Modulation of TMEM16 proteins: a novel therapeutic approach to Cystic Fibrosis therapy

Cystic Fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. CFTR is the apical cAMP activated Cl- channel of epithelial cells. Mutations of this protein cause disruption of the electrolyte balance, leading to the formation of thick and sticky mucus in the lungs, and a variety of other problems. Cystic Fibrosis is a multifaceted disease and, despite being monogenic, its treatment is very challenging. Current therapies aim to modulate defective CFTR but are beneficial only to patients harboring a restricted number of mutations including F508del and G551D, thus excluding about 15% of the patients carrying so-called “unrescuable” mutations. Therefore, bypass therapies are of great importance to many patients who could benefit from them regardless of their genotype. This implies that with the increase in the number of mutations that are sensitive to a given therapy, the costs would diminish and the accessibility to the treatment should increase accordingly. TMEM16A (Anoctamin 1; ANO1) is the major contributor to Ca2+ activated Cl- secretion in the airways and is regarded as the ideal druggable channel to bypass defective CFTR dependent Cl- secretion. In the present work we demonstrate that TMEM16A facilitates compartmentalized Ca2+ store release in membrane microdomains. Modulation of intracellular Ca2+ by TMEM16A is necessary for proper function as well as membrane insertion of CFTR. We showed that cAMP-dependent and Ca2+ activated Cl- currents in airway epithelial cells strongly overlap due to crosstalk of intracellular signaling molecules. Intracellular Ca2+ rise mediated by TMEM16A triggers so-called Store Operated cAMP signaling (SOcAMPs) and activates Ca2+ regulated adenylyl cyclase 1 (ADCY1) and the exchange protein directly activated by cAMP (EPAC1). This crosstalk is compartmentalized and supports activation of both, TMEM16A and CFTR. Our tissue specific knockout model for TMEM16A showed a peculiar phenotype due to intracellular mucus accumulation. We found that TMEM16A is essential for ATP-dependent constitutive exocytosis of mucus in airways and intestine. Moreover, we demonstrate that TMEM16A is upregulated in inflammatory airway disease. This upregulation occurs in epithelial cells as well as airway smooth muscle. We concluded that the effect of TMEM16A on intracellular Ca2+ signaling contributes to mucus hypersecretion and airways bronchoconstriction. As further shown, inhibition of TMEM16 proteins has beneficial effects in inflammatory airway disease by inhibiting mucus release and promoting bronchorelaxation and in addition, shows anti-inflammatory effects. TMEM16 inhibitors are therefore suggested as novel potential drugs for the treatment of CF lung disease and other inflammatory airway diseases

Comparison of Shigella GMMA and glycoconjugate four-component formulations in animals

Shigellosis is leading bacterial cause of diarrhea with high prevalence in children younger than 5 years in low- and middle-income countries, and increasing number of reports of Shigella cases associated to anti-microbial resistance. No vaccines against Shigella are still licensed, but different candidates based on the O-antigen portion of lipopolysaccharides are in clinic. Generalized Modules for Membrane Antigens (GMMA) have been proposed as an alternative delivery system for the O-antigen, and a 4-component vaccine candidate (altSonflex1-2-3), containing GMMA from S. sonnei and S. flexneri 1b, 2a and 3a is being tested in a phase 1/2 clinical trial, with the aim to elicit broad protection against the most prevalent Shigella serotypes. Here, the 4-component GMMA vaccine candidate has been compared to a more traditional glycoconjugate formulation for the ability to induce functional antibodies in mice and rabbits. In mice, in the absence of Alhydrogel, GMMA induce higher IgG antibodies than glycoconjugates and stronger bactericidal titers against all Shigella serotypes. In the presence of Alhydrogel, GMMA induce O-antigen specific IgG levels similar to traditional glycoconjugates, but with a broader range of IgG subclasses, resulting in stronger bactericidal activity. In rabbits, GMMA elicit higher functional antibodies than glycoconjugates against S. sonnei, and similar responses to S. flexneri 1b, 2a and 3a, independently from the presence of Alhydrogel. Different O-antigen based vaccines against Shigella are now in clinical stage and it will be of particular interest to understand how the preclinical findings in the different animal models translate in humans

Pinofuranoxins A and B, Bioactive Trisubstituted Furanones Produced by the Invasive Pathogen Diplodia sapinea

Two new bioactive trisubstituted furanones, named pinofuranoxins A and B (1 and 2), were isolated from Diplodia sapinea, a worldwide conifer pathogen causing severe disease. Pinofuranoxins A and B were characterized essentially by NMR and HRESIMS spectra, and their relative and absolute configurations were assigned by NOESY experiments and computational analyses of electronic circular dichroism spectra. They induced necrotic lesions on Hedera helix L., Phaseolus vulgaris L., and Quercus ilex L. Compound 1 completely inhibited the growth of Athelia rolfsii and Phytophthora cambivora, while 2 showed antioomycetes activity against P. cambivora. In the Artemia salina assay both toxins showed activity inducing larval mortality

