Biology subjects

Lee, C.-L.

Publications and source records attributed to Lee, C.-L..

12 recordsLinked to original sources

bioRxiv2026

Serum pro-N-cadherin: a biomarker of cardiac fibrosis and diastolic dysfunction in irradiated non-human primates

BackgroundThe delayed effects of radiation exposure on the heart often manifest as cardiac fibrosis and diastolic dysfunction, which can develop years after exposure. However, no FDA-approved serological biomarker is available to assess the risk of individuals for developing radiation-related heart disease (RRHD). ObjectivesSerum pro-N-cadherin (PNC) has shown promise as a marker for predicting the onset of heart failure in the general population. We hypothesize that serum PNC levels will correlate with the risk of RRHD following radiation exposure. MethodsWe examined male non-human primates (NHPs) exposed to total-body irradiation (TBI) and unirradiated controls from the Wake Forest University radiation late effects cohort. NHPs exhibited cardiac fibrosis scores ranging from less severe (F0-1) to more severe (F2-3). Cardiac tissue samples collected at necropsy, median 6.8 years post-irradiation, were stained for PNC by immunohistochemistry. PNC was quantified in longitudinal serum samples collected 2, 1 and 0 years before necropsy. The associations of serum PNC levels with cardiac fibrosis scores and echocardiographic parameters were examined. ResultsHistological examinations showed aberrant localization of PNC in NHPs with cardiac fibrosis. Elevated serum PNC levels significantly correlated with severe cardiac fibrosis (AUC = 0.81, p = 0.006) and echocardiogram parameters of diastolic dysfunction. Cardiac fibrosis was the only measured comorbidity with a significant difference in serum PNC. ConclusionsOur results demonstrate that serum PNC significantly correlates with cardiac fibrosis and diastolic dysfunction in irradiated NHPs. These findings pave the way for future clinical studies to develop serum PNC as a biomarker of RRHD in humans. HIGHLIGHTSO_LIRadiation-related heart disease is an often under-recognized complication of radiation exposure and radiation therapy, which has no FDA-approved biomarkers for assessing risk. C_LIO_LIOur results reveal that serum pro-N-cadherin is a biomarker of cardiac fibrosis and diastolic dysfunction in non-human primates that survived radiation exposure. C_LIO_LIThis study lays the foundation for further research into the development of serum pro-N-cadherin as a biomarker for assessing the risk of radiation-related heart disease in humans. C_LI

physiology↗

bioRxiv2025

Lgr5+ Stem Cells Maintain Apex Position in Cell Hierarchy of the Intestinal Epithelium During Homeostasis and Injury

The cellular origin of intestinal epithelial homeostasis and regeneration has been a subject of continued debate, with recent models challenging the primacy of WNT-dependent Lgr5 crypt base columnar (CBC) cells as the central intestinal stem cell population. Here, we revisit this question through quantitative integration of single-cell transcriptomic, chromatin accessibility, spatial, and lineage-tracing analyses across the proximal-to-distal axis of the small intestinal epithelium. Our data show that under homeostatic conditions, Lgr5 cells exclusively sustain epithelial self-renewal in nearly all crypt-villus units along the entire length of the small intestine, a process for which R-spondin is indispensable. Following irradiation or chemotoxic injury, surviving Lgr5 cells and their progeny reprogram into transient fetal-like cell states that initiate epithelial repair. Crucially, successful regeneration depends on the reactivation of canonical WNT/{beta}-catenin signaling, as evidenced by increased TCF motif accessibility and upregulation of WNT target genes in newly forming Lgr5+ stem cells. Accordingly, pharmacological inhibition of WNT signaling blocks the reconstitution of Lgr5 cells and crypt regeneration, leading to epithelial collapse. These findings reconcile prior controversies by demonstrating the central role of Lgr5 CBC cells in epithelial self-renewal and regeneration following injury.

