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The unit for the concentration of peptide in the proliferation study was incorrectly reported in the Results and Methods sections and the Figure 4 legend. The corrected sentences, with sections indicated, are below. Results Evaluation of Tα-1 sequence and its effect on proliferation and apoptosis of MCF-7 breast cancer cells. Therefore, we plated MCF-7 at low density on 96-well plates suitable for confocal high-content imaging and evaluated cell proliferation for 72 hours following treatment with Tα-1 (100 μM) or scrambled peptide (100 μM). Methods Proliferation study. MCF-7 cells were plated at low density (10,000 cells/well) on 96-well plates suitable for high-content imaging. After 6 hours, cells were treated with the scrambled peptide (100 μM) or with Tα-1 (100 μM). Figure 4 legend (A) Dot plot showing proliferation of MCF-7 cells after 72-hour treatment with Tα-1 (100 μM) or scrambled peptide (100 μM, control). (B) Dot plot showing the number of apoptotic MCF-7 cells after 72-hour treatment with Tα-1 (100 μM) or scrambled peptide (100 μM, control). The article has been updated to reflect these changes. The authors regret the errors.

More than 2000 CFTR mutations have been identified so far, but only few of them are clearly defined as CF-causing based on functional studies. We present a case of a rare mutation, the G970D, that has been shown using transfected cDNA in HEK293 cells to be sensitive to Ivacaftor. However, a similar missense mutant, G970R in the same codon, was found to be sensitive to potentiators in vitro, but not in vivo due to splicing alteration. Thus, we used several basic research methodologies to evaluate if this patient was eligible for treatment with Ivacaftor. Materials and methods 1) nasal epithelial cells (HNEC) were collected from patients to evaluate the effect of mutations on splicing by RT-PCR assay, 2) HNEC were expanded and polarized for evaluation of CFTR function by Ussing chamber system 3) the use of a minigene system was used to confirm in vitro the splicing pattern. Results Firstly, we used in silico tool to predict the physio-pathological effect of mutations, confirming that the G970R completely abolishes the canonical 5’ splice donor site of exon 17 resulting in a likely retention of intron 17. On the contrary, the G970D predicted not to affect splicing. This prediction was confirmed by RT-PCR analysis of mRNA extracted from HNEC cells and from in vitro minigene assay. Finally, the functional behavior of CFTR, from HNEC bearing the G970D, was evaluated by short-circuit recordings. The cells responded to cAMP agonist with an increase in trans-epithelial current and this current was nearly doubled by stimulation with Ivacaftor. Moreover, in cells that were also incubated with VX-809 (1 μM) for 24 hours, CFTR function was significantly enhanced, with a proportional increase of both cAMP- and potentiator-dependent responses. Conclusions Our results show that the G970R actually disrupts the RNA splicing, thus leading to a severely altered CFTR protein. This event explains the lack of success of treatment with potentiator. In contrast, the G970D does not alter RNA splicing. The resulting G970D mutation affects the gating and trafficking of the CFTR channel that can be targeted with VX-770 and VX-809. These results represent the evidence needed to justify the treatment of the patient with these drugs. Finally, our study is an interesting example of the precision medicine approach by emphasizing the role of appropriate in vitro studies, in this case focused on RNA analysis, to fully characterize the effects of rare CFTR mutations. We thank Telethon Foundation (TMLGCBX16TT) and Ministero della Salute (Rome, Italy) L.548/93 for the regional research funding quote 2008-2015.

