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Biology subjects

Heath, G. R.

Publications and source records attributed to Heath, G. R..

4 recordsLinked to original sources

afMLevel: Deep learning U-Net Models for Levelling Atomic Force Microscopy Images and Movies

AFM experiments can now produce hundreds of frames per session and this rises to thousands in the case of video-rate high-speed AFM, but every image must still be levelled to remove tilt, bowing and line noise before it can be measured. Current automatic routines rely on user-chosen masks and parameters that work for some sample types but fail on others, so levelling often remains a manual, subjective bottleneck. Here, we present two U-Net models developed using machine learning for levelling AFM images without altering the sample topography. MLMask segments features so that they are excluded from conventional plane and line background fits, and MLBackground predicts the noise and artifact background directly, which is then subtracted. Both were trained on 2,001 experimental images from multiple instruments and sample types, using manually levelled images as ground truth. On 164 unseen images, MLMask routines brought 70-80% of images below a mean squared error of 0.1 relative to manual levelling, compared with 60-70% for the best multi-plane method, with a lower mean error (0.1-0.15 vs 0.15-0.3), and both models significantly outperformed fourteen classical routines (p < 0.01). Applied frame by frame, the models process 512x512 pixel HS-AFM movies at 4.7 frames.s-1. We provide afMLevel as an openly-available package for the use of other researchers. By removing the need to choose levelling parameters image by image, this package together with its models and paired training data, provides a foundation for automated AFM analysis pipelines.

biophysics↗

High-speed AFM imaging of the Entropic Disassembly of DNA Origami for Single-Molecule Biosensing

Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis, but their small size and instability hinder direct detection. The detection of small RNA, such as miRNA, using solid-state nanopores typically involves the binding of miRNA to a larger carrier molecule to generate detectable signals. However, this approach is prone to RNAse degradation in the environment leading to accidental digestion of miRNA prior to analysis. Here, we present an alternative approach based on DNA origami disassembly driven by toehold-mediated strand displacement (TMSD). Specifically, we designed a symmetric DNA origami dimer that undergoes TMSD-driven separation into monomers using miRNAs as invading strands. We visualized the real-time dynamics of dimer separation at high resolution using high-speed atomic force microscopy (HS-AFM), directly capturing nanoscale mechanical dynamics of the TMSD process that are inaccessible to ensemble or fluorescence-based measurements. Following the TMSD, single molecule nanopore sensing enables quantitative endpoint analysis of dimer separation by measuring the ratio of dimers to monomers. This direct read-out method enabled the multiplexed detection of miRNAs. Due to the near irreversible process of the TMSD, we performed the detection of miRNA in crude RNA tissue extracts under the presence of RNAse and showed that our approach allowed the robust detection of small RNA that is unaffected by the complex degrading environment.

biophysics↗

NanoLocz: Image analysis platform for AFM, high-speed AFM and localization AFM

NanoLocz is an open-source computer program designed for high-throughput automatic processing and single-particle analysis of Atomic Force Microscopy (AFM) image data. High-Speed AFM and Localization AFM (LAFM) enable the study of single molecules with increasingly higher spatiotemporal resolution. However, efficient and rapid analysis of the images and movies produced by these techniques can be challenging, often requiring the use of multiple image processing software applications and scripts. Here, we introduce NanoLocz, an AFM and high-speed AFM analysis program that facilitates various single-particle analysis workflows through a simple, interactive interface. Workflows include but are not limited to: single-particle tracking, single-particle topographic feature analysis, single-molecule LAFM, time-resolved LAFM, and simulation LAFM. The source code and installation instructions for NanoLocz are available at https://github.com/George-R-Heath/NanoLocz.

biophysics↗

BRCA1-BARD1 combines multiple chromatin recognition modules to bridge nascent nucleosomes

Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4AK20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structural, and single molecule imaging approaches. We find that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed-AFM to provide real-time visualization of BRCA1-BARD1 complex recognising chromatin, we show a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiqiutin ligase catalytic activity. Multivalent interactions across nucleosomes explains how BRCA1-BARD1 can recognize chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.

molecular biology↗