Biology subjects

London, J. A.

Publications and source records attributed to London, J. A..

7 recordsLinked to original sources

bioRxiv2025

A Python framework for magnetic tweezers real-time image processing and microscope control

Magnetic tweezers are a popular biophysical instrument for manipulating and measuring single molecules. Most groups rely on custom-built setups tailored to specific experiments, making it challenging to implement and share software. Typically, image acquisition and hardware control are automated via LabVIEW, while real-time video processing is implemented in C++/CUDA libraries. Live processing can eliminate the need to store raw video, enabling high throughput, fast acquisition rates, and simplified experimental workflows. However, no open-source general-purpose software framework currently unifies these capabilities for magnetic tweezers experiments. Here, we introduce MagTrack and MagScope open-source Python-based tools designed to fill this gap. MagTrack is an image-processing library that efficiently determines bead positions from magnetic-tweezers videos using CPU or GPU computation. MagScope is a comprehensive software framework offering a graphical user interface, real-time hardware control, data acquisition, and video processing. It is built on a multiprocessing architecture for responsive, high-throughput computation. Together, MagTrack and MagScope offer a fully customizable, end-to-end, open-source Python alternative to proprietary or fragmented systems, enabling laboratories to adapt and extend the framework according to their experimental needs.

biophysics↗

bioRxiv2025

GT61 β-1,2-xylosyltransferases define a conserved xylan modification in gymnosperm and Arabidopsis primary cell walls

Plant primary and secondary cell walls differ in molecular composition, structure, and mechanical properties. While secondary wall xylan has been extensively characterised, the structure of xylan in primary walls remains less well understood, particularly in gymnosperms. Here, we identify a previously uncharacterised {beta}-1,2-linked xylosyl side chain in conifer and Arabidopsis thaliana xylan. Using enzymatic fingerprinting, NMR, and mass spectrometry, we show that this structure is positioned two xylose residues away from glucuronic acid substitutions, forming an evenly patterned substituted xylan. This spacing pattern is consistent with xylan-cellulose interaction, suggesting a structural role in primary wall architecture. This modification, found in primary wall-rich tissues of diverse conifer species, including needles and pro-embryogenic mass (PEM), is also present in Arabidopsis callus. We demonstrate that conifer Group III GT61 glycosyltransferases introduce this modification with consistent positional specificity. In Arabidopsis, three closely related GT61 enzymes act redundantly to generate the same structure, and their combined loss results in its complete absence. These findings uncover a conserved primary wall xylan modification in seed plants and define the GT61 enzymes responsible for its biosynthesis, opening new avenues to explore how xylan structure contributes to primary wall function. Significance StatementXylan structure is well characterised in secondary walls, but its primary wall counterpart remains poorly understood. We identified a conserved {beta}-1,2-xylosyl modification on xylan in the primary walls of conifers and Arabidopsis. This side chain is positioned at a defined position from a glucuronic acid substitution and is introduced by GT61 glycosyltransferases that cluster in one phylogenetic subclade. Our findings revealed a previously unrecognised xylan structural pattern and the biosynthetic enzymes responsible for its addition. This work expands the current understanding of primary wall architecture across seed plants.

plant biology↗

bioRxiv2024

A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but does not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also opens new avenues for exploring the intricate dynamics of protein-DNA interactions under the influences of supercoiling.

biophysics↗

bioRxiv2023

Massively parallel single molecule tracking of sequence-dependent DNA mismatch repair in vivo

Whether due to mutagens or replication errors, DNA mismatches arise spontaneously in vivo. Unrepaired mismatches are sources of genetic variation and point mutations which can alter cellular phenotype and cause dysfunction, diseases, and cancer. To understand how diverse mismatches in various sequence contexts are recognized and repaired, we developed a high-throughput sequencing-based approach to track single mismatch repair outcomes in vivo and determined the mismatch repair efficiencies of 5682 distinct singly mispaired sequences in E. coli. We found that CC mismatches are always poorly repaired, whereas local sequence context is a strong determinant of the hypervariable repair efficiency of TT, AG, and CT mismatches. Single molecule FRET analysis of MutS interactions with mismatched DNA showed that well-repaired mismatches have a higher effective rate of sliding clamp formation. The hypervariable repair of TT mismatches can cause selectively enhanced mutability if a failure to repair would result in synonymous codon change or a conservative amino acid change. Sequence-dependent repair efficiency in E. coli can explain the patterns of substitution mutations in mismatch repair-deficient tumors, human cells, and C. elegans. Comparison to biophysical and biochemical analyses indicate that DNA physics is the primary determinant of repair efficiency by its impact on the mismatch recognition by MutS.

biophysics↗

bioRxiv2021

The Formation of a Stable Sliding Clamp Discriminates MSH2-MSH3 and MSH2-MSH6 Mismatch Interaction

MutS homologs (MSH) are highly conserved core components of DNA mismatch repair (MMR). Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Once on DNA the MSH sliding clamps provide a platform for the assembly of MMR strand-specific excision components beginning with the highly conserved MutL homologs (MLH/PMS). Previous studies with short mismatch-containing oligonucleotides revealed an MSH ATP hydrolysis (ATPase) cycle that included mismatch recognition, the formation of an ATP-bound sliding clamp and dissociation from the end of a mismatched DNA that ultimately recovers the mismatch binding conformation. We found that ATP-bound MSH complexes on blocked-end or very long DNA are extremely stable under a range of ionic conditions. These observations underpinned the development of a high-throughput fluorescence resonance energy transfer (FRET) system capable of clearly distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 activities that is suitable for chemical inhibitor screens.

biochemistry↗

bioRxiv2020

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current gold standards for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated enzymes. One sentence summarySulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation.

biochemistry↗

bioRxiv2020

A single bacterial sulfatase is required for metabolism of colonic mucin O-glycans and intestinal colonization by a symbiotic human gut bacterium

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

microbiology↗