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At least 19 recordsLinked to original sources

PubMed2026

Seeing and Feeling DNA Methylation: Single-Molecule Biophysics Meets Machine Learning.

DNA methylation at 5-methylcytosine (5mC) is crucial for embryonic development and cellular function, while aberrant patterns strongly drive disease onset and progression. Its reversible nature offers substantial therapeutic potential, emphasizing the need for precise, context-specific genome wide 5mC mapping. Conventional techniques such as bisulfite sequencing and ensemble biosensor assays are hindered by DNA degradation, amplification bias, high cost, and inability to resolve single-molecule structural and mechanical effects of methylation. This review examines advances in single-molecule biophysical methods (nanopore sensing, smFRET, optical/magnetic tweezers, and AFM) that provide direct, label-free/minimally invasive 5mC detection, along with quantitative insights into DNA conformation, mechanics, and protein-DNA interactions. These techniques complement traditional methylome mapping by linking genomic localization to molecular mechanisms. Emerging machine-learning approaches are revolutionizing analysis, particularly in nanopore sensing, while promising applications in smFRET, tweezers, and AFM address throughput and reproducibility challenges. Their convergence promises scalable, high-resolution epigenetic profiling, advancing precision epigenomics toward clinical application.

DNA Methylation↗

PubMed2026

Biophysical mechanisms underlying the generation and maintenance of rule-learning engram.

Training rodents in a particularly difficult olfactory-discrimination task results with acquisition of high-skill to perform the task superbly, termed 'rule-learning'. We show that rule-learning occurs abruptly, in a "light-bulb moment". Using whole-cell patch-clamp recordings from the piriform cortex (PC) of Fos2A-iCreER/TRAP2 mice, we target activated-neurons, expressing immediate early genes (IEG). We notice, from the onset of training, IEG-positive neurons from trained animals display enhanced intrinsic excitability. Subsequently, synaptic excitation and inhibition are enhanced in these neurons, in a coordinated, cell-wide process. Additionally, in parallel, we detect the density of IEG-expressing neurons sharply declines. Double labeling with TRAP and c-Fos reveal that nearly two-thirds of the rule-memory cell ensemble neurons are activated from the beginning of training. Silencing TRAP-expressing neurons using inhibitory DREADD leads to a complete loss of rule memory. Hence, we propose that rule learning occurs at a discrete moment and is developed through a gradual process that stabilizes the memory of the rule.

Animals↗

PubMed2025

Biophysical metabolic modeling of complex bacterial colony morphology.

Microbial colony growth is shaped by the physics of biomass propagation and nutrient diffusion and by the metabolic reactions that organisms activate as a function of the surrounding environment. While microbial colonies have been explored using minimal models of growth and motility, full integration of biomass propagation and metabolism is still lacking. Here, building upon our framework for computation of microbial ecosystems in time and space (COMETS), we combine dynamic flux balance modeling of metabolism with collective biomass propagation and demographic fluctuations to provide nuanced simulations of E. coli colonies. Simulations produced realistic colony morphology, consistent with our experiments. They characterize the transition between smooth and furcated colonies and the decay of genetic diversity. Furthermore, we demonstrate that under certain conditions, biomass can accumulate along "metabolic rings" that are reminiscent of coffee-stain rings but have a completely different origin. Our approach is a key step toward predictive microbial ecosystems modeling. A record of this paper's transparent peer review process is included in the supplemental information.

Models, Biological↗

PubMed2025

Trajectory inference from single-cell genomics data with a process time model.

