Biology subjects

Miller, D.

Publications and source records attributed to Miller, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Impaired loading following denervation elicits dynamic, differential growth trajectories of musculoskeletal tissues

Physical forces dictate the function and maintenance of adult musculoskeletal tissues. Yet, the impact of biophysical cues on the growth and maturation of these tissues, especially dense connective tissues, remains unclear. Here, we investigated the effects of neonatal mechanical loading on early postnatal growth of the musculoskeletal tissues of the lower limb, with particular focus on the Achilles tendon. We performed sciatic nerve resection (SNR) on one-day-old mice to denervate the hamstrings and distal hindlimb muscles. Sciatic Nerve Resection resulted in severe and sustained alterations in gait, accompanied by an altered transcriptional landscape in distal musculoskeletal tissues, with marked reductions in bone and tendon growth and smaller, fibrotic hindlimb musculature. Sciatic nerve resection impaired cumulative growth in the Achilles tendon, shifting cell proliferation and matrix-driven growth trajectories and altering mechanotransduction gene programs toward inflammation and matrix catabolism with age. This work highlights the essential role of mechanical loading in driving mechanosignaling and postnatal growth trajectories in musculoskeletal tissues of the developing limb, with implications for overall musculoskeletal health in adulthood.

bioengineering↗

bioRxiv2026

Projected Gradient Descent for Hallucination-Based Protein Binder Design

Gradient-based hallucination is a popular approach to protein binder design that backpropagates a loss defined on a structure predictor's output to guide optimisation in a continuous relaxation of sequence space, yet the choice of optimiser for this task has received little systematic evaluation. In this work we evaluate simplex-projected gradient descent (PGD) as an alternative to the commonly used gradient descent on softmax-reparameterised sequence logits. By updating amino-acid probabilities directly, PGD avoids softmax-induced gradient attenuation and allows amino acids suppressed early in optimisation to re-enter the design. Using an ESMFold2-based pipeline on ten targets, we find that PGD minimises the design loss in fewer iterations and produces designs with higher predicted interface confidence compared to its softmax counterpart. Simulating selection in real campaigns by considering the upper tail of the ipSAE distribution across a pool of 1,024 designs per target, we show that PGD significantly improves on six of the ten targets, with no significant degradation on any, and produces more structurally diverse designs on every target. Additionally, we find that pairing shorter PGD runs with candidate screening raises the total number of in silico hits per GPU-hour by 29\% relative to default-length trajectories. These results support PGD as a simple and effective alternative for hallucination-based binder design. Code and design data are publicly available.

bioinformatics↗

bioRxiv2026

Transposon end recognition and pairing by I-F3 CRISPR-associated transposase

To develop gene therapy tools based on CRISPR-associated transposons (CASTs), it is essential to define how transposon ends are recognized and paired during transposition. Tn7-like transposons typically contain asymmetric left- and right-end sequences that flank and define DNA cargo. However, how the transposase recognizes these different sequences and assembles them into a paired end complex for cut-and-paste transposition remains unknown. Here we present the cryo-EM structure of type I-F3 (VchCAST) CAST transposase TnsB in complex with transposon DNA ends and host factor IHF, along with biochemistry, molecular dynamics, and in vivo analyses. Our structure reveals the stoichiometry and architecture of the assembly, as well as the DNA distortions required to accommodate transposon end asymmetry. Molecular dynamics suggests that these distortions are required for the coordinated assembly of the complex. Physical pairing of asymmetric left and right ends results in a novel protein-protein interface that is required for transposition efficiency. Our findings explain how transposases regulate the pairing of transposon end sequences for high-fidelity recognition, reveal a model of transposon end synapsis, and suggest future avenues to engineer DNA cargo for genome-editing applications.

biochemistry↗

bioRxiv2026

Protocol for scalable generation of uniform 3D porcine oviduct epithelial cell spheroids

