Biology subjects

Miller, E.

Publications and source records attributed to Miller, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Spatial overlap and phenotypic similarity predict hybridisation in birds

Hybridisation is widespread in birds and has important eco-evolutionary implications. Historically, much research has focused on the consequences of hybridisation. To date, however, far less attention has been paid to the causes of hybridisation and, in particular, why particular pairs of species form hybrids. Using large-scale comparative analyses, we find that similarity in plumage, vocalisations, and morphometric traits, as well as evolutionary relatedness, drive hybridisation in North American and European birds. In passerines, the avian order including songbirds, similarity in vocalisations and morphology are the key phenotypic predictors of hybridisation, whereas in non-passerines, species pairs with similar plumage and morphology are more likely to hybridise. We also find that the likelihood of hybrid formation increases with the magnitude of range and habitat overlap, demonstrating that hybridisation is not just a product of occasional contact between closely related species. Our findings have important conservation implications in this age of rapid anthropogenic change and species reassembly.

evolutionary biology↗

bioRxiv2026

Human Metalloenzymes Are Disproportionately Linked to Disease: The HEAM Structural Annotation Resource

Catalytic metal-binding sites are established drug targets for many human diseases, yet systematic study of how metal function relates to disease is limited by the lack of a resource combining structural localization of human metal-binding sites with catalytic annotation at scale. We introduce HEAM (the Human Enzymatic Annotation of Metalloproteins), a structurally annotated dataset of 1,933 human metalloenzymes and 1,171 non-catalytic metalloproteins. HEAM incorporates annotated metals into protein structures at their corresponding binding sites and classifies each site as catalytic or non-catalytic using the machine learning classifier MAHOMES II. HEAM more than quadruples the human site-level coverage of the largest prior metalloenzyme database. Using HEAM, we show that human metalloproteins are significantly enriched in KEGG disease pathways relative to the human proteome, and that metalloenzymes specifically are significantly enriched in ClinVar pathogenic variants -- a distinction obscured in prior, catalytically unresolved metalloprotein datasets. These pathogenic variants are concentrated in metabolic disorders, indicating that metabolic dysfunction is a substantial contributor to metalloenzyme-linked disease. By pairing 3D structural annotation with catalytic classification, HEAM provides both a foundation for structure-guided drug design against metal-binding sites and a resource for determining how catalytic and non-catalytic metal function contribute to human disease.

biophysics↗

bioRxiv2026

BAYESIAN STATE-SPACE MODEL FOR JOINT INFERENCE OF OSCILLATORY DYNAMICS AND POINT-PROCESS COUPLING

Under a range of behavioral and physiological conditions, spike times and local field potential (LFP) oscillations exhibit phase coupling within specific frequency bands. Classical measures such as spike-field coherence (SFC) and the phase-locking value (PLV) quantify this coupling but estimate the LFP spectrum independently of spike timing. We introduce Joint SSMT, a Bayesian state-space framework that jointly infers LFP spectrograms and spike-field coupling strength. The model treats narrowband LFP activity as a latent process evolving in continuous time, with spike trains linked to the complex spectral state through a Bernoulli-logistic model. In simulations, Joint SSMT accurately recovers coupling strength, denoises the spectrogram, and uses spike timing to resolve fine temporal structure in the LFP. Applied to propofol anesthesia data, the model identifies coupling at a specific slow-oscillation frequency where SFC and PLV report only broad low-frequency coupling. We extend Joint SSMT to trial-structured experiments and apply it to primate recordings during an associative learning task, revealing frequency-specific coupling in hippocampus and prefrontal cortex. We also derive closed-form expressions for SFC and PLV as functions of the generative model parameters. Across simulations and two primate datasets, Joint SSMT provides more frequency-specific coupling estimates with principled uncertainty quantification than classical PLV and SFC.

