Search Results (554)

Objectives: Ketamine has demonstrated rapid and sustained antidepressant effects; however, its clinical utility is limited by the risk of addiction and systemic side effects. This study aimed to develop ketamine-loaded nanodroplets (Ket-NDs) with high encapsulation efficiency (EE) and stability for targeted low-dose intravenous (IV) administration in a mice model of depression. Low-intensity focused ultrasound (LIFU) was employed to induce transcranial, region-specific drug release in the lateral habenula (LHb). Methods: Ket-NDs were synthesized using a thin-film hydration method with sonication and emulsification, incorporating perfluoropentane as the core material. Characterization was performed using light microscopy, cryogenic scanning electron microscopy (cryo-SEM), transmission electron microscopy, and dynamic light scattering (DLS). Drug EE and loading efficiency (LE) were quantified by reversed-phase high-performance liquid chromatography. A chronic restraint stress model was established, and Ket-NDs were administered intravenously followed by LIFU targeting the LHb. Antidepressant efficacy and biosafety were systematically evaluated. Results: (1) Ket-NDs exhibited uniform spherical morphology and a narrow size distribution, as confirmed by DLS (particle size: 139.75 ± 9.43 nm; Polydispersity index: 0.225 ± 0.025) and cryo-SEM analysis (number-average diameter: 109.5 ± 10.4 nm). The zeta potential was −15.93 ± 5.906 mV, and the formulation remained stable under 4 °C storage. (2) Ket-NDs demonstrated high EE (78.25 ± 16.13%) and LE (15.55 ± 4.49%). (3) In depressive mice, IV administration of Ket-NDs followed by LIFU targeting the LHb significantly improved behavioral outcomes: increased locomotor activity in the open field test, elevated sucrose preference index, and reduced immobility time in the tail suspension test. (4) Safety assessments revealed no significant organ toxicity or brain tissue damage in ultrasound-exposed regions. Conclusions: In summary, this study developed stable Ket-NDs. When combined with LIFU, they enable precise regional drug delivery to the brain, showcasing a promising treatment strategy for depression with reduced systemic side effects. Full article

The COVID-19 pandemic, driven by SARS-CoV-2, continues to challenge global health due to emerging variants and the potential risk posed by related sarbecoviruses. Neutralizing antibodies targeting the spike (S) glycoprotein, particularly the receptor-binding domain (RBD), play a crucial role in viral neutralization and vaccine design. Although broadly neutralizing anti-RBD antibodies have been identified, the nature of cross-reactive humoral responses induced by natural infection with ancestral SARS-CoV-2 strains remains incompletely understood. Here, we isolated 105 S-specific monoclonal antibodies (mAbs) from individuals recovered from prototype SARS-CoV-2 infection. Of these, 30 mAbs cross-recognized SARS-CoV-1, including 25 RBD-directed mAbs, of which 12 displayed cross-neutralizing activity against both viruses. Among them, mAb 12C2 potently neutralized SARS-CoV-1 and multiple SARS-CoV-2 variants, likely through mechanisms that include inhibition of membrane fusion and potential destabilization of the S trimer. Cryo-electron microscopy revealed that 12C2 engages the outer face of the RBD, overlapping with the epitope recognized by the broadly neutralizing antibody S309 derived from SARS-CoV-1 convalescent. Collectively, these findings demonstrate that ancestral SARS-CoV-2 infection can elicit robust cross-neutralizing antibody responses and provide valuable insights for the design of broadly protective antibodies and vaccines. Full article

Parkinson’s disease, associated with mutations in the GBA1 gene (GBA1-PD), is the most common genetic form of Parkinson’s disease (PD), marked by clinical heterogeneity influenced by mutation type. Extracellular vesicles (EVs), key mediators of intercellular communication, are implicated in PD pathogenesis through the transport of pathological proteins and lipids. In this study, we analyzed blood plasma-derived EVs from GBA1-PD patients carrying p.N370S and p.L444P mutations and from healthy controls using cryo-electron microscopy, lipidomics, and proteomics. EVs from GBA1-PD patients were significantly larger than those from controls, with the largest size and most multilayered vesicles observed in p.N370S carriers. Lipidomic profiling identified 237 lipid species; of these, 186 lipids were altered in p.N370S and 24 in p.L444P versus controls. Mutation carriers showed distinct lipid signatures, with p.L444P samples enriched predominantly in sphingolipids, while p.N370S carriers exhibited more extensive lipid remodeling across multiple classes, including triglycerides, cholesteryl esters, and phospholipids. Notably, Cer 23:0 was elevated across all GBA1-PD groups. Proteomic analysis revealed enrichment in pathways related to lipid transport, immune regulation, and vesicle-mediated processes. Overall, GBA1-PD patients share a distinct lipidomic EV signature, with mutation-specific patterns reflecting differing mechanisms of lysosomal dysfunction. These findings support the potential of EV profiling to unravel disease heterogeneity and identify biomarkers. Full article

