novel drawing/reaching tasks best captured impairment in dexterity (|rho9HPT| ≤0.81) and FARS-ADL upper limb items (|rhoADLul| ≤0.64), particularly by kinematic analysis of smoothness (SPARC). Target hit rate, a composite of speed and endpoint precision, almost perfectly discriminated ataxia and controls (AUC: 0.97). Selected measures in all tasks discriminated between mild, moderate, and severe impairment (SARA upper limb composite: 0-2/>2-4/>4-6) and correlated with severity in the trial-relevant mild ataxia stage (SARA ≤10, n = 20). INTERPRETATION: Q-Motor assessment captures multiple features of impaired upper limb movements in degenerative ataxia. Validation with key clinical outcome domains provides the basis for evaluation in longitudinal studies and clinical trial settings.

Protocol for a randomised controlled unblinded feasibility trial of HD-DRUM: a rhythmic movement training application for cognitive and motor symptoms in people with Huntington's disease.

INTRODUCTION: Huntington's disease (HD) is an inherited neurodegenerative disease causing progressive cognitive and motor decline, largely due to basal ganglia (BG) atrophy. Rhythmic training offers promise as therapy to counteract BG-regulated deficits. We have developed HD-DRUM, a tablet-based app to enhance movement synchronisation skills and improve cognitive and motor abilities in people with HD. This paper outlines a randomised controlled unblinded trial protocol to determine the feasibility of a larger effectiveness trial for HD-DRUM. Additionally, the trial investigates cognitive and motor function measures, along with brain microstructure, aiming to advance our understanding of the neural mechanisms underlying training effects. METHODS, DESIGN AND ANALYSIS: 50 individuals with HD, confirmed by genetic testing, and a Total Functional Capacity (TFC) score of 9-13, will be recruited into a two-arm randomised controlled feasibility trial. Consenting individuals with HD will be randomised to the intervention group, which entails 8 weeks of at-home usage of HD-DRUM or a usual-activity control group. All participants will undergo cognitive and motor assessments, alongside ultra-strong gradient (300 mT/m) brain microstructural MRI before and after the 8-week period. The feasibility assessment will encompass recruitment, retention, adherence and acceptability of HD-DRUM following prespecified criteria. The study will also evaluate variations in cognitive and motor performance and brain microstructure changes resulting from the intervention to determine effect size estimates for future sample size calculations. ETHICS AND DISSEMINATION: The study has received favourable ethical opinion from the Wales Research Ethics Committee 2 (REC reference: 22/WA/0147) and is sponsored by Cardiff University (SPON1895-22) (Research Integrity, Governance and Ethics Team, Research & Innovation Services, Cardiff University, second Floor, Lakeside Building, University Hospital of Wales, Cardiff, CF14 4XW). Findings will be disseminated to researchers and clinicians in peer-reviewed publications and conference presentations, and to participants, carers and the general public via newsletters and public engagement activities. Data will be shared with the research community via the Enroll-HD platform. TRIAL REGISTRATION NUMBER: ISRCTN11906973.

Safety and efficacy of laquinimod for Huntington's disease (LEGATO-HD): a multicentre, randomised, double-blind, placebo-controlled, phase 2 study.