While previous in vitro porcine (Sus scrofa) oviduct spheroid models often rely on manual selection of the formed aggregates, here, we establish a scalable and reproducible system for generating oviduct spheroids. We describe steps for isolating primary secretory and ciliated oviduct epithelial cells and their assembling them into spheroids by forced aggregation using the AggreWell platform. This enables the mass production of uniformly sized spheroids ready for use in a range of downstream applications, such as sperm-oviduct co-incubation experiments. For complete details on the use and execution of this protocol, please refer to Molina et al.1 Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/731731v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@20f94aorg.highwire.dtl.DTLVardef@ce80eeorg.highwire.dtl.DTLVardef@172c631org.highwire.dtl.DTLVardef@31f420_HPS_FORMAT_FIGEXP M_FIG C_FIG Before you beginThis protocol describes the isolation of primary porcine oviduct epithelial cells and their use in generating a three-dimensional (3D) spheroid model in vitro. The protocol is specifically designed to enrich and preserve ciliated oviduct epithelial cells, which are critical for physiologically relevant sperm-oviduct interaction studies yet are frequently underrepresented in conventional in vitro oviduct models. To minimize the de-differentiation of ciliated cells during culture, a specialized Incubation Medium is employed to maintain epithelial cell viability and ciliary function. The relatively brief spheroid formation period (48 h) further supports preservation of ciliary structure and activity prior to downstream functional applications. This protocol consists of four major steps. Steps 1 and 2 cover oviductal epithelial cell isolation through mechanical and enzymatic digestion, step 3 describes the spheroid formation and culture conditions, and step 4 applies the spheroid model to sperm co-incubations or immunofluorescent staining. Although optimized for the porcine isthmus, this protocol may also be suitable for the cells from the ampulla and adaptable to other large animal species.

Cell Biology↗

bioRxiv2026

Spatial engineering of posterior organizers in cerebral organoids via controlled morphogen exposure within hydrogels

Cerebral cortex organoids are powerful in vitro models that recapitulate key features of human development. However, conventional methods produce cortical organoids with spontaneous, spatially disorganized cortical regions due to limited control over morphogen distribution within local environments. Here, we present a spatially engineered hydrogel platform that drives localized posterior organizer formation in cortical organoids through controlled, localized exposure to morphogens. Using a combination of bulk photopolymerization, thermal crosslinking, and digital light processing (DLP) approaches, we fabricated hydrogels with stiffness-controlled layers that preferentially deliver morphogens to one side of the organoid, selectively inducing posterior organizer formation on the exposed face. We further validated this platform by delivering fluorescently tagged dextran, used as molecular weight-matched model morphogens, to visualize spatiotemporal delivery dynamics at the organoid interface. As a proof of principle, we also demonstrated that DLP fabrication enables the printing of dual morphogen hubs, serving as a model for establishing two opposing gradients within a single organoid. Together, this hydrogel platform enables systematic spatial patterning of cell populations in organoids, more faithfully recapitulating the spatial organization and cellular diversity of native tissues and advancing higher-fidelity models for studying human development and disease.

bioengineering↗

bioRxiv2026

High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

T cells adopt diverse states in human disease, with tumor-specific CD8 T cells serving as the quintessential example. These cells are critical for immunotherapy response, yet acquire hypofunctional states of exhaustion that impair treatment efficacy. While significant heterogeneity is observed among exhausted CD8 T cells (TEX), the mechanisms directing their unique phenotypes remain unresolved, limiting our efforts to augment TEX cell function. Here, we profile TEX cells in human head and neck squamous cell carcinoma (HNSCC) tumors and define a major mechanism regulating their cell state. With single-cell RNA and T cell receptor (TCR) sequencing of 106,667 tumor-infiltrating CD8 T cells, we define and validate three fundamental TEX subsets, each harboring dominant clonotypes: a progenitor subset (TPEX) and two intra-tumoral populations marked by intermediate (TEX-int) or high expression of inhibitory receptors, including killer cell lectin-like receptors, such as KLRC1/NKG2A (TEX-KLR). Using an in vitro model, we show that TCR avidity-dependent NFAT signaling governs an axis between the TEX-int and TEX-KLR states across human cancers, with high-avidity signaling driving the TEX-KLR state. Additionally, we uncover that interleukin-12 signaling selectively antagonizes high-avidity TEX-KLR-specific genes, despite broadly potentiating activation and cytotoxicity. Finally, we find that TEX-int and TEX-KLR cells are associated with anti-PD-1 response in HNSCC, with both of these cell types enriched near viable cancer cells following treatment. Together, these findings reveal that TCR avidity orchestrates tumor-infiltrating TEX cell states through NFAT signaling, a tunable axis that may be targeted to improve TEX cell function.

