Cell Biology

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21679


Recent Submissions

Now showing 1 - 4 of 4
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    Measuring Intracellular Mucin Viscosity in Human Bronchial epithelial cells with Cystic Fibrosis
    (2017-03-14) Ponomarchuk, Olga; Castillo, Marlius; Rebik, Jonathan; Brochiero, Emmanuelle; GrygorczYk, Ryszard; Dzyuba, Sergei; Gryczynski, Ignacy; Gryczynski, Zygmunt; Borejdo, Julian; Fudala, Rafal; Requena, Sebastian
    Background: Cystic fibrosis (CF) is a genetic disease which causes mucus to be abnormally thick and viscous. The thick mucus harbors bacteria and particulates and is unable to be cleared by the mucociliary system resulting in respiratory disease. Understanding mucus pathology is critical to understanding and treating diseases like CF. While many factors that influence CF mucus to be unusually thick are known, the question remains if there are differences in the viscosity of the mucus before secretion. Before being secreted, mucus exists as granules in the cell known as mucin. In this work, we examine the viscosity of intracellular mucin of human bronchial epithelial cells with and without cystic fibrosis. Methods: We use a simple fluorescent phenyl-BODIPY rotor molecule which is readily uptaken into mucin granules and exhibits dramatic changes in its fluorescent lifetime as a function of its environments viscosity. To measure the distribution of viscosities in intracellular mucin, we use time-resolved fluorescent microscopy to image the non-CF and CF cells and measure the fluorescent lifetime of the probe in intracellular mucin. We employ a machine learning algorithm to analyze the pictures and use a combination of Python and ImageJ to compute the size and viscosity distribution of intracellular mucin granules. Results: Our results show that our molecular rotor is readily uptaken into mucin granules of human epithelial cells. The changes in fluorescent lifetime are substantial enough to determine the apparent viscosity distribution of intracellular mucin granules. The non-CF cells have a single normally distributed peak in the viscosity distribution centered at 560 cP. The CF cells have a bimodal distribution with a peak at 560 cP and an additional peak at 210 cP. The origin and implications this second low viscosity group of mucin granules in is unclear but may provide biophysical insight into CF mucus pathology. Conclusions: Our phenyl-BODIPY molecular rotor in combination with fluorescent lifetime imaging microscopy is a promising method to study the intracellular viscosity distribution of cells. Our results suggest that there is a distinct difference in the viscosity of mucin granules in non-CF cells versus CF cells. We believe our work will provide a new tool for investigators to study intracellular mucin and examine a variety of mucus related diseases.
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    Red/Green Astrocytes Mimic CNS Viral Reservoirs in Post ART HAND: Implications for Meth Abuse
    (2017-03-14) Ghorpade, Anuja; Edara, Venkata Viswanadh
    Hypothesis: Though anti-retroviral therapy (ART) has increased the life expectancy of HIV-1 infected individuals, the quest for eradication of latent viral reservoirs continues. Methamphetamine (Meth) abuse and HIV-1 infection increase neuroinflammation through cellular and molecular mechanisms such as gliosis, viral replication, oxidative stress, and excitotoxicity. Multiple studies have validated astrocytes as a major reservoir of HIV-1 in the CNS. We hypothesized that astrocyte HIV-1 reservoirs contribute to HIV-associated neurocognitive disorders (HAND) pathogenesis, and are mediated by Meth abuse during HIV-1 infection. Materials and Methods: A doubly labeled fluorescent reporter Red/Green-HIV-1 (R/G-HIV-1) was used to model latency in primary human astrocytes. Active (mCherry+/GFP+) and latently infected (mCherry+/GFP-) astrocytes were enriched using fluorescence activated cell sorting. Results: Pseudotyped R/G-HIV-1-infected astrocytes established latency over a period of 21 days. These studies were also conducted with pre- and/or post-Meth treatment. Latently-infected astrocytes were devoid of late viral proteins such as p24, indicating a functionally silent HIV-1 LTR. Vorinostat, an HDAC inhibitor, reactivated the silenced HIV-1 LTR in a mixed population of pseudotyped R/G-HIV-1-infected astrocytes. Conclusions: Our data suggests R/G-HIV-1 could be used as a relevant model of latency in astrocytes since it mimics virus reactivation in inflammation leading to viral proteins expression. We anticipate that healthy versus latently infected astrocytes respond differentially to inflammation. Investigating the underlying mechanisms will help in assessing the role of HIV-1 astrocyte reservoirs in HAND pathogenesis.
