Biology subjects

Amatngalim, G. D.

Publications and source records attributed to Amatngalim, G. D..

9 recordsLinked to original sources

bioRxiv2026

Personalized multi-assay profiling of respiratory motile ciliopathies and mRNA therapy

IntroductionImpaired motile cilia function contributes to many respiratory disorders, but therapies targeting this cellular defect are currently lacking. Personalized airway epithelial models combined with quantitative, complementary ciliary assays can pave the way for the development of such therapies. However, existing airway epithelial cultures often show variable ciliogenesis, and ciliary function is frequently assessed using a single assay that does not capture the phenotypic heterogeneity of ciliary dysfunction. Here, we established a personalized, multi-assay in vitro platform using human nasal epithelial cells (HNECs) to assess ciliary function and therapeutic response, using primary ciliary dyskinesia (PCD) as a model disease. MethodsHNECs from 8 healthy individuals and 13 individuals with PCD carrying distinct disease-associated variants were obtained by nasal brushing. Cells were differentiated under optimized conditions, including {gamma}-secretase/Notch and BMP pathway inhibitors and a low liquid-liquid interface, to generate highly ciliated 2D epithelial cultures. Ciliary function was assessed using ciliary beat frequency, bead transport, and apical-out nasal organoid rotation assays. Therapeutic rescue was assessed in HNECs harboring DNAI1 alterations using DNAI1 mRNA-loaded lipid nanoparticles. ResultsOptimized differentiation yielded reproducibly multiciliated HNEC cultures. The multi-assay platform distinguished healthy from PCD-derived HNECs and revealed individual- and genotype-specific patterns of ciliary dysfunction not captured by a single assay. Basolateral administration of DNAI1 mRNA-loaded lipid nanoparticles resulted in partial, dose-dependent recovery of ciliary function in DNAI1-deficient HNECs. ConclusionThis study establishes a standardized, individual-specific multi-assay nasal epithelial platform for functional phenotyping of motile cilia and preclinical evaluation of emerging therapies, with demonstrated utility in PCD.

cell biology↗

bioRxiv2025

Rab27-dependent egress of SARS-CoV-2 via secretory amphisomes

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, hijacks host cellular machinery to replicate and spread. Understanding how SARS-CoV-2 reorganizes host cell architecture to accommodate this is essential for elucidating its pathogenesis and identifying therapeutic targets. While the molecular mechanisms of SARS-CoV-2 entry are well characterized, the pathways governing viral egress remain incompletely understood. Conventional approaches such as transcriptomics and electron microscopy have provided valuable insights but lack the combined spatial and molecular resolution needed to map these processes within intact cells. Here, we apply Ten-fold Robust Expansion Microscopy (TREx) to visualize SARS-CoV-2-induced remodeling of the endolysosomal system in multiciliated cells of primary human airway epithelial tissue. This approach reveals Golgi fragmentation and the formation of enlarged virus-containing organelles. Analysis of endolysosomal markers in Vero E6 cells shows that these structures are positive for CD63, Rab7, and LC3, consistent with amphisome identity. Moreover, pharmacological inhibition of Rab27-dependent amphisome-plasma membrane fusion with Nexinhib20 reduces viral infection, implicating secretory autophagy as a pathway for SARS-CoV-2 egress. These findings establish expansion microscopy as a powerful tool for spatial virology and uncover a Rab27-mediated amphisome fusion mechanism as a druggable route for SARS-CoV-2 release.

cell biology↗

bioRxiv2025

Immortalization of human nasal and bronchial airway epithelial cells for genome editing applications

In vitro air-liquid interface culture of airway epithelial cells is used as a model system to study respiratory diseases. This culture system not only overcomes the need for animal models or continuous biopsies from individuals but also enables studies of pathophysiology associated with the disease in a patient background. Human airway basal cells serve as progenitor cells for a functional pseudostratified airway epithelium composed mainly of multiciliated and secretory cells. However, due to the limited ability of basal cells to proliferate and differentiate, the long-term use of primary material in culture is restricted. This challenges research that requires genome editing. Here, we describe airway stem cells from nasal and bronchial origin immortalized by hTERT overexpression followed by polyclonal expansion. We demonstrate that this diverse panel of cell lines shows differentiation patterns similar to primary stem cells and can be used for lentiviral and CRISPR/Cas9 genome editing. These cell lines and optimized protocols facilitate airway biology research and disease phenotyping.

cell biology↗

bioRxiv2024

Cigarette smoke induces PPP1R15A via oxidative stress to modulate inflammatory cytokine production by bronchial epithelial cells

Cigarette smoke is the principal cause of chronic obstructive pulmonary disease (COPD) in western societies. It induces pulmonary inflammation and activates multiple stress signaling pathways in bronchial epithelial cells, including the integrated stress response (ISR). The ISR is triggered by phosphorylation of the translation initiation factor eIF2 in response to a variety of insults and is functionally antagonised by the eIF2 phosphatase subunit PPP1R15A. Deletion of PPP1R15A exaggerates the ISR and in many models protects against a cellular stress. However, loss of PPP1R15A was recently shown to exaggerate pulmonary fibrosis in bleomycin-challenged mice. We therefore wished to understand the role of PPP1R15A in the pulmonary response to cigarette smoke, which can cause both COPD and pulmonary fibrosis. Whole cigarette smoke rapidly activated the ISR in mucociliary differentiated human primary bronchial epithelial cells with those from COPD patients exhibiting higher levels of ISR. Acute exposure to cigarette smoke condensate induced a pro-inflammatory cytokine response accompanied by PPP1R15A expression. In this model, PPP1R15A induction was independent of eIF2 phosphorylation but was suppressed by antioxidants suggesting a response to oxidative stress. In vivo studies in mice revealed that PPP1R15A deficiency enhanced the secretion of proinflammatory cytokines in mice, suggesting a regulatory role for PPP1R15A in modulating the inflammatory response to smoke. Taken together, these data suggest that PPP1R15A is induced by cigarette smoke by an ISR-independent oxidative stress mechanism and ameliorates the pro-inflammatory cytokine response to cigarette smoke.

