Browsing by Author "Liu, Jing"
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Item A Computational Framework for Investigating mRNA Localization Patterns in Pancreatic Beta-Cells During Type 1 Diabetes Progression(2024-12) Chang, Hok Wai; Petrache, Horia; Liu, Jing; Wassall, Stephen; Vemuri, Gautam; Syed, FarooqSpatial transcriptomics improves transcriptomic studies by incorporating RNA localization information, which provides a more profound insight into cellular functions, interactions between cells, and their reactions to external stimuli. Single-molecule fluorescent in situ hybridization (smFISH) is a commonly utilized technique in spatial transcriptomics that allows for the accurate visualization of mRNA distribution in cells. This method aids in the quantitative evaluation of mRNA localization patterns by utilizing various physical properties, thereby illuminating processes such as transcription, nuclear export, and localized translation. Nevertheless, existing computational approaches for analyzing smFISH images often have constraints, concentrating primarily on cellular expression or specific biological contexts while overlooking broader physical analysis. In my PhD research, I created STProfiler, a comprehensive tool aimed at an unbiased physical examination of mRNA distribution. STProfiler includes an image analysis workflow that processes raw biological images to effectively detect mRNA and nuclei. It also employs machine learning techniques to biologically interpret mRNA spatial characteristics and categorize cells based on these features. My dissertation illustrates the use of STProfiler in multiple studies investigating the transcriptomic profiles of β-cells during the progression of type 1 diabetes (T1D), uncovering spatial transcriptomic diversity in β-cells. These investigations involve analyzing mRNA clusters and stress granules in pancreatic β-cells, measuring the physical characteristics of mRNAs linked to cellular stress and inflammation in mice developing T1D, evaluating the rise in HLA-DMB mRNA spliced variant in T1D, and exploring miRNA as a potential biomarker for T1D. Furthermore, STProfiler has also proven beneficial in tissue-wide spatial transcriptomics by creating masks for nuclei and cells from biological images and assigning mRNA transcripts to develop subcellular expression profiles. This capability allows for more thorough bioinformatic evaluations. In summary, STProfiler serves as a robust tool for both cell- and tissue-level spatial transcriptomics, offering an unbiased platform for researchers to investigate complex transcriptomic variations within cells.Item Advancing super-resolution microscopy for quantitative in-vivo imaging of chromatin nanodomains(2024-12) Seitz, Clayton; Liu, Jing; Vemuri, Gautam; Cheng, Ruihua; Wassall, Stephen; Petrache, HoriaSingle molecule localization microscopy (SMLM) techniques, such as direct stochastic optical reconstruction microscopy (dSTORM), can be used to produce a pointillist representation of fluorescently-labeled biological structures at diffraction-unlimited precision. This class of techniques permits localization of fluorescent molecules in the cell with nanometer precision and thus is commonly referred to as nanoscopy. Conventional nanoscopy utilizes the deactivation of standard fluorescent tags, followed by spontaneous or photoinduced reactivation, to resolve fluorophores at distances below the diffraction limit. While powerful, this approach has limited throughput and requires localization in sparse scenes. This dissertation introduces fluorescence nanoscopy and covers its innovation and application as discussed in the following papers: Quantum enhanced localization microscopy with a single photon avalanche diode array [ 1 ] leverages recent advancements in single photon avalanche diode array technology to count fluorescent emitters using a widefield microscope. Uncertainty-aware localization microscopy by variational diffusion [ 2 ] describes a novel algorithm that applies a diffusion model in order to model a posterior distribution on high resolution localization microscopy images, given low resolution inputs. role of the BRD4 phosphoswitch in the maintenance of chromatin nanodomains via super resolution microscopy and molecular dynamics simulation. We demonstrate that BRD4 phosphorylation regulates chromatin packing and mobility in mammalian nuclei.Item ARF6 is a Novel Target for Immunotherapy in Triple Negative Breast Cancer(2024-07) Moulana, Fathima Ishara; Lu, Xiongbin; Pollok, Karen; Hopewell, Emily; Liu, JingTriple negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes with poor clinical outcomes due to lack of effective treatments owing to its hormone receptor negative status. Immune checkpoint blockade (ICB) therapy, which prevents the exhaustion of CD8+ T cells, has shown promise in treating these patients. However, only a small proportion respond, possibly due to resistance and immune evasion mechanisms by the tumor