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Quantifying interpretation reproducibility in Vision Transformer models with TAVAC.
Get PDFDeep learning algorithms can extract meaningful diagnostic features from biomedical images, promising improved patient care in digital pathology. Vision Transformer (ViT) models capture long-range spatial relationships and offer robust prediction power and better interpretability for image classification tasks than convolutional neural network models. However, limited annotated biomedical imaging datasets can cause ViT models to overfit, leading to false predictions due to random noise. To address this, we introduce Training Attention and Validation Attention Consistency (TAVAC), a metric for evaluating ViT model overfitting and quantifying interpretation reproducibility. By comparing high-attention regions between training and testing, we tested TAVAC on four public image classification datasets and two independent breast cancer histological image datasets. Overfitted models showed significantly lower TAVAC scores. TAVAC also distinguishes off-target from on-target attentions and measures interpretation generalization at a fine-grained cellular level. Beyond diagnostics, TAVAC enhances interpretative reproducibility in basic research, revealing critical spatial patterns and cellular structures of biomedical and other general nonbiomedical images
Instrumented swim test for quantifying motor impairment in rodents.
Get PDFSwim tests are highly effective for identifying vestibular deficits in rodents by offering significant vestibular motor challenges with reduced proprioceptive input, unlike rotarod and balance beam tests. Traditional swim tests rely on subjective assessments, limiting objective quantification and reproducibility. We present a novel instrumented swim test using a miniature motion sensor with a 3D accelerometer and 3D gyroscope affixed to the rodent’s head. This setup robustly quantifies six-dimensional motion—three translational and three rotational axes—during swimming with high temporal resolution. We demonstrate the test’s capabilities by comparing head movements of Gpr156-/- mutant mice, which have impaired otolith organ development, to their heterozygous littermates. Our results show axis-specific differences in head movement probability distribution functions and dynamics that identify mice with the Gpr156 mutation. Axis-specific power spectrum analyses reveal selective movement alterations within distinct frequency ranges. Additionally, our spherical visualization and 3D analysis quantifies swimming performance based on head vector distance from upright. We use this analysis to generate a single classifier metric—a weighted average of an animal’s head deviation from upright during swimming. This metric effectively distinguishes animals with vestibular dysfunction from those with normal vestibular function. Overall, this instrumented swim test provides quantitative metrics for assessing performance and identifying subtle, axis- and frequency-specific deficits not captured by existing systems. This novel quantitative approach can enhance understanding of rodent sensorimotor function including enabling more selective and reproducible studies of vestibular-motor deficits
Alternative splicing is coupled to gene expression in a subset of variably expressed genes.
Get PDFNumerous factors regulate alternative splicing of human genes at a co-transcriptional level. However, how alternative splicing depends on the regulation of gene expression is poorly understood. We leveraged data from the Genotype-Tissue Expression (GTEx) project to show a significant association of gene expression and splicing for 6874 (4.9%) of 141,043 exons in 1106 (13.3%) of 8314 genes with substantially variable expression in nine GTEx tissues. About half of these exons demonstrate higher inclusion with higher gene expression, and half demonstrate higher exclusion, with the observed direction of coupling being highly consistent across different tissues and in external datasets. The exons differ with respect to multiple characteristics and are enriched for hundreds of isoform-specific Gene Ontology annotations suggesting an important regulatory mechanism. Notably, splicing-expression coupling of exons with roles in JUN and MAP kinase signalling could play an important role during cell division
Interruption of glucagon signaling augments islet non-alpha cell proliferation in SLC7A2- and mTOR-dependent manners.
Get PDFOBJECTIVE: Dysregulated glucagon secretion and inadequate functional beta cell mass are hallmark features of diabetes. While glucagon receptor (GCGR) antagonism ameliorates hyperglycemia and elicits beta cell regeneration in pre-clinical models of diabetes, it also promotes alpha and delta cell hyperplasia. We sought to investigate the mechanism by which loss of glucagon action impacts pancreatic islet non-alpha cells, and the relevance of these observations in a human islet context.
METHODS: We used zebrafish, rodents, and transplanted human islets comprising six different models of interrupted glucagon signaling to examine their impact on delta and beta cell proliferation and mass. We also used models with global deficiency of the cationic amino acid transporter, SLC7A2, and mTORC1 inhibition via rapamycin, to determine whether amino acid-dependent nutrient sensing was required for islet non-alpha cell growth.
RESULTS: Inhibition of glucagon signaling stimulated delta cell proliferation in mouse and transplanted human islets, and in mouse islets. This was rapamycin-sensitive and required SLC7A2. Likewise, gcgr deficiency augmented beta cell proliferation via SLC7A2- and mTORC1-dependent mechanisms in zebrafish and promoted cell cycle engagement in rodent beta cells but was insufficient to drive a significant increase in beta cell mass in mice.
CONCLUSIONS: Our findings demonstrate that interruption of glucagon signaling augments islet non-alpha cell proliferation in zebrafish, rodents, and transplanted human islets in a manner requiring SLC7A2 and mTORC1 activation. An increase in delta cell mass may be leveraged for future beta cell regeneration therapies relying upon delta cell reprogramming
RREB1 regulates neuronal proteostasis and the microtubule network.
