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INTRODUCTION One method for assessing the in vitro response to CFTR-modulating compounds is by analysis of epithelial monolayers in an Ussing chamber,where the apical and basolateral surfaces are isolated and the potential difference,short-circuit current,and transepithelial resistance can be monitored. The effect of a chloride ion gradient across airway epithelia on transepithelial chloride transport and the magnitude of CFTR modulator efficacy were examined. METHODS CFTR-mediated changes in the potential difference and transepithelial currents of primary human nasal epithelial cell cultures were quantified in Ussing chambers with either symmetrical solutions or reduced chloride solutions in the apical chamber. CFTR activity in homozygous F508del CFTR epithelia was rescued by treatment with VX-661,C4/C18,4-phenylbutyrate (4-PBA) for 24 hr at 37°C or by incubation at 29°C for 48 hr. RESULTS Imposing a chloride gradient increased CFTR-mediated and CaCC-mediated ion transport. Treatment of F508del CFTR homozygous cells with CFTR modulating compounds increased CFTR activity,which was significantly more evident in the presence of a chloride gradient. This observation was recapitulated with temperature-mediated F508del CFTR correction. CONCLUSIONS Imposing a chloride gradient during Ussing chamber measurements resulted in increased CFTR-mediated ion transport in expanded non-CF and F508del CFTR homozygous epithelia. In F508del CFTR homozygous epithelia,the magnitude of response to CFTR modulating compounds or low temperature was greater when assayed with a chloride gradient compared to symmetrical chloride,resulting in an apparent increase in measured efficacy. Future work may direct which methodologies utilized to quantify CFTR modulator response in vitro are most appropriate for the estimation of in vivo efficacy. View Publication

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein,we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence,aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2,Calu-3,and primary bronchial epithelial cell air-liquid interface cultures) and against four virus isolates. In conclusion,therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe,systemic disease. View Publication

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo,miniaturization of respiratory drug discovery assays is of pivotal importance. Thus,a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture,a pseudostratified epithelium containing basal,club,goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition,ciliary beating frequency,and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements,together with dextran permeability measurements,confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor $\beta$1 (TGF-$\beta$1) and tumor necrosis factor $\alpha$ (TNF-$\alpha$) in a concentration dependent manner. Thus,the miniaturized cellular model system enables the recapitulation of a physiologically responsive,differentiated small airway epithelium,and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery,for instance,in respect of fibrotic distal airway/lung diseases. View Publication

NAD is a multifunctional molecule involved in both metabolic processes and signaling pathways. Such signalling pathways consume NAD which is replenished via one of several biosynthesis pathways. We show that influx of NAD across the plasma membrane may be able to contribute to the homeostasis of intracellular NAD levels. Indeed,extracellular application of NAD was able to replete NAD levels that had been lowered pharmacologically using the novel drug FK866 and was also able to rescue cells from FK866-induced cell death. A marked lag between the drop in NAD levels and cell death prompted us to investigate the mechanism of cell death. We were unable to find evidence of apoptosis as assessed by immunoblotting for the Caspase 3 activation fragment and immunostaining for cytochrome C and AIF translocation. We,therefore,investigated whether autophagy was initiated by FK866. Indeed,we were able to observe the formation of LC3-positive vesicles that had fused with lysosomes in FK866-treated but not control cells. Furthermore,this autophagic phenotype could be reverted by the addition of NAD to the extracellular medium. View Publication

Cellular immunotherapeutics targeting the human papillomavirus (HPV)-16 E6 and E7 proteins have achieved limited success in HPV-positive oropharyngeal cancer (OPC). Here we have conducted proteome-wide profiling of HPV-16-specific T cell responses in a cohort of 66 patients with HPV-associated OPC and 22 healthy individuals. Unexpectedly,HPV-specific T cell responses from OPC patients were not constrained to the E6 and E7 antigens; they also recognized E1,E2,E4,E5,and L1 proteins as dominant targets for virus-specific CD8+ and CD4+ T cells. Multivariate analysis incorporating tumor staging,treatment status,and smoking history revealed that treatment status had the most significant impact on HPV-specific CD8+ and CD4+ T cell immunity. Specifically,the breadth and overall strength of HPV-specific T cell responses were significantly higher before the commencement of curative therapy than after therapy. These data provide the first glimpse of the overall human T cell response to HPV in a clinical setting and offer groundbreaking insight into future development of cellular immunotherapies for HPV-associated OPC patients. View Publication

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here,we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1,and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1),which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD,OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD. View Publication

BACKGROUND Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy,a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties. METHODOLOGY/PRINCIPAL FINDINGS Using an automated cell-based assay,we screened a collection of {\textgreater}3,500 chemicals and identified three approved drugs (perhexiline,niclosamide,amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2,which also contains mTOR as a catalytic subunit,suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline,niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2,a negative regulator of mTORC1,was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline,niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions,by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2,an upstream regulator of mTORC1. By contrast,transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition,sustained inhibition of cell growth and no selective cell killing in starvation. CONCLUSION/SIGNIFICANCE The observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis,diabetes,cardiovascular disease and cancer. View Publication

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells,while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells,accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression,resulting in gefitinib tolerance (P {\textless} 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P {\textless} 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance,by EMT introduction and ABCG2 upregulation,and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs. View Publication