Single-cell transcriptomics experiments provide gene expression snapshots of heterogeneous cell populations across cell states. These snapshots have been used to infer trajectories and dynamic information even without intensive, time-series data by ordering cells according to gene expression similarity. However, while single-cell snapshots sometimes offer valuable insights into dynamic processes, current methods for ordering cells are limited by descriptive notions of "pseudotime" that lack intrinsic physical meaning. Instead of pseudotime, we propose inference of "process time" via a principled modeling approach to formulating trajectories and inferring latent variables corresponding to timing of cells subject to a biophysical process. Our implementation of this approach, called Chronocell, provides a biophysical formulation of trajectories built on cell state transitions. The Chronocell model is identifiable, making parameter inference meaningful. Furthermore, Chronocell can interpolate between trajectory inference, when cell states lie on a continuum, and clustering, when cells cluster into discrete states. By using a variety of datasets ranging from cluster-like to continuous, we show that Chronocell enables us to assess the suitability of datasets and reveals distinct cellular distributions along process time that are consistent with biological process times. We also compare our parameter estimates of degradation rates to those derived from metabolic labeling datasets, thereby showcasing the biophysical utility of Chronocell. Nevertheless, based on performance characterization on simulations, we find that process time inference can be challenging, highlighting the importance of dataset quality and careful model assessment.

Single-Cell Analysis↗

PubMed2026

Stem Cell Differentiation Disperses Transcriptional Clusters via a Conserved Surface-Condensate Trajectory.

Stem cells exhibit exceptionally prominent transcriptional clusters, which dissolve with progressing differentiation. Although these clusters are assigned central roles in embryonic gene regulation, their formation and loss during differentiation remain poorly understood. This study reveals that these prominent clusters disperse along a conserved trajectory in mouse embryonic stem cells, fruit fly testes, and zebrafish embryos. Imaging and lattice simulations show that these clusters form via surface condensation on H3K27ac-marked super-enhancer regions, which act as genomic scaffolds. Upon differentiation, partial loss of these active epigenetic marks and transcription-driven unfolding lead to dispersal of the prominent clusters. The block copolymer-based lattice simulations explain this process as a conserved trajectory through a three-dimensional state space, governed by surface condensation principles that extend beyond canonical liquid-liquid phase separation. This work marks surface condensation as a biophysical mechanism for the dynamic organization of stem cell-specific transcriptional hubs and demonstrates evolutionary conservation in several organisms. By uncovering a conserved biophysical mechanism for transcriptional organization in development, our work illustrates how polymer properties can contribute to the control of cell identity and fate.

Animals↗

PubMed2026

Zea mays Drought-Overly Sensitive1/TUBA4 Is Wilty3, and Transcriptome Co-Expression Analysis of Shoot Meristem Mutant Tissues Reveals Wilty2/TUB6:Wi3 Interactions Associated With Stem Vascular Bundle Development.

Plant vasculature is essential for the transport of water, nutrients, and signaling molecules across organs, while also providing critical mechanical support for growth and development. Disruptions in vascular bundle formation can therefore lead to severe physiological and developmental defects. In maize, ethyl methanesulfonate (EMS)-induced dominant nonallelic Wilty mutants exhibit a pronounced wilting phenotype even under well-watered conditions, indicating underlying defects in vascular function. In this study, we characterized the Wi3 mutant, identified as ZmDrought-Overly-Sensitive1/DOS1, and compared it with the previously described Wi2 mutant to uncover shared mechanisms underlying their phenotypes. We provide evidence, by bulk segregant resequencing linkage disequilibrium of SNPs adjacent to the causal Wilty SNPs in respective ß- and α-tubulin genes, for the personal communication from Gerry Neuffer that Wi2/ß-tub6 provenance is from ACR-related stock, whereas Wi3/α-tub4 allele is from Mo17, not B73 as claimed by the authors who cloned Dos1. Histochemical staining and Fourier-transform infrared (FTIR) spectroscopy of vascular bundles in Wi3 indicated apparent alterations in cellulose and lignin content consistent with those observed in Wi2. Transcriptome analysis of shoot meristems further indicated that similar sets of genes and pathways are differentially expressed in both mutants, suggesting convergence on common biological pathways. Using bulk-segregant whole-genome resequencing, we identified alpha-tubulin4 (TUA4) as the causal gene in Wi3 (ZmDOS1), harboring a C-to-T substitution within the N-terminal GTPase-binding domain. This mutation results in a glutamic acid196-to-lysine substitution. Given that α- and β-tubulin subunits heterodimerize, and in many plants and animal mutant alleles are dominant-negative gains-of-function, we infer Wi2, Wi3, and likely Wi4, based on very similar FTIR biophysical difference spectra, may act as effectors of vascular bundle cell wall deposition, potentially involving vesicle trafficking as recently shown for asymmetric cell divisions in maize stomatal development. Together, these findings highlight the functional interdependence of tubulin subunits and provide a plausible mechanistic framework for the striking biophysical, transcriptomic, and phenotypic similarities observed between Wi2, Wi3/ZmDOS1, and Wi4 mutants.

bulk segregant analysis↗

PubMed2024

Role of RNA G-Quadruplexes in the Japanese Encephalitis Virus Genome and Their Recognition as Prospective Antiviral Targets.