neuroscience↗

bioRxiv2026

Tension TRAAKer: a chemigenetic fluorescent membrane tension reporter

Mechanical force transduction is essential to survival, underlying biological processes as fundamental as morphogenesis, somatosensation, audition, and interoception; and driving pathologies as diverse as hypertension and cancer metastasis. Exogenous forces are translated to intracellular signals through transient changes in membrane tension which are currently not possible to directly monitor in situ. To remedy this, we have designed and validated Tension TRAAKer, a chemigenetic fluorescent membrane tension reporter for the visualization of tension induction, propagation, and dissipation in living cells. Tension TRAAKer is derived from inserting a tension-sensitive nonconductive variant of the mechanosensitive potassium ion channel TRAAK into a self-labelling HaloTag. Increasing membrane tensions effect conformational changes in the TRAAK channel that are optically monitored by a HaloTag-conjugated fluorogenic (environment-sensitive) dye. EGFP incorporation C-terminal to the HaloTag enables unambiguous tension reporting in mobile membranes via dual-color ratiometric imaging that controls for variations in sensor density. Tension TRAAKer reports membrane tension changes rapidly, reversibly, and with spatiotemporal precision--its fluorescence scaling to both stimulus magnitude and area, with consistent effect sizes observed between diverse cell types. It better distinguishes among elevated membrane tensions than do available indirect chemical reporters, with the additional advantage of being readily genetically targetable. We thus expect Tension TRAAKer to be a powerful tool for the study of membrane tension across biological systems and disease states.

molecular biology↗

bioRxiv2026

Granular Extracellular Matrix (gECM) Hydrogels Enable Distinct Composition and Mechanics Across Tissue Types for Translation

Biomaterials-based tissue engineering aims to recapitulate native tissue architecture and function for both clinical repair and advanced in vitro models. While improvements in biomaterials have been made, including granular hydrogels and ECM-derived scaffolds, current biomaterials lack intentional design choices for effective translation, including regulatory considerations, practical extrusion delivery, and biomimetic characteristics. Here, we develop and characterize a library of granular ECM (gECM) biomaterials for five key tissues (cartilage, bone, skin, liver, and kidney), in which ECM particles are densely packed within a hyaluronic acid hydrogel. We optimize tissue processing methods that preserve proteomic content and structure while also aligning with scale-up manufacturing and regulatory guidelines. We show that gECM hydrogels can be molded, extruded, and 3D-printed while retaining their shape, and they stabilize at physiological temperature and pH. Lastly, we demonstrate that bulk gECM mechanics are driven by tissue type, and gECM hydrogels support viability, proliferation, and tissue-specific cellular activity. Together, these findings establish gECM hydrogels as a translational and biomimetic platform for clinical tissue repair and complex in vitro models.

bioengineering↗

bioRxiv2026

Flowable Grafts Made from Granular Extracellular Matrix (gECM) Hydrogels Promote Integrative Repair of Articular Cartilage in a Large-Animal Model

Focal injuries to articular cartilage in load-bearing joints fail to heal and often progress to degeneration, underscoring the need for repair strategies that result in restored cartilage structure and function rather than fibrocartilage formation. Granular extracellular matrix (gECM) hydrogels, flowable grafts composed of densely-packed matrix particles, offer a promising approach but lack long-term functional validation in large-animal models. Here, we developed a flowable gECM hydrogel composed of decellularized cartilage microparticles incorporated within a thiol-functionalized hyaluronan matrix. Proteomic analysis confirmed enrichment of cartilage-specific gECM matrisome components. When implanted into critical-sized femoral condyle defects in a goat model and evaluated 12 months post-implantation, both gECM hydrogel and microdrilling (surgical controls) achieved >80% defect filling. However, in contrast to microdrilling, gECM repair tissue exhibited surface tribological (friction, adhesion) and compressive mechanical properties comparable to native cartilage, with a similar proteoglycan-to-collagen ratio, enrichment of type II collagen, minimal type I collagen (typical of a fibrous scar), improved quantitative MRI metrics, and evidence of lateral cartilage integration and subchondral bone remodeling. Together, these findings demonstrate that a flowable gECM hydrogel supports integrative, cartilage-like repair in a load-bearing joint, supporting advancement of this approach toward clinical translation. One Sentence SummaryA granular ECM hydrogel implanted in a goat condyle provided a robust repair, filling the defect tissue with integrated, hyaline-like cartilage at 12 months.