cell biology↗

bioRxiv2023

Mis-splicing Drives Loss of Function of p53E224D Point Mutation

BackgroundTp53 is the most commonly mutated gene in cancer. Canonical Tp53 DNA damage response pathways are well characterized and classically thought to underlie the tumor suppressive effect of Tp53. Challenging this dogma, mouse models have revealed that p53 driven apoptosis and cell cycle arrest are dispensable for tumor suppression. Here, we investigated the inverse context of a p53 mutation predicted to drive expression of canonical targets, but is detected in human cancer. MethodsWe established a novel mouse model with a single base pair mutation (GAG>GAC, p53E221D) in the DNA-Binding domain that has wild-type function in screening assays, but is paradoxically found in human cancer in Li-Fraumeni syndrome. Using mouse p53E221D and the analogous human p53E224D mutant, we evaluated expression, transcriptional activation, and tumor suppression in vitro and in vivo. ResultsExpression of human p53E224D from cDNA translated to a fully functional p53 protein. However, p53E221D/E221D RNA transcribed from the endogenous locus is mis-spliced resulting in nonsense mediated decay. Moreover, fibroblasts derived from p53E221D/E221D mice do not express a detectable protein product. Mice homozygous for p53E221D exhibited increased tumor penetrance and decreased life expectancy compared to p53 WT animals. ConclusionsMouse p53E221D and human p53E224D mutations lead to splice variation and a biologically relevant p53 loss of function in vitro and in vivo.

genomics↗

bioRxiv2023

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced GI injury. Through single-cell RNA-sequencing of the irradiated mouse intestine, we find that p53 target genes are specifically enriched in stem cells of the regenerating epithelium, including revival stem cells that promote animal survival after GI damage. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce revival stem cells. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells that is controlled by an Mdm2-mediated negative feedback loop. These results suggest that p53 suppresses severe radiation-indued GI injury by promoting intestinal epithelial cell reprogramming. One-Sentence SummaryAfter severe radiation injury to the intestine, transient p53 activity induces revival stem cells to promote regeneration.

cell biology↗

bioRxiv2023

A Weakly Supervised U-Net Model for Precise Whole Brain Immunolabeled Cell Detection

Cell segmentations low precision due to the intensity differences hinders widespread use of whole brain microscopy imaging. Previous studies used ResNet or CNN to account for this problem, but are unapplicable to immunolabeled signals across samples. Here we present a semiauto ground truth generation and weakly-supervised U-Net-based Deep-learning precise segmentation pipeline for whole brain immunopositive c-FOS signals, which reveals the distinct neural activity maps with different social motivations.

neuroscience↗

bioRxiv2022

Human Early Syncytiotrophoblasts Are Highly Susceptible to SARS-CoV-2 Infection

The ongoing and devastating pandemic of coronavirus disease 2019 (COVID-19) has led to a global public health crisis. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and can potentially pose a serious risk to maternal and neonatal health. Cases of abnormal pregnancy and vertical transmission of SARS-CoV-2 from mother to foetus have been reported but no firm conclusions are drawn. Trophoblasts are the major constituents of the placenta to protect and nourish the developing foetus. However, direct in vivo investigation of trophoblasts susceptibility to SARS-CoV-2 and of COVID-19 and pregnancy is challenging. Here we report that human early syncytiotrophoblasts (eSTBs) are highly susceptible to SARS-CoV-2 infection in an angiotensin-converting enzyme 2 (ACE2)-dependent manner. From human expanded potential stem cells (hEPSCs), we derived bona fide trophoblast stem cells (TSCs) that resembled those originated from the blastocyst and the placenta in generating functional syncytiotrophoblasts (STBs) and extravillus trophoblasts (EVTs) and in low expression of HLA-A/B and amniotic epithelial (AME) cell signature. The EPSC-TSCs and their derivative trophoblasts including trophoblast organoids could be infected by SARS-CoV-2. Remarkably, eSTBs expressed high levels of ACE2 and produced substantially higher amounts of virion than Vero E6 cells which are widely used in SARS-CoV-2 research and vaccine production. These findings provide experimental evidence for the clinical observations that opportunistic SARS-CoV-2 infection during pregnancy can occur. At low concentrations, two well characterized antivirals, remdesivir and GC376, effectively eliminated infection of eSTBs by SARS-CoV-2 and middle east respiratory syndrome-related coronavirus (MERS-CoV), and rescued their developmental arrest caused by the virus infection. Several human cell lines have been used in coronavirus research. However, they suffer from genetic and/or innate immune defects and have some of the long-standing technical challenges such as cell transfection and genetic manipulation. In contrast, hEPSCs are normal human stem cells that are robust in culture, genetically stable and permit efficient gene-editing. They can produce and supply large amounts of physiologically relevant normal and genome-edited human cells such as eSTBs for isolation, propagation and production of coronaviruses for basic research, antivirus drug tests and safety evaluation.

cell biology↗

bioRxiv2022

Temporary knockdown of p53 during focal limb irradiation increases the development of sarcomas