Get PDFTranscription factors play vital roles in neuron development; however, little is known about the role of these proteins in maintaining neuronal homeostasis. Here, we show that the transcription factor RREB1 (Ras-responsive element-binding protein 1) is essential for neuron survival in the mammalian brain. A spontaneous mouse mutation causing loss of a nervous system–enriched Rreb1 transcript is associated with progressive loss of cerebellar Purkinje cells and ataxia. Analysis of chromatin immunoprecipitation and sequencing, along with RNA sequencing data revealed dysregulation of RREB1 targets associated with the microtubule cytoskeleton. In agreement with the known role of microtubules in dendritic development, dendritic complexity was disrupted in Rreb1- deficient neurons. Analysis of sequencing data also suggested that RREB1 plays a role in the endomembrane system. Mutant Purkinje cells had fewer numbers of autophagosomes and lysosomes and contained P62- and ubiquitin-positive inclusions. Together, these studies demonstrate that RREB1 functions to maintain the microtubule network and proteostasis in mammalian neurons
AAV8 gene therapy reverses cardiac pathology and prevents early mortality in a mouse model of Friedreich\u27s ataxia.
Get PDFFriedreich\u27s ataxia (FRDA) is an autosomal-recessive disorder primarily attributed to biallelic GAA repeat expansions that reduce expression of the mitochondrial protein frataxin (FXN). FRDA is characterized by progressive neurodegeneration, with many patients developing cardiomyopathy that progresses to heart failure and death. The potential to reverse or prevent progression of the cardiac phenotype of FRDA was investigated in a mouse model of FRDA, using an adeno-associated viral vector (AAV8) containing the coding sequence of th
IDHwt glioblastomas can be stratified by their transcriptional response to standard treatment, with implications for targeted therapy.
Get PDFBACKGROUND: Glioblastoma (GBM) brain tumors lacking IDH1 mutations (IDHwt) have the worst prognosis of all brain neoplasms. Patients receive surgery and chemoradiotherapy but tumors almost always fatally recur.
RESULTS: Using RNA sequencing data from 107 pairs of pre- and post-standard treatment locally recurrent IDHwt GBM tumors, we identify two responder subtypes based on longitudinal changes in gene expression. In two thirds of patients, a specific subset of genes is upregulated from primary to recurrence (Up responders), and in one third, the same genes are downregulated (Down responders), specifically in neoplastic cells. Characterization of the responder subtypes indicates subtype-specific adaptive treatment resistance mechanisms that are associated with distinct changes in the tumor microenvironment. In Up responders, recurrent tumors are enriched in quiescent proneural GBM stem cells and differentiated neoplastic cells, with increased interaction with the surrounding normal brain and neurotransmitter signaling, whereas Down responders commonly undergo mesenchymal transition. ChIP-sequencing data from longitudinal GBM tumors suggests that the observed transcriptional reprogramming could be driven by Polycomb-based chromatin remodeling rather than DNA methylation.
CONCLUSIONS: We show that the responder subtype is cancer-cell intrinsic, recapitulated in in vitro GBM cell models, and influenced by the presence of the tumor microenvironment. Stratifying GBM tumors by responder subtype may lead to more effective treatment
Utility of long-read sequencing for All of Us.
Get PDFThe All of Us (AoU) initiative aims to sequence the genomes of over one million Americans from diverse ethnic backgrounds to improve personalized medical care. In a recent technical pilot, we compare the performance of traditional short-read sequencing with long-read sequencing in a small cohort of samples from the HapMap project and two AoU control samples representing eight datasets. Our analysis reveals substantial differences in the ability of these technologies to accurately sequence complex medically relevant genes, particularly in terms of gene coverage and pathogenic variant identification. We also consider the advantages and challenges of using low coverage sequencing to increase sample numbers in large cohort analysis. Our results show that HiFi reads produce the most accurate results for both small and large variants. Further, we present a cloud-based pipeline to optimize SNV, indel and SV calling at scale for long-reads analysis. These results lead to widespread improvements across AoU
NLGN4X TCR transgenic T cells to treat gliomas.
Get PDFBACKGROUND: Neuroligin 4 X-linked (NLGN4X) harbors a human leukocyte antigen (HLA)-A*02-restricted tumor-associated antigen, overexpressed in human gliomas, that was found to induce specific cytotoxic T cell responses following multi-peptide vaccination in patients with newly diagnosed glioblastoma.
METHODS: T cell receptor (TCR) discovery was performed using droplet-based single-cell TCR sequencing of NLGN4X-tetramer-sorted T cells postvaccination. The identified TCR was delivered to Jurkat T cells and primary human T cells (NLGN4X-TCR-T). Functional profiling of NLGN4X-TCR-T was performed by flow cytometry and cytotoxicity assays. Therapeutic efficacy of intracerebroventricular NLGN4X-TCR-T was assessed in NOD scid gamma (NSG) major histocompatibility complex (MHC) I/II knockout (KO) (NSG MHC I/II KO) mice bearing NLGN4X-expressing experimental gliomas.
RESULTS: An HLA-A*02-restricted vaccine-induced T cell receptor specifically binding NLGN4X131-139 was applied for preclinical therapeutic use. Reactivity, cytotoxicity, and polyfunctionality of this NLGN4X-specific TCR are demonstrated in various cellular models. Intracerebroventricular administration of NLGN4X-TCR-T prolongs survival and leads to an objective response rate of 44.4% in experimental glioma-bearing NSG MHC I/II KO mice compared to 0.0% in control groups.
CONCLUSION: NLGN4X-TCR-T demonstrate efficacy in a preclinical glioblastoma model. On a global scale, we provide the first evidence for the therapeutic retrieval of vaccine-induced human TCRs for the off-the-shelf treatment of glioblastoma patients.Keywords cell therapy | glioblastoma | T cell receptor | tumor antigen