G-quadruplexes (GQs) have been primarily studied in the context of cancer and neurodegenerative pathologies. However, recent research has shifted focus to their existence and functional roles in viral genomes, revealing GQ-regulated key pathways in various human pathogenic viruses. While GQ structures have been reported in the genomes of emerging and re-emerging viruses, RNA viruses have been understudied compared to DNA viruses, including notable examples such as human immunodeficiency virus-1, hepatitis C virus, Ebola virus, Nipah virus, Zika virus, and SARS-CoV-2. The flavivirus family, comprising the Japanese encephalitis virus (JEV), poses a significant global threat due to recurring outbreaks yet lacks approved antivirals. In this study, we identified and characterized eight putative G-quadruplex-forming motifs within essential genes involved in genome replication, assembly, and internalization in the host cell, conserved across different JEV isolates. The formation and stability of these motifs were validated through a multitude of biophysical and cell-based assays. The interaction and binding affinity of these motifs with the known GQ-binding ligand BRACO-19 were supported by biophysical assays, confirming the capability of these motifs to form GQ structures. Notably, BRACO-19 also exerted antiviral properties through reduction of viral replication and infectious virus titers as well as inhibition of viral protein expression, as evaluated by the cell-based assays. This comprehensive molecular characterization of G-quadruplex structures within the JEV genome highlights their potential as promising antiviral targets for intervention strategies against JEV infection through GQ-specific ligands.

G-Quadruplexes↗

PubMed2026

Loss of SUMOylation drives aberrant PRC1 clustering and 3D genome rewiring independent of H3K27me3.

Polycomb repressive complex 1 (PRC1) forms nuclear condensates that organize target chromatin domains. SUMOylation modulates PRC1 clustering, but its impact on condensate properties and 3D genome architecture remains unclear. Here, we show that depletion of small ubiquitin-like modifier (SUMO) in Drosophila wing imaginal discs transforms PRC1 condensates into large structures with reduced molecular dynamics. Biophysical modeling suggests that the changes in PRC1 self-interactions are responsible for the formation of large PRC1 condensates when SUMO is depleted. Interestingly, this biophysical reorganization occurs without global loss of the H3K27me3 mark. Instead, Hi-C reveals widespread rewiring of topologically associating domain (TAD) interactions. PRC1-bound TADs lose specific long-range contacts with each other while gaining ectopic interactions with active chromatin. These topological shifts correlate with gene misregulation independently of changes in Polycomb histone modifications. Our results establish SUMOylation as a critical regulator of PRC1 condensates, demonstrating that post-translational control of biomolecular condensation modulates 3D genome architecture and transcriptional output through mechanisms separable from histone mark deposition.

Animals↗

PubMed2025

The challenge of chromatin model comparison and validation: A project from the first international 4D Nucleome Hackathon.