Current gene therapy treatments utilizing adeno-associated virus (AAV) vectors are based on capsids of primate origin. However, pre-existing neutralizing anti-AAV antibodies, that are present in a significant portion of the population, can lead to vector inactivation and reduced therapeutic efficacy. Advances in DNA sequencing have facilitated the discovery of many AAVs from non-primate species, including isolates from pigs, which exhibit up to 50% capsid protein sequence divergence, compared to primate AAV serotypes. In this study, AAVs isolated from porcine tissues (AAVpo.1 and AAVpo.6) were selected for structural characterization due to their low capsid protein VP1 sequence identity compared to each other and to AAV9. The AAV vectors were produced via the standard triple transfection system in HEK293 cells using AAV2 rep to package AAV2-ITR vector genomes and were purified by iodixanol density gradient ultracentrifugation. The capsid structures of AAVpo.1 and AAVpo.6 were determined using cryo-electron microscopy and then compared to each other in addition to the AAV5 and AAV9 structures. Given that porcine AAVpo.6 has been reported to infect human cells and the ability to cross the blood–brain barrier, the functional characterization was focused on the identification of a potential glycan receptor utilized by the porcine capsids. Additionally, the porcine AAV capsid reactivity to human derived anti-AAV antibodies was assessed to evaluate the potential for these capsids to be used as alternative vectors for gene therapy, particularly for patients with pre-existing immunity to primate-derived AAV serotypes. Full article

Detergent solubilisation remains the most commonly used but potentially problematic method to extract membrane proteins from lipid bilayers for Cryo-EM studies. Although recent advances have introduced excellent alternatives—such as amphipols, nanodiscs and SMALPs—the use of detergents is often necessary for intermediate steps. In this paper, we share our experiences working with detergent-solubilised samples within the modern Cryo-EM structural pipeline from the perspective of an EM specialist. Our aim is to inform novice users about potential challenges they may encounter. Drawing on specific examples from a variety of biological membrane systems, including Magnesium channels, lipopolysaccharide biosynthesis, and the human major facilitator superfamily transporters, we describe how the intrinsic properties of detergent-extracted samples can affect protein purification, Cryo-EM grid preparation (including the formation of vitreous ice) and the reconstitution of proteins into micelles. We also discuss how these unique characteristics can impact different stages of structural analysis and lead to complications in single-particle averaging software analysis. For each case, we present our insights into the underlying causes and suggest possible mitigations or alternative approaches. Full article

Neurodegenerative diseases such as Alzheimer’s disease (AD), frontotemporal lobar degeneration (FTLD), and α-synucleinopathies—including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA)—are characterized by the accumulation of misfolded protein aggregates. Advances in positron emission tomography (PET) imaging have enabled in vivo visualization of these pathologies, particularly tau and α-synuclein fibrils, facilitating early diagnosis and differential classification. Tau PET tracers such as ¹⁸F-florzolotau have demonstrated robust imaging of both AD-type and 4-repeat tauopathies, including atypical parkinsonian syndromes in FTLD such as progressive supranuclear palsy and corticobasal degeneration. Cryo-electron microscopy has elucidated the molecular interactions underlying tracer binding, highlighting hydrophobic grooves in cross-βstructures as binding components commonly present in multiple tau fibril types. For α-synucleinopathies, new tracers with a modified cross-β-binding scaffold, including ¹⁸F-SPAL-T-06 and ¹⁸F-C05-05, have shown promise in detecting MSA-related pathology and, more recently, midbrain pathology in PD and DLB. However, sensitive detection of pathologies in early PD/DLB stages remains a challenge. The integration of high-resolution PET technologies and structurally optimized ligands may enable earlier and more accurate detection of protein aggregates, supporting both clinical decision-making and the development of targeted disease-modifying therapies. Full article

Natural product-derived drugs represent a cornerstone of modern pharmacotherapy, with many serving as essential therapeutic agents across diverse medical conditions. Recent advances in structural biology have provided unprecedented insights into the molecular mechanisms underlying their biological activities. This review presents a comprehensive structural analysis of five representative natural product-derived drugs: digoxin, simvastatin, morphine, paclitaxel, and penicillin. Through an examination of high-resolution crystal structures and cryo-electron microscopy (cryo-EM) data, we elucidate how these compounds interact with their respective protein targets and modulate biological functions. The structural data reveal diverse binding mechanisms—ranging from competitive inhibition and covalent modification to allosteric modulation via conformational selection and induced fit—demonstrating how natural products achieve their therapeutic effects through precise molecular recognition. These structural insights provide a molecular foundation for understanding natural product pharmacology and offer valuable guidance for structure-based drug design approaches in developing next-generation therapeutics. Full article