bioengineering↗

bioRxiv2026

Migratory bird and marine mammal surveillance fails to find evidence for an HPAI H5N1 2.3.4.4b incursion into Australia in 2025

The panzootic caused by high pathogenicity avian influenza (HPAI) H5N1 clade 2.3.4.4b has been devastating for animals, globally. Despite global spread, the virus remains absent in Oceania. Herein we report the results of our fourth year of enhanced migratory bird surveillance, coinciding with the spring migration of wild birds in 2025; none of the 847 migratory wild birds or 38 marine mammals were positive for HPAI H5N1, although we did detect LPAI. Surveillance remains a critical tool for HPAI H5N1 response, with early detection and rapid response being critical to mitigate the impacts of this virus on animal, environment and human health.

microbiology↗

bioRxiv2026

clootl: an R package for accessing and manipulating a complete, versioned avian phylogeny

Birds are frequently used as a focal taxon for evolutionary and ecological studies. Thousands of papers a year are published using birds as study systems. Hundreds more are published clarifying the evolutionary relationships among clades or regionally circumscribed sets of bird species. Up to date phylogenies are essential for informing and guiding avian studies, controlling for the expectation of shared trait evolution, and for science communication, among other applications. However, employing up to date phylogenies to address these questions has proven challenging for a number of reasons. First, individually published phylogenies are often hard to access in a usable manner. For example, sequences are usually made available, and an image of the phylogeny is published, but the actual phylogeny data product is often not digitally available. Second, published phylogenies often do not include all taxa of interest or have branch lengths in units of time. Third, taxonomic mismatches between phylogenies and existing datasets can complicate analyses. We address these issues by sharing an R package, clootl, that wraps together a new, complete, dated bird phylogeny with easy to use tools to extract trees for taxa of interest and sample over uncertainty. The phylogeny incorporates information from more than 300 individually published bird phylogenies. The R package includes tools to help appropriately cite these input studies. This software will enable users to smoothly integrate accurate evolutionary information into any analyses on birds.

evolutionary biology↗

bioRxiv2026

Expression of soluble Type IV Minor Pilins and isolation of a Neisseria gonorrhoeae PilI-PilJ subcomplex

Type IV pili and type II secretion systems assemble dynamic fibers used by bacteria and archaea for diverse functions. The pilus fiber is made up of major and minor pilin subunits containing a hydrophobic -helical spine and a globular head. Purifying minor pilins is complicated by the hydrophobic -helical spine, frequently present disulfide bonds, and low abundance within the fiber. These challenges have impeded structural and functional studies of pilin protomers. Here, we describe a method for expression and purification of soluble type IV pilin proteins from Escherichia coli. Signal peptidase I cleavage sites are engineered into the -helix of the pilin proteins. This allows their globular domains to be purified from the periplasmic fraction. We used this method to obtain the Neisseria gonorrhoeae minor pilins PilI and PilK in soluble form. In a third case, where the minor pilin PilJ could not be obtained on its own, coexpression with PilI and purification of a PilI-PilJ heterodimer was possible. We suggest that PilI and PilJ form an obligate heterodimer that is essential for their function. ImportanceType IV pili are essential to many bacteria responsible for disease. They can be found in both Gram-negative and Gram-positive bacteria, as well as archaea, making them likely present in the last common ancestor of all life on Earth. Despite their significance in a variety of species, there are large gaps in our understanding of the structure of these diverse biological machines. One roadblock to this research has been the difficulty of purifying the minor pilin proteins that serve different functions in the fiber. Here, we describe a novel method for the purification of these proteins and demonstrate the ability of this method to identify a protein-protein interaction between two minor pilins of Nesseria gonorrhoeae.

microbiology↗

bioRxiv2025

The Two Frontiers Project Field Handbook and OpenTools: Standardizing microbial fieldwork for biobank-scale sequencing and culturomics