The computational modeling of chromatin structure is highly complex due to the hierarchical organization of chromatin, which reflects its diverse biophysical principles, as well as inherent dynamism, which underlies its complexity. Chromatin structure modeling can be based on diverse approaches and assumptions, making it essential to determine how different methods influence the modeling outcomes. We conducted a project at the NIH-funded 4D Nucleome Hackathon on March 18-21, 2024, at The University of Washington in Seattle, USA. The hackathon provided an amazing opportunity to gather an international, multi-institutional and unbiased group of experts to discuss, understand and undertake the challenges of chromatin model comparison and validation. Here we give an overview of the current state of the 3D chromatin field and discuss our efforts to run and validate the models. We used distance matrices to represent chromatin models and we calculated Spearman correlation coefficients to estimate differences between models, as well as between models and experimental data. In addition, we discuss challenges in chromatin structure modeling that include: 1) different aspects of chromatin biophysics and scales complicate model comparisons, 2) large diversity of experimental data (e.g., population-based, single-cell, protein-specific) that differ in mathematical properties, heatmap smoothness, noise and resolutions complicates model validation, 3) expertise in biology, bioinformatics, and physics is necessary to conduct comprehensive research on chromatin structure, 4) bioinformatic software, which is often developed in academic settings, is characterized by insufficient support and documentation. We also emphasize the importance of establishing guidelines for software development and standardization.

Chromatin↗

PubMed2026

Shared ligand-blocking mechanism but distinct conformational modulation by α5-targeting antibodies BIIG2 and MINT1526A.

Integrins are heterodimeric receptors important for cell adhesion and signaling. Integrin α5β1 is a key mediator of angiogenesis and its dysregulation is associated with tumor progression and metastasis. Despite numerous efforts, α5β1-targeting therapeutics have been unsuccessful due to poor efficacy and off-target effects. A contributing factor is our limited understanding of how integrin conformation influences interactions with therapeutics. Using cell-based functional assays, patient-derived xenografts, biophysics, X-ray crystallography, and electron microscopy, we shed light on these relationships by characterizing two anti-α5β1 antibodies, BIIG2 and MINT1526A. We show that both antibodies bind α5β1 with nanomolar affinity, reduce tube formation in vitro, and bind overlapping epitopes that block fibronectin binding. However, using electron microscopy, we reveal that while BIIG2 binding does not substantially alter the conformational states, MINT1526A preferentially recognizes the bent conformation and restricts the conformational ensemble. These insights can guide which aspects to prioritize to improve the design of future integrin-targeted therapeutics.

angiogenesis↗

PubMed2026

Unraveling G-Quadruplex and i-Motif Coexistence Within a Double-Stranded DNA.

DNA can transiently fold into variable arrangements, which are expected to exploit regulatory functions. Guanine-rich sequences can fold into G-quadruplexes (G4s), while the complementary strand adopts potentially i-Motif (iM) arrangements. Their concomitant formation at the same genomic site is still under debate. However, recently, single-molecule analyses have shown the simultaneous G4 and iM presence within a double-stranded (ds) DNA context, addressing them as synergic blockers of replication fork progression. While these findings point to a functional interplay between G4 and iM, a deeper understanding of the factors enabling their coexistence remains unclear. In this work, we unravel the equilibria governing G4- and iM-folding within dsDNA, adopting an extensive biophysical approach allowing analysis of an optimized modular system, scalable across constructs of increasing molecular complexity. Our findings corroborate the simultaneous formation model and further clarify the thermodynamic determinants driving duplex denaturation and the favorable folding of stable G4 and iM structures.

G-Quadruplexes↗

PubMed2026

Biomaterial-Integrated Electroporation for Therapeutic Delivery: From Gene Editing to Tumor Ablation and Immune Modulation.

Electroporation has evolved from a membrane-permeabilization method into a versatile therapeutic platform for intracellular delivery, locoregional tumor intervention, and bioelectrically regulated treatment. Depending on pulse intensity and duration, electroporation operates in two distinct modes: reversible electroporation (RE), which transiently permeabilizes the plasma membrane to enable delivery of nucleic acids, proteins, and small molecules while preserving cell viability, and irreversible electroporation (IRE), which causes permanent membrane damage for non-thermal tissue ablation. Increasingly, the therapeutic scope of electroporation is being expanded through integration with biomaterials, including nanocarriers, hydrogels, soft conductors, and micro/nanoengineered bioelectronic interfaces. These material-assisted strategies improve cargo protection, field confinement, local retention, tissue conformity, and spatiotemporal control, thereby extending electroporation beyond conventional transfection toward gene editing, engineered cell manufacturing, electrochemotherapy, tumor ablation, immune modulation, and transdermal or localized delivery. In this Review, we summarize the biophysical principles of RE and IRE, discuss how biomaterials reshape electroporation performance across therapeutic settings, compare the design logic of major biomaterial-assisted electroporation platforms, and highlight key translational challenges, including pulse-material compatibility, manufacturing scalability, in vivo dosimetry, and regulatory complexity.

