Predictive models for missense variant pathogenicity offer little functional interpretation for intrinsically disordered regions (IDRs), since they mostly leverage conservation and coevolution across homologous sequences. In our study, we use molecular dynamics (MD) simulations to model biophysics of IDRs for improved interpretation of variant effects. We develop MDmis, a method that uses biophysical features extracted from MD simulations of IDRs to predict pathogenicity. We find that pathogenic variants in IDRs longer than 800 residues manifest differently, being strongly associated with a propensity for transient order and depleted solvent access, compared to those in IDRs <= 800 residues in length. Using MD simulations of proteins with single missense variants, we identify evidence for local structural changes, such as an increase in solvent accessible surface area, and global structural changes, such as increase in overall compaction, in IDRs >800aa. Lastly, MDmis, when combined with conservation information, can aid predictive accuracy, especially for pathogenic variants in IDRs >800aa. Overall, extracting information from MD simulations can help elucidate biophysical behaviors affected by pathogenic variants in IDRs and understand the drivers of predictive performance in different models.
This chapter outlines a protocol for preparing an oligomeric amyloid-β peptide (AβO) for solid-state NMR structural studies. This protocol was developed for the 42-amino acid isoform Aβ(1-42), which is a focus due to its pathological link to Alzheimer's disease (AD). This peptide is highly aggregation-prone and would rapidly form fibrils without special efforts to direct aggregation towards AβO. Our protocol includes separation of Aβ(1-42) monomers from fibril seeds that are typically present in synthetic preparations, exposure of monomers to detergent micelles of sodium dodecyl sulfate (SDS), and separation of oligomers from monomers. Separation of distinct aggregated states of Aβ(1-42) is performed using size-exclusion chromatography. Removal of SDS is performed by dialysis. Solid-state NMR rotors can be loaded via ultracentrifugation or lyophilization.
Pseudomonas aeruginosa is increasingly becoming resistant to multiple drugs and is held responsible for high rate of mortality and morbidity across the globe. This study aims to examine proteins from outer membrane such as OprE, OprF, OprC, and OprG, for the multi-epitope vaccine design. In this article, the prediction of epitopes for helper T-cells, cytotoxic T-cells, and B-cell epitopes was carried out by the application of various immune-informatics tools. All the predicted epitopes were aligned and assembled into a peptide sequence along with linkers and adjuvant sequences. Further, secondary structure and three-dimensional structure were predicted for the multiepitope construct. The vaccine construct designs were evaluated and validated for allergenicity, toxicity, and antigenicity. The validation of the predicted structure was carried out by determining its physiochemical properties by the application of ProtParam. Protein docking and molecular-dynamic simulation confirmed strong and stable interaction between the vaccine construct and Toll-like receptor-4. The vaccine construct was cloned into pET-29b vector and expressed in Escherichia coli. The designed multiepitope vaccine construct was overexpressed and purified by the application of Ni-NTA affinity chromatography and subjected to SDS-PAGE analysis. The circular dichroism spectroscopy analysis revealed it to be stable and structured. The hemolysis assay demonstrated minimal RBC toxicity suggesting that it was safe to use. The designed vaccine construct could activate both humoral and cellular immune responses as demonstrated by the advanced immunoinformatic approach making it a promising vaccine construct for protection against P. aeruginosa.