BACKGROUND: Laquinimod modulates CNS inflammatory pathways thought to be involved in the pathology of Huntington's disease. Studies with laquinimod in transgenic rodent models of Huntington's disease suggested improvements in motor function, reduction of brain volume loss, and prolonged survival. We aimed to evaluate the safety and efficacy of laquinimod in improving motor function and reducing caudate volume loss in patients with Huntington's disease. METHODS: LEGATO-HD was a multicentre, double-blind, placebo-controlled, phase 2 study done at 48 sites across ten countries (Canada, Czech Republic, Germany, Italy, Netherlands, Portugal, Russia, Spain, UK, and USA). Patients aged 21-55 years with a cytosine-adenosine-guanine (CAG) repeat length of between 36 and 49 who had symptomatic Huntington's disease with a Unified Huntington's Disease Rating Scale-Total Motor Score (UHDRS-TMS) of higher than 5 and a Total Functional Capacity score of 8 or higher were randomly assigned (1:1:1:1) by centralised interactive response technology to laquinimod 0·5 mg, 1·0 mg, or 1·5 mg, or to matching placebo, administered orally once daily over 52 weeks; people involved in the randomisation had no other role in the study. Participants, investigators, and study personnel were masked to treatment assignment. The 1·5 mg group was discontinued before recruitment was finished because of cardiovascular safety concerns in multiple sclerosis studies. The primary endpoint was change from baseline in the UHDRS-TMS and the secondary endpoint was percent change in caudate volume, both comparing the 1·0 mg group with the placebo group at week 52. Primary and secondary endpoints were assessed in the full analysis set (ie, all randomised patients who received at least one dose of study drug and had at least one post-baseline UHDRS-TMS assessment). Safety measures included adverse event frequency and severity, and clinical and laboratory examinations, and were assessed in the safety analysis set (ie, all randomised patients who received at least one dose of study drug). This trial is registered with ClinicalTrials.gov, NCT02215616, and EudraCT, 2014-000418-75, and is now complete. FINDINGS: Between Oct 28, 2014, and June 19, 2018, 352 adults with Huntington's disease (179 [51%] men and 173 [49%] women; mean age 43·9 [SD 7·6] years and 340 [97%] White) were randomly assigned: 107 to laquinimod 0·5 mg, 107 to laquinimod 1·0 mg, 30 to laquinimod 1·5 mg, and 108 to matching placebo. Least squares mean change from baseline in UHDRS-TMS at week 52 was 1·98 (SE 0·83) in the laquinimod 1·0 mg group and 1·2 (0·82) in the placebo group (least squares mean difference 0·78 [95% CI -1·42 to 2·98], p=0·4853). Least squares mean change in caudate volume was 3·10% (SE 0·38) in the 1·0 mg group and 4·86% (0·38) in the placebo group (least squares mean difference -1·76% [95% CI -2·67 to -0·85]; p=0·0002). Laquinimod was well tolerated and there were no new safety findings. Serious adverse events were reported by eight (7%) patients on placebo, seven (7%) on laquinimod 0·5 mg, five (5%) on laquinimod 1·0 mg, and one (3%) on laquinimod 1·5 mg. There was one death, which occurred in the placebo group and was unrelated to treatment. The most frequent adverse events in all laquinimod dosed groups (0·5 mg, 1·0 mg, and 1·5 mg) were headache (38 [16%]), diarrhoea (24 [10%]), fall (18 [7%]), nasopharyngitis (20 [8%]), influenza (15 [6%]), vomiting (13 [5%]), arthralgia (11 [5%]), irritability (ten [4%]), fatigue (eight [3%]), and insomnia (eight [3%]). INTERPRETATION: Laquinimod did not show a significant effect on motor symptoms assessed by the UHDRS-TMS, but significantly reduced caudate volume loss compared with placebo at week 52. Huntington's disease has a chronic and slowly progressive course, and this study does not address whether a longer duration of laquinimod treatment could have produced detectable and meaningful changes in the clinical assessments. FUNDING: Teva Pharmaceutical Industries.

Light-induced Trpin/Metout Switching During BLUF Domain Activation in ATP-bound Photoactivatable Adenylate Cyclase OaPAC.

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in an energetically unfavorable conformation for the conversion to cAMP. However, FTIR spectroscopic experiments confirm that this conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the early events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Early changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180° rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via α-helix 3 and 4 in the linker region between sensor and effector domain is discussed.

SARS-CoV-2 Mpro responds to oxidation by forming disulfide and NOS/SONOS bonds.

The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Initiation and modulation of Tau protein phase separation by the drug suramin.

Tau is an intrinsically disordered neuronal protein in the central nervous system. Aggregated Tau is the main component of neurofibrillary tangles observed in Alzheimer's disease. In vitro, Tau aggregation can be triggered by polyanionic co-factors, like RNA or heparin. At different concentration ratios, the same polyanions can induce Tau condensates via liquid-liquid phase separation (LLPS), which over time develop pathological aggregation seeding potential. Data obtained by time resolved Dynamic Light Scattering experiments (trDLS), light and electron microscopy show that intermolecular electrostatic interactions between Tau and the negatively charged drug suramin induce Tau condensation and compete with the interactions driving and stabilizing the formation of Tau:heparin and Tau:RNA coacervates, thus, reducing their potential to induce cellular Tau aggregation. Tau:suramin condensates do not seed Tau aggregation in a HEK cell model for Tau aggregation, even after extended incubation. These observations indicate that electrostatically driven Tau condensation can occur without pathological aggregation when initiated by small anionic molecules. Our results provide a novel avenue for therapeutic intervention of aberrant Tau phase separation, utilizing small anionic compounds.