immunology↗

bioRxiv2026

ConvergeCELL: An end-to-end platform from patient transcriptomics to therapeutic hypotheses

Translating transcriptomic data into therapeutic hypotheses remains fragmented and labor-intensive. Here we present ConvergeCELL, a platform combining a patient representation model trained on over 20 million cells across 4,479 patients, an interpretability framework for gene discovery, and a large language model-driven workflow that classifies candidates along an evidence hierarchy and constructs mechanism-of-action hypotheses. Validated on held-out cohorts spanning lupus, multiple myeloma, and sepsis across single-cell and bulk modalities, ConvergeCELL recovers known disease-associated genes at or above differential expression, machine-learning, and patient-level foundation model (PaSCient) baselines. The advantage is most pronounced for clinically validated, disease-specific drug targets: ConvergeCELL ranks TNFSF13B (Belimumab; lupus), TNFRSF17/BCMA (Belantamab; myeloma), and CXCR4 (Plerixafor; myeloma) within the top 0.3% of its gene rankings - significantly outcompeting alternative approaches. ConvergeCELL delivers an end-to-end translational workflow with state-of-the-art performance on both disease-associated gene recovery and patient-level disease classification. The pretrained ConvergeCELL patient representation model and bulk distillation module are publicly available on Hugging Face (huggingface.co/ConvergeBio/virtual-cell-patient) under the Apache 2.0 license.

bioinformatics↗

bioRxiv2026

Comparative anatomy of the giraffe distal limb

Giraffe in human care are known to experience significant clinical issues related to their feet. To characterize normal foot anatomy, we analyzed six sets of front and hind feet from wild Angolan giraffe and one calf in human care. We used computed tomography, three-dimensional reconstruction, sagittal sections, and gross dissection to acquire as much gross anatomical detail as possible. Significant anatomical findings include the deep digital flexor tendon that is very gracile as it crosses the fetlock and proximal phalanges but widens significantly before inserting onto the entire palmar/plantar surface of the distal phalanx. Significant subcutaneous abaxial veins were noted at the level of the fetlock in both front and hind feet. The digital cushion was found to be a complex structure consisting of two distinct regions, one underneath the distal phalanx, characterized by multiple transversely oriented small adipose compartments separated by dense connective tissue septa, and a significantly larger portion within the heel bulb, consisting of two sagittally oriented fat bodies encased in a dense connective tissue capsule and divided by a thick septum. The proportion of adipose tissue volume in the heel bulbs compared to the distal phalanx decreases with age. The thickness of the sole was found to be much greater than that of the wall and the sole appears to be the major weight supporting structure of the foot. In particular, the heel is greatly expanded in giraffe relative to other ruminants and was found to consist of softer material than the rest of the sole. Data presented here provide an overview of normal giraffe foot anatomy, which can be compared with data from giraffe in human care to better understand, guide treatment and prevention of abnormal anatomical conditions.

zoology↗

bioRxiv2026

Fully Phased Telomere-to-Telomere Assemblies for Thoroughbred Horse and Donkey Haplotypes derived from a Mule Illuminate the Peculiar Evolution of Equid Centromeres

We present telomere-to-telomere genome assemblies of a Thoroughbred horse and a donkey derived from their mule offspring. Now adopted and annotated by NCBI as reference genomes, these assemblies resolve previously inaccessible regions, including satellite arrays, duplications, and telomeres. Equids are known to exhibit an uncoupling between satellite DNA and centromeric function. The completeness of these assemblies enabled annotation of both satellite-based and satellite-free centromeres, as well as non-centromeric satellite loci, revealing notable centromeric plasticity. They also allowed detailed characterization of the variable binding domains of CENP-A--the epigenetic determinant of centromere identity--and CENP-B, whose association with CENP-A, previously considered typical based on a few model organisms, is absent in equids. Comparative analyses of satellite repeats and centromere positions provide new insights into the accelerated karyotypic reshuffling in equid evolution. These assemblies represent foundational resources for equid genomics and support ongoing initiatives such as the Equine Pangenome Project.