Regenerating cementum remains a major unmet challenge in periodontal and peri-implant therapy, underscoring the need to understand how cementoblasts respond to engineered surface cues. This study examined the manner in which titanium nanosurfaces integrating anisotropic nanopatterns with three-dimensional (3D) nanospike architectures regulate mechanotransduction and matrix mineralization in human cementoblast-like cells (hCEM). Titanium surfaces with isotropic, anisotropic, and 3D anisotropic nanospike architectures were fabricated and characterized through quantitative analyses of nanoscale geometry and topographical organization. Surface chemistry and crystallinity were characterized using Fourier transform infrared spectroscopy, grazing-incidence X-ray diffraction, and X-ray photoelectron spectroscopy. hCEM cultures on each surface were evaluated for extracellular calcium (Ca) and phosphate (P) levels, Ca/P ratios, extracellular matrix crystallinity, cytomorphology, and phosphate metabolism-associated gene expression. Mechanotransduction activity was assessed through focal adhesion-Hippo pathway signaling. Relationships between nanoscale architecture, cell stimulation, morphology, and mineralization were examined using correlation and path analyses. Despite comparable wettability and oxide chemistry to that of other nanosurfaces, 3D anisotropic nanospike surfaces produced the highest mineralization and exhibited the highest Ca/P ratios, clear hydroxyapatite signatures, pronounced extracellular nodules, and coordinated activation of phosphate metabolism gene profiles. These surfaces induced prominent nanoscale vertex-cell interactions and distinct cytomorphological responses. Mineralization did not show association with vertical roughness, hydroxyl content, or crystallographic features but positively correlated (r = 0.94) with composite nanoscale architectural metrics capturing spatial heterogeneity and vertex density. The finding that anisotropic 3D nanospike architectures are associated with enhanced matrix mineralization in human cementoblast-like cells under osteogenic conditions provides mechanistic insight into how nanoscale architecture modulates mineralization responses and may inform the design of cementum-targeted bioactive titanium surfaces.
Antibody folding and aggregation are major challenges in the development of relevant reagents and therapeutics. Antibodies face a biophysical trade-off; the immense diversity in complementarity-determining regions (CDRs), which is crucial for broad antigen recognition, comes at the cost of folding stability. How CDR sequences influence antibody folding remains poorly understood because of their sequence diversity and lack of large-scale data. Here we develop a high-throughput 'deep loop profiling' approach to quantify folding fitness across millions of diverse CDRs. Machine learning models trained on this dataset predict folding propensity directly from sequence and identify interpretable residue-level rules that reveal CDR1 and CDR2 as key folding determinants. Using these insights, we rescue two unstable nanobodies, including an aggregation-prone SARS-CoV-2 binder and a G-protein-coupled receptor-targeting intrabody, and build next-generation synthetic libraries enriched for biophysically optimized nanobodies. This approach provides a scalable framework for understanding and engineering folding competence in antibody-based scaffolds.
All organisms employ strategies to cope with changing environmental conditions. In budding yeast, nutrient deprivation induces a reversible non-proliferative state known as quiescence, characterized by extensive remodeling of gene expression, metabolism, and cellular biophysical properties. Yeast cells survive prolonged periods of starvation-induced quiescence, provided they can respire in the early stages of glucose withdrawal, and blocking respiration causes premature aging and markedly reduced survival and cytoplasmic diffusion. We find that respiration is required to initiate a quiescence-specific adaptive program. Induction of such a program prior to glucose withdrawal bypasses the need for respiration, rescuing survival and biophysical properties to the levels of respiration-competent cells. This rescue relies on proteomic adaptation and is mediated by Ras/PKA inactivation and Msn2/4-dependent activation of the environmental stress response, leading to modulation of cytoplasmic diffusion. Together, this enables long-term survival in quiescence even in the absence of respiration, underscoring the role of the stress response and the modulation of cytoplasmic properties in quiescence and aging.
Immune checkpoint inhibitors elicit responses in merely 20-40% of patients with microsatellite instability-high or mismatch repair-deficient colorectal cancer (CRC), making immunotherapy resistance a formidable clinical challenge. The immunosuppressive tumor microenvironment, characterized by regulatory T-cell accumulation and metabolic reprogramming, substantially drives this treatment failure. To determine whether neferine, a bioactive alkaloid derived from the traditional Chinese medicine formulation Shenling Baizhu Tang (SLBZT), enhances anti-PD-1 (aPD-1) efficacy in CRC liver metastasis by modulating the CYP2E1-PPARα metabolic axis. A CRC liver metastasis model was established via intrasplenic injection of MC38-Luc cells into C57BL/6 J mice. The animals were administered aPD-1 alone or in combination with low- or high-dose SLBZT. Tumor burden was evaluated via in vivo imaging and histopathology. Integrated transcriptomics and metabolomics, CRISPR-Cas9-mediated CYP2E1 knockout, T-cell coculture assays, alongside computational and biophysical analyses, were employed to elucidate the active components and underlying mechanisms. Evaluated functional outcomes included tumor burden, immune phenotyping, CYP2E1/PPARα signaling, and homovanillic acid (HVA) levels. High-dose SLBZT markedly augmented aPD-1-mediated suppression of CRC liver metastasis without compromising systemic tolerability. Multi-omics profiling coupled with genetic validation revealed that CYP2E1-driven lipid metabolic reprogramming and its downstream metabolite HVA serve as key mediators of regulatory T-cell expansion and CD8⁺ T-cell exhaustion. Both SLBZT and neferine diminished HVA accumulation, restored effector T-cell function, and potentiated aPD-1 efficacy. Furthermore, biophysical and computational analyses confirmed the direct inhibition of CYP2E1 by neferine. Neferine sensitizes tumors to PD-1 blockade by reprogramming lipid metabolism and remodeling the immune microenvironment via the CYP2E1-PPARα axis, highlighting its translational potential as a metabolic immunoadjuvant to overcome immunotherapy resistance in CRC.
