Biology subjects

Whan, R. M.

Publications and source records attributed to Whan, R. M..

3 recordsLinked to original sources

bioRxiv2021

Molecular dynamics and theratyping in airway and gut organoids reveal R352Q-CFTR conductance defect

A significant challenge to making targeted CFTR modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q - with potential role contributing to intrapore chloride ion permeation - in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using ion transport assay and forskolin-induced swelling (FIS) assay respectively. Two CFTR potentiators (VX-770, GLPG1837) and a corrector (VX-809) were tested. Data from R352Q-CFTR were compared to that of participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function which was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics (MD) simulations of R352Q-CFTR were carried out which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico MD simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

molecular biology↗

bioRxiv2021

Ageing impairs the airway epithelium defence response to SARS-CoV-2

Age-dependent differences in the clinical response to SARS-CoV-2 infection is well-documented1-3 however the underlying molecular mechanisms involved are poorly understood. We infected fully differentiated human nasal epithelium cultures derived from healthy children (1-12 years old), young adults (26-34 years old) and older adults (56-62 years old) with SARS-COV-2 to identify age-related cell-intrinsic differences that may influence viral entry, replication and host defence response. We integrated imaging, transcriptomics, proteomics and biochemical assays revealing age-related changes in transcriptional regulation that impact viral replication, effectiveness of host responses and therapeutic drug targets. Viral load was lowest in infected older adult cultures despite the highest expression of SARS-CoV-2 entry and detection factors. We showed this was likely due to lower expression of hijacked host machinery essential for viral replication. Unlike the nasal epithelium of young adults and children, global host response and induction of the interferon signalling was profoundly impaired in older adults, which preferentially expressed proinflammatory cytokines mirroring the "cytokine storm" seen in severe COVID-194,5. In silico screening of our virus-host-drug network identified drug classes with higher efficacy in older adults. Collectively, our data suggests that cellular alterations that occur during ageing impact the ability for the host nasal epithelium to respond to SARS-CoV-2 infection which could guide future therapeutic strategies.

immunology↗

bioRxiv2020

Novel antioxidant therapy with the immediate precursor to glutathione, γ-glutamylcysteine (GGC), ameliorates LPS-induced cellular stress in an in vitro cystic fibrosis model

IntroductionGlutathione deficiency and chronic bacterial inflammation exacerbates the oxidative stress damage to airways in cystic fibrosis. Improvements to current antioxidant therapeutic strategies are needed. Dietary supplement, {gamma}-glutamylcysteine (GGC), the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against Pseudomonas aeruginosa derived lipopolysaccharide (LPS), a prominent factor in mediating both bacterial virulence and host responses, in CF remains unassessed. MethodsPrimary F508del/F508del mucociliary differentiated bronchial and nasal epithelial cells were created to model LPS-induced oxidative stress and inflammation of CF. The proteomic signature of GGC treated cells was resolved by qLC-MS/MS. Parameters including cell redox state (glutathione, ROS), anti-inflammatory mediators (IL-8, IDO-1) and cellular health (membrane integrity, stress granule formation and cell viability) were assayed. ResultsProteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced ROS and stress granule formation, while significantly increasing intracellular glutathione levels and improving epithelial cell barrier integrity. Moreover, we compared the effect of GGC with thiols NAC and glutathione on cell viability. GGC was the only thiol that increased cell viability; protecting cells against LPS induced cell death. Both therapeutic and prophylactic treatments were successful. ConclusionTogether, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress related damage to airways in Cystic Fibrosis.

cell biology↗