From medicines to materials, our planets microbial diversity comprises an enormous wellspring of biotechnological potential. For centuries, microbiologists have developed tools for interrogating microbial function, ranging from microscopy and culturing to, more recently, metagenomics. However, deploying these tools during fieldwork requires substantial forward planning, interdisciplinary technical expertise, and plans for navigating permitting and the ethical implications of bioprospecting. To address these challenges, we built The Two Frontiers Project Handbook and OpenTools Resource, which aggregates our expertise in high-throughput sampling, sequencing, and culturing of microbes from thousands of samples. We provide our full suite of fieldwork methods as well as relevant software and hardware. We lay our standards for team roles and construction, general expedition planning, sample transport, permitting, and numerous other key aspects of executing a successful field campaign. The version-controlled resource is available at https://two-frontiers-project.github.io/ and is open for non-commercial use.

microbiology↗

bioRxiv2025

Ephaptic coupling can explain variability in neural activity

The waxing and waning cortical oscillatory power correlates with function and disease. This cross-trial variability has been thought to be due to neuromodulation, uncertainty encoding, and/or changes in cortical excitability. Here, we report evidence that it is also due to fluctuations in ephaptic influences of mesoscale electric fields. We analyzed LFP data from the PFC recorded during a spatial delay saccade task. We constructed a model that describes the electric field close to the cortical patch given the neural activity that generates it. This revealed that field-to-neuron interactions (ephaptic coupling strength) was stronger that neuron-to-field and it correlated trial by trial changes in oscillatory power. This suggests a form of circular causality where neural activity and extracellular electric fields continuously shape each other. These results further suggest that mesoscale ephaptic effects help drive the formation of memory ensembles, a prediction of the cytoelectric coupling hypothesis.

neuroscience↗

bioRxiv2025

Surveillance of migratory shorebirds and seabirds in 2024 in Australia reveals incursions of a diversity of low pathogenicity avian influenza viruses, but not high pathogenicity avian influenza H5N1

The current panzootic of high pathogenicity avian influenza (HPAI) H5N1 has been catastrophic for wildlife, and following a significant sweep, clade 2.3.4.4b is found in every region aside from Oceania. Herein, we report the results of our third year of targeted surveillance of incoming migratory seabirds and shorebirds into Australia. We did not find evidence of HPAI H5N1 in any of the birds tested, and there were no reports of HPAI H5N1 in wildlife tested through other surveillance schemes in 2024. Unlike previous years, we detected a diversity of low pathogenicity avian influenza (LPAI) viruses in shorebirds. Through phylogenetic analysis we revealed that the H3N7 and H4N7 viruses recovered from Red-necked Stints were complex mosaic viruses, comprising segments of Eurasian, Australian shorebird, and Australian waterfowl segments. A H1N7 virus detected comprised a wholly Eurasian introduction, confirming this route for avian influenza viruses into Australian ecosystems. These results provide further evidence for the key role of long-distance migratory shorebirds in introducing novel LPAI viruses into Oceania. While our focus on northern migration routes remained appropriate for HPAI H5N1 surveillance in 2024, the continued spread of HPAI H5N1 to sub-Antarctic Islands demands consideration of a potential southern incursion route for Oceania in future.

microbiology↗

bioRxiv2025

Maternal Influenza A Virus Infection Induces Antiviral and Immune Dysregulation in the Placenta and Fetus Without Vertical Transmission

