Biology subjects

Brown, C. M.

Publications and source records attributed to Brown, C. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Cardiolipin modulation of MmpL3 in mycobacteria

The complex cell envelope of mycobacteria is characterized by the presence of a unique outer membrane (OM) rich in mycolic acids (MAs). These long-chain, branched fatty acids confer extreme hydrophobicity to the OM, in part rendering the mycobacterial envelope impermeable to antibiotics and host defences. How MAs are transported from the inner membrane (IM) to the OM is largely unknown. The integral membrane protein MmpL3 plays an essential role in this process, but the mechanism(s) by which it exploits the proton motive force to flip and/or release MAs at the IM, in the form of trehalose monomycolates (TMMs), remains elusive. Here, we reconstitute and quantify the proton translocation activity of MmpL3 in artificial lipid bilayers, and discover a novel role for the phospholipid species, cardiolipin (CL), in regulating MmpL3 function. We find that mutations in conserved residues, or binding of known inhibitors in the central channel of MmpL3 do not diminish proton translocation activity. Instead, the specific presence of CL abolishes proton translocation by MmpL3 in vitro. Furthermore, we establish that an MmpL3 variant containing substitutions in a CL-binding site predicted in silico is no longer modulated by CL in vitro, and is unable to support growth of Mycobacterium smegmatis. Our work provides previously unappreciated insights into lipid regulation of MmpL3 activity in mycobacteria, and expands the guiding principles for the development of anti-mycobacterial inhibitors targeting this essential transporter. SignificanceMycobacterial species, including the human pathogen Mycobacterium tuberculosis, are surrounded by a double-membrane cell envelope that makes them intrinsically resistant to many antibiotics. Specifically, the outer membrane (OM) contains unique lipids called mycolic acids (MAs), whose transport pathway across the envelope is poorly understood. In this study, we characterized the biochemical activity of the essential MA transporter MmpL3, and uncovered a novel mechanism for lipid-mediated functional regulation. Our work highlights the importance of protein-lipid interactions in defining transporter activity, provides insights into MA transport and OM assembly in mycobacteria, and sets the stage for the development of anti-mycobacterial strategies.

biochemistry↗

bioRxiv2026

The Martini 3 Metabolome

Metabolites are ubiquitous in all living cells and are essential mediators of biochemical processes, serving either as substrates or as cofactors to enable the reactions. Capturing this diversity in computational workflows is important for allowing realistic simulations of the cytoplasm. Coarse-grained molecular dynamics enables the simulation of large scale systems up to the level of whole-cells, but is limited by the availability of refined parameters for all possible components in the system. In this work, we describe the parameterization of 186 common metabolites found in bacteria and eukaryotes within the framework of the Martini 3 force field. To showcase the behavior of Martini metabolites in a biological setting, we report simulations of protein-ligand binding and membrane permeation. The establishment of a Martini metabolome enables high-throughput simulations of metabolites interacting with other biomolecules, and opens the way for simulations of realistic cellular environments.

biophysics↗

bioRxiv2026

Ligand Binding Free Energy Landscapes at the Tubulin Colchicine Site from Coarse-Grained Metadynamics

Accessing deeply buried binding sites remains a major challenge in structure-based drug discovery, where accurate description of both protein dynamics and ligand binding pathways is required. Funnel metadynamics enables simulation of complete binding processes but is computationally demanding at the all-atom resolution. By adopting the Martini 3 force field, coarse-grained funnel metadynamics (CG-FMD) substantially reduces computational requirements while retaining enhanced sampling capabilities. In this work, we assess the capability of CG-FMD to model ligand recognition at the deeply buried colchicinoids site of the tubulin {beta}-heterodimer, a multisite protein of strategic importance. We investigated the binding of colchicine, podophyllotoxin and combretastatin-A4, recovering free energy profiles with improved statistical convergence compared to AA-FMD and comparable to experimental references. In particular CG-FMD binding free energies present mean absolute errors between 3 and 10 kJ mol-1. These results propose CG-FMD as an efficient, physics-based framework for probing ligand binding to challenging sites.

biophysics↗

bioRxiv2026

Community Web Portal for Open Collaboration in the Martini Force Field Initiative