Questo capitolo presenta la sintesi di uno studio a carattere multidisciplinare, condotto dai membri di numerose unità operative afferenti al progetto “GEOBASI” su campioni di suolo del sito “pilota” di Muravera, ai fini di cogliere le dinamiche interne del suolo e quelle a scala territoriale, e fornire un contributo all’individuazione di appropriati strumenti di indagine e di intervento. I risultati, di seguito riportati, sono scaturiti dalle varie analisi di laboratorio, condotte con un approccio metodologico integrato e multiscala su campioni appartenenti a ciascuno dei diversi orizzonti genetici di cui si compone ogni profilo pedologico ed, ove possibile, anche del substrato roccioso. In tal modo si è cercato di garantire, a partire dal campionamento, un rigore di metodi in grado di tenere in dovuto conto la specificità delle proprietà e dei meccanismi di cui il suolo è sede. La caratterizzazione della composizione chimica degli elementi totali, sia maggiori sia in tracce, tramite spettrometria di fluorescenza ai raggi X è stata curata dalla UO di Siena (Protano G., Nannoni F., Pizzetti E., Riccobono F., Rossi S.), con un contributo specifico dell’UO afferente all’ENEA per quanto riguarda l’arsenico. I risultati ottenuti mediante XRF sono stati messi in relazione con i principali litotipi presenti nell’area di studio e con i diversi orizzonti pedologici. Sono stati inoltre confrontati con quelli derivanti dall’estrazione degli elementi pseudototali in acqua regia da parte dell’UO di Pavia (Sacchi E., Garzetti F., Fornelli Genot S., Vannucci R.), che discute la validità di applicabilità dei due diversi metodi, mettendo altresì in luce le difficoltà di applicazione della normativa vigente in materia di determinazione delle soglie di contaminazione di diverse matrici ambientali. I contributi successivi del presente capitolo analizzano attraverso molteplici sfaccettature ed approcci analitici il variegato tema della mobilità degli elementi nel sistema suolo. Si passa dall’analisi della frazione biodisponibile dei metalli tramite estrazione in EDTA al confronto con i tenori pseudototali (Sacchi E., Fornelli Genot S., Garzetti F., Vannucci R. dell’UO di Pavia) ed allo studio dei processi di mobilità, frazionamento e speciazione attraverso le estrazioni sequenziali (UO dell’ENEA: Angelone M., Armiento G., Caci E., Carucci V., Cremisini C., Crovato C.), attraverso procedure sempre applicate ancora al campione totale. Attraverso tecniche d’indagine micromorfologica e microanalitica in microscopia ottica ed elettronica e l’ausilio di analisi spettrometriche con l’utilizzo di sorgente laser si evidenziano alcuni meccanismi di ritenzione o mobilizzazione degli elementi con carattere “puntuale” all’interno di determinati profili e/o orizzonti pedologici (Scarciglia F., De Rosa R., Barca D., Apollaro C., Caruso A.M., Vecchio G. dell’UO di Cosenza e Vacca A. dell’unità di Cagliari). Vengono poi proposti un’analisi della distribuzione spaziale di alcuni isotopi radioattivi nonché l’applicazione di un metodo per stimare i tassi di erosione o accumulo sulla base dell’attività del 137Cs (UO di Roma Tre: Tuccimei P., Salzano R., Soligo M., Primavera P., Delitala M.C., Taddeucci A.) ed uno studio di dettaglio sul rapporto isotopico del ferro con la proposta di una metodologia innovativa (Castorina F., Masi U., Voltaggio M. dell’UO di Roma “La Sapienza”). Questo lavoro mette in luce le difficoltà di applicazione della normativa vigente, qualora si proceda alla misura ed alla discussione delle concentrazioni e distribuzioni degli elementi nei suoli dell’area rappresentata nel Foglio 549 Muravera della Carta Geologica d’Italia a scala 1:50.000. L’approccio multidisciplinare adottato mostra in modo chiaro i limiti dei metodi d’indagine sinora proposti dalla legislazione ambientale ed applicati dagli operatori del territorio: i limiti, cioè, sia di un campionamento del solo orizzonte di superficie, senza tener conto invece dei differenti orizzonti genetici (ovvero prodotti da specifici processi di pedogenesi) di cui consta un profilo di suolo, sia della sola analisi degli elementi totali, a scapito della valutazione dei contributi offerti dalle diverse sub-“matrici” costituenti ogni singolo orizzonte (scheletro minerale, matrice pedogenetica s.s., pellicole di argilla illuviale, sostanza organica, segregazioni di ossidi di ferro, ecc.).