Effective control of calcium carbonate (CaCO₃) scale formation is crucial to improve the performance and economic efficiency of water systems. This study investigates the impact of various scale inhibitors on the nucleation and crystallization processes of CaCO₃. Calcium carbonate particles were synthesized by mixing CaCl₂·2H₂O and NaHCO₃ solutions, in the presence of various scale inhibitors that had not previously been investigated using the experimental techniques employed in this study. Particle size distribution and zeta potential were analyzed using dynamic light scattering (DLS), while Ca⁺² consumption and pH changes were monitored with ion-selective electrodes. Crystal morphology was evaluated using scanning electron microscopy (SEM) and cryo-transmission electron microscopy (cryo-TEM). We demonstrated that, in all samples, approximately 98% of the CaCO₃ particles (sized between 400 and 840 nm) are formed within the first 30 min of synthesis, and these particles then aggregate to form larger particles (840–1100 nm in size). Due to the solution’s high supersaturation, the inhibitors influence calcium consumption only after 5 min of synthesis. All inhibitors, especially DTPMP, decrease calcium consumption and particle size during synthesis. The zeta potential and morphology of the particles in the samples containing inhibitors differed from those in the control group. Cryo-TEM observations revealed distinct nanometric precursor phases in the calcite crystallization process without inhibitors and different nanostructures when scale inhibitors were used. Moreover, conchoidal fractures were observed in the nanoparticles formed in the presence of DTPMP. This study demonstrates the effectiveness of various inhibitors in reducing calcium consumption in solution and altering the morphology of CaCO₃ crystals, thereby preventing calcium carbonate (CaCO₃) scale formation. Full article

Proteins that span cellular membranes represent around 30% of the proteome and over 50% of drug targets. A variety of synthetic and naturally-occurring small organic molecules interact with membrane proteins and up- and down-regulate protein function. The atomic details of these regulatory molecules offer important information about protein function and aid the discovery, refinement and optimization of new drugs. X-ray crystallography and cryo-electron microscopy (cryo-EM) are not always able to resolve the structures of small molecules in their physiological sites on membrane proteins, particularly if the molecules are unstable or are reactive enzyme substrates. Solid-state nuclear magnetic resonance (SSNMR) is a valuable technique for filling in missing details on the conformations, dynamics and binding environments of small molecules regulators of membrane proteins. SSNMR does not require diffracting crystals possessing long-range order and can be performed on proteins within their native membranes and with freeze-trapping to maintain sample stability. Here, work over the last two decades is described, in which SSNMR methods have been developed to report on interactions of the ATP substrate with the Na,K-ATPase (NKA), an ion-transporting enzyme that maintains cellular potential in all animals. It is shown how a combination of SSNMR measurements on membranous NKA preparations in the frozen and fluid states have provided unique information about the molecular conformation and local environment of ATP in the high-affinity nucleotide site. A combination of chemical shift analysis using density functional theory (DFT) calculations, dipolar coupling measurements using REDOR and measurements of the rates of proton spin diffusion is appraised collectively. The work described herein highlights the methods developed and challenges encountered, which have led to a detailed and unrivalled picture of ATP in its high-affinity binding site. Full article

Cryo-electron microscopy single-particle analysis (cryo-EM SPA) has advanced three-dimensional protein structure determination, yet resolving intrinsically disordered proteins and regions (IDPs/IDRs) remains challenging due to conformational heterogeneity. This research evaluates cryo-EM’s capacity to map dynamic regions, assesses the adaptability of disorder prediction tools, and explores optimization strategies for dynamic structure prediction. Cryo-EM SPA datasets from 2000 to 2024 were categorized into different periods, forming a database integrating sequence data and disorder indices. Established prediction tools—AlphaFold2 (pLDDT), flDPnn, and IUPred—were evaluated for transferability, while a multi-level CLTC hybrid model (combining CNN, LSTM, Transformer, and CRF architectures) was developed to link local conformational fluctuations with global sequence contexts. Analyses revealed consistent advancements in average resolution and model counts over the past decade, although mapping disordered regions remained technically demanding. Both the adapted AlphaFold pLDDT and the CLTC model demonstrated efficacy in predicting structurally variable and poorly resolved regions. A subset of the cryo-EM missing residues exhibited intermediate conformational features, suggesting classification ambiguities potentially influenced by experimental conditions. These findings systematically outline the evolving capabilities of cryo-EM in resolving dynamic regions, benchmark the adaptability of computational tools, and introduce a hybrid model to enhance prediction accuracy. This study provides a framework for addressing conformational heterogeneity, contributing to methodological advancements in structural biology. Full article