molecular biology↗

bioRxiv2026

Autophagy Cholesterol Axis Remodeling Supports Malignant Progression and Chemoresistance in Glioma

Glioma progression and resistance to temozolomide (TMZ) remain major clinical challenges. Here, we investigated whether dysregulated autophagy and cholesterol metabolism are coordinately remodeled during glioma progression and TMZ resistance. Tissue microarray analysis of astrocytoma and glioblastoma specimens revealed progressive autophagosome accumulation, reflected by increased LC3{beta} puncta, coupled with impaired autophagic flux compared with adjacent normal brain tissue. These alterations intensified with tumor grade and were associated with upregulation of farnesyl diphosphate synthase (FDPS), linking malignant progression to cholesterol pathway remodeling. TMZ-resistant (R) glioblastoma cells exhibited epithelial-to-mesenchymal transition, mitotic quiescence, and mitochondrial remodeling consistent with a therapy-tolerant phenotype. Bioenergetic profiling demonstrated reduced respiratory reserve, diminished ATP-linked respiration, and elevated proton leak, indicating constrained metabolic flexibility. In parallel, impaired autophagy flux was associated with suppression of de novo cholesterol synthesis and transcriptional downregulation of SREBP-2 and LDL-R. Comprehensive lipidomic profiling revealed marked cholesterol metabolic reprogramming in R cells, characterized by accumulation of specific cholesteryl esters, including CE 22:5, CE 22:6, CE 22:4, and CE 20:4, despite reduced cholesterol biosynthesis. Pharmacologic inhibition of the mevalonate pathway with simvastatin significantly altered cholesteryl ester profiles but failed to restore autophagy flux or sensitize R cells to TMZ-induced apoptosis, even under combined TMZ-simvastatin treatment. Lay AbstractAs gliomas progress from astrocytoma to glioblastoma, autophagy becomes dysregulated and cholesterol metabolism is rewired. This coordinated remodeling supports tumor survival, metabolic plasticity, and resistance to temozolomide therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/697885v2_ufig1.gif" ALT="Figure 1000"> View larger version (79K): org.highwire.dtl.DTLVardef@1183dd2org.highwire.dtl.DTLVardef@82e20dorg.highwire.dtl.DTLVardef@c6c8dforg.highwire.dtl.DTLVardef@adb427_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsAutophagy flux blockade intensifies during progression from astrocytoma to glioblastoma Dysregulated autophagy is coupled to altered cholesterol metabolism in malignant gliomas TMZ-resistant glioblastoma cells undergo epithelial-to-mesenchymal transition and mitotic quiescence Resistant cells exhibit constrained bioenergetic capacity and mitochondrial remodeling Impaired autophagy suppresses de novo cholesterol synthesis and lipid recycling Lipidomics reveals accumulation of long-chain cholesteryl esters in TMZ-resistant cells Statin-based cholesterol inhibition fails to resensitize glioblastoma cells to temozolomide

cancer biology↗

bioRxiv2025

Intrinsic and non-cell autonomous roles for a neurodevelopmental syndrome-linked transcription factor