Influenza A virus (IAV) infection during pregnancy is associated with stillbirth and preterm birth, but the degree to which IAV alters placental and fetal immunity is poorly understood. The objective of our study was to determine the immunologic impact of maternal IAV infection on the placenta and fetus in a pigtail macaque (Macaca nemestrina) model. Pregnant pigtail macaques were inoculated with 107 plaque forming units (PFU) of IAV [A/California/07/2009 (H1N1)] and underwent necropsy 5 days post-infection (N=11). Results were compared to uninfected historical controls (N=16). IAV inoculation induced maternal pneumonia in all cases. Stillbirth occurred in 18% (2/11) of IAV-infected pregnancies, but not in controls. While vertical transmission was not observed, low-level IAV viral RNA was detected in two placentas. In the placenta, maternal IAV infection was associated with increased IL-1{beta}, IL-18, and IFN-{beta} levels, and an upregulated type I interferon (IFN) transcriptional response. IAV infection was also associated with significantly higher frequencies of intermediate and non-classical monocytes, plasmacytoid dendritic cells, CD4 T cells, and NKT cells in the fetus (lung, lymph node, blood). Although placental immune and transcriptional perturbations were rarely correlated with maternal IAV disease indicators (e.g., maternal lung viral load/IFN-/IFN-{beta}/IL-6), there were consistent and significant correlations between these metrics and perturbed immune cell populations in the fetus (CD4+ and CD8+ T cells, plasmacytoid dendritic cells, monocyte sub-populations). Maternal IAV infection disrupted both placental and fetal immune environments, but only fetal immune alterations correlated with maternal lung disease severity. One Sentence SummaryMaternal influenza A virus infection in pregnant pigtail macaques dysregulates placental and fetal immunity, with disease severity correlating strongly with fetal, but not placental, immune perturbations.

systems biology↗

bioRxiv2025

Spatial genomics of the cardiac sarcomere

Structure-function mapping of proteins has improved our understanding of disease mechanisms and protein domains while human population genetics has provided a window into variational tolerance that can be modeled at a structural level. Here, to reveal novel insight into the cardiac sarcomere, the motor unit of the heart, we develop a novel analytical framework to integrate data from over 17,000 patients with hypertrophic cardiomyopathy (HCM) and combine it with two population-scale genomic databases that incorporate data from more than 800,000 individuals. We integrate in silico genetic predictions of gene variant pathogenicity with 3 dimensional integrative spatial scanning across multiple structural models of cardiac motor proteins. Results reveal both recognized and novel regions of structural variant intolerance across the critical genes of the cardiac sarcomere including novel insights into protein function. We discuss the structural relevance of variant enrichment in the context of sarcomere organization and destabilization of sequestered myosin leading to hypercontractility seen in HCM, by incorporating the recently defined high-resolution structure of human cardiac myosin filament. We extend and validate these findings through pathogenic variant class enrichment and reveal novel associations with the earlier onset of disease in a large clinical cohort. In summary, our study provides a multi-dimensional framework for integrating structural, genomic, and modeling data to reveal novel insight into the cardiac sarcomere.

genomics↗

bioRxiv2025

Membrane potential and feedback dynamics regulate CatSper-mediated progesterone signaling in human sperm

Activation of the sperm-specific Ca2+ channel CatSper by progesterone evokes rapid changes in intracellular Ca2+ in human sperm that are required for fertilization. However, the mechanisms regulating the progesterone-induced Ca2+ signals have remained elusive. Here, we used quantitative kinetic fluorimetry with fast voltage-sensitive fluorescent indicators to investigate how progesterone affects the membrane potential (Vm) of human sperm. Additionally, we employed the FASTM technique to simultaneously record at millisecond time resolution changes in both Vm and intracellular Ca2+. We show that progesterone evokes a rapid pulse-like depolarization and repolarization. The depolarization is caused by Ca2+ influx through CatSper, which pulls Vm away from a resting membrane potential (Vrest) of -65 mV set by the sperm-specific K+ channel Slo3. We further show that Vm- and Ca2+-dependent mechanisms limit the CatSper-mediated Ca2+ influx, thereby promoting repolarization and enabling K+ efflux through Slo3 channels to restore Vrest. Our findings demonstrate that non-genomic progesterone signaling in human sperm is regulated by negative feedback on CatSper and involves a dynamic interplay between CatSper and Slo3 in controlling Vm. We anticipate that our novel kinetic, quantitative Vm recording and Vm/Ca2+-multiplexing techniques will reveal additional molecular mechanisms underlying CatSper-mediated Ca2+ signaling in human sperm both in health and disease.

