Biology subjects

Detweiler, A. M.

Publications and source records attributed to Detweiler, A. M..

11 recordsLinked to original sources

bioRxiv2026

Single-cell splice isoform usage reveals distinct axes of cellular identity and senescence

Alternative splicing greatly expands the diversity of gene products encoded by the human genome. Single-cell transcriptomic atlases have characterized human cell types through gene-level expression, but short-read sequencing has limited the ability to resolve full-length isoforms and their functional consequences. Here, we present a cross-tissue single-cell long-read isoform atlas spanning 26 human tissues. We identify hundreds of thousands of novel isoforms along with their cell-type-specific usage, and discover that over one-third of expressed isoforms are absent from existing reference databases. We further demonstrate that isoform usage is a structured, measurable axis of cellular identity that is distinct from gene expression. Applying this framework to cellular senescence, we resolve p16INK4a and p14ARF transcripts from the CDKN2A locus in individual cells and uncover cell-type-dependent isoform remodeling associated with the p16INK4a senescence program. This isoform-resolved single-cell atlas offers a versatile framework to dissect the cellular logic of isoform regulation in senescence and beyond.

genomics↗

bioRxiv2026

Lifelong Invertebrate Chordate Central Nervous System Regeneration is Driven by a Migrating Pool of Conserved Neural Stem Cells Program

Adult central nervous systems (CNS) are generally constrained by limited cellular replacement. How regenerating organisms integrate structural maintenance with continuous, cyclic renewal remains unresolved. Here, we leverage the colonial chordate Botryllus schlosseri which completely resorbs and regenerates its entire CNS de novo weekly. Combining indexed single-cell sorting, Smart-seq3 sequencing, spatial mapping, and temporal deconvolution, our studies suggest that this continuous CNS renewal is mediated by an intrinsic pool of lineage-restricted neural stem cells. Single-cell analysis resolved a conserved SOX2/NOTCH1/NEUROG1 prospective progenitor continuum alongside mature excitability-defined neurons. Brain aging drives a phase-specific desynchronization of this regenerative program, inducing early hyper-activation followed by a sudden collapse. Finally, syngeneic transplantation and in vivo tracking show that neural-complex-derived cells persist after vascular delivery and localize to developing bud and neural-associated territories, with injury-associated accumulation near the neural gland. This work establishes a spatiotemporal framework for lifelong chordate CNS renewal and aging.

cell biology↗

bioRxiv2025

Germline stem cell isolation, lineage tracing, and aging in a protochordate

Germline stem cells (GSCs), the source of gametes, are the only stem cells capable of passing genes to future generations and are therefore considered units of natural selection. Yet, the factors that influence GSC fitness, and thus govern GSC competition, which exist in both protochordates and mammals, remain poorly understood. We studied how aging affects GSC fitness in the protochordate Botryllus schlosseri, an evolutionary crosspoint between invertebrates and vertebrates. GSCs were isolated and distinguished from developing and mature gametes using flow cytometry and scRNA-Seq, facilitated by a new PacBio genome assembly. Moreover, their function was validated through a novel lineage tracing approach that combines membrane-labeled GSC transplantation with scRNA-Seq. Leveraging our method to isolate them, single-cell transcriptomics showed significant age-related changes between young and old GSCs. Spermatids and sperm, however, showed minimal changes, suggesting that reproductive aging is governed by GSCs rather than by gametes. Reduced expressions of markers like DDX4 and PIWIL1 in aged GSCs mirrored trends in mammalian datasets, pointing to a conserved GSC-driven aging mechanism across chordate evolution. This study provides new techniques that lay the foundation to investigate further drivers of GSC fitness and highlights fertility-related genes as promising targets for therapies to preserve reproductive health.

cell biology↗

bioRxiv2025

Electrical Stimulation Rejuvenates Tunicates: Altered Stem Cell and Immune Activity

Applicable methods of rejuvenating organisms and improving resistance to environmental stimuli are needed. During attempts to synchronize heart rates in unhealthy colonial chordates, we observed morphological rejuvenation. While the importance of endogenously generated bioelectric currents in development is well-established1,2, and exogenously applied current has shown promise in regenerative medicine3,4,5,6,7,8,9, a model that robustly increases longevity and fertility while providing detailed mechanistic insights has not been reported. Here, we report the establishment of such a model using pulsatile electrical current (PEC) in Botryllus schlosseri, an established colonial chordate model10,11,12,13,14,15. PEC treatment significantly improved survival, morphological integrity, stem cell mediated regeneration, and gonad production in Botryllus. Transcriptomic analysis revealed pathway changes associated with cellular metabolism, cell cycle, stem cell activity, DNA repair, and immune modulation. Notably, PEC-induced expression patterns resemble the exercise-induced macrophage-associated transcriptional response previously observed across several mammalian species16,17. This transcriptomic signature correlated with an increase in immune-cell-containing populations. These findings demonstrate that PEC can improve longevity, vitality, and reproduction in an established model renowned for defining broadly applicable biological principles. These studies offer insights into novel strategies for promoting healthy aging and organismal survival.

