Biology subjects

Khan, P.

Publications and source records attributed to Khan, P..

8 recordsLinked to original sources

bioRxiv2024

Cultured alveolar basal cells display similar characteristics and functional properties to airway basal cells

RationaleBasal cells (BC) appear ectopically within the lung parenchyma of interstitial lung disease (ILD) patients, potentially through migration of airway BC or though trans-differentiation of alveolar epithelial type 2 (AT2) cells. The exact origin and function of these ectopic alveolar BC remains elusive. By comparing ectopic alveolar to "classical" airway BC, we aimed to get a better understanding of the origin and characteristics of alveolar BC in ILD. MethodsAlveolar and airway BC were isolated from transbronchial and airway mucosal biopsies, respectively, from the same ILD patients and expanded in culture. Samples were analyzed by single cell RNA sequencing (scRNA-seq), TaqMan RT-PCR, and immunochemistry. ResultsscRNA-seq analysis revealed several differences in gene expression that suggested a shift to a more mesenchymal-like phenotype and a decrease in keratinization genes in alveolar compared to airway BC. Specific AT2 cell marker genes were not expressed in either BC type. While the morphology, wound repair and proliferation capacities of BC from both origins were not significantly different, alveolar BC formed significantly fewer organoids, expressing more MUC5B. After instillation into bleomycin-injured mice, alveolar and airway BC showed similar engraftment, differentiation capacity and effects on fibrosis. ConclusionDespite similar overall functionality in vitro and after instillation into bleomycin-injured mice, alveolar and airway BC differed in their transcriptomes and in their capacities to form and to differentiate in organoids. Our data provide no evidence to support their potential derivation from AT2 cells. Take home messageAlveolar and airway basal cells differ in their transcriptomes and in their capacities to form and to differentiate in organoids, although with no indication of an AT2 cell origin.

cell biology↗

bioRxiv2023

The use of cultured human alveolar basal cells to mimic honeycomb formation in idiopathic pulmonary fibrosis

Honeycomb cysts (HC) within the alveolar region are distinct histopathological features in the lungs of idiopathic pulmonary fibrosis (IPF) patients. HC are lined with basal cells (BC), or with a bronchiolar-like epithelium composed of basal-, ciliated- and secretory epithelial cells. By using cultured IPF patient-derived alveolar BC, we aimed to establish in vitro- and in vivo models to mimic HC formation in IPF. In order to do so, we cultured the cells (1) on an air liquid interface (ALI) or (2) in a three dimensional (3D) organoid model in vitro, and (3) investigated the cells behavior after instillation into bleomycin-challenged mice in vivo. Under the here tested in vitro- and in vivo conditions, alveolar BC differentiate and formed HC-like structures, which closely resemble HC within the IPF lung. These models therefore represent powerful tools to study HC formation, and its potential therapeutic inhibition in IPF.

cell biology↗

bioRxiv2022

High-Contrast PET imaging with -NT160, a Class-IIa Histone Deacetylase (Class-IIa HDAC) Probe for In Vivo Imaging of Epigenetic Machinery in the Central Nervous System

We utilized positron emission tomography (PET) imaging in vivo to map the spatiotemporal biodistribution/expression (protein density) of class-IIa histone deacetylases (class-IIa HDACs) in the brain. Herein, we report an improved radiosynthesis of [18F]-NT160 using 4-hydroxy-TEMPO which led to a significant improvement in radiochemical yield and molar activity. PET imaging with [18F]-NT160, a highly potent class-IIa HDAC inhibitor with sub-nM affinity for HDAC4 and 5 isoforms, led to high-quality and high-contrast images among various brain regions. [18F]-NT160 displayed excellent pharmacokinetic and imaging characteristics: brain uptake is high in gray matter regions, leading to high-quality PET images; tissue kinetics are appropriate for an 18F tracer and specific binding for class-IIa HDACs is demonstrated by self-blockade. Higher uptake with [18F]-NT160 was observed in the hippocampus, thalamus, and cortex while there was relatively lower uptake in the cerebellum and striatum. Overall, our current studies with [18F]-NT160 will likely facilitate the development and clinical translation of class-IIa HDACs of the next generation of PET tracers for imaging and targeted therapy of cancer and the diseases of the central nervous system (CNS).