BackgroundApproximately half of all cancer patients receive radiotherapy and, as cancer survivorship rates increase with more effective therapies, the very low rate of radiation-associated sarcomas is rising. Radiation-associated sarcomas are life-threatening cancers, and radiation exposure is a primary risk factor for sarcoma development. During radiotherapy or other genotoxic cancer therapy for p53 mutant cancers, pharmacological inhibition of p53 has been proposed to ameliorate acute injury of normal tissues. However, enhancing the survival of normal cells that sustain DNA damage by temporarily inhibiting p53 has the potential to increase the risk of cancer development. Here, we use in vivo shRNA technology to examine the consequences of temporarily reducing p53 expression on radiation-induced sarcoma development. MethodsWe utilized a mouse model of radiation-induced sarcoma where mice express a doxycycline (dox)-inducible p53 shRNA to temporarily and reversibly reduce p53 expression. Mice were placed on a dox diet 10 days prior to receiving 30 or 40 Gy hind limb irradiation in a single fraction and then returned to normal chow. Mice were examined weekly for sarcoma development and scored for radiation-induced normal tissue injuries. Radiation-induced sarcomas were harvested and subjected to RNA sequencing. ResultsFollowing single high-dose irradiation, 21% of temporary p53 knockdown animals developed a sarcoma in the radiation field compared to 2% of control animals. Mice with more severe acute injuries in the first 3 months after irradiation had a significantly increased risk of developing late persistent wounds in the soft tissue and bone. Chronic radiation-induced wounds were associated with sarcomagenesis. Examination of muscle stem cells by flow cytometry following hind limb irradiation indicated p53 knockdown preserves muscle stem cells in the irradiated limb, supporting the notion that temporary p53 knockdown at the time of irradiation reduces death of cells with DNA damage which may then persist to develop into a sarcoma. We performed RNA sequencing on 16 radiation-induced sarcomas compared to normal muscle controls. Gene set enrichment analysis revealed upregulation in the sarcomas of genes related to translation, epithelial mesenchymal transition (EMT), inflammation, and the cell cycle versus downregulation of genes related to myogenesis and tumor metabolism. Furthermore, genes with increased copy number such as Met and Cdk4 were overexpressed in tumors. ConclusionsTemporary reduction of p53 during high-dose irradiation increases late effects including tissue injuries and sarcoma development.

cancer biology↗

bioRxiv2021

The p53 transactivation domain 1-dependent response to acute DNA damage in endothelial cells protects against radiation-induced cardiac injury

Thoracic radiation therapy can cause endothelial injury in the heart, leading to cardiac dysfunction and heart failure. Although it has been demonstrated that the tumor suppressor p53 functions in endothelial cells to prevent the development of radiation-induced myocardial injury, the key mechanism(s) by which p53 regulates the radiosensitivity of cardiac endothelial cells is not completely understood. Here, we utilized genetically engineered mice that express mutations in p53 transactivation domain 1 (TAD1) (p5325,26) or mutations in p53 TAD1 and TAD2 (p5325,26,53,54) specifically in endothelial cells to study the p53 transcriptional program that protects cardiac endothelial cells from ionizing radiation in vivo. p5325,26,53,54 loses the ability to drive transactivation of p53 target genes after irradiation while p5325,26 can induce transcription of a group of non-canonical p53 target genes, but not the majority of classic radiation-induced p53 targets critical for p53-mediated cell cycle arrest and apoptosis. After 12 Gy whole-heart irradiation, we found that both p5325,26 and p5325,26,53,54 sensitized mice to radiation-induced cardiac injury, in contrast to wild-type p53. Histopathological examination suggested that mutation of TAD1 contributes to myocardial necrosis after whole-heart irradiation, while mutation of both TAD1 and TAD2 abolishes the ability of p53 to prevent radiation-induced heart disease. Taken together, our results show that the transcriptional program downstream of p53 TAD1, which activates the acute DNA damage response after irradiation, is necessary to protect cardiac endothelial cells from radiation injury in vivo.

genetics↗

bioRxiv2020

Activation of Notch1 drives the development of radiation-induced thymic lymphoma in p53 wild-type mice

Mouse models of radiation-induced thymic lymphoma are widely used to study the development of radiation-induced blood cancers and to gain insights into the biology of human T-lymphoblastic leukemia/lymphoma. Here, we aimed to determine key oncogenic drivers for the development of radiation-induced thymic lymphoma by performing whole-exome sequencing using tumors and paired normal tissues from mice with and without irradiation. Thymic lymphomas from irradiated wild-type (WT), p53+/- and KrasLA1 mice were not observed to harbor significantly higher numbers of non-synonymous somatic mutations compared to thymic lymphomas from unirradiated p53-/- mice. However, we observed distinct patterns of recurrent mutations in genes that control the Notch1 signaling pathway based on the mutational status of p53. Preferential activation of Notch1 signaling in p53 WT lymphomas was also observed at the RNA and protein level. Using reporter mice for activation of the Notch1 signaling we observed that TBI enriched Notch1hi CD44+ thymocytes, which are capable of self-renewal in vivo. Mechanistically, genetic inhibition of Notch1 signaling in thymocytes prevented the formation of radiation-induced thymic lymphoma in p53 WT mice. Taken together, our results demonstrate a critical role of activated Notch1 signaling in driving multi-step carcinogenesis of thymic lymphoma following total-body irradiation in p53 WT mice.