Humans↗

PubMed2025

Single-Molecule Nanopore Detection of Non-Canonical Thymine-Melamine Hydrogen Bonding Base Pair in DNA Abasic Site.

The binding of small molecules to DNA may represent a mutagenic process capable of inducing genomic structural alterations and functional impairment. Melamine (MA), a toxic small molecule, exhibits a hydrogen-bonding interface structurally analogous to adenine, enabling to form non-canonical thymine-melamine (T-MA) base pairs like Watson-Crick pairing. This property allows MA to program DNA nanostructure formation. Given MA's documented biological consequences, such as kidney disease, reproductive toxicity, and central nervous system dysfunction, sensitive detection of MA-DNA interactions has become critically important. However, such subtle structural changes remain challenging to identify because of the paucity of effective detection approaches in a high-resolution manner. To overcome this limitation, nanopore measurement is employed to identify T-MA hydrogen bonding base pairing in DNA. Results demonstrate that nanopore enables unambiguous identification of T-MA hydrogen bonding via mechanically unzipping thymine-melamine-thymine (T-MA-T) triplets in DNA structures. The approach achieves single-base-pair resolution, as evidenced by nucleotide substitutions flanking the abasic site in complex DNA structures. In addition, nanopore-based kinetic analysis reveals an enhanced intramolecular stability in MA-binding DNA compared to those consisting of complete canonical DNA pairs. This research establishes a powerful platform for high-resolution interrogation of DNA-small molecule interactions and quantitative biophysical characterization of mutagenic modifications at the nanoscale.

Single Molecule Imaging↗

PubMed2025

Fluorescence Loss After Photoactivation (FLAPh): A Pulse-Chase Cellular Assay for Understanding Kinetics and Dynamics of Viral Inclusions.

Influenza A virus (IAV) relies on host cellular machinery for replication. Upon infection, the eight genomic segments, independently packed as viral ribonucleoproteins (vRNPs), are released into the cytosol before nuclear import for viral replication. After nucleocytoplasmic transport, the resulting progeny vRNPs reach the cytosol, accumulating in highly mobile and dynamic viral inclusions that display liquid properties. Being sites postulated to support IAV genome assembly, the biophysical properties of IAV inclusions may be critical for function. In agreement, imposing liquid-to-solid transitions was demonstrated to impact viral replication negatively. Therefore, screening for host factors or compounds able to alter the material properties may provide the molecular basis for how influenza genomic complex forms as well as identify novel antivirals. Conventional techniques employed to investigate biomolecular condensates' material properties include fluorescence correlation spectroscopy, raster image correlation spectroscopy, single molecule or microrheology particle tracking, and Fluorescence Recovery After Photobleaching (FRAP). These approaches allow measuring molecular dynamics in systems that do not move very much. However, the analysis of highly mobile intracellular condensates, such as IAV inclusions, poses significant challenges as these structures not only constantly move within the cell but also exchange material, fusing, and dividing, rendering the quantitation of internal rearrangements and diffusion coefficients of molecules within condensates inaccurate. As an alternative, we opted for measuring the kinetics and the exchange of material between IAV inclusions using the Fluorescence Loss After Photoactivation (FLAPh) technique. It involves pulse photoactivation of individual or pools of viral inclusions in the cell, and chasing over time in photoactivated and non-photoactivated regions. This approach is suitable for quantifying the movement and spatial distribution of components within inclusions over time, enabling the determination of both the distance and speed from a specific cellular location. As a result, this method allows the quantification of decay profiles, half-lives, decay constant rate, and mobile and immobile fractions in viral inclusions. It, therefore, enables high throughput screenings for compounds or host factors that affect this dynamism and indirectly allows assessing the material properties of IAV inclusions.

Humans↗

PubMed2026

Identification and validation of natural dengue virus NS1 inhibitors with promising antiviral potential.