cell biology↗

bioRxiv2024

Small molecule-directed differentiation of submerged-cultured human nasal airway epithelia for respiratory disease modelling

Submerged cultures of undifferentiated or transformed epithelial cells are widely used in respiratory research due to their ease of use and scalability. However, these systems fail to capture the cellular diversity of the human airway epithelium. In this study, we developed an in vitro model where cryopreserved human nasal epithelial cells, collected by brushings, are differentiated under submerged conditions on standard plastic cultureware. By applying small-molecule inhibitors targeting Notch and BMP signaling, we achieved efficient differentiation of cultures containing basal, secretory, and ciliated cells. This approach supports scalable culturing of both 2D epithelial monolayers and 3D organoids, validated as (personalized) disease models for primary ciliary dyskinesia, cystic fibrosis, and respiratory syncytial virus infection. This model offers a cost-effective, scalable platform that combines the simplicity of traditional cultures with the cellular complexity of the human airway epithelium, providing a valuable tool for respiratory disease research.

cell biology↗

bioRxiv2023

Establishment and characterization of a new Pseudomonas aeruginosa infection model using 2D airway organoids and dual RNA sequencing

Pseudomonas aeruginosa is a Gram-negative bacterium that is notorious for infections in the airway of cystic fibrosis (CF) subjects. Often, these infections become chronic, leading to higher morbidity and mortality rates. Bacterial quorum sensing (QS) coordinates the expression of virulence factors and the formation of biofilms at a population level. QS has become the focus of attention for development of alternatives to antimicrobials targeting P. aeruginosa infections. However, a better understanding of the bacteria-host interaction, and the role of QS in infection, is required. In this study, we set up a new P. aeruginosa infection model, using 2D airway organoids derived from healthy and CF individuals. Using dual RNA-sequencing, we dissected their interaction, focusing on the role of QS. As expected, P. aeruginosa induced epithelial inflammation. However, QS signaling did not affect the epithelial airway cells. The epithelium influenced several infection-related processes of P. aeruginosa, including metabolic changes, induction of type 3 and type 6 secretion systems (T3SS and T6SS), and increased expression of antibiotic resistance genes, including mexXY efflux pump and several porins. Interestingly, the epithelium influenced the regulation by QS of the type 2 (T2SS) and T6SS. Finally, we compared our model with in vivo P. aeruginosa transcriptomic datasets, from samples directly isolated from the airways of CF subjects. This shows that our model recapitulates important aspects of in vivo infection, like enhanced denitrification, betaine/choline metabolism, increased antibiotic resistance, as well as an overall decrease of motility-related genes. This relevant infection model is interesting for future investigations, helping to reduce the burden of P. aeruginosa infections in CF.

microbiology↗

bioRxiv2022

Drug repurposing for Cystic Fibrosis: identification of drugs that induce CFTR-independent fluid secretion in nasal organoids

Individuals with Cystic Fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but [~]19% of pwCF cannot benefit from CFTR modulators [1]. Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here we developed a medium-throughput 384-wells screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR dependent epithelial fluid secretion. From a [~]1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including {beta}2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in Ussing chamber, and fluid secretion was not affected by TMEM16A as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for mediumscale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

cell biology↗

bioRxiv2021

Optical nanoscopy reveals SARS-CoV-2-induced remodeling of human airway cells

A better understanding of host cell remodeling by the coronavirus SARS-CoV-2 is urgently needed to understand viral pathogenesis and guide drug development. Expression profiling and electron microscopy have frequently been used to study virus-host interactions, but these techniques do not readily enable spatial, sub-cellular and molecular analysis of specific cellular compartments. Here, we use diffraction-unlimited fluorescence microscopy to analyze how SARS-CoV-2 infection exploits and repurposes the subcellular architecture of primary human airway cells. Using STED nanoscopy, we detect viral entry factors along the motile cilia of ciliated cells and visualize key aspects of the viral life cycle. Using Tenfold Robust Expansion (TREx) microscopy, we analyze the extensively remodeled three-dimensional ultrastructure of SARS-CoV-2-infected ciliated cells and uncover Golgi fragmentation, emergence of large and atypical multivesicular bodies enclosing viral proteins, ciliary clustering, and remodeling of the apical surface. These results demonstrate a broadly applicable strategy to study how viruses reorganize host cells with spatial and molecular specificity and provide new insights into SARS-CoV-2 infection in primary human cell models.

cell biology↗

bioRxiv2021

CFTR modulator response measurements in subjects with cystic fibrosis using 2D differentiated nasal epithelia converted into spheroids

Cystic Fibrosis (CF) is caused by genetic defects that impair the cystic fibrosis transmembrane conductance regulator (CFTR) channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described application of 3D airway organoids in CFTR function assays have not been fully optimal. In this report we therefore describe an alternative method of culturing nasal brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1{beta} and interleukine-1{beta}, which enabled consistent detection of CFTR modulator responses in nasal airway organoids cultures from subjects with CF.

cell biology↗