Non-specific membrane disruption induced by the amyloidogenic aggregation of β-amyloid (Aβ) peptides is considered an underlying molecular mechanism of Alzheimer's disease (AD). Therefore, elucidating the membrane interruptive intermediate states of Aβ aggregates is a crucial step towards the understanding of molecular basis of AD pathology. However, such intermediate states are heterogeneous, low-abundant, and insoluble, bringing challenges for the application of high-resolution techniques. The solid-state nuclear magnetic resonance (ssNMR) spectroscopy remains the most feasible technique to characterize the structural features and molecular dynamics of the Aβ-membrane intermediate systems. Despite the capability, specific quantitative and sensitivity-enhanced ssNMR approaches, as well as membrane biophysical and/or cell-based biophysical assays, should be combined to maximize the biological relevance of the intermediate structural characterizations. In this methodology chapter, we will review the experimental and data analysis protocols that have been established in our laboratory.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) underpins nearly all primary production yet remains a slow, error-prone enzyme because it catalyzes both carboxylation and oxygenation, the latter initiating photorespiration and reducing net carbon gain. Many organisms mitigate these limitations not by improving Rubisco selectivity for CO2 directly, but by modifying its local environment using CO₂-concentrating mechanisms (CCMs). In algae, a prominent biophysical CCM strategy is the pyrenoid: a phase-separated, Rubisco-rich condensate coupled to bicarbonate transport, local carbonic anhydrase activity, and diffusion barriers that elevate CO₂ at Rubisco active sites. Although pyrenoids have been most intensively studied in algal models, a pyrenoid-based CCM has evolved independently in a single land-plant lineage-the hornworts-providing a powerful comparative system for understanding how chloroplast organization can be tuned to terrestrial CO₂-delivery constraints. Here we synthesize a century of hornwort pyrenoid research in ecological, phylogenetic, and mechanistic context. We summarize bryophyte anatomical and microhabitat features that impose strong CO₂ diffusion limitation, and compare hornwort and algal pyrenoids in ultrastructure, molecular parts lists, and regulation. We highlight emerging models for hornwort pyrenoid formation, inorganic-carbon delivery, CO₂ generation and recapture, and recent biochemical/structural work revealing distinctive hornwort Rubisco properties and biogenesis. Finally, we discuss how hornwort pyrenoids complement efforts to engineer algal pyrenoid components into C₃ crops, and propose modular, hybrid engineering strategies that leverage hornwort compatibility with the embryophyte chloroplast, while selectively importing algal modules. Together, hornwort pyrenoids illustrate both the convergent logic and the lineage-specific solutions of biophysical CO₂ concentration, and they open avenues for mechanistic discovery and photosynthesis engineering.
The development of 3D scaffolds addresses a critical gap in neural tissue modeling by mimicking the physiological architecture in brain cell culture. This is a key challenge to improve the reliability of in vitro assays and reduce animal testing, but also mandatory for the successful engineering of neural tissues in the future. Recently various types of applicable scaffolds have been applied for 3D cell cultures, which are typically porous hydrogels or fibrous mats produced by electrospinning. These scaffold materials oftentimes have a 3D structure limiting the ingrowth of cells and the diffusion of biophysical factors throughout the material. In this study, we show the capability of Aerohydrogels, fabricated via initiated chemical vapor deposition, to allow biophysical communication throughout the material in a spatially divided brain cell coculture of microglia and astrocytes. The recently developed Aerohydrogels have an ultralow density and mechanically stable 3D hollow fibrous structure. The analysis of Interleukin inflammatory pathways shows the protective influence of astrocytes within the coculture by intercellular communication. The findings support the great applicability of the newly applied Aerohydrogels in 3D brain cell coculture and relevant analysis methods like live cell imaging, cell viability assays, or gene expression. This successful establishment allows more dedicated future applications and optimization of Aerohydrogels in neural tissue modeling.