cancer biology↗

bioRxiv2019

Neuronal TORC1 modulates longevity via AMPK and cell nonautonomous regulation of mitochondrial dynamics in C. elegans

Target of rapamycin complex 1 (TORC1) and AMP-activated protein kinase (AMPK) antagonistically modulate metabolism and aging. However, how they coordinate to determine longevity and if they act via separable mechanisms is unclear. Here, we show that neuronal AMPK is essential for lifespan extension from TORC1 inhibition, and that TORC1 suppression increases lifespan cell non autonomously via distinct mechanisms from global AMPK activation. Lifespan extension by null mutations in genes encoding raga-1 (RagA) or rsks-1 (S6K) is fully suppressed by neuronal-specific rescues. Loss of RAGA-1 increases lifespan via maintaining mitochondrial fusion. Neuronal RAGA-1 abrogation of raga-1 mutant longevity requires UNC-64/syntaxin, and promotes mitochondrial fission cell nonautonomously. Finally, deleting the mitochondrial fission factor DRP-1 renders the animal refractory to the pro-aging effects of neuronal RAGA-1. Our results highlight a new role for neuronal TORC1 in cell nonautonomous regulation of longevity, and suggest TORC1 in the central nervous system might be targeted to promote healthy aging.

genetics↗

bioRxiv2019

Photon-counting cine-cardiac CT in the mouse

The maturation of photon-counting detector (PCD) technology promises to enhance routine CT imaging applications with high-fidelity spectral information. In this paper, we demonstrate the power of this synergy and our complementary reconstruction techniques, performing 4D, cardiac PCD-CT data acquisition and reconstruction in a mouse model of atherosclerosis, including calcified plaque. Specifically, in vivo cardiac micro-CT scans were performed in four ApoE knockout mice, following their development of calcified plaques. The scans were performed with a prototype PCD (DECTRIS, Ltd.) with 4 energy thresholds. Projection sampling was performed with 10 ms temporal resolution, allowing the reconstruction of 10 cardiac phases at each of 4 energies (40, 3D volumes per mouse scan). Reconstruction was performed iteratively using the split Bregman method with constraints on spectral rank and spatio-temporal gradient sparsity. The reconstructed images represent the first in vivo, 4D PCD-CT data in a mouse model of atherosclerosis. Robust regularization during iterative reconstruction yields high-fidelity results: an 8-fold reduction in noise standard deviation for the highest energy threshold (relative to algebraic reconstruction), while absolute spectral bias measurements remain below 13 Hounsfield units across all energy thresholds and scans. Qualitatively, image domain material decomposition results show clear separation of iodinated contrast and soft tissue from calcified plaque in the in vivo data. Quantitatively, spatial, spectral, and temporal fidelity are verified through a water phantom scan and a realistic MOBY phantom simulation experiment: spatial resolution is robustly preserved by iterative reconstruction (10% MTF: 2.8-3.0 lp/mm), left-ventricle, cardiac functional metrics can be measured from iodine map segmentations with [~]1% error, and small calcifications (615 m) can be detected during slow moving phases of the cardiac cycle. Given these preliminary results, we believe that PCD technology will enhance dynamic CT imaging applications with high-fidelity spectral and material information.

bioengineering↗

bioRxiv2018

Targeting redox regulatory site of protein kinase B impedes neutrophilic inflammation

Neutrophil activation has a pathogenic effect in inflammatory diseases. Protein kinase B (PKB)/AKT regulates diverse cellular responses. However, the significance of AKT in neutrophilic inflammation is still not well understood. Here, we identified CLLV-1 as a novel AKT inhibitor. CLLV-1 inhibited respiratory burst, degranulation, chemotaxis, and AKT phosphorylation in activated human neutrophils and dHL-60 cells. Significantly, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Additionally, CLLV-1 ameliorated lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. CLLV-1 acts as a covalent allosteric AKT inhibitor by targeting AKT Cys310 to restrain inflammatory responses in human neutrophils and LPS-induced ALI in vivo. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.

pharmacology and toxicology↗