Biology subjects

Doty, R.

Publications and source records attributed to Doty, R..

7 recordsLinked to original sources

bioRxiv2026

Cell-type Specific Alteration of Dicer1 Accelerates Tumor Progression in Mouse Models of KRAS-driven Lung Adenocarcinoma

MicroRNAs (miRNAs) have been widely implicated in cancer initiation and progression, yet examination of the effects of global miRNA disruption on these processes has been limited. We developed novel genetically engineered mouse models of Kras-driven pulmonary adenocarcinoma (LUAD) with cell-type-specific disruption of miRNA biosynthesis via Dicer1 allele deletion, which exhibit significant differences in tumor progression rates and expected survival. Dicer1 is an RNase III enzyme that is required for the biogenesis of mature, functional miRNAs. Lung tumor progression was accelerated, and expected survival was decreased only when we initiated tumors and deleted one allele of Dicer1 in club cells and mutated Dicer1 in alveolar type 2 (AT2) cells. Reversing the cell types by inducing tumorigenesis, deleting one Dicer1 allele in AT2 cells, and mutating Dicer1 in club cells modestly accelerated tumor progression and had no effect on expected survival. Collectively, our results demonstrate that Dicer1 disruption accelerates lung cancer progression in a cell-type-dependent and non-cell-autonomous manner, and our mice represent tools for investigating the roles of miRNAs and miRNA-mediated intercellular communication in tumor progression. SummaryKras-driven mouse models show that Dicer1 mutations accelerate lung adenocarcinoma (LUAD) progression in a cell-type-dependent manner and suggest that the influence of miRNA-mediated intercellular communication is unidirectional and non-cell-autonomous.

cancer biology↗

bioRxiv2026

Modeling gene regulatory perturbations via deep learning from high-throughput reporter assays

Assessing likely variant effects on phenotypes is of critical importance in diagnostic settings, and while much progress has been made in interpreting genic mutations based on our understanding of coding sequence, noncoding variants can be much more challenging to reliably interpret based on DNA sequence alone. High-throughput reporter assays such as STARR-seq and MPRA have shown utility in experimentally measuring regulatory effects of noncoding variants present in samples but provide no readout for variants not present in the assay inputs. However, whole-genome reporter assays provide copious data that can be used to train predictive models for prioritizing variants not directly observed in the experiment. We describe a retrainable predictive modeling framework, BlueSTARR, for this task, and present results of training several models with this framework on whole-genome STARR-seq data from two cell lines and one drug treatment. Using these models, we uncover a global signature across the human genome consistent with purifying selection against both loss-of-function and gain-of-function regulatory variants, with the latter showing a significant bias consistent with selection against gains of cis regulatory function in closed chromatin proximal to genes. By testing the model on synthetic enhancers with binding motifs for transcription factors GR and AP-1, we find that when trained on drug perturbation data, the model is able to learn distance-dependent and treatment-dependent binding patterns and their resulting reporter gene activation. These results demonstrate that lightweight, easily retrainable models such as ours have utility in probing latent signals present in novel experimental data. Finally, we find only modest differences in performance between different deep-learning architectures when trained on this single data modality, and while somewhat greater predictive accuracy can be achieved with much larger models trained at great expense on many terabytes of data, there is still copious room for improvement even for industrial strength, state-of-the-art models.

bioinformatics↗

bioRxiv2022

RNA Immunoprecipitation and Sequencing of Isolated RNAs (RIP-SIR) Identifies Endogenous miRNA-Target Interactions

MicroRNAs (miRNAs) are crucial post-transcriptional regulators of gene expression in species ranging from plants to mammals. Unraveling the complex networks of miRNA-mediated regulation of gene expression requires the ability to identify the mRNA targets of individual miRNAs in native cellular contexts. Here we describe RIP-SIR, an unbiased genome-wide method for identifying interactions between endogenous miRNAs and their targets in almost any tissue or cell type.

molecular biology↗

bioRxiv2021

Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology

NADK2 encodes the mitochondrial isoform of NAD Kinase, which phosphorylates nicotinamide adenine dinucleotide (NAD). Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here we describe two chemically-induced mouse mutations in Nadk2, S326L and S330P, which cause a severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5 weeks of age and muscle atrophy by 11 weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on brain, muscle, liver, and spinal cord at the same ages. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency, and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNAseq and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, such as translation, and those processes that are gene-specific such as glycolysis and acetylcholine binding. These findings improve our understanding of the pathophysiology of both Nadk2 and Pla2g6 mutations, as well as pathways common to neuromuscular/neurodegenerative diseases.

genetics↗

bioRxiv2020

Long title: The occurrence of tarsal injuries in male mice of C57BL/6N substrains in multiple international mouse facilities

Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneous-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed.

genetics↗

bioRxiv2019

REAVER: Improved Analysis of High-resolution Vascular Network Images Revealed Through Round-robin Rankings of Accuracy and Precision

Alterations in vascular networks, including angiogenesis and capillary regression, play key roles in disease, wound healing, and development. Imaging of microvascular networks can reveal their spatial structures, but effective study of network architecture requires methods to accurately quantify them using a variety of metrics. We present REAVER (Rapid Editable Analysis of Vessel Elements Routine), a freely available open source tool that researchers can use to analyze and quantify high resolution fluorescent images of blood vessel networks, and assess its performance compared to alternative state-of-the-art image analysis software programs. Top performing programs for each metric are identified by assigning a rank based on statistical multiple comparisons of accuracy and precision, modeled as matches in a round-robin style tournament. This comparison method yields a clearly defined and consistent standard for characterizing program performance, avoiding the use of non-standard ad hoc interpretations of multiple comparisons between programs. Using this comparison method and a dataset of manually analyzed images as a ground-truth, we show that REAVER was the top ranked program for both accuracy and precision for all metrics quantified, including vessel length density, vessel area fraction, mean vessel diameter, and branchpoint count. REAVER can be used to quantify differences in blood vessel architectures between study groups, which makes it particularly useful in experiments designed to evaluate the effects of different external perturbations (e.g. drugs or disease states).

bioinformatics↗

bioRxiv2019

Pericyte Bridges in Homeostasis and Hyperglycemia: Reconsidering Pericyte Dropout and Microvascular Structures

Diabetic retinopathy threatens the vision of a third of diabetic patients. Progression of the disease is attributed to the dropout of pericytes, a cell type that enwraps and stabilizes the microvasculature. In tandem with this presumptive pericyte dropout, there is enriched formation of structures assumed to be remnants of collapsed or regressed vessels, previously classified as acellular capillaries, string vessels, and basement membrane bridges. Instead of endothelial cells, we show that pericytes colocalize with basement membrane bridges, and both bridging structures are enriched by cell-specific knockout of KLF4 and reversibly enriched with elevation of Ang-2, PDGF-BB, and blood sugar. Our data suggests that pericyte counts from retinal digests have misclassified pericyte bridges as endothelial structures and have exaggerated the role of pericyte loss in DR progression. In vivo imaging of corneal limbal vessels demonstrates pericyte migration off-vessel, implicating pericyte movement in formation of pericyte bridges and pathogenesis of diabetic retinopathy.

cell biology↗