neuroscience↗

bioRxiv2022

MiR-1253 Potentiates Cisplatin Response in Pediatric Group 3 Medulloblastoma by Regulating Ferroptosis

Medulloblastomas (MB) are the most common malignant pediatric brain tumor and a leading cause of childhood mortality. Aggressive tumors belonging to group 3 (G3MB) are distinguished by a marked reduction in programmed cell death by ferroptosis. These aggressive tumors also enrich iron transport and glutathione metabolism. A highly enriched pathway in these tumors is iron-sulfur (Fe-S) cluster binding corresponding to significant upregulation of ABCB7, a poor prognostic feature linked with accelerated mortality. This study elucidated whether repressing ABCB7 activates ferroptosis to mitigate G3MB aggressiveness and whether this pathway is pharmacologically targetable. In silico and in vitro analyses confirmed upregulation of ABCB7 and GPX4, the central regulator of ferroptosis, in G3MB cell lines and tumors. Repressing ABCB7 (miR-1253OE, siABCB7, sh-ABCB7) induced iron overload, elicited oxidative stress, and triggered lipid peroxidation, leading to an abrogation of medullosphere formation and cell death by ferroptosis. Intriguingly, fractionation studies revealed that ABCB7 repression abrogated GPX4 expression, most likely by GSH depletion. Repressing ABCB7 induced mitochondrial dysfunction and reduced oxidative phosphorylation. Cisplatin, a chemotherapeutic mainstay of G3MB, induces cell death by DNA crosslinking. In ABCB7 repressed cell lines, the IC50 of cisplatin was halved, resulting in augmented oxidative stress and lipid peroxidation, culminating in a higher index of ferroptosis. Artesunate, an anti-malarial drug capable of triggering ferroptosis, was shown to synergize with cisplatin, reducing tumor burden and significantly prolonging survival. Taken together, the current study illustrates how targeting iron transport can augment ferroptosis in G3MBs. It further identifies an FDA-approved drug capable of recapitulating these effects and potentiating mainstay chemotherapy.

cancer biology↗

bioRxiv2021

Glutathione regulates subcellular iron homeostasis via transcriptional activation of iron responsive genes in Arabidopsis

While glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants, its involvement in modulating subcellular Fe homeostasis remained elusive. In this study, we report that the GSH depleted mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with significant down-regulation of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4 and the chloroplast Fe importer, AtPIC1. Moreover, the pad2-1 mutant accumulated higher Fe content in vacuoles and lower in chloroplasts compared with Col-0 under Fe limited condition. Exogenous GSH treatment could enhance the chloroplast Fe content in Col-0 plants but failed to do so in the nramp3nramp4 double mutant demonstrating the role of GSH in modulating subcellular Fe homeostasis. Pharmacological experiments, mutant analysis and promoter assay revealed that this regulation involved transcriptional activation of the transporter genes by a GSH-GSNO module. The Fe responsive bHLH transcription factors (TFs), AtbHLH29, AtbHLH38 and AtbHLH101 were identified to interact with the promoters of these genes which were in turn activated via S-nitrosylation. Together, the present study delineates the role of GSH-GSNO module in regulating subcellular Fe homeostasis by transcriptional activation of the Fe transporters AtNRAMP3, AtNRAMP4 and AtPIC1 via S-nitrosylation of the bHLH TFs during Fe deficiency. Summary statementGlutathione regulates subcellular iron homeostasis under iron deficiency via GSNO dependent transcriptional activation of AtNRAMP3, AtNRAMP4 and AtPIC1 genes by S-nitrosylation of the iron responsive bHLH transcription factors, AtbHLH29, AtbHLH38 and AtbHLH101.

