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Cell-based high-content approach for SARS-CoV-2 neutralization identifies unique monoclonal antibodies and PI3K pathway inhibitors.
Get PDFThe sudden rise of the SARS-CoV-2 virus and the delay in development of effective therapeutics for mitigation made evident a need for ways to screen compounds that can block infection and prevent further pathogenesis and spread. However, identifying effective drugs that are efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, but as the SARS-CoV-2 mutated, these antibodies became ineffective. Small-molecule antivirals were identified using pseudovirus constructs to recapitulate infection in nonhuman cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce viral infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e., PIK3i, mTORi, and multiple RTKi) that could be effective against the SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells
Cross-Task Translation of Motor Recovery in Mouse Models of Stroke
No full textThe mouse model has been used to study stroke recovery through a variety of behavioral assays, but results are not easily translatable across tasks. This project connects data from two such assays examining motor deficits after stroke: the string-pull task, in which a mouse pulls on a string to reach a reward, and the pasta-reach task, in which the mouse reaches through a glass window to retrieve a piece of pasta. DeepLabCut pose estimation and DeepEthogram behavior labeling algorithms are used to track the mouse’s movement from videos, and kinematic features including speed, acceleration, and joint angles were extracted from the data. We used machine learning to predict different phases of stroke (pre-stroke, acute, or chronic) and found that joint and wrist angles were universally important in making these predictions. We then created a framework for translation across the string-pull and pasta-reach tasks using CycleGAN, a generative and adversarial neural network. We found that angles of pull were most translatable across the two tasks. In the future, we hope to use this pipeline to translate shared behavioral features in mice to those in humans, since it is difficult to gather standardized data on human stroke
Senescence-associated Transcriptomic Signatures and Spatial Localization of Immune Cells in Male and Female Mammalian Hearts
No full textAging remodels the cardiac immune landscape and contributes to sex- and cell-specific risks for cardiovascular disease. In this study, we characterized transcriptomic profiles of macrophages and dendritic cells from young and aged male and female murine hearts, focusing on p21+ senescence-associated populations, using integrated single-cell, single-nucleus, and spatial transcriptomics. Male aged immune cells exhibited differential gene expression and pathways associated with inflammation, calcification, and maladaptive tissue remodeling, marked by enhanced pro-inflammatory and apoptotic pathways alongside suppression of contractile and metabolic functions. In contrast, female immune cells showed elevated insulin-like growth factor (IGF) signaling and TGF-β pathway activity, reflecting metabolic robustness and a balanced senescent state characterized by pro-survival and profibrotic signaling. Spatial analyses revealed sex- and cell-specific colocalizations supporting these divergent pathways, including male-enriched survival and Hippo pathway suppressive interactions and female-enriched IGF-axis signaling in valvular regions. In regards to senescence-associated profiling, p21+ senescent cells displayed stronger growth arrest and stress response signatures than general aged cells, with males favoring pathogenic calcification and inflammation, while females emphasized proteostasis and DNA damage responses. This integrated molecular and spatial characterization unveils fundamental sex/cell-specific and senescence-dependent differences in cardiac immune aging, offering new insights for therapeutic strategies against age-related cardiovascular diseases
Examining and optimizing the embryoid body model for early embryonic germ layer development in diverse hiPSC lineages
No full textEmbryoid bodies are a useful tool in the fields of developmental biology and genetics due to their utility as an in vivo model for early embryonic processes and method by which specialized transient cell types may be acquired. Preliminary work in our lab seeking to develop embryoid bodies from the KOLF2.2J iPSC line has indicated a disproportionate propensity in this lineage for neuroectoderm differentiation under neutral (non-directed) conditions, presenting a challenge to comprehensively recapitulate the development of all three germ layers through the embryoid body model in this cell line. We sought to assess the effect of altered growth conditions on embryoid bodies derived from this cell line, as well as a set of genetically diverse iPSC lines, to assess the effect of germ layer development through embryoid body formation. Through the modulation of growth conditions and embryoid body development protocols, we observe a conserved high propensity for ectoderm differentiation, specifically through the expression of the OTX2, PAX6, and TUBB3 markers across cell lines. However, preliminary results gathered through this investigation also indicate a cell-line specific role of low oxygen in enhancing expression of endoderm and mesoderm markers, such as FOXA2 and MIXL1. This work lays the foundation for reliable and reproducible embryoid body development across newly developed iPSC lineages representing diverse human populations, which may be applied and further developed towards the modulation of differentiation propensity towards the acquisition of an expanded panel of cell types in these lineages and others
Biological databases in the age of generative artificial intelligence.