The interplay between cells and their surrounding microenvironment drives multiple cellular functions, including migration, proliferation, and cell fate transitions. The nucleus is a mechanosensitive organelle; however, the morphological and functional changes of the nucleus induced by a three-dimensional (3D) extracellular environment remain unclear. Here, we report that leukemia Jurkat cells selected after 3D growth conditions retain persistent nuclear changes even after being released from confinement. These altered cells showed aberrant nuclear wrinkling, visualized by the lamin B1 distribution and mediated by disrupted actin dynamics and protein kinase C (PKC)β signaling. Moreover, these cells presented changes in chromatin compaction, transcription, apoptosis, and in vivo dissemination. By combining biomechanical techniques and single-nucleus analysis, we have determined that these cells exhibit a distinct nuclear mechanical behavior and biophysical signature compared with control cells. Together, these findings demonstrate that 3D microenvironments alter leukemia cell biology by promoting persistent changes in chromatin organization, morphology, and mechanical response of the nucleus.
The immunological synapse (IS) is a nanoscale platform that coordinates T cell activation, cytoskeletal polarization, Ca2+ signaling, and the directed secretion of lytic granules. In cancers, an acidic tumor microenvironment (TME; extracellular pHe ~ 6.4-6.8) imposes a biophysical and metabolic stress that could destabilize this interface. Experimental studies indicate that even modest acidification could significantly reduce integrin-dependent adhesion strength, delay actin clearance at the synapse, and suppress store-operated Ca2+ entry, thereby leading to marked decreases in cytokine production and cytotoxic granule release. In parallel, tumor cells often maintain relative intracellular alkalinity by enhancing proton export via transporters such as NHE1, MCT1/4, and CAIX, thereby reinforcing cortical actin "shielding," metabolic resilience, and resistance to perforin-mediated killing. These asymmetric pH adaptations may therefore establish a hidden checkpoint at the IS that favors tumor survival. We synthesize current evidence on pH-dependent regulation of actin dynamics, integrin activation, mitochondrial function, and Ca2+ channels (Orai1/STIM1); highlight key methodological gaps, including the lack of approaches combining real-time intra- and extracellular pH and Ca2+ imaging; and discuss enabling technologies such as microfluidic platforms, genetically encoded pH sensors, and multiparametric single-cell assays. Finally, we outline therapeutic strategies aimed at modulating pH (buffers, inhibitors of NHE1, MCTs, V-ATPases, or CAIX) or engineering pH-resistant effector cells and consider how these approaches could synergize with immune checkpoint blockade, CAR-T cells, and bispecific antibodies. Viewing acidosis as a druggable checkpoint reframes the IS as a bidirectional, pH-tuned system and suggests testable paths to restore antitumor immunity.
MoonProt 4.0 (http://moonlightingproteins.org) is an updated open-access database storing manually-curated annotations for moonlighting proteins. Moonlighting proteins exhibit two or more physiologically relevant distinct biochemical or biophysical functions performed by a single polypeptide chain. Here we describe an expansion in the database since our report published in 2021. With the assistance of five undergraduate annotators, we have added approximately 200 protein entries to give a total of over 700 moonlighting proteins. The new entries include more examples from plants, more transmembrane proteins and additional combinations of functions. The MoonProt Database collection of proteins with multiple functions serves as a resource for developing algorithms for predicting protein functions and provides examples of the evolution of new functions on a protein scaffold that can be valuable in developing novel methods for designing proteins with added functions.