LASSI-L detects early cognitive changes in pre-motor manifest Huntington's disease: a replication and validation study.

Background and objectives: Cognitive decline is an important early sign in pre-motor manifest Huntington's disease (preHD) and is characterized by deficits across multiple domains including executive function, psychomotor processing speed, and memory retrieval. Prior work suggested that the Loewenstein-Acevedo Scale for Semantic Interference and Learning (LASSI-L)-a verbal learning task that simultaneously targets these domains - could capture early cognitive changes in preHD. The current study aimed to replicate, validate and further analyze the LASSI-L in preHD using larger datasets. Methods: LASSI-L was administered to 50 participants (25 preHD and 25 Healthy Controls) matched for age, education, and sex in a longitudinal study of disease progression and compared to performance on MMSE, Trail A & B, SCWT, SDMT, Semantic Fluency (Animals), and CVLT-II. Performance was then compared to a separate age-education matched-cohort of 25 preHD participants. Receiver operating curve (ROC) and practice effects (12 month interval) were investigated. Group comparisons were repeated using a preHD subgroup restricted to participants predicted to be far from diagnosis (Far subgroup), based on CAG-Age-Product scaled (CAPs) score. Construct validity was assessed through correlations with previously established measures of subcortical atrophy. Results: PreHD performance on all sections of the LASSI-L was significantly different from controls. The proactive semantic interference section (PSI) was sensitive (p = 0.0001, d = 1.548), similar across preHD datasets (p = 1.0), reliable on test-retest over 12 months (spearman rho = 0.88; p = <0.00001) and associated with an excellent area under ROC (AUROC) of 0.855. In the preHD Far subgroup comparison, PSI was the only cognitive assessment to survive FDR < 0.05 (p = 0.03). The number of intrusions on PSI was negatively correlated with caudate volume. Discussion: The LASSI-L is a sensitive, reliable, efficient tool for detecting cognitive decline in preHD. By using a unique verbal learning test paradigm that simultaneously targets executive function, processing speed and memory retrieval, the LASSI-L outperforms many other established tests and captures early signs of cognitive impairment. With further longitudinal validation, the LASSI-L could prove to be a useful biomarker for clinical research in preHD.

A new approach to digitized cognitive monitoring: validity of the SelfCog in Huntington's disease.

Cognitive deficits represent a hallmark of neurodegenerative diseases, but evaluating their progression is complex. Most current evaluations involve lengthy paper-and-pencil tasks which are subject to learning effects dependent on the mode of response (motor or verbal), the countries' language or the examiners. To address these limitations, we hypothesized that applying neuroscience principles may offer a fruitful alternative. We thus developed the SelfCog, a digitized battery that tests motor, executive, visuospatial, language and memory functions in 15 min. All cognitive functions are tested according to the same paradigm, and a randomization algorithm provides a new test at each assessment with a constant level of difficulty. Here, we assessed its validity, reliability and sensitivity to detect decline in early-stage Huntington's disease in a prospective and international multilingual study (France, the UK and Germany). Fifty-one out of 85 participants with Huntington's disease and 40 of 52 healthy controls included at baseline were followed up for 1 year. Assessments included a comprehensive clinical assessment battery including currently standard cognitive assessments alongside the SelfCog. We estimated associations between each of the clinical assessments and SelfCog using Spearman's correlation and proneness to retest effects and sensitivity to decline through linear mixed models. Longitudinal effect sizes were estimated for each cognitive score. Voxel-based morphometry and tract-based spatial statistics analyses were conducted to assess the consistency between performance on the SelfCog and MRI 3D-T1 and diffusion-weighted imaging in a subgroup that underwent MRI at baseline and after 12 months. The SelfCog detected the decline of patients with Huntington's disease in a 1-year follow-up period with satisfactory psychometric properties. Huntington's disease patients are correctly differentiated from controls. The SelfCog showed larger effect sizes than the classical cognitive assessments. Its scores were associated with grey and white matter damage at baseline and over 1 year. Given its good performance in longitudinal analyses of the Huntington's disease cohort, it should likely become a very useful tool for measuring cognition in Huntington's disease in the future. It highlights the value of moving the field along the neuroscience principles and eventually applying them to the evaluation of all neurodegenerative diseases.