developmental biology↗

bioRxiv2025

Comparative analysis of human and mouse ovaries across age

Mouse is a tractable model for human ovarian biology, however its utility is limited by incomplete understanding of how transcription and signaling differ interspecifically and with age. We compared ovaries between species using 3D-imaging, single-cell transcriptomics, and functional studies. In mice, we mapped declining follicle numbers and oocyte competence during aging; in human ovaries, we identified cortical follicle pockets and density changes. Oocytes had species-specific gene expression patterns during growth that converged toward maturity. Age-related transcriptional changes were greater in oocytes than granulosa cells across species, although mature oocytes change more in humans. We identified ovarian sympathetic nerves and glia; nerve density increased in aged human ovaries and, when ablated in mice, perturbed folliculogenesis. This comparative atlas defines shared and species-specific hallmarks of ovarian biology.

genomics↗

bioRxiv2025

Nitroxoline treatment of Balamuthia mandrillaris triggers genomic crisis, transcriptional collapse, and incomplete encystment via copper and iron chelation

Balamuthia mandrillaris is a free-living amoeba that can infect the central nervous system to cause granulomatous amebic encephalitis (GAE), a highly fatal disease with mortality rates up to 90%. GAE is typically treated with a six-drug regimen with limited efficacy and significant toxicity. Our lab previously identified nitroxoline, a quinolone antibiotic, as a promising therapeutic complement to the existing regimen, and in 2021 nitroxoline was first successfully used to treat a human patient. In this study, we elucidate nitroxolines mechanism of action using a combination of genomics, transcriptomics, chemical complementation, growth assays, and electron microscopy. In service of this goal, an annotated draft genome of B. mandrillaris strain CDC:V039 was generated using long read sequencing, yielding over 32,000 predicted protein coding sequences, the majority of which were present on telomere-to-telomere predicted chromosomal contigs. Leveraging this resource, comparative transcriptomics were used to characterize encystment responses to three different cellular stressors: nitroxoline, galactose, and hypoxia. Our findings reveal that nitroxoline-induced cysts undergo transcriptional collapse and genomic and structural destabilization via copper and iron chelation, and that preformed cysts are also disrupted by nitroxoline. These data provide insight into nitroxolines multifaceted impact on B. mandrillaris growth and cellular processes and advance our understanding of the transcriptional landscape associated with stress induced encystment. Our findings support the continued clinical study of nitroxoline as a complement to the existing regimen. Author SummaryBalamuthia mandrillaris, one of three known brain-eating amoebas, causes a rare but usually fatal disease called granulomatous amoebic encephalitis. Until 2025, treatment options were limited, with most drugs proving ineffective against lab grown Balamuthia and toxic to humans. We previously discovered that the antibacterial drug nitroxoline prevented Balamuthia from destroying tissue by triggering encystment, whereby amoeba transition into dormant cysts. Nitroxoline was later used to successfully treat a patient, but it was unclear how the drug worked and whether the induced cysts could reawaken and cause the infection to return. To understand this process, we studied how Balamuthia responds to nitroxoline and compared it to two other stressors: low oxygen and excess sugar. Using genomic, chemical, and imaging tools, we found that Balamuthia activates different sets of genes depending on the type of stress, arguing against a single, universal encystment program. We also found that nitroxoline prevented formation of viable cysts and killed existing ones by depriving them of essential copper and metal ions. These findings improve our understanding of how Balamuthia becomes a cyst, offers clues for therapeutic intervention, and supports the continued use of nitroxoline to treat this deadly infection in combination with the existing drug regimen.

microbiology↗

bioRxiv2023

Metagenomics for pathogen detection during a wildlife mortality event in songbirds

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria comprising the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds, and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events. Article Summary LineThe causative agent of a mass mortality event in passerines remains inconclusive after metagenomic high-throughput sequencing with results prompting further investigation into non-pathogenic causes.

genomics↗

bioRxiv2021

DiMeLo-seq: a long-read, single-molecule method for mappingprotein-DNA interactions genome-wide