AO_SCPLOWBSTRACTC_SCPLOWThe Martini coarse-grained force field is widely used for biomolecular simulations by a large and rapidly expanding community worldwide. Over time, the development of Martini parameters, tools, and documentation has become increasingly dispersed across numerous research groups, leading to fragmentation and making it challenging for users and developers to keep track of the latest models, software, and best practices. Consequently, the development of Martini as a genuinely community-driven process has grown into a bottleneck. In response, the Martini Force Field Initiative (MFFI) has been established as an open-science effort to coordinate and support the collaborative development of all Martini resources. Here, we introduce the MFFI web portal, a platform designed around five core pillars: (i) avoiding reliance on a single group or local server; (ii) minimizing long-term maintenance overhead; (iii) reducing technical barriers for contributions; (iv) providing a unified home for parameters, tools, tutorials, example workflows, and research outputs; and (v) enabling timely dissemination of updates to the community. To achieve this, we use Quarto to generate a static website authored in Markdown, lowering the technical barrier to making contributions, and serverless architectures on Amazon Web Services for scalable, event-triggered backend operations. The source code is hosted in a public GitHub repository under an MIT license, with automated deployment via GitHub Actions and a contribution model based on pull requests for quality control. This design creates a sustainable, low-maintenance, and collaborative infrastructure that consolidates Martini resources and supports transparency. More broadly, our design exemplifies a transferable pattern for building open, community-oriented platforms for molecular modeling and computational science.

biophysics↗

bioRxiv2025

Knowledge-informed multimodal cfDNA analysis improves sensitivity and generalization in cancer detection

Liquid biopsy offers a minimally invasive opportunity to detect and monitor cancers through analysis of cell-free DNA (cfDNA). However, current approaches face challenges of limited sensitivity at low tumor fractions, technical variability, and poor generalization across cohorts. Tumor-informed targeted methods offer high specificity but suffer from low sensitivity due to random sampling, tumor evolution and adaptation (including resistance mechanisms), and other sources of heterogeneity. Conversely, tumor-naive genome-wide methods can increase sensitivity but often sacrifice specificity, particularly at low tumor fractions. We developed Fragmentomics Analysis for Tumor Evaluation with AI (Fate-AI), a multimodal framework that integrates fragmentomic and methylation-derived features from low-pass whole-genome sequencing (LPWGS) and cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq). It employs a knowledge-informed strategy to select recurrently altered genomic regions and tissue-specific methylation loci to combine the advantages of tumor-naive approaches with the specificity of tumor-informed approaches. This approach derives robust per-sample normalized features that mitigate batch effects and enhance cross-cohort reproducibility. We evaluated Fate-AI on a total of 1,219 plasma samples spanning ten cancer types and healthy controls from multiple laboratories and sequencing centers, including 432 newly profiled cases (280 with both cfMeDIP-seq and LPWGS) together with 787 samples from four independent public datasets. Fate-AI achieved superior sensitivity and specificity compared to state-of-the-art methods, detecting tumor-derived signals at fractions as low as 10-5 in experimental dilutions. Fate-AI scores correlated with disease stage and tracked longitudinal progression, anticipating relapse months before clinical progression. Furthermore, Fate-AI enabled tissue-of-origin classification, with AUCs ranging from 0.84 to 0.97 across six cancer types. Collectively, our results demonstrate that Fate-AI provides a sensitive, generalizable, and clinically actionable platform for early detection, minimal residual disease monitoring, and tissue-of-origin classification, supporting its potential as a liquid biopsy framework in precision oncology.

cancer biology↗

bioRxiv2025

TS2CG as a membrane builder

Molecular dynamics (MD) simulations excel at capturing biological processes at the molecular scale but rely on a well-defined initial structure. As MD simulations now extend to whole-cell-level modeling, new tools are needed to efficiently build initial structures. Here, we introduce TS2CG version 2, designed to construct coarse-grained membrane structures with any desired shape and lateral organization. This version enables precise placement of lipids and proteins based on curvature preference, facilitating the creation of large, near-equilibrium membranes. Additional features include controlled pore generation and the placement of specific lipids at membrane edges for stabilization. Moreover, a Python interface allows users to extend functionality while maintaining the high performance of the C++ core. To demonstrate its capabilities, we showcase challenging simulations, including a Mobius strip membrane, a vesicle with lipid domain as continental plates (Martini globe), and entire mitochondrial membranes exhibiting lipid heterogeneity due to curvature, along with a comprehensive set of tutorials.