plant biology↗

bioRxiv2020

In vitro culture of aberrant basal-like cells from fibrotic lung tissue

RationaleIn idiopathic pulmonary fibrosis (IPF) atypical epithelial cells are present in the alveolar compartment. Their origin and contribution to IPF pathogenesis is unknown. We recently cultured a distinct population of cells, which readily grew from fibrotic lung tissue, but only rarely from non-fibrotic tissue. Here we aimed to characterize these fibrosis-enriched cells and determine transcriptomic differences between cells derived from IPF and patients with other interstitial lung diseases (ILD). MethodsCells were cultured from peripheral lung tissue of ILD patients and analysed by bulk or single cell RNA sequencing (scRNA-seq), TaqMan-PCR, immunofluorescence (IF), immunoblotting or electron microscopy (EM). ResultsscRNA-seq demonstrated an overall homogeneity and epithelial origin of the cells. The majority of cells expressed basal cell markers (Cytokeratin (KRT) 5 and 17, TP63), of which a fraction co-expressed mesenchymal cell markers (VIM, FN1, CDH2), alveolar (SLC34A2, ABCA3, LPCAT1, EMP2, HOPX) and/or secretory epithelial cell markers (SCGB1A1, MUC4). Interestingly, most of the cells showed closest transcriptomic similarity to recently described aberrant basal-like cells. Cells derived from IPF versus other ILD patients revealed significant transcriptomic differences with an up-regulation of fibrosis-associated and a down-regulation of inflammatory pathways in IPF cells. ConclusionWe here confirm the presence of aberrant basal-like cells in fibrotic lung tissue and, importantly, are the first to describe their in vitro characteristics and a way of culturing these cells in vitro. Cultured basal-like cells co-express epithelial and mesenchymal markers, suggesting a partial epithelial to mesenchymal transition (EMT). A subset of cells co-express alveolar, ciliated or secretory epithelial cell markers, possibly indicating differentiation towards these cell linages. Furthermore, cultured basal-like cells display a disease-specific transcriptome, possibly induced by their specific microenvironment. Our findings will contribute to a better understanding of the cells origin and their potential contribution to IPF pathogenesis.

cell biology↗

bioRxiv2020

Assessment of NR4A Ligands that Directly Bind and Modulate the Orphan Nuclear Receptor Nurr1

ABSTRACTNurr1/NR4A2 is an orphan nuclear receptor transcription factor implicated as a potential drug target for neurological disorders including Alzheimer’s and Parkinson’s diseases. Previous studies identified small molecule modulators of NR4A nuclear receptors including Nurr1 and Nur77/NR4A1; it remains unclear whether these ligands affect Nurr1 through direct binding or indirect non-binding mechanisms. We assessed a panel of twelve ligands reported to affect NR4A activity for Nurr1-dependent and Nurr1-independent transcriptional effects and binding to the Nurr1 ligand-binding domain (LBD). Most of the NR4A ligands show Nurr1-independent effects on transcription in a cell type-specific manner, suggesting they may function through binding to effector proteins whose downstream activities influence Nurr1 function. Protein NMR spectroscopy structural footprinting data show that 4-amino-7-chloroquinoline derivatives (amodiaquine and chloroquine) and cytosporone B directly bind the Nurr1 LBD. In contrast, other NR4A ligands including commercially available compounds such as C-DIM12, celastrol, camptothecin, IP7e, isoalantolactone, and TMPA do not bind the Nurr1 LBD. Interestingly, previous crystal structures indicate that cytosporone B analogs bind to surface pockets in the Nur77 LBD, but protein NMR data indicate cytosporone B likely binds to the Nurr1 orthosteric pocket. These findings should influence medicinal chemistry efforts that desire to optimize Nurr1-binding ligands as opposed to ligands that function through binding to Nurr1 effector proteins.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which Nucleic Acid Sequence Based Amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon, fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform employing a cell phone camera module, compatible with field detection.

synthetic biology↗