Dengue infection remains a major global public health challenge, with no specific antiviral therapy currently available. The dengue virus non-structural protein 1 (NS1) exists in both intracellular and secreted forms playing a pivotal role in viral replication, immune evasion, and pathogenesis, particularly by contributing to endothelial disruption and vascular leakage during severe disease, thereby making it a promising therapeutic target. In silico screening identified berberine, betulinic acid, and ursolic acid as top candidates, exhibiting high binding affinities and stable interactions within the NS1 binding pocket. These computational predictions were further validated by biophysical assays, which demonstrated strong and specific binding interactions between the purified NS1 protein and the selected compounds. All three compounds significantly reduced viral genome levels, with the highest inhibition observed for berberine (60%), and followed by betulinic acid (40%) and ursolic acid (28%). Consistently, berberine showed the most potent inhibition of both intracellular and extracellular NS1. Overall, these findings highlight the inhibitory potential of natural compounds against DENV NS1 and provide a strong foundation for the development of NS1-targeted antivirals as a novel therapeutic strategy against dengue infection.

Antiviral Agents↗

PubMed2026

How advances in chromosome conformation capture (3C) methods are reshaping our understanding of gene regulation in hematopoiesis.

The three-dimensional organization of the DNA within the nucleus plays a key role in regulating gene expression. Over the past two decades, advances in chromosome conformation capture (3C) technologies, in tandem with other methods, have shown that the genome forms a complex structure at multiple scales. Early studies identified large-scale structures such as chromosome territories, compartments and topologically associating domains (TADs). As the resolution of 3C techniques has improved, it has become possible to identify contacts between regulatory elements in detail and more recently, it has become possible to define intricate structures within cis-regulatory elements. In this chapter, we review the development of 3C-based methodologies and discuss the strengths and limitations of the different approaches. We examine how these technologies have refined our understanding of genome organization and gene regulation. Recent high-resolution studies reveal that chromatin architecture extends beyond classical domain structures to include nanoscale organization. Integration of 3C data with super-resolution imaging and molecular dynamics simulations supports a model in which genome folding is governed by the biophysical properties of chromatin.

Animals↗

PubMed2026

Lysine methylation is an endogenous post-translational modifications of tau protein in human brain and a modulator of aggregation propensity.

Tau protein undergoes a broad range of post-translational modifications in the brain, influencing its structure, solubility, and propensity to aggregate. This chapter presents an integrated methodological framework for characterizing tau methylation and evaluating its impact on tau biology. We describe procedures for isolating soluble and filamentous tau from post-mortem human brain tissue while preserving modifications for proteomic analysis. These approaches support precise mapping of methylation sites alongside other co-occurring modifications. To model methylation under controlled conditions, we outline protocols for recombinant tau expression, purification, and chemical reductive methylation, including radiolabeled assays for determining modification stoichiometry. We then detail biophysical assays used to assess how methylation alters tau conformation and aggregation propensity. This methodological framework supports experimentation seeking insight into mechanisms relevant to Alzheimer's disease and related tauopathies.

Humans↗

PubMed2025

Structural and thermodynamic impact of oncogenic mutations on the nucleosome core particle.

The nucleosome core particle is essential for chromatin structure and function, serving as the fundamental unit of eukaryotic chromatin. Oncogenic mutations in core histones disrupt chromatin dynamics, altering DNA repair and transcription processes. Here, we investigate the molecular consequences of two mutations-H2BE76K and H4R92T-using 36 μs of all-atom molecular dynamics simulations and experimental biophysical assays. These mutations destabilize the H2B-H4 interface by disrupting critical salt bridges and hydrogen bonds, reducing binding free energy at this interface. Principal-component analysis reveals altered helix conformations and increased interhelical distances in mutant systems. Thermal stability assays and differential scanning calorimetry confirm that these mutations lower the dimer dissociation temperature and reduce enthalpy compared with the wild-type. Taken together, our results elucidate how these mutations compromise nucleosome stability and propose mechanisms through which they could modulate chromatin accessibility and gene dysregulation in cancer.

Nucleosomes↗