cells. A primary mechanism by which tumor cells evade immune surveillance is by reduced tumor antigen presentation, as indicated by a decreased level of antigen-MHC-I (major histocompatibility complex-I) on the surface of tumor cells. The dynamics of tumor antigens on the cell surface and how cell endocytosis contributes to antigen presentation and their recycling is little known. Here we sought to study the roles of two proteins: clathrin and ADP-Ribosylation Factor 6 (ARF6) which are essential for clathrin-mediated endocytosis and clathrin-independent endocytosis respectively, on the surface turnover of fluorophore-conjugated antigenic peptide bound to MHC-I. We employed Total Internal Reflection Fluorescence Microscopy (TIRFM) and Single Molecule Tracking (SMT) to determine the dynamics of tumor antigen endocytosis on the surface of EO771 murine TNBC cells. We found that the inhibition of ARF6 remarkably impaired the endocytosis of the antigen-MHC-I foci, leading to extended stay of the foci on the cell membrane, while inhibition of clathrin did not, suggesting that clathrin-independent endocytosis is the primary route for MHC-I-mediated antigen endocytosis. Consistent with this finding, reduced ARF6 levels promoted in vitro tumor cell killing by CD8+ T cells and suppressed tumor growth in mice when combined with ICB therapy. We further investigated the effect of pharmacological inhibition of ARF6 in murine TNBC cells and splenic CD8+ T cells using a commercially available ARF6 inhibitor NAV-2729. Treatment with NAV-2729 increased surface MHC-I levels and enhanced the secretion of T cell functional markers such as IFN-, TNF- and IL-2, suggesting the possibility of in vivo administration of ARF6 inhibitors in combination with ICB therapy. Collectively, these data suggest that ARF6 is a novel target for the combined treatment with ICB therapy to improve its efficacy in TNBC patients.Item DNA damage reduces heterogeneity and coherence of chromatin motions(National Academy of Science, 2022) Locatelli, Maëlle; Lawrimore, Josh; Lin, Hua; Sanaullah, Sarvath; Seitz, Clayton; Segall, Dave; Kefer, Paul; Moreno, Naike Salvador; Lietz, Benton; Anderson, Rebecca; Holmes, Julia; Yuan, Chongli; Holzwarth, George; Bloom, Kerry S.; Liu, Jing; Bonin, Keith; Vidi, Pierre-Alexandre; Physics, School of ScienceChromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.Item Enhancing anti-tumor potential: low-intensity vibration suppresses osteosarcoma progression and augments MSCs' tumor-suppressive abilities(2024) Xiong, Xue; Huo, Qingji; Li, Kexin; Cui, Changpeng; Chang, Chunyi; Park, Charles; Ku, BonHeon; Hong, Chin-Suk; Lim, HeeChang; Pandya, Pankita H.; Saadatzadeh, M. Reza; Bijangi-Vishehsaraei, Khadijeh; Lin, Chien-Chi; Kacena, Melissa A.; Pollok, Karen E.; Chen, Andy; Liu, Jing; Thompson, William R.; Li, Xue-Lian; Li, Bai-Yan; Yokota, Hiroki; Health Sciences, School of Health and Human SciencesRationale: Osteosarcoma (OS), a common malignant bone tumor, calls for the investigation of novel treatment strategies. Low-intensity vibration (LIV) presents itself as a promising option, given its potential to enhance bone health and decrease cancer susceptibility. This research delves into the effects of LIV on OS cells and mesenchymal stem cells (MSCs), with a primary focus on generating induced tumor-suppressing cells (iTSCs) and tumor-suppressive conditioned medium (CM). Methods: To ascertain the influence of vibration frequency, we employed numerical simulations and conducted experiments to determine the most effective LIV conditions. Subsequently, we generated iTSCs and CM through LIV exposure and assessed the impact of CM on OS cells. We also explored the underlying mechanisms of the tumor-suppressive effects of LIV-treated MSC CM, with a specific focus on vinculin (VCL). We employed cytokine array, RNA sequencing, and Western blot techniques to investigate alterations in cytokine profiles, transcriptomes, and tumor suppressor proteins. Results: Numerical simulations validated LIV frequencies within the 10-100 Hz range. LIV induced notable morphological changes in OS cells and MSCs, confirming its dual role in inhibiting OS cell progression and promoting MSC conversion into iTSCs. Upregulated VCL expression enhanced MSC responsiveness to LIV, significantly bolstering CM's efficacy. Notably, we identified tumor suppressor proteins in LIV-treated CM, including procollagen C endopeptidase enhancer (PCOLCE), histone H4 (H4), peptidylprolyl isomerase B (PPIB), and aldolase A (ALDOA). Consistently, cytokine levels decreased significantly in LIV-treated mouse femurs, and oncogenic transcript levels were downregulated in LIV-treated OS cells. Moreover, our study demonstrated that combining LIV-treated MSC CM with chemotherapy drugs yielded additive anti-tumor effects. Conclusions: LIV effectively impeded the progression of OS cells and facilitated the transformation of MSCs into iTSCs. Notably, iTSC-derived CM demonstrated robust anti-tumor