Molecular studies of genome regulation often rely on the ability to map where specific proteins interact with genomic DNA. Existing techniques for mapping protein-DNA interactions genome-wide rely on DNA amplification methods followed by sequencing with short reads, which dissociates joint binding information at neighboring sites, removes endogenous DNA methylation information, and precludes the ability to reliably map interactions in repetitive regions of the genome. To address these limitations, we created a new protein-DNA mapping method, called Directed Methylation with Long-read sequencing (DiMeLo-seq), which methylates DNA near each target proteins DNA binding site in situ, then leverages the ability to distinguish methylated and unmethylated bases on long, native DNA molecules using long-read, single-molecule sequencing technologies. We demonstrate the optimization and utility of this method by mapping the interaction sites of a variety of different proteins and histone modifications across the human genome, achieving a single-molecule binding site resolution of less than 200 bp. Furthermore, we mapped the positions of the centromeric histone H3 variant CENP-A in repetitive regions that are unmappable with short reads, while simultaneously analyzing endogenous CpG methylation and joint binding events on single molecules. DiMeLo-seq is a versatile method that can provide multimodal and truly genome-wide information for investigating protein-DNA interactions.

genomics↗

bioRxiv2020

Identification of a polymorphism in the N gene of SARS-CoV-2 that adversely impacts detection by a widely-used RT-PCR assay

We identify a mutation in the N gene of SARS-CoV-2 that adversely affects annealing of a commonly used RT-PCR primer; epidemiologic evidence suggests the virus retains pathogenicity and competence for spread. This reinforces the importance of using multiple targets, preferably in at least 2 genes, for robust SARS-CoV-2 detection. Article Summary LineA SARS-CoV-2 variant that occurs worldwide and has spread in California significantly affects diagnostic sensitivity of an N gene assay, highlighting the need to employ multiple viral targets for detection.

molecular biology↗

bioRxiv2019

Negative plant-microbiome feedback limits productivity in aquaponics

The demand for food will outpace productivity of conventional agriculture due to projected growth of the human population, concomitant with shrinkage of arable land, increasing scarcity of freshwater, and a rapidly changing climate. Efforts to increase conventional agricultural output come with significant environmental impacts stemming from deforestation and excessive use of chemicals, including soil salinization, erosion, and nutrient runoffs. While aquaponics has potential to sustainably supplement food production with minimal environmental impact, there is a need to better characterize the complex interplay between the various components (fish, plant, microbiome) of these systems to optimize scale up and productivity. For instance, much of our knowledge of beneficial and detrimental microbial communities vis-a-vis crop productivity comes from studies on plant-microbiome interactions in soil. Here, we investigated how the practice of continued transfer of microbial communities from pre-existing systems might promote or impede productivity of aquaponics. Specifically, we monitored plant growth phenotypes, water chemistry, and microbiome composition of rhizospheres, biofilters, and fish feces over 61-days of lettuce (Lactuca sativa) growth in aquaponic systems inoculated with bacteria that were either commercially sourced or originating from a pre-existing aquaponic system. Strikingly, L. sativa plant and root growth was significantly reduced across all replicates inoculated with the established microbiome. Further analyses revealed the reduced productivity was potentially a consequence of plant-specific pathogen enrichment, including Pseudomonas, through transfer of microbiomes from pre-existing systems - a phenomenon consistent with negative feedbacks in soil ecology. These findings underscore the need for diagnostic tools to monitor microbiome composition, detect negative feedbacks early, and minimize pathogen accumulation in aquaponic systems.

microbiology↗

bioRxiv2017

Metagenomics reveals niche partitioning within the phototrophic zone of a microbial mat

Hypersaline photosynthetic microbial mats are stratified microbial communities known for their taxonomic and metabolic diversity and strong light-driven day-night environmental gradients. In this study of the upper photosynthetic zone of hypersaline microbial mats of Elkhorn Slough, California (USA), we show how reference-based and reference-free methods can be used to meaningfully assess microbial ecology and genetic partitioning in these complex microbial systems. Mapping of metagenome reads to the dominant Cyanobacteria observed in the system, Coleofasciculus (Microcoleus) chthonoplastes, was used to examine strain variants within these metagenomes. Highly conserved gene subsystems indicate a core genome for the species, and a number of variant genes and subsystems suggest strain level differentiation, especially for carbohydrate utilization. Metagenome sequence coverage binning was used to assess ecosystem partitioning of remaining microbes. Functional gene annotation of these bins (primarily of Proteobacteria, Bacteroidetes, and Cyanobacteria) recapitulated the known biogeochemical functions in microbial mats using a genetic basis, and also revealed evidence of novel functional diversity within the Gemmatimonadetes and Gammaproteobacteria. Combined, these two approaches show how genetic partitioning can inform biogeochemical partitioning of the metabolic diversity within microbial ecosystems.

microbiology↗