biophysics↗

bioRxiv2024

An integrative modelling approach to the mitochondrial cristae

Mitochondria are implicated in many cellular functions such as energy production and apoptosis but also disease pathogenesis. To effectively perform these roles, the mitochondrial inner membrane has invaginations known as cristae that dramatically increase the surface area. This works to provide more space for membrane proteins that are essential to the roles of mitochondria. While separate components of this have been studied computationally, it remains a challenge to combine elements into an overall model. Here we present a workflow to create a comprehensive model of a crista junction from a human mitochondrion. Our coarse-grained representation of a crista shows how various experimentally determined features of organelles can be combined with molecular modelling to give insights into the interactions and dynamics of complicated biological systems. This work is presented as an initial living model for this system, intended to be built upon and improved as our understanding, methodology and resources develop.

biochemistry↗

bioRxiv2024

Structural insights into terminal arabinosylation biosynthesis of the mycobacterial cell wall arabinan

The emergence of drug-resistant strains exacerbates the global challenge of tuberculosis caused by Mycobacterium tuberculosis (Mtb). Central to the pathogenicity of Mtb is its complex cell envelope, which serves as a barrier against both immune system and pharmacological attacks. Two key components of this envelope, arabinogalactan (AG) and lipoarabinomannan (LAM) are complex polysaccharides that contain integral arabinan domains important for cell wall structural and functional integrity. The arabinofuranosyltransferase AftB terminates the synthesis of these arabinan domains by catalyzing the addition of {beta}-(1[->]2)-linked terminal arabinofuranose residues. Here, we present the cryo-EM structures of Mycobacterium chubuense AftB in its apo and donor substrate analog-bound form, determined to 2.9 [A] and 3.4 [A] resolution, respectively. Our structures reveal that AftB has a GT-C fold transmembrane (TM) domain comprised of eleven TM helices and a periplasmic cap domain. AftB has an irregular tube-shaped cavity that bridges the two proposed substrate binding sites. By integrating structural analysis, biochemical assays, and molecular dynamics simulations, we elucidate the molecular basis of the reaction mechanism of AftB and propose a model for catalysis.

biochemistry↗

bioRxiv2024

Mechanistic studies of mycobacterial glycolipid biosynthesis by the mannosyltransferase PimE

Tuberculosis (TB), exceeded in mortality only by COVID-19 among global infectious diseases, is caused by Mycobacterium tuberculosis (Mtb). The pathogenicity of Mtb is largely attributed to its complex cell envelope, which includes a class of glycolipids called phosphatidyl-myo-inositol mannosides (PIMs), found uniquely in mycobacteria and its related corynebacterineae. These glycolipids maintain the integrity of the mycobacterial cell envelope, regulate its permeability, and mediate host-pathogen interactions. PIMs consist of a phosphatidyl-myo-inositol core decorated with one to six mannose residues and up to four acyl chains. The mannosyltransferase PimE catalyzes the transfer of the fifth PIM mannose residue from a polyprenyl phosphate-mannose (PPM) donor. This step in the biosynthesis of higher-order PIMs contributes to the proper assembly and function of the mycobacterial cell envelope; however, the structural basis for substrate recognition and the catalytic mechanism of PimE remain poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of PimE from Mycobacterium abscessus captured in its apo form and in a product-bound complex with the reaction product Ac1PIM5 and the by-product polyprenyl phosphate (PP), determined at 3.0 [A] and 3.5 [A], respectively. The structures reveal the active site within a distinctive binding cavity that accommodates both donor and acceptor substrates/products. Within the cavity, we identified residues involved in substrate coordination and catalysis, which we confirmed through in vitro enzymatic assays and further validated by in vivo complementation experiments. Molecular dynamics simulations were applied to identify the access pathways and the dynamics involved in substrate binding. Integrating structural, biochemical, genetic, and computational experiments, our study provides comprehensive insights into how PimE functions, opening potential avenues for development of novel anti-TB therapeutics.