Transcription factors (TFs) are essential for neuronal identity, yet their potential non-cell-autonomous functions remain largely unexplored. Here, we uncover both cell- and non-cell-autonomous roles for the conserved terminal selector UNC-3 in C. elegans motor neurons (MNs). UNC-3 is an ortholog of human EBF3, mutations in which cause a severe neurodevelopmental syndrome. Single-cell RNA sequencing of cholinergic MNs, which express unc-3, and downstream GABA MNs, which do not, revealed that unc-3 loss disrupts neuronal identity in distinct ways across MN classes. Four cholinergic MN classes lose their molecular identity entirely, whereas the AS class retains it partially, illuminating terminal selector-driven neuronal diversification processes. Integrated transcriptomic and genomic analyses uncovered a dual cell-autonomous role for UNC-3 as both a direct activator and repressor of neuron-type- specific genes in cholinergic MNs, including repression of alternate neurotransmitter programs. Unexpectedly, unc-3 loss also caused widespread transcriptional, morphological, and connectivity defects in downstream GABA MNs. Mechanistically, these non-cell-autonomous effects are mediated by cholinergic neurotransmission and include activation of the pro-regenerative bZIP TF CEBP-1 (C/EBP) and dysregulation of UNC-6/Netrin signaling. These findings redefine terminal selectors as both intrinsic and extrinsic regulators of neuronal identity and circuit assembly, offering a mechanistic framework for understanding EBF3 syndrome pathogenesis.

developmental biology↗

bioRxiv2025

NKTR-255, a polymer-conjugated IL-15, synergizes with CAR-T cell therapy to activate endogenous anti-tumor immunity and improve tumor control

CAR-T cells have yet to show widespread efficacy in solid tumors due in part to their poor persistence and loss of function in the tumor microenvironment. Further, heterogenous expression of most CAR target antigens in solid tumors can lead to escape of antigen-null tumors that resist CAR-T killing. Strategies to cooperatively boost both CAR-T and endogenous anti-tumor immunity could curb tumor escape and may be critical for achieving durable efficacy in cancer patients. NKTR-255 is a polymer-conjugated IL-15 with extended half-life that can boost endogenous T and NK cells, as well as CD19 CAR-T activity in B cell malignancies. However, whether NKTR-255 is sufficient to overcome CAR-T dysfunction in the suppressive solid tumor microenvironment, and how NKTR-255 and CAR-Ts together re-shape endogenous anti-tumor immunity, is not known. Using an autochthonous mouse model of ROR1+ lung adenocarcinoma, we show that NKTR-255 significantly boosted accumulation, reduced exhaustion, and improved function of tumor-infiltrating CAR-T cells. Compared with NKTR-255 or CAR-T treatment alone, combination of NKTR-255 and CAR-T therapy synergistically increased tumor-infiltrating CD11b+ cytotoxic NK cells, activated dendritic cells, and endogenous tumor-specific T cells that preserved a PD-1+Tcf1+ stem-like phenotype. Consequently, NKTR-255 and CAR-T combination therapy induced complete elimination of ROR1+ tumor and significantly improved survival, with enhanced tumor control dependent on activity of both CAR-Ts and endogenous T cells. Altogether, our data suggest that combining NKTR-255 with CAR-T therapy is a promising strategy to enhance both CAR-T and endogenous anti-tumor immunity to promote coordinated control of aggressive tumors.

immunology↗

bioRxiv2025

Design and fabrication of demountable 3D microphysiological systems for modeling barrier function and underlying tissue interactions

Several recent advances in microphysiological systems (MPSs) or organ-on-chip technology have demonstrated its potential for replacing traditional in vitro and animal models in the coming years. Despite the physiological relevance and cost-effectiveness of organ chips, there are several hurdles that must be overcome for widespread adoption for biological studies. Many shortcomings of manufacturing and scalability have been overcome by a transition from PDMS to thermoplastics. However, challenges have arisen in these sealed, brittle systems related to end-point tissue analyses, harvest, and high-resolution imaging, which is particularly difficult for multi-layer organ chips. Here, we present low-cost organ chips that are fluidically sealed but demountable, fabricated using a cut-and-assemble method without the need for cleanroom technologies. We have validated the capabilities of this method by demonstrating the culture of human aortic smooth muscle cells and induced pluripotent stem cell-derived neural cells, encapsulated in gelatin methacryloyl (GelMA) hydrogel on chip, for up to 27 days. The 3D culture layer of the organ chip was removed, and high-resolution images were obtained via immunostaining. Furthermore, these organ chips facilitate rapid redesign and manufacture for alternative tissue and/or interface systems. To our knowledge, this is the first innervated organ chip with multiple removable cell culture layers, as well as the first humanized nerve-artery model that includes a three-dimensional hydrogel culture. In future work, these unique features of our platform can be utilized for investigating the crosstalk mechanisms between different cell types in co-culture. Impact StatementWe present here a new method for fabricating low-cost demountable organ-on-a-chip platforms. This method leverages our recent cut & assemble method for layered 3D organ chips comprised of gas impermeable thermoplastics.