cell biology↗

bioRxiv2025

The Pdgfd-Pdgfrb axis orchestrates tumor-nerve crosstalk in pancreatic cancer

Nerves are an integral component of the tumor microenvironment, contributing to cancer progression, metastasis, morbidity, and mortality. In pancreatic ductal adenocarcinoma (PDAC), worse clinical outcomes are associated with perineural invasion (PNI), a process by which cancer cells surround and invade nerves. Here, we employed whole-transcriptome and single-cell spatial transcriptomics to identify candidate tumor-nerve interactions that promote PNI. We discovered that Pdgfd signaling promotes key features of nerve invasion. Mechanistically, Pdgfd stimulated cancer cell invasiveness, neurite outgrowth, and direct physical engagement with glia. Pharmacological blockade of this axis reduced each of these processes in vitro as well as PNI in vivo. Thus, Pdgfd-Pdgfrb signaling mediates PNI by coordinating multifaceted cancer-neuron-glia interactions and represents a promising therapeutic strategy aimed at disrupting harmful cancer-nerve crosstalk.

cancer biology↗

bioRxiv2025

An orally available PfPKG inhibitor blocks sporozoite infection of the liver

Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides, climate-driven outbreaks, and limited chemoprotective options. Here, we report the characterization of RUPB-61, the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG). RUPB-61 prevents infection by P. falciparum and P. cynomolgi sporozoites, including the formation of hypnozoites by the latter. A single oral dose blocks liver infection by P. berghei sporozoites in vivo, demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling. The compound retains activity against field isolates resistant to chloroquine, mefloquine, cycloguanil, sulfadoxine and pyrimethamine, suggesting low likelihood of cross-resistance to existing antimalarials. Structural studies and free energy-based modeling guided-compound design prospectively validated the predictive accuracy of an in silico model of PfPKG interactions with this chemotype. While selectivity profiling identified off-target activity against human kinases, structural modeling provides a clear path for optimization. These results establish PfPKG inhibitors as promising candidates for chemoprevention and support further preclinical development of the RUPB-61 chemotype. Author SummaryMalaria remains a serious global health threat, made worse by growing resistance to existing drugs and the spread of insecticide-resistant mosquitoes. A critical gap in our prevention toolkit is the lack of safe, long-acting oral drugs that stop malaria parasites from infecting the liver - the first and obligatory step of every new infection. Here, we report the characterization of RUPB-61, a compound that blocks an enzyme that the malaria parasite needs to invade and survive in the liver. We show that a single oral dose completely prevents liver infection in mice, and that the compound remains active against parasites that are resistant to several currently used antimalarial drugs. Using X-ray crystallography and computer-based modeling, we determined how RUPB-61 binds to its target in atomic detail, providing a roadmap for designing improved versions. While the compound shows some unwanted interactions with human proteins, our structural data offer clear strategies to solve this challenge. Together, these findings provide a solid foundation for developing a new class of once-weekly oral malaria prevention drugs.

microbiology↗

bioRxiv2025

Ex vivo recapitulation of intramuscular mRNA vaccination with naive and recall antigens using a human Lymphoid Follicle Chip platform

Predicting the efficacy and toxicity of intramuscular mRNA vaccines remains challenging. Here, we describe an ex vivo human cell-based model that replicates immune responses to lipid nanoparticle (LNP)-based mRNA vaccines that require intramuscular injection. Vaccines are administered into a biomimetic muscle module containing human skeletal myoblasts and antigen-presenting cells (APCs) to mimic intramuscular vaccination, followed by transfer of the APCs and soluble factors to a microfluidic human lymphoid follicle chip (LF Chip) to mimic lymphatic drainage. Non-replicating mRNA vaccines directly induce antigen expression in APCs, whereas self-amplifying mRNA vaccines require muscle cell-APC contact within the intramuscular vaccination module. Transfer of APCs and soluble factors to the LF Chip induces LF expansion, de novo antigen-specific IgG production against a naive antigen (rabies virus glycoprotein), and cytokine release, with responses varying depending on LNP type. Vaccination of LF chips against SARS- COV-2 Spike recall antigen using the Moderna Spikevax vaccine generates neutralizing antibodies and induces somatic hypermutation. This biomimetic platform offers an all-human alternative for evaluating vaccine-induced immunity, potentially obviating the need for non-human primates and accelerating vaccine development.

immunology↗