Questo capitolo presenta la sintesi di uno studio a carattere multidisciplinare, condotto dai membri di numerose unità operative afferenti al progetto “GEOBASI” su campioni di suolo del sito “pilota” di Muravera, ai fini di cogliere le dinamiche interne del suolo e quelle a scala territoriale, e fornire un contributo all’individuazione di appropriati strumenti di indagine e di intervento. I risultati, di seguito riportati, sono scaturiti dalle varie analisi di laboratorio, condotte con un approccio metodologico integrato e multiscala su campioni appartenenti a ciascuno dei diversi orizzonti genetici di cui si compone ogni profilo pedologico ed, ove possibile, anche del substrato roccioso. In tal modo si è cercato di garantire, a partire dal campionamento, un rigore di metodi in grado di tenere in dovuto conto la specificità delle proprietà e dei meccanismi di cui il suolo è sede. La caratterizzazione della composizione chimica degli elementi totali, sia maggiori sia in tracce, tramite spettrometria di fluorescenza ai raggi X è stata curata dalla UO di Siena (Protano G., Nannoni F., Pizzetti E., Riccobono F., Rossi S.), con un contributo specifico dell’UO afferente all’ENEA per quanto riguarda l’arsenico. I risultati ottenuti mediante XRF sono stati messi in relazione con i principali litotipi presenti nell’area di studio e con i diversi orizzonti pedologici. Sono stati inoltre confrontati con quelli derivanti dall’estrazione degli elementi pseudototali in acqua regia da parte dell’UO di Pavia (Sacchi E., Garzetti F., Fornelli Genot S., Vannucci R.), che discute la validità di applicabilità dei due diversi metodi, mettendo altresì in luce le difficoltà di applicazione della normativa vigente in materia di determinazione delle soglie di contaminazione di diverse matrici ambientali. I contributi successivi del presente capitolo analizzano attraverso molteplici sfaccettature ed approcci analitici il variegato tema della mobilità degli elementi nel sistema suolo. Si passa dall’analisi della frazione biodisponibile dei metalli tramite estrazione in EDTA al confronto con i tenori pseudototali (Sacchi E., Fornelli Genot S., Garzetti F., Vannucci R. dell’UO di Pavia) ed allo studio dei processi di mobilità, frazionamento e speciazione attraverso le estrazioni sequenziali (UO dell’ENEA: Angelone M., Armiento G., Caci E., Carucci V., Cremisini C., Crovato C.), attraverso procedure sempre applicate ancora al campione totale. Attraverso tecniche d’indagine micromorfologica e microanalitica in microscopia ottica ed elettronica e l’ausilio di analisi spettrometriche con l’utilizzo di sorgente laser si evidenziano alcuni meccanismi di ritenzione o mobilizzazione degli elementi con carattere “puntuale” all’interno di determinati profili e/o orizzonti pedologici (Scarciglia F., De Rosa R., Barca D., Apollaro C., Caruso A.M., Vecchio G. dell’UO di Cosenza e Vacca A. dell’unità di Cagliari). Vengono poi proposti un’analisi della distribuzione spaziale di alcuni isotopi radioattivi nonché l’applicazione di un metodo per stimare i tassi di erosione o accumulo sulla base dell’attività del 137Cs (UO di Roma Tre: Tuccimei P., Salzano R., Soligo M., Primavera P., Delitala M.C., Taddeucci A.) ed uno studio di dettaglio sul rapporto isotopico del ferro con la proposta di una metodologia innovativa (Castorina F., Masi U., Voltaggio M. dell’UO di Roma “La Sapienza”). Questo lavoro mette in luce le difficoltà di applicazione della normativa vigente, qualora si proceda alla misura ed alla discussione delle concentrazioni e distribuzioni degli elementi nei suoli dell’area rappresentata nel Foglio 549 Muravera della Carta Geologica d’Italia a scala 1:50.000. L’approccio multidisciplinare adottato mostra in modo chiaro i limiti dei metodi d’indagine sinora proposti dalla legislazione ambientale ed applicati dagli operatori del territorio: i limiti, cioè, sia di un campionamento del solo orizzonte di superficie, senza tener conto invece dei differenti orizzonti genetici (ovvero prodotti da specifici processi di pedogenesi) di cui consta un profilo di suolo, sia della sola analisi degli elementi totali, a scapito della valutazione dei contributi offerti dalle diverse sub-“matrici” costituenti ogni singolo orizzonte (scheletro minerale, matrice pedogenetica s.s., pellicole di argilla illuviale, sostanza organica, segregazioni di ossidi di ferro, ecc.).