bioengineering↗

bioRxiv2025

Complex HPV-human DNA structures revealed by large-scale DNA analyses in an HPV-cancer derived cell line

Most human papillomavirus (HPV)-associated cancers harbor viral DNA integrated into the human genome as extrachromosomal circles, intrachromosomal segments, or both. Distinguishing intrachromosomal from identical-sequence extrachromosomal DNA (ecDNA) by sequencing alone is challenging, and the architecture of large-scale HPV-human DNA structures remains incompletely understood. To address this, we applied complementary genomic tools, spanning single-nucleotide to megabase resolution, to the HPV16-positive oropharyngeal cancer-cell line UM-SCC-47. These revealed that an initial integration event formed a 23 kb extrachromosomal heterocatemer circle comprising 7.5 kb of HPV16 DNA and 16 kb of the human TP63 gene. Subsequent genomic rearrangements generated heterocatemer tandem arrays extending to 0.6 megabases, plus additional large-scale rearrangements involving the HPV-TP63 structures, as revealed by long-read DNA sequencing and optical genome mapping. Fluorescent in situ Hybridization (FISH) showed that the heterocatemers were intrachromosomally localized at chromosome 3 at the TP63 locus in 100% of the cells. Long-read RNA sequencing further showed that these intrachromosomal templates produced spliced, polyadenylated transcripts. A subset of cells also harbored HPV16 ecDNA derived from the intrachromosomal HPV-TP63 DNAs. These findings define previously unrecognized higher-order architecture of integrated HPV DNA and highlight the power of FISH for distinguishing intrachromosomal from extrachromosomal DNA structures. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/680684v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@f3d584org.highwire.dtl.DTLVardef@470811org.highwire.dtl.DTLVardef@162c88corg.highwire.dtl.DTLVardef@5c80dd_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

CellSpliceNet: Interpretable Multimodal Modeling ofAlternative Splicing Across Neurons in C. elegans

Alternative splicing profoundly diversifies the transcriptome and proteome, but decoding its regulatory mechanisms remains a challenge. We introduce CellSpliceNet, an interpretable transformer-based multimodal deep learning framework designed to predict splicing outcomes across the neurons of C. elegans. By integrating four complementary data modalities, namely long-range genomic sequence, local regions of interest (ROIs) in the RNA sequence, secondary structure, and gene expression, CellSpliceNet captures the complex interplay of factors that influence splicing decisions within the cellular context. CellSpliceNet employs modality-specific transformer embeddings, incorporating structural representations guided by mutual information and scattering graph embeddings. To this end, a novel and carefully designed multimodal multi-head attention mechanism preserves the integrity of each modality while facilitating selective cross-modal interactions, notably allowing gene expression data to inform sequence and structural predictions. Attention-based pooling within each modality highlights biologically critical elements, such as canonical intron-exon splice boundaries and accessible single-stranded RNA loop structures within the exon. Quantitative comparisons with current state-of-the-art methods demonstrated CellSpliceNet s superior predictive accuracy (Spearman{rho} = 0.88) and high accuracy across diverse neuron subtypes. Furthermore, CellSpliceNet elucidates a hierarchical, neuron-specific splicing code by preferentially weighting upstream enhancer motifs (e.g., GGAAGAAC) and identifying neuron-class-specific splicing-factor signatures, including smu-1, unc-75, and hrp-1. Thus, CellSpliceNet not only advances the frontiers of alternative splicing predictive capabilities but also provides mechanistic insights into the multimodal regulation of alternative splicing.

genetics↗

bioRxiv2025

Modeling Rotational Fluoroquinolone Therapy as a Novel Treatment for Ophthalmic MRSA Infections