properties and the augmentation of MSC responsiveness to LIV via VCL. Furthermore, the enrichment of tumor suppressor proteins within LIV-treated MSC CM and the reduction of cytokines within LIV-treated isolated bone underscore the pivotal tumor-suppressive role of LIV within the bone tumor microenvironment.Item Enhancing anti-tumor potential: low-intensity vibration suppresses osteosarcoma progression and augments MSCs' tumor-suppressive abilities(Ivyspring, 2024-01-27) Xiong, Xue; Huo, Qingji; Li, Kexin; Cui, Changpeng; Chang, Chunyi; Park, Charles; Ku, BonHeon; Hong, Chin-Suk; Lim, HeeChang; Pandya, Pankita H.; Saadatzadeh, M. Reza; Bijangi-Vishehsaraei, Khadijeh; Lin, Chien-Chi; Kacena, Melissa A.; Pollok, Karen E.; Chen, Andy; Liu, Jing; Thompson, William R.; Li, Xue-Lian; Li, Bai-Yan; Yokota, Hiroki; Anatomy, Cell Biology and Physiology, School of MedicineRationale: Osteosarcoma (OS), a common malignant bone tumor, calls for the investigation of novel treatment strategies. Low-intensity vibration (LIV) presents itself as a promising option, given its potential to enhance bone health and decrease cancer susceptibility. This research delves into the effects of LIV on OS cells and mesenchymal stem cells (MSCs), with a primary focus on generating induced tumor-suppressing cells (iTSCs) and tumor-suppressive conditioned medium (CM). Methods: To ascertain the influence of vibration frequency, we employed numerical simulations and conducted experiments to determine the most effective LIV conditions. Subsequently, we generated iTSCs and CM through LIV exposure and assessed the impact of CM on OS cells. We also explored the underlying mechanisms of the tumor-suppressive effects of LIV-treated MSC CM, with a specific focus on vinculin (VCL). We employed cytokine array, RNA sequencing, and Western blot techniques to investigate alterations in cytokine profiles, transcriptomes, and tumor suppressor proteins. Results: Numerical simulations validated LIV frequencies within the 10-100 Hz range. LIV induced notable morphological changes in OS cells and MSCs, confirming its dual role in inhibiting OS cell progression and promoting MSC conversion into iTSCs. Upregulated VCL expression enhanced MSC responsiveness to LIV, significantly bolstering CM's efficacy. Notably, we identified tumor suppressor proteins in LIV-treated CM, including procollagen C endopeptidase enhancer (PCOLCE), histone H4 (H4), peptidylprolyl isomerase B (PPIB), and aldolase A (ALDOA). Consistently, cytokine levels decreased significantly in LIV-treated mouse femurs, and oncogenic transcript levels were downregulated in LIV-treated OS cells. Moreover, our study demonstrated that combining LIV-treated MSC CM with chemotherapy drugs yielded additive anti-tumor effects. Conclusions: LIV effectively impeded the progression of OS cells and facilitated the transformation of MSCs into iTSCs. Notably, iTSC-derived CM demonstrated robust anti-tumor properties and the augmentation of MSC responsiveness to LIV via VCL. Furthermore, the enrichment of tumor suppressor proteins within LIV-treated MSC CM and the reduction of cytokines within LIV-treated isolated bone underscore the pivotal tumor-suppressive role of LIV within the bone tumor microenvironment.Item A guide for single-particle chromatin tracking in live cell nuclei(Wiley, 2022) Zhang, Mengdi; Seitz, Clayton; Chang, Garrick; Iqbal, Fadil; Lin, Hua; Liu, Jing; Physics, School of ScienceThe emergence of labeling strategies and live cell imaging methods enables the imaging of chromatin in living cells at single digit nanometer resolution as well as milliseconds temporal resolution. These technical breakthroughs revolutionize our understanding of chromatin structure, dynamics and functions. Single molecule tracking algorithms are usually preferred to quantify the movement of these intranucleus elements to interpret the spatiotemporal evolution of the chromatin. In this review, we will first summarize the fluorescent labeling strategy of chromatin in live cells which will be followed by a systematic comparison of live cell imaging instrumentation. With the proper microscope, we will discuss the image analysis pipelines to extract the biophysical properties of the chromatin. Finally, we expect to give practical suggestions to broad biologists on how to select methods and link to the model properly according to different investigation purposes. This article is protected by copyright. All rights reserved.Item Histone Deacetylase Inhibitors Prevent Cytokine-Induced β Cell Dysfunction Through Restoration of Stromal Interaction Molecule 1 Expression and Activation of Store-Operated Calcium Entry(bioRxiv, 2023-12-08) Lee, Chih-Chun; Kono, Tatsuyoshi; Syed, Farooq; Weaver, Staci A.; Sohn, Paul; Wu, Wenting; Chang, Garrick; Liu, Jing; Slak Rupnik, Marjan; Evans-Molina, Carmella; Pediatrics, School of MedicineHistone deacetylase inhibitors (HDIs) modulate β cell function in preclinical models of diabetes; however, the mechanisms