Biology subjects

Inman, J.

Publications and source records attributed to Inman, J..

8 recordsLinked to original sources

bioRxiv2026

Comprehensive interaction profiling and machine learning prediction of bacteriophage infectivity across clinically diverse Pseudomonas aeruginosa

The rise of antibiotic-resistant bacterial infections has driven renewed interest in bacteriophage therapy, where viruses that specifically kill bacteria are used as targeted antimicrobials. Pseudomonas aeruginosa, a WHO critical-priority pathogen that causes severe infections in hospitalized and immunocompromised patients, presents a major challenge for phage therapy because of its extraordinary genetic diversity. Phages effective against one bacterial strain often fail against others, and existing cross-resistance-profiling approaches require iterative empirical testing of each new patient isolate. To establish a genome-based framework for rapid phage-isolate matching, we assembled a collection of 95 genomically diverse P. aeruginosa phages representing 20 genera and tested each against 99 genetically diverse clinical isolates, generating 9,405 infection outcome measurements. Bacterial O-antigen serotype emerged as the dominant determinant of strain susceptibility, while defense systems, anti-defense systems, and prophage burden contributed smaller strain-specific effects. The full curated multivariate model explained 47% of strain-susceptibility variance. Machine-learning models integrating these features and pangenome-derived gene clusters reached a per-strain AUROC of 0.86. In an in vivo proof-of-concept test against a single held-out strain, the ML-designed cocktail produced a [~]12-fold greater median CFU reduction than the expert-designed cocktail (q = 0.045), with both cocktails substantially reducing burden relative to the untreated control ([~]113-fold for ML, [~]9-fold for CG; both q < 10{square}3). SHAP analysis of the model identified bacterial surface-architecture genes (LPS biosynthesis, outer membrane proteins, type IV pili) as the dominant predictors, with defense-system content modulating which specific phages succeed against a strain rather than uniformly damping susceptibility. Together, these results establish a genome-based framework for predicting phage susceptibility in genetically diverse clinical isolates.

microbiology↗

bioRxiv2025

Modeling Friedreich's ataxia with Bergmann glia-enriched human cerebellar organoids

The human cerebellum is a specialized brain region that is involved in various neurological and psychiatric diseases but has been challenging to study in vitro due its complex neurodevelopment and cellular diversity. Despite the progress in generating neural tissues from human induced pluripotent stem cells (iPSCs), an organoid model that recapitulates the key features of cerebellar development has not been widely established. Here, we report the generation of a 60-day method for human cerebellar organoids (hCBOs) that is characterized by induction of rhombomere 1 (R1) cellular identity followed by derivation of neuronal and glial cell types of the cerebellum. In contrast to forebrain organoids with multiple neural rosettes and inside-out neuronal migration, hCBOs develop a SOX2+ cerebellar plate on the outermost surface of organoids with outside-in neuronal migration, which is a characteristic hallmark of cerebellar histogenesis. These hCBOs produced various other cell types including granule neurons, Purkinje cells, Golgi neurons, and deep cerebellar nuclei. By using a glial induction strategy, we generate Bergmann glial cells (BGCs) within the hCBOs that not only serve as scaffolds for granule cells migration but also enhance electrophysiological response of the hCBOs. Furthermore, by generating hCBOs from patients with Friedreichs ataxia (FRDA), we revealed abnormal disease-specific phenotypes that could be reversed by histone deacetylase (HDAC) inhibitors and gene editing by CRISPR-Cas9. Taken together, our advanced hCBO model provides new opportunities to investigate the molecular and cellular mechanisms of cerebellar ontogenesis and utilize patient-derived iPSCs for translational research.

developmental biology↗

bioRxiv2024

Scalable Hypothalamic Arcuate Neuron Differentiation from Human Pluripotent Stem Cells Suitable for Modeling Metabolic and Reproductive Disorders

The hypothalamus, composed of several nuclei, is essential for maintaining our bodys homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.