Methicillin-resistant Staphylococcus aureus (MRSA) increasingly undermines the effectiveness of topical fluoroquinolone monotherapy in ophthalmic infections. We present a theoretical evaluation of rotational fluoroquinolone therapy, in which moxifloxacin and trovafloxacin are alternated to enhance bacterial suppression and mitigate resistance. Experimental time-kill data were fit using Bayesian parameter estimation via Markov Chain Monte Carlo (MCMC) to derive drug-specific growth and kill parameters, which were integrated into spatiotemporal pharmacodynamic models. These models incorporate radial anterior chamber geometry, intraocular diffusion, and aqueous humor pharmacokinetics governed by a circadian clearance function. Structural docking simulations of topoisomerase IV mutants reveal that common resistance mutations disrupt moxifloxacin binding while preserving trovafloxacin affinity, supporting a collateral-sensitivity mechanism. Under high-resistance conditions, rotational therapy improves bacterial clearance compared to monotherapy by increasing heterogeneity in antimicrobial exposure fronts and reducing overall bacterial burden. By alternating fluoroquinolones with complementary resistance profiles, rotational therapy leverages physiological clearance rhythms and spatial drug gradients to enhance anterior-segment efficacy, providing a quantitative foundation for its use as a rational alternative to monotherapy in ocular MRSA management.

microbiology↗

bioRxiv2025

Novel in situ seeding immunodetection assay uncovers neuronal-driven alpha-synuclein seeding in Parkinson's disease

Aggregates of alpha-synuclein (-syn) and tau propagate through template-induced misfolding in the brains of Parkinsons (PD) and Alzheimers disease (AD) patients. Prion-like seeding is crucial in disease initiation and progression and represents a major target for drug-modifying therapies. The detection of aggregated -syn and tau seeding activity with seeding amplification assays (SAAs) have remarkable diagnostic and research potential. However, current SAAs rely on bulk tissue homogenates or fluids, losing critical spatial and cellular resolution. Here, we report our novel in situ seeding immunodetection (isSID) assay that enables the visualization of -syn and tau seeding activities with unprecedented morphological detail in intact biological samples. Using the isSID assay, we confirm seeding activity in the pathological aggregates of PD and AD, among others, while uncovering neuron-driven -syn seeding events that precede the onset of clinical symptoms in PD. Our findings provide new fundamental insights into the pathogenesis underlying neurodegeneration and establish an invaluable tool for studying protein aggregation dynamics, with potential applications in biomarker discovery, diagnostics and drug testing.

neuroscience↗

bioRxiv2025

Machine learning-predicted chromatin organization landscape across pediatric tumors

Structural variants (SVs) are increasingly recognized as important contributors to oncogenesis through their effects on 3D genome folding. Recent advances in whole-genome sequencing have enabled large-scale profiling of SVs across diverse tumors, yet experimental characterization of their individual impact on genome folding remains infeasible. Here, we leveraged a convolutional neural network, Akita, to predict disruptions in genome folding caused by somatic SVs identified in 61 tumor types from the Childrens Brain Tumor Network dataset. Our analysis reveals significant variability in SV-induced disruptions across tumor types, with the most disruptive SVs coming from lymphomas and sarcomas, metastatic tumors, and germline cell tumors. Dimensionality reduction of disruption scores identified five recurrently disrupted regions enriched for high-impact SVs across multiple tumors. Some of these regions are highly disrupted despite not being highly mutated, and harbor tumor-associated genes and transcriptional regulators. To further interpret the functional relevance of high-scoring SVs, we integrated epigenetic data and developed a modified Activity-by-Contact scoring approach to prioritize SVs with disrupted genome contacts at active enhancers. This method highlighted highly disruptive SVs near key oncogenes, as well as novel candidate loci potentially implicated in tumorigenesis. These findings highlight the utility of machine learning for identifying novel SVs, loci, and genetic mechanisms contributing to pediatric cancers. This framework provides a foundation for future studies linking SV-driven regulatory changes to cancer pathogenesis.

genomics↗