underlying these beneficial effects have not been determined. In this study, we investigated the impact of the HDI sodium butyrate (NaB) on β cell function and calcium (Ca2+) signaling using ex vivo and in vitro models of diabetes. Our results show that NaB significantly improved glucose-stimulated insulin secretion in islets from human organ donors with type 2 diabetes and in cytokine-treated INS-1 β cells. Consistently, NaB partially rescued glucose-stimulated Ca2+ oscillations in mouse islets treated with proinflammatory cytokines. Because the oscillatory phenotype of Ca2+ in the β cell is governed by changes in endoplasmic reticulum (ER) Ca2+ levels, next we explored the relationship between NaB and store-operated calcium entry (SOCE), a rescue mechanism that acts to refill ER Ca2+ levels through STIM1-mediated gating of plasmalemmal Orai channels. We found that NaB treatment preserved basal ER Ca2+ levels and restored SOCE in IL-1β-treated INS-1 cells. Furthermore, we linked these changes with the restoration of STIM1 levels in cytokine-treated INS-1 cells and mouse islets, and we found that NaB treatment was sufficient to prevent β cell death in response to IL-1β treatment. Mechanistically, NaB counteracted cytokine-mediated reductions in phosphorylation levels of key signaling molecules, including AKT, ERK1/2, glycogen synthase kinase-3α (GSK-3α), and GSK-3β. Taken together, these data support a model whereby HDI treatment promotes β cell function and Ca2+ homeostasis under proinflammatory conditions through STIM1-mediated control of SOCE and AKT-mediated inhibition of GSK-3.Item Histone deacetylase inhibitors protect against cisplatin-induced acute kidney injury by activating autophagy in proximal tubular cells(Nature Publishing group, 2018-02-23) Liu, Jing; Livingston, Man J.; Dong, Guie; Tang, Chengyuan; Su, Yunchao; Wu, Guangyu; Yin, Xiao-Ming; Dong, Zheng; Pathology and Laboratory Medicine, School of MedicineHistone deacetylase inhibitors (HDACi) have therapeutic effects in models of various renal diseases including acute kidney injury (AKI); however, the underlying mechanism remains unclear. Here we demonstrate that two widely tested HDACi (suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA)) protect the kidneys in cisplatin-induced AKI by enhancing autophagy. In cultured renal proximal tubular cells, SAHA and TSA enhanced autophagy during cisplatin treatment. We further verified the protective effect of TSA against cisplatin-induced apoptosis in these cells. Notably, inhibition of autophagy by chloroquine or by autophagy gene 7 (Atg7) ablation diminished the protective effect of TSA. In mice, TSA increased autophagy in renal proximal tubules and protected against cisplatin-induced AKI. The in vivo effect of TSA was also abolished by chloroquine and by Atg7 knockout specifically from renal proximal tubules. Mechanistically, TSA stimulated AMPK and inactivated mTOR during cisplatin treatment of proximal tubule cells and kidneys in mice. Together, these results suggest that HDACi may protect kidneys by activating autophagy in proximal tubular cells.Item Imaging the Dynamics of Chromatin at Single-Nucleosome Resolution(2024-12) Iqbal, Mohamed Fadil; Vemuri, Gautam; Liu, Jing; Petrache, Horia; Decca, Ricardo; Wassall, StephenDNA is organized into chromatin – a complex polymeric structure which stores information and controls gene expressions. Advancements in microscopy have enabled us to see chromatin in motion – which was previously thought to be static, and these motions contribute to various cellular functions. In my thesis I will demonstrate the molecular tools and biophysical approaches our lab has developed to uncover the mysteries of chromatin dynamics and structures at the single nucleosome resolution; I will also discuss how these new discoveries in chromatin enable us to explore its role in cell functions. This dissertation will first describe the technology advancement of live-cell image analysis; particularly, I will discuss the utilization of AI to improve the spatial and temporal resolution of chromatin imaging. Then I will show complex nature of chromatin where depending on the temporal scale of observation we see a different behavior and how computer simulations can see these differences. Following that, I will introduce our investigation on the role of chromatin motion in DNA damage and repair. Afterwards, I will discuss how the cell regulates its chromatin dynamics in response to the metabolism indicators AMPK (AMP-activated protein kinase). I will also show how chromatin motion and structure behave without the presence of key proteins such as RAD51 that aid in DNA damage. Finally I will go over future directions and improvements we can do to our current techniques to improve our understanding of chromatin’s role is various biological functions. We expect that the exploration of the spatiotemporal dynamics in live cells will facilitate the diagnosis, treatment, and prevention of cancers.