Sindbis virus (SINV), a prototype of the Alphavirus genus (family Togaviridae), is a globally distributed arbovirus causing febrile rash and debilitating arthritis in humans. Viral structural proteins—capsid (C), E1, and E2—are fundamental to the virion’s architecture, mediating all stages from assembly to host cell entry and pathogenesis, thus representing critical targets for study. This review consolidates the historical and current understanding of SINV structural biology, tracing progress from early microscopy to recent high-resolution cryo-electron microscopy (cryo-EM) and X-ray crystallography. We detail the virion’s precise T = 4 icosahedral architecture, composed of a nucleocapsid core and an outer glycoprotein shell. Key functional roles tied to protein structure are examined: the capsid’s dual capacity as a serine protease and an RNA-packaging scaffold that interacts with the E2 cytoplasmic tail; the E1 glycoprotein’s function as a class II fusion protein driving membrane fusion; and the E2 glycoprotein’s primary role in receptor binding, which dictates cellular tropism and serves as the main antigenic target. Furthermore, we connect these molecular structures to viral evolution and disease, analyzing how genetic variation among SINV genotypes, particularly in the E2 gene, influences host adaptation, immune evasion, and the clinical expression of arthritogenic and neurovirulent disease. In conclusion, the wealth of structural data on SINV offers a powerful paradigm for understanding alphavirus biology. However, critical gaps persist, including the high-resolution visualization of dynamic conformational states during viral entry and the specific molecular determinants of chronic disease. Addressing these challenges through integrative structural and functional studies is paramount. Such knowledge will be indispensable for the rational design of next-generation antiviral therapies and broadly protective vaccines against the ongoing threat posed by SINV and related pathogenic alphaviruses. Full article

Rice blast, caused by Magnaporthe oryzae, significantly threatens global rice production. Disease control is complicated by the pathogen’s high genetic diversity, which is driven by heterothallic recombination between opposite mating types that underlies variation. However, mechanisms governing sexual reproduction in this fungus remain poorly characterized, largely due to the absence of reliable methods for scalable ascospore progeny production. In this study, we established two novel mating methods, namely Conidial Mixing Mating (CMM) and Hyphal Segments Mixed Mating (HMM). Both methods employed optimized suspensions (5 × 10⁴ conidia/mL or equivalent hyphal density) mixed at 1:1 ratios, incubated under standardized conditions: 20 °C with a 12 h/12 h photoperiod. We characterized perithecia, asci, and ascospore morphology using fluorescence microscopy, paraffin sectioning, cryo-scanning electron microscopy, and transmission electron microscopy. Furthermore, both methods enabled phenotypic characterization of sexual reproduction-deficient mutants, including ΔMopmk1 and ΔMoopy2. In conclusion, we established two efficient methods for investigating M. oryzae sexual reproduction, providing foundational tools to advance studies of sexual mechanisms, pathogenicity evolution, and genetic variation. Full article

In recent years, electron diffraction (ED) and structural imaging have undergone a major resurgence in the scientific community, driven by continuous advancements in transmission electron microscopy (TEM) instrumentation, such as Cs correctors, direct detection cameras and automation, and the development or expansion of analytical methods, such as cryo-EM, beam precession, 4D Scanning Electron Diffraction, 3D electron diffraction, 4D-STEM, and ptychography [...] Full article

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application. Full article

Recent major advancements in cryo-electron microscopy (cryo-EM) have enabled high-resolution structural analysis, accompanied by developments in image processing software packages for single-particle analysis (SPA). SPA facilitates the 3D reconstruction of proteins and macromolecular complexes from numerous individual particles. In this study, we systematically evaluated the impact of symmetry parameters and particle quantity on the 3D reconstruction efficiency using the dihydrolipoyl acetyltransferase (E2) inner core of the pyruvate dehydrogenase complex (PDC). We specifically examined how inappropriate symmetry constraints can introduce structural artifacts and distortions, underscoring the necessity for accurate symmetry determination through rigorous validation methods such as directional Fourier shell correlation (FSC) and local-resolution mapping. Additionally, our analysis demonstrates that efficient reconstructions can be achieved with a moderate particle number, significantly reducing computational costs without compromising structural accuracy. We further contextualize these results by discussing recent developments in SPA workflows and hardware optimization, highlighting their roles in enhancing reconstruction accuracy and computational efficiency. Overall, our comprehensive benchmarking provides strategic insights that will facilitate the optimization of SPA experiments, particularly in resource-limited settings, and offers practical guidelines for accurately determining symmetry and particle quantity during cryo-EM data processing. Full article