Cyclic adenosine 3′,5′-monophosphate (cAMP)–elevating agents, such as β 2 -adrenergic receptor (β 2 -AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by β 2 -ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a β 2 -AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL-dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM-MSC) lines from wild type and Rankl−/− mice, and found that Rankl−/− BM-MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl−/− BM-MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM-MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL-defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL-dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics.

The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high (~3%) ionocyte abundance in ex vivo nasal samples, with no difference between CF and control individuals. In bronchi, ionocytes instead appeared very rarely as previously reported, thus suggesting a possible proximal–distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture, which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure, we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions, thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern.

We identified a small molecule that, at picomolar concentrations, rescues mutant CFTR chloride channel from protein degradation., F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC50 of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies.

NEW FINDINGS What is the central question of this study? What is the precise subcellular localization of the epithelial sodium channel (ENaC) in human airway epithelium? What is the main finding and its importance? ENaC protein has an unexpected localization in the peripheral region of the apical membrane of bronchial epithelial cells, very close to tight junctions. This may be important for the mechanism of Na+ absorption ABSTRACT: The epithelial sodium channel (ENaC) has a key role in absorbing fluid across the human airway epithelium. Altered activity of ENaC may perturb the process of mucociliary clearance, thus impairing the innate defence mechanisms against microbial agents. The proteins forming ENaC are present on the apical membrane of the epithelium. However, their precise localization is unknown. In the present study, we used two antibodies recognizing the α and β ENaC subunits. Both antibodies revealed a restricted localization of ENaC in the peripheral region of the apical membrane of cultured bronchial epithelial cells, close to but not overlapping with tight junctions. In contrast, the cystic fibrosis transmembrane conductance regulator chloride channel was more diffusely expressed on the whole apical membrane. Modulation of ENaC activity by aprotinin or elastase resulted in a decrease or increase in the peripheral localization, respectively. Our results suggest that sodium absorption is mainly occurring close to tight junctions where this cation may be rapidly expelled by the Na+ /K+ pump present in lateral membranes. This arrangement of channels and pumps may limit Na+ build-up in other regions of the cells.

Key points Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4 stimulation leads to CFTR channel activation. Abstract TMEM16A is a Ca2+ -activated Cl- channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl- channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl- secretory processes.

Gnathodiaphyseal dysplasia (GDD) is an autosomal dominant syndrome characterized by frequent bone fractures at a young age, bowing of tubular bones and cemento-osseus lesions of the jawbones. Anoctamin 5 (ANO5) belongs to the anoctamin protein family that includes calcium-activated chloride channels. However, recent data together with our own experiments reported here add weight to the hypothesis that ANO5 may not function as calcium-activated chloride channel. By sequencing the entire ANO5 gene coding region and untranslated regions in a large Italian GDD family, we found a novel missense mutation causing the p.Thr513Ile substitution. The mutation segregates with the disease in the family and has never been described in any database as a polymorphism. To date, only two mutations on the same cysteine residue at position 356 of ANO5 amino-acid sequence have been described in GDD families. As ANO5 has also been found to be mutated in two different forms of muscular dystrophy, the finding of this third mutation in GDD adds clues to the role of ANO5 in these disorders.