Sustaining agricultural productivity while maintaining ecological integrity requires understanding the spatial dynamics of ecosystem services (ES). In the Canadian prairies-an intensively modified agricultural region-the degradation of natural habitats has impacted ES flows crucial for food security. We investigated how landscape structure, acting as a structural proxy for potential internal ES flows, mediated by landscape structure, influence crop yield at the Soil Landscape of Canada (SLC) scale, an ecologically meaningful delineation based on natural features. Our primary objective was to determine the relative importance of landscape composition versus configuration in predicting agricultural productivity. We conducted a biophysical assessment of key ES (pollination, carbon storage, habitat quality, soil erosion control) for the year 2020. We quantified landscape composition and configuration metrics at the SLC scale to represent the structural potential for ES flow pathways. Generalized additive models (GAMs) were used to analyze the non-linear effects of these variables on a composite crop yield index. Our findings reveal that landscape configuration-notably connectivity (positive linear effect) and crop diversity (complex non-linear effect)-significantly predicts crop yield, often exerting greater influence than the mere amount of natural habitat. A secondary analysis showed that yield in specific crops like canola, which depends on pollination, responded positively to natural habitat extent. The models explained a substantial portion of yield variance (Adjusted R2 ≈ 0.66-0.67). Our analysis highlights that agricultural output is not solely a function of field-level inputs but is deeply embedded within, and responsive to the landscape matrix at SLC scale and the ecological processes it mediates. Strategically enhancing landscape cohesion and crop diversity may therefore offer greater yield benefits than focusing on increasing isolated natural habitat, guiding a shift towards spatially explicit, multifunctional landscape planning. The online version contains supplementary material available at 10.1007/s10980-026-02333-y.
Antibody-drug conjugates (ADCs) are transforming oncology by enabling targeted delivery of potent cytotoxic agents to tumors. However, challenges such as resistance and limited response to established ADCs highlight the need for improved linkers and novel payloads driven by rational multivariate design and extensive synthetic optimization. We describe a discovery chemistry campaign for novel camptothecin-based linker-payloads enabling high drug-to-antibody ratio (DAR) ADCs with good biophysical properties, good in vitro potency, and strong in vivo efficacy. Starting from exatecan, amine-containing cytotoxic analogs were synthesized and evaluated for potency and permeability. Optimization of protease-cleavable p-aminobenzyl carbamate (PABC) linkers with a focus on stability and polarity leads to the discovery of promising α-amino-isobutyramido-exatecan constructs. The corresponding high-DAR ADCs showed low aggregation, good in vitro potency, and robust target-mediated efficacy in vivo across two antibody-antigen pairs. These results demonstrate the potential of these linker-payloads and the broad applicability of this design strategy.
Disruption of hypoxia-inducible factor (HIF) signaling is implicated in multiple diseases, including clear cell renal cell carcinoma (ccRCC), where HIF-2α functions as a key oncogenic driver. Here, we report the design and structure-activity relationships of a novel class of N-aryl-substituted tetrahydroquinolines that bind the HIF-2α PAS-B domain with high affinity. Structural and biophysical studies revealed that ligand binding induces localized conformational perturbations at the dimerization interface between HIF-2α and HIF-1β. Notably, incorporation of cis-vicinal difluorination was critical for productive engagement of the lipophilic pocket, with hyperconjugative effects contributing to improved binding efficiency and facial polarization enhancing physicochemical properties. These features translated into improved biochemical and cellular activity, including inhibition of hypoxia-regulated gene expression. Insights from this campaign ultimately informed the discovery of casdatifan, a clinical-stage HIF-2α inhibitor, underscoring the translational potential of this novel chemotype.
The radiation-induced adaptive response (RAR), also referred to as radioadaptation, describes modifications of biological radiation sensitivity following prior exposure to low doses or low dose-rates of ionizing radiation. Despite extensive experimental evidence, RAR remains difficult to reproduce consistently and lacks a unified quantitative and methodological framework. The objective of this study was to develop a systematic biophysical approach enabling coherent analysis and comparison of RAR experiments performed under different irradiation protocols. We formulated a dose- and time-dependent adaptive response function characterized by transient, memory-like dynamics. On this basis, we derived analytical expressions describing RAR under multiple irradiation schemes, including priming-challenge protocols, radiation training, constant low dose-rate exposure, and variable dose-rate scenarios. A unified relative endpoint parameter was introduced to quantify the magnitude of the adaptive response across experimental designs. The proposed framework yields explicit expressions for the adaptive response parameter under diverse exposure conditions and demonstrates how RAR magnitude depends on dose, dose-rate, and time interval between exposures. The methodology enables consistent normalization of experimental endpoints, facilitates parameter estimation from empirical data, and clarifies conditions under which adaptive effects are expected to emerge or vanish. This work provides a coherent and transferable methodological foundation for quantitative RAR research. The framework improves comparability between experimental studies and supports mechanistic interpretation of low dose adaptive effects, while remaining primarily applicable to controlled experimental systems rather than population-level radiation risk assessment.