The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study,we describe a highly selective and potent inhibitor of Vps34,termed VPS34-IN1,that inhibits Vps34 with 25 nM IC50 in vitro,but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes,within 1 min,without affecting the ability of class I PI3K to regulate Akt. Moreover,we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3),the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain,might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid {\~{}}50-60{\%} loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore,class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by {\~{}}40{\%}. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity {\~{}}80-90{\%}. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product,PtdIns(3,4,5)P3 into PtdIns(3)P,via the sequential actions of the PtdIns 5-phosphatases [SHIP1/2 (Src homology 2-domain-containing inositol phosphatase 1/2)] and PtdIns 4-phosphatase [INPP4B (inositol polyphosphate 4-phosphatase type II)]. VPS34-IN1 will be a useful probe to delineate physiological roles of the Vps34. Monitoring SGK3 phosphorylation and activity could be employed as a biomarker of Vps34 activity,in an analogous manner by which Akt is used to probe cellular class I PI3K activity. Combining class I (GDC-0941) and class III (VPS34-IN1) PI3K inhibitors could be used as a strategy to better analyse the roles and regulation of the elusive class II PI3K. View Publication

The stimulator of interferon genes (STING) pathway has been proposed as a key regulator of gastrointestinal homeostasis and inflammatory responses. Although STING reportedly protects against gut barrier damage and graft-versus-host disease (GVHD) after major histocompatibility complex (MHC)-mismatched allogeneic hematopoietic stem cell transplantation (aHSCT),its effect in clinically relevant MHC-matched aHSCT is unknown. Studies here demonstrate that STING signaling in nonhematopoietic cells promoted MHC-matched aHSCT-induced GVHD and that STING agonists increased type I interferon and MHC I expression in nonhematopoietic mouse intestinal organoid cultures. Moreover,mice expressing a human STING allele containing three single-nucleotide polymorphisms associated with decreased STING activity also developed reduced MHC-matched GVHD,demonstrating STING's potential clinical importance. STING-/- recipients experienced reduced GVHD with transplant of purified donor CD8+ T cells in both MHC-matched and MHC-mismatched models,reconciling the seemingly disparate results. Further examination revealed that STING deficiency reduced the activation of donor CD8+ T cells early after transplant and promoted recipient MHC class II+ antigen-presenting cell (APC) survival. Therefore,APC persistence in STING pathway absence may account for the increased GVHD mediated by CD4+ T cells in completely mismatched recipients. In total,our findings have important implications for regulating clinical GVHD by targeting STING early after aHSCT and demonstrate that an innate immune pathway has opposing effects on the outcome of aHSCT,depending on the donor/recipient MHC disparity. View Publication

As SARS-CoV-2 infections and death counts continue to rise,it remains unclear why some individuals recover from infection,whereas others rapidly progress and die. Although the immunological mechanisms that underlie different clinical trajectories remain poorly defined,pathogen-specific antibodies often point to immunological mechanisms of protection. Here,we profiled SARS-CoV-2-specific humoral responses in a cohort of 22 hospitalized individuals. Despite inter-individual heterogeneity,distinct antibody signatures resolved individuals with different outcomes. Although no differences in SARS-CoV-2-specific IgG levels were observed,spike-specific humoral responses were enriched among convalescent individuals,whereas functional antibody responses to the nucleocapsid were elevated in deceased individuals. Furthermore,this enriched immunodominant spike-specific antibody profile in convalescents was confirmed in a larger validation cohort. These results demonstrate that early antigen-specific and qualitative features of SARS-CoV-2-specific antibodies point to differences in disease trajectory,highlighting the potential importance of functional antigen-specific humoral immunity to guide patient care and vaccine development. View Publication

Perhexiline,2-(2,2-dicyclohexylethyl)piperidine,was originally developed as an anti-anginal drug in the 1970s. Despite its success,its use diminished due to the occurrence of poorly understood side effects including neurotoxicity and hepatotoxicity in a small proportion of patients. Recently,perhexiline's mechanism of action and the molecular basis of its toxicity have been elucidated. Perhexiline reduces fatty acid metabolism through the inhibition of carnitine palmitoyltransferase,the enzyme responsible for mitochondrial uptake of long-chain fatty acids. The corresponding shift to greater carbohydrate utilization increases myocardial efficiency (work done per unit oxygen consumption) and this oxygen-sparing effect explains its antianginal efficacy. Perhexiline's side effects are attributable to high plasma concentrations occurring with standard doses in patients with impaired metabolism due to CYP2D6 mutations. Accordingly,dose modification in these poorly metabolizing patients identified through therapeutic plasma monitoring can eliminate any significant side effects. Herein we detail perhexiline's pharmacology with particular emphasis on its mechanism of action and its side effects. We discuss how therapeutic plasma monitoring has led to perhexiline's safe reintroduction into clinical practice and how recent clinical data attesting to its safety and remarkable efficacy led to a renaissance in its use in both refractory angina and chronic heart failure. Finally,we discuss the application of pharmacogenetics in combination with therapeutic plasma monitoring to potentially broaden perhexiline's use in heart failure,aortic stenosis,and other cardiac conditions. View Publication