biochemistry↗

bioRxiv2024

Hexokinase-I directly binds to a charged membrane-buried glutamate of mitochondrial VDAC1 and VDAC2

Binding of hexokinase HKI to mitochondrial voltage-dependent anion channels (VDACs) has far-reaching physiological implications. However, the structural basis of this interaction is unclear. Combining computer simulations with experiments in cells, we here show that complex assembly relies on intimate contacts between the N-terminal -helix of HKI and a charged membrane-buried glutamate on the outer wall of VDAC1 and VDAC2. Protonation of this residue blocks complex formation in silico while acidification of the cytosol causes a reversable release of HKI from mitochondria. Membrane insertion of HKI occurs adjacent to the bilayer-facing glutamate where a pair of polar channel residues mediates a marked thinning of the cytoplasmic leaflet. Disrupting the membrane thinning capacity of VDAC1 dramatically impairs its ability to bind HKI in silico and in cells. Our data reveal key topological and mechanistic insights into HKI-VDAC complex assembly that may benefit the development of therapeutics to counter pathogenic imbalances in this process.

biochemistry↗

bioRxiv2023

Photosymbiosis reduces the environmental stress response under a heat challenge in a facultatively symbiotic coral

The symbiosis between corals of the order Scleractinia and dinoflagellates of the family Symbiodiniaceae is sensitive to environmental stress. The oxidative bleaching hypothesis posits that extreme temperatures lead to accumulation of photobiont-derived reactive oxygen species ROS, which exacerbates the coral environmental stress response (ESR). To understand how photosymbiosis modulates coral ESRs, these responses must be explored in hosts in and out of symbiosis. We leveraged the facultatively symbiotic coral Astrangia poculata, which offers an opportunity to uncouple the ESR across its two symbiotic states (symbiotic, aposymbiotic). Colonies of both symbiotic states were exposed to three temperature treatments for 15 days: i) control (static 18{degrees}C), ii) heat challenge (increasing from 18 to 32{degrees}C), and iii) cold challenge (decreasing from 18 to 6{degrees}C) after which host gene expression was profiled. Cold challenged corals elicited widespread differential expression, however, there were no differences between symbiotic states. In contrast, symbiotic colonies exhibited greater gene expression plasticity under heat challenge, including enrichment of cell cycle pathways involved in controlling photobiont growth. Counter to the oxidative bleaching hypothesis, this plasticity did not include signatures of stress, and rather a dampened ESR under heat challenge was observed, suggesting that photobionts reduce the hosts ESR under elevated temperatures in A. poculata.

ecology↗

bioRxiv2022

Polarity and mixed-mode oscillations may underlie different patterns of cellular migration

In mesenchymal cell motility, several migration patterns have been observed, including directional, exploratory and stationary. Two key members of the Rho-family of GTPases, Rac and Rho, along with an adaptor protein called paxillin, have been particularly implicated in the formation of such migration patterns and in regulating adhesion dynamics. Together, they form a key regulatory network that involves the mutual inhibition exerted by Rac and Rho on each other and the promotion of Rac activation by phosphorylated paxillin. Although this interaction is sufficient to generating wave-pinning that underscores cellular polarization comprised of cellular front (high active Rac) and back (high active Rho), it remains unclear how they interact collectively to induce other modes of migration detected in Chinese hamster Ovary (CHO-K1) cells. We previously developed a 6D reaction-diffusion model describing the interactions of these three proteins (in their active/phosphorylated and inactive/unphosphorylated forms) along with other auxiliary proteins, to decipher their role in generating wave-pinning. In this study, we explored, through computational modeling and image analysis, how differences in timescales within this molecular network can potentially produce the migration patterns in CHO-K1 cells and how switching between them could occur. To do so, the 6D model was reduced to an excitable 4D spatiotemporal model possessing three different timescales. The model produced not only wave-pinning in the presence of diffusion, but also mixed-mode oscillations (MMOs) and relaxation oscillations (ROs). Implementing the model using the Cellular Potts Model (CPM) produced outcomes in which protrusions in cell membrane changed Rac-Rho localization, resulting in membrane oscillations and fast directionality variations similar to those seen in CHO-K1 cells. The latter was assessed by comparing the migration patterns of CHOK1 cells with CPM cells using four metrics: instantaneous cell speed, exponent of mean square-displacement (called -value), directionality ratio and protrusion rate. Variations in migration patterns induced by mutating paxillins serine 273 residue was also captured by the model and detected by a machine classifier, revealing that this mutation alters the dynamics of the system from MMOs to ROs or nonoscillatory behaviour through variation in the concentration of an active form of an adhesion protein called p21-Activated Kinase 1 (PAK). These results thus suggest that MMOs and adhesion dynamics are the key ingredients underlying CHO-K1 cell motility.