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    Polymeric Nanoparticle-Mediated Gene Delivery to Human Astrocytes
    (2017-03-16) Labhasetwar, Vinod; Ghorpade, Anuja; Joshi, Chaitanya
    Purpose: Astrocyte tissue-inhibitor of metalloproteinases-1 (TIMP-1) protects neurons during HIV-1-induced apoptosis. However, TIMP-1 levels decrease during chronic inflammation typical of HIV-associated neurocognitive disorders (HAND). We propose that astrocyte-targeted TIMP-1-gene delivery could be a suitable therapeutic for HAND. Nanoparticle (NP)-mediated gene delivery is a viable approach since genes can be delivered to specific brain cell types and NPs are less immunogenic than viral vectors. To test this hypothesis, obtaining a safe and effective gene delivery system is essential. Methods: We tested the gene delivery potential of arginine-based polyethylenimine (PEI) analogs (AnPn) and poly-lactic-co-glycolic-acid (PLGA) in vitro (primary human cells) and in vivo (mice) using a luciferase-reporter plasmid (pLuc). Immunocytochemisty and immunohistochemistry were carried out using antibodies specific to glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), and luciferase. Results: PLGA NPs delivered pLuc across astrocyte plasma membrane but failed to induce protein expression. In parallel, A5P50, a PEI analog, efficiently expressed pLuc in astrocytes. Yet, its neuronal biocompatibility was not optimal. Combining low doses of AnPn with pLuc-loaded-PLGA NPs lead to high gene expression in all cell types including astrocytes. Live imaging indicated that AnPn facilitated PLGA-released-pLuc delivery across the nuclear membrane by an unknown mechanism. Consequently, optimally biocompatible PEI analogs were also synthesized and tested. Conclusions: Our data indicate that AnPn-PLGA combination and new AnPn analogs overcome both neuronal biocompatibility and astrocyte-specific gene expression issues promising clinical translations for HAND treatment in future.
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    Interleukin-1β and Abacavir Induce Astrocyte Endoplasmic Reticulum Stress During HIV-1-Associated Neurocognitive Disorders
    (2017-03-14) Ghorpade, Anuja; Nooka, Shruthi
    Purpose: Globally 69% of HIV-1-positive individuals suffer from HIV-1-associated neurocognitive disorders (HAND) despite effective anti-retroviral therapy (ART). Persistent glial-mediated inflammation, BBB disruption, increased oxidative stress, and viral protein expression together lead to HIV-1 disease progression. ARV drugs, while successfully controlling viral load, likely induce cellular stress responses, oxidative stress, inflammation, and mitochondrial damage. Recently, endoplasmic reticulum (ER) stress has been linked to many neurological diseases, including HAND. Astrocyte elevated gene (AEG)-1, a HIV-1 inducible gene, upregulation in Huntington’s disease model along with ER stress markers, recommends its possible role in HIV-1/ART triggered ER stress. We hypothesize that HAND-relevant inflammatory stimuli and ARV drugs induce astrocyte ER stress and AEG-1 expression that further mediates cellular stress responses in post-ART HAND. Materials and Methods: Cultured human astrocytes were treated with HIV-1DJV, interleukin (IL)-1β and ARV drugs. Astrocytes were transfected with GCaMP6s plasmid. ER stress markers gene expression and protein levels were determined by RT-PCR, western blot analysis and immunocytochemistry. Confocal imaging and mPTP assay was also performed. Results: HIV-1, IL-1β and ARV drugs abacavir and lamivudine, upregulated ER stress markers, and activated unfolded protein response (UPR) pathways i.e., PERK, ATF6, and IRE1α in astrocytes. IL-1β and abacavir treated astrocytes indicated phosphorylation of eIF2α. ARV drugs and ER stress compounds induced astrocyte AEG-1 levels that correlated to PERK and BiP expression. Intracellular calcium signaling changes in response to IL-1β and abacavir were observed in astrocytes transfected with a genetically encoded calcium indicator, GCaMP6s. IL-1β and abacavir also increased calnexin levels in astrocytes. Further, confocal analysis and mPTP assay showed AEG-1 colocalization with calnexin and mitochondrial damage with ER stress. Conclusions: In summary, our study highlights that ARV drugs and IL-1β induced AEG-1 expression, ER stress, cellular calcium overload, and mitochondrial damage in astrocytes. Therefore, identifying novel mechanisms mediated by astrocytes via ER stress and UPR signaling may have broader implications in neuroAIDS management.