Get PDFSUMMARY: Modern biological research critically depends on public databases. The introduction and propagation of errors within and across databases can lead to wasted resources as scientists are led astray by bad data or have to conduct expensive validation experiments. The emergence of generative artificial intelligence systems threatens to compound this problem owing to the ease with which massive volumes of synthetic data can be generated. We provide an overview of several key issues that occur within the biological data ecosystem and make several recommendations aimed at reducing data errors and their propagation. We specifically highlight the critical importance of improved educational programs aimed at biologists and life scientists that emphasize best practices in data engineering. We also argue for increased theoretical and empirical research on data provenance, error propagation, and on understanding the impact of errors on analytic pipelines. Furthermore, we recommend enhanced funding for the stewardship and maintenance of public biological databases.
AVAILABILITY AND IMPLEMENTATION: Not applicable
Tipping the balance: innate and adaptive immunity in mitochondrial disease.
Get PDFMitochondrial diseases (MtD) provide a unique window into the complex interplay between metabolism and immune function. These rare disorders, caused by defects in oxidative phosphorylation, result in bioenergetic deficiencies that disrupt multiple organ systems. While traditionally studied for their metabolic impact, MtD also profoundly affect the immune system, altering both innate and adaptive responses. This review explores how mitochondrial dysfunction shapes immune dysregulation, influencing thymocyte maturation, regulatory T cells, and B cell function while also driving innate immune activation through mitochondrial DNA instability and type I interferon signaling. Additionally, MtD highlight an emerging overlap between inborn errors of metabolism and inborn errors of immunity, revealing shared pathways that connect mitochondrial dysfunction to immune deficiencies and inflammatory disease. Studying MtD not only advances our understanding of immunometabolism but also provides critical insights into more common inflammatory and autoimmune conditions, offering potential therapeutic targets that extend beyond rare mitochondrial disorders
The multilayered transcriptional architecture of glioblastoma ecosystems.
Get PDFIn isocitrate dehydrogenase wildtype glioblastoma (GBM), cellular heterogeneity across and within tumors may drive therapeutic resistance. Here we analyzed 121 primary and recurrent GBM samples from 59 patients using single-nucleus RNA sequencing and bulk tumor DNA sequencing to characterize GBM transcriptional heterogeneity. First, GBMs can be classified by their broad cellular composition, encompassing malignant and nonmalignant cell types. Second, in each cell type we describe the diversity of cellular states and their pathway activation, particularly an expanded set of malignant cell states, including glial progenitor cell-like, neuronal-like and cilia-like. Third, the remaining variation between GBMs highlights three baseline gene expression programs. These three layers of heterogeneity are interrelated and partially associated with specific genetic aberrations, thereby defining three stereotypic GBM ecosystems. This work provides an unparalleled view of the multilayered transcriptional architecture of GBM. How this architecture evolves during disease progression is addressed in the companion manuscript by Spitzer et al
TRP Channels in Excitotoxicity.
No full textGlutamate excitotoxicity is a central mechanism contributing to cellular dysfunction and death in various neurological disorders and diseases, such as stroke, traumatic brain injury, epilepsy, schizophrenia, addiction, mood disorders, Huntington\u27s disease, Alzheimer\u27s disease, Parkinson\u27s disease, multiple sclerosis, pathologic pain, and even normal aging-related changes. This detrimental effect emerges from glutamate binding to glutamate receptors, including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, N-methyl-d-aspartate receptors, kainate receptors, and GluD receptors. Thus, excitotoxicity could be prevented by targeting glutamate receptors and their downstream signaling pathways. However, almost all the glutamate receptor antagonists failed to attenuate excitotoxicity in human patients, mainly due to the limited understanding of the underlying mechanisms regulating excitotoxicity. Transient receptor potential (TRP) channels serve as ancient cellular sensors capable of detecting and responding to both external and internal stimuli. The study of human TRP channels has flourished in recent decades since the initial discovery of mammalian TRP in 1995. These channels have been found to play pivotal roles in numerous pathologic conditions, including excitotoxicity. In this review, our focus centers on exploring the intricate interactions between TRP channels and glutamate receptors in excitotoxicity
TET2-Mutant Clonal Hematopoiesis Promotes Heart Failure With Preserved Ejection Fraction.
No full textPatient-Specific In Vivo Gene Editing to Treat a Rare Genetic Disease.
No full textBase editors can correct disease-causing genetic variants. After a neonate had received a diagnosis of severe carbamoyl-phosphate synthetase 1 deficiency, a disease with an estimated 50% mortality in early infancy, we immediately began to develop a customized lipid nanoparticle-delivered base-editing therapy. After regulatory approval had been obtained for the therapy, the patient received two infusions at approximately 7 and 8 months of age. In the 7 weeks after the initial infusion, the patient was able to receive an increased amount of dietary protein and a reduced dose of a nitrogen-scavenger medication to half the starting dose, without unacceptable adverse events and despite viral illnesses. No serious adverse events occurred. Longer follow-up is warranted to assess safety and efficacy. (Funded by the National Institutes of Health and others.)