cell biology↗

bioRxiv2022

Fan cells in lateral entorhinal cortex directly influence medial entorhinal cortex through synaptic connections in layer 1

Standard models for spatial and episodic memory suggest that the lateral entorhinal cortex (LEC) and medial entorhinal cortex (MEC) send parallel independent inputs to the hippocampus, each carrying different types of information. Here, we evaluate the possibility that information is integrated between divisions of the entorhinal cortex prior to reaching the hippocampus. We demonstrate that fan cells in layer 2 (L2) of LEC that receive neocortical inputs, and that project to the hippocampal dentate gyrus, also send axon collaterals to layer 1 (L1) of the MEC. Activation of fan cell inputs evokes monosynaptic glutamatergic excitation of stellate and pyramidal cells in L2 of the MEC, typically followed by inhibition that contains fast and slow components mediated by GABAA and GABAB receptors, respectively. Fan cell inputs also directly activate interneurons in L1 and L2 of MEC, with synaptic connections from L1 interneurons accounting for slow feedforward inhibition of L2 principal cell populations. The relative strength of excitation and inhibition following fan cell activation differs substantially between neurons and is largely independent of anatomical location. Our results demonstrate that the LEC, in addition to directly influencing the hippocampus, can activate or inhibit major hippocampal inputs arising from the MEC. Thus, local circuits in the superficial MEC may combine spatial information with sensory and higher order signals from the LEC, providing a substrate for integration of what and where components of episodic memories.

neuroscience↗

bioRxiv2022

Tissue-Like 3D Standard and Protocols for Microscope Quality Management

This article outlines a global study conducted by the Association of Biomedical Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). The results present a novel 3D tissue-like biologically relevant standard sample that is affordable and straightforward to prepare. Detailed sample preparation and instrument specific image acquisition protocols and image analysis methods are presented and made available to the community. The standard consists of sub-resolution and large well characterized relative intensity fluorescence microspheres embedded in a 120 {micro}m thick 3D gel with a refractive index of 1.365. The standard allows the evaluation of several properties as a function of depth. These include: 1) microscope resolution with automated analysis of the point spread function (PSF), 2) automated signal-to-noise- ratio analysis, 3) calibration and correction of fluorescence intensity loss, and 4) quantitative relative intensity. Results demonstrate expected refractive index mismatch dependent losses in intensity and resolution with depth but the relative intensities of different objects at similar depths were maintained. This is a robust standard showing reproducible results across laboratories, microscope manufacturers and objective lens types (e.g. magnification, immersion medium). Thus, these tools will be valuable for the global community to benchmark fluorescence microscopes and will contribute to improved rigor and reproducibility.

cell biology↗

bioRxiv2022

From Molecular Dynamics to Supramolecular Organization: The Role of PIM Lipids in the Originality of the Mycobacterial Plasma Membrane

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that claims ~1.5 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. There is much to be understood about the Mtb cell envelope, a complicated barrier that antibiotics need to negotiate to enter the cell. Within this envelope, the plasma membrane is the ultimate obstacle and is proposed to be comprised of over 50% mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs), whose role in the membrane structure remains elusive. Here we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to drive biophysical properties of the Mycobacterial plasma membrane. To validate the model, we tested known anti-tubercular drugs and replicated previous experimental results. Our results shed new light into the organization of the Mycobacterial plasma membrane and provides a working model of this complex membrane to use for in silico studies. This opens the door for new methods to probe potential antibiotic targets and further understand membrane protein function. O_FIG O_LINKSMALLFIG WIDTH=169 HEIGHT=200 SRC="FIGDIR/small/498153v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@102b357org.highwire.dtl.DTLVardef@4c30f8org.highwire.dtl.DTLVardef@22f32aorg.highwire.dtl.DTLVardef@8a6f65_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

US Dog Importations during the COVID-19 Pandemic: Do we have an erupting problem?