developmental biology↗

bioRxiv2024

Optical Torque Calculations and Measurements for DNA Torsional Studies

The angular optical trap (AOT) is a powerful instrument for measuring the torsional and rotational properties of a biological molecule. Thus far, AOT studies of DNA torsional mechanics have been carried out using a high numerical aperture oil-immersion objective, which permits strong trapping, but inevitably introduces spherical aberrations due to the glass-aqueous interface. However, the impact of these aberrations on torque measurements is not fully understood experimentally, partly due to a lack of theoretical guidance. Here, we present a numerical platform based on the finite element method to calculate forces and torques on a trapped quartz cylinder. We have also developed a new experimental method to accurately determine the shift in the trapping position due to the spherical aberrations by using a DNA molecule as a distance ruler. We found that the calculated and measured focal shift ratios are in good agreement. We further determined how the angular trap stiffness depends on the trap height and the cylinder displacement from the trap center and found full agreement between predictions and measurements. As further verification of the methodology, we showed that DNA torsional properties, which are intrinsic to DNA, could be determined robustly under different trap heights and cylinder displacements. Thus, this work has laid both a theoretical and experimental framework that can be readily extended to investigate the trapping forces and torques exerted on particles with arbitrary shapes and optical properties. SIGNIFICANCEWe developed a simulation platform based on the finite element method for force and torque calculation for particles in an angular optical trap (AOT), with considerations of tightly focused Gaussian beam, spherical aberrations, and optically anisotropic particles. Experimental measurements of focal shift ratio, force, and torque under multiple conditions were in good agreement with predictions from the simulations. We also demonstrated that intrinsic DNA torsional properties can be robustly measured under different AOT measurement conditions, strongly validating our simulations and calibrations. Our platform can facilitate trapping particle design for single-molecule assays using the AOT.

biophysics↗

bioRxiv2023

Chromatinization Modulates Topoisomerase II Processivity

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress in vivo. While cellular processes constantly create different degrees of torsional stress, how this stress feeds back to control type IIA topoisomerase function remains obscure. Using a suite of single-molecule approaches, we examined the torsional impact on supercoiling relaxation of both naked DNA and chromatin by eukaryotic topoisomerase II (topo II). We observed that topo II was at least [~] 50-fold more processive on plectonemic DNA than previously estimated, capable of relaxing > 6000 turns. We further discovered that topo II could relax supercoiled DNA prior to plectoneme formation, but with a [~]100-fold reduction in processivity; strikingly, the relaxation rate in this regime decreased with diminishing torsion in a manner consistent with the capture of transient DNA loops by topo II. Chromatinization preserved the high processivity of the enzyme under high torsional stress. Interestingly, topo II was still highly processive ([~] 1000 turns) even under low torsional stress, consistent with the predisposition of chromatin to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function, capable of enhancing function even under low torsional stress.

biophysics↗

bioRxiv2018

High-Performance Image-Based Measurements of Biological Forces and Interactions in a Dual Optical Trap

Optical traps enable nanoscale manipulation of individual biomolecules while measuring molecular forces and lengths. This ability relies on the sensitive detection of optically trapped particles, typically accomplished using laser-based interferometric methods. Recently, precise and fast image-based particle tracking techniques have garnered increased interest as a potential alternative to laser-based detection, however successful integration of image-based methods into optical trapping instruments for biophysical applications and force measurements has remained elusive. Here we develop a camera-based detection platform that enables exceptionally accurate and precise measurements of biological forces and interactions in a dual optical trap. In demonstration, we stretch and unzip DNA molecules while measuring the relative distances of trapped particles from their trapping centers with sub-nanometer accuracy and precision, a performance level previously only achieved using photodiodes. We then use the DNA unzipping technique to localize bound proteins with extraordinary sub-base-pair precision, revealing how thermal DNA fluctuations allow an unzipping fork to sense and respond to a bound protein prior to a direct encounter. This work significantly advances the capabilities of image tracking in optical traps, providing a state-of-the-art detection method that is accessible, highly flexible, and broadly compatible with diverse experimental substrates and other nanometric techniques.

biophysics↗

bioRxiv2018

Supragingival plaque microbiome ecology and functional potential in the context of health and disease

To address the question of how microbial diversity and function in the oral cavities of children relates to caries diagnosis, we surveyed the supragingival plaque biofilm microbiome in 44 juvenile twin pairs. Using shotgun sequencing, we constructed a genome encyclopedia describing the core supragingival plaque microbiome. Caries phenotypes contained statistically significant enrichments in specific genome abundances and distinct community composition profiles including strain-level changes. Metabolic pathways that are statistically associated with caries include several sugar-associated phosphotransferase systems, antimicrobial resistance, and metal transport. Numerous closely-related previously-uncharacterized microbes had substantial variation in central metabolism, including the loss of biosynthetic pathways resulting in auxotrophy, changing the ecological role. We also describe the first complete Gracilibacteria genomes from the human microbiome. Caries is a microbial community metabolic disorder that cannot be described by a single etiology and our results provide the information needed for next generation diagnostic tools and therapeutics for caries.

microbiology↗

bioRxiv2018

Helicase Promotes Replication Re-initiation from an RNA Transcript

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind the DNA, resulting in a gap in the newly replicated DNA. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances the fork, the DNAP also moves forward processively, via its association with the helicase. The presence of the DNAP, in turn, increases both helicases processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicases novel role in replication re-initiation, independent of replication restart proteins or primase.

biophysics↗