Viruses are complex supramolecular assemblies that propagate their genetic material from cell to cell, thereby relying on host cell mechanisms. Employing a combination of passive and active strategies, they efficiently package, transport and release nucleic acids. While structural and biochemical techniques offer insights into certain, static aspects of the viral life cycle, recent advancements in biophysical approaches now allow for direct measurement of their inherent dynamic activities in the research field commonly referred to as physical virology. One of these methods is optical tweezers, enabling the precise measurement of force and position at the single-molecule level over time. Over the past decades, the ability to optically trap beads and to manipulate biomolecules has revolutionised medical and biophysical research. In this paper, we provide a comprehensive analysis of optical tweezers, exploring its integration with imaging modalities and review its diverse applications in the study of viruses and viral components. In particular we focus on studies that use optical tweezers to study virus-cell interactions, genome packaging using molecular motors and co-assembly of viral assembly proteins with their nucleic acid.
The small intestine possesses a complex architecture and microenvironment. Current in vitro three-dimensional models fail to fully replicate the architectural, biophysical and biochemical cues in both healthy and pathological intestine tissues. In this study, we designed and engineered a biomimetic villi-crypt scaffold-on-chip via digital light processing (DLP) 3D-printing. The fabricated villi-crypt scaffold-on-chip model is specifically designed to emulate physiological mechanical properties and enable advanced investigation of intestinal epithelial architecture, cellular functions, and interactions with fluid flow, while also being compatible with downstream proteomic analysis. Using gelatin methacryloyl (GelMA) and poly(ethylene glycol) diacrylate (PEGDA), we fabricated high-fidelity villi and crypt-like structures with tuned mechanical properties and enhanced long-term stability. By optimizing the GelMA-PEGDA composition, we achieved precise microarchitecture with minimal swelling or deformation. Computational fluid dynamic studies demonstrated the consistency of the villi-crypt scaffold-on-chip model with the physiological shear forces observed in the intestinal epithelium. Among the tested formulations, the Villi-(Rigid)-Crypt scaffold exhibits superior structural stability and a more physiologically relevant intestinal-like environment, maintaining its integrity in culture. In contrast, the Villi-(Flexible)-Crypt scaffold presents superior flexibility while still supporting cell growth. Proteomic analysis revealed that the different mechanical properties of the fabricated biomimetic villi-crypt scaffold-on-chip models can modulate cells functions towards barrier formation, epithelial polarization, and metabolic activity, or even expression of mucus-associated and adhesion proteins. These results confirm the model's relevance for in vitro studies of intestinal epithelial function and dynamics, offering a powerful tool for drug screening and modeling.
The intrinsic programmability of nucleic acids has positioned them as versatile molecular building blocks for constructing nanodevices with significant diagnostic and therapeutic potential. However, the clinical translation of these constructs is severely hindered by major pharmacokinetic (PK) and biophysical limitations, including susceptibility to enzymatic degradation, short circulation half-life, and inefficient cellular uptake. Chemical modification, encompassing nucleobase engineering, backbone and sugar-ring alterations, terminal conjugation, and higher-order structural reinforcement, provides a powerful strategy to overcome these barriers by enhancing in vivo stability, prolonging circulation, improving cellular internalization, and enabling stimulus-responsive cargo release. In this review, we summarize recent advances in chemically modified nucleic acid nanodevices, focusing on how specific chemical designs modulate physicochemical properties, improve pharmacokinetics, enable organ- or cell-selective targeting, and enable spatiotemporally controlled molecular release. We further highlight their emerging applications in precision drug delivery, high-sensitivity biosensing, and integrated theranostics. Finally, we critically discuss persistent translational challenges, including batch-to-batch scalability, immunogenicity, and long-term nanotoxicity, and propose forward-looking solutions, such as AI-assisted design, to pave the way for industrial adoption and clinical implementation.