Dog importation data from 2018-2020 were evaluated to ascertain whether the dog importation patterns in the United States changed during the COVID-19 pandemic, specifically with regard to denial of entry. Dog denial of entry reports from January 1, 2018, to December 31, 2020, stored within the Centers for Disease Control and Prevention (CDC) Quarantine Activity Reporting System, were reviewed. Basic descriptive statistics were used to analyze the data. Reason for denial, country of origin, and month of importation were all examined to determine which countries of origin resulted in the largest number of denials, and whether there was a seasonal change in importations during the COVID-19 pandemic (2020), compared to previous years (2018 and 2019). During 2020, CDC denied entry to 458 dogs. This represents a 52% increase in dogs denied entry compared to the averages in 2018 and 2019. Dogs were primarily denied entry for falsified rabies vaccination certificates (56%). Three countries exported 74% of all dogs denied entry into the United States, suggesting that targeted interventions may be needed for certain countries. Increased attempts to import inadequately vaccinated dogs from countries with canine rabies in 2020 may have been due to the increased demand for domestic pets during the COVID-19 pandemic. Educational messaging should highlight the risk of rabies and the importance of making informed pet purchases from foreign entities to protect pet owners, their families, and the public.

scientific communication and education↗

bioRxiv2021

MethodsJ2: A Software Tool to Improve Microscopy Methods Reporting

Proper reporting of metadata is essential to reproduce microscopy experiments, interpret results and share images. Experimental scientists can report details about sample preparation and imaging conditions while imaging scientists have the expertise required to collect and report the image acquisition, hardware and software metadata information. MethodsJ2 is an ImageJ/Fiji based software tool that gathers metadata and automatically generates text for the methods section of publications.

bioinformatics↗

bioRxiv2021

Micro-Meta App: an interactive software tool to facilitate the collection of microscopy metadata based on community-driven specifications

For the information content of microscopy images to be appropriately interpreted, reproduced, and meet FAIR (Findable Accessible Interoperable and Reusable) principles, they should be accompanied by detailed descriptions of microscope hardware, image acquisition settings, image pixel and dimensional structure, and instrument performance. Nonetheless, the thorough documentation of imaging experiments is significantly impaired by the lack of community-sanctioned easy-to-use software tools to facilitate the extraction and collection of relevant microscopy metadata. Here we present Micro-Meta App, an intuitive open-source software designed to tackle these issues that was developed in the context of nascent global bioimaging community organizations, including BioImaging North America (BINA) and QUAlity Assessment and REProducibility in Light Microscopy (QUAREP-LiMi), whose goal is to improve reproducibility, data quality and sharing value for imaging experiments. The App provides a user-friendly interface for building comprehensive descriptions of the conditions utilized to produce individual microscopy datasets as specified by the recently proposed 4DN-BINA-OME tiered-system of Microscopy Metadata model. To achieve this goal the App provides a visual guide for a microscope-user to: 1) interactively build diagrammatic representations of hardware configurations of given microscopes that can be easily reused and shared with colleagues needing to document similar instruments. 2) Automatically extracts relevant metadata from image files and facilitates the collection of missing image acquisition settings and calibration metrics associated with a given experiment. 3) Output all collected Microscopy Metadata to interoperable files that can be used for documenting imaging experiments and shared with the community. In addition to significantly lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training users that have limited knowledge of the intricacies of light microscopy experiments. To ensure wide-adoption by microscope-users with different needs Micro-Meta App closely interoperates with MethodsJ2 and OMERO.mde, two complementary tools described in parallel manuscripts.

bioinformatics↗