Pharmacological Inhibition and Activation of the Ca2+ Activated Cl− Channel TMEM16A

TMEM16A is a Ca2+ activated Cl- channel with important functions in airways, intestine, and other epithelial organs. Activation of TMEM16A is proposed as a therapy in cystic fibrosis (CF) to reinstall airway Cl- secretion and to enhance airway surface liquid (ASL). This CFTR-agnostic approach is thought to improve mucociliary clearance and lung function in CF. This could indeed improve ASL, however, mucus release and airway contraction may also be induced by activators of TMEM16A, particularly in inflamed airways of patients with asthma, COPD, or CF. Currently, both activators and inhibitors of TMEM16A are developed and examined in different types of tissues. Here we compare activation and inhibition of endogenous and overexpressed TMEM16A and analyze potential off-target effects. The three well-known blockers benzbromarone, niclosamide, and Ani9 inhibited both TMEM16A and ATP-induced Ca2+ increase by variable degrees, depending on the cell type. Niclosamide, while blocking Ca2+ activated TMEM16A, also induced a subtle but significant Ca2+ store release and inhibited store-operated Ca2+ influx. Niclosamide, benzbromarone and Ani9 also affected TMEM16F whole cell currents, indicating limited specificity for these inhibitors. The compounds Eact, cinnamaldehyde, and melittin, as well as the phosphatidylinositol diC8-PIP2 are the reported activators of TMEM16A. However, the compounds were unable to activate endogenous TMEM16A in HT29 colonic epithelial cells. In contrast, TMEM16A overexpressed in HEK293 cells was potently stimulated by these activators. We speculate that overexpressed TMEM16A might have a better accessibility to intracellular Ca2+, which causes spontaneous activity even at basal intracellular Ca2+ concentrations. Small molecules may therefore potentiate pre-stimulated TMEM16A currents, but may otherwise fail to activate silent endogenous TMEM16A

Evaluation of potential transfer of the pathogen Saprolegnia parasitica between farmed salmonids and wild fish

Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 634429. This publication reflects the views only of the author, and the European Commission cannot be held responsible for any use, which may be made of the information contained therein. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors would like to thank the fish farmers/staff, veterinarians, biologists and local authorities who collaborated in the sample collection.Peer reviewedPublisher PD

Where have the children with epilepsy gone? An observational study of seizure-related accesses to emergency department at the time of COVID-19

Purpose: The COVID-19 pandemic and related lockdown measures drastically changed health care and emergency services utilization. This study evaluated trends in emergency department (ED) access for seizure-related reasons in the first 8 weeks of lockdown in Italy. Methods: All ED accesses of children (<14 years of age) at two university hospitals, in Turin and Rome, Italy, between January 6, 2020 and April 21, 2020, were examined and compared with the corresponding periods of 2019. Results: During the COVID-19 lockdown period (February 23-April 21, 2020), there was a 72 % decrease in all pediatric ED accesses over the corresponding 2019 period (n = 3,395 vs n = 12,128), with a 38 % decrease in seizure-related accesses (n = 41 vs n = 66). The observed decrease of seizure-related ED accesses was not accompanied by significant changes in age, sex, type of seizure, or hospitalization rate after the ED visit. Conclusion: The COVID-19 lockdown was accompanied by a sudden decrease in seizure-related hospital emergency visits. School closure, social distancing, reduced risk of infection, and increased parental supervision are some of the factors that might have contributed to the findin

One hour-post-load plasma glucose ≥155 mg/dl in healthy glucose normotolerant subjects is associated with subcortical brain MRI alterations and impaired cognition. A pilot study

Background: Glucose alterations are associated with impaired cognition. The 1-h-post-load plasma glucose ≥155mg/dl in non-diabetic subjects confers an increased risk of cardiovascular events and diabetes. This pilot study aimed to investigate whether the 1-h-post-load plasma glucose ≥155 mg/dl negatively affects the subcortical regions of the brain and the cognitive functions. Methods: We enrolled 32 non-diabetic subjects. Patients were divided into two groups based on 1-h- post-load plasma glucose value > or < 155 mg/dl: normal glucose tolerance (NGT) 1-h-high and NGT 1-h-low subjects. All subjects underwent 3 Tesla MRI and standard neuropsychological tests. Results: NGT 1-h-high subjects showed significantly lower values of both right (4.9 ± 0.9 vs. 5.1 ± 0.9ml) and left (4.8 ± 1.1 vs. 5.1 ± 1.1ml) hippocampal hemisphere volume, while right hemisphere hippocampal diffusivity was lower in the NGT 1-h-high group (10.0 ± 0.6 vs. 10.6 ± 0.5 10−4 mm2s−1). NGT 1-h-high subjects also showed a poorer memory performance. In particular, for both Rey Auditory Verbal Learning Task (RAVLT)—immediate-recall and Free and Cued Selective Reminding Test (FCSRT)—delayed total recall, we found lower cognitive test scores in the NGT-1 h-high group (26.5 ± 6.3 and 10.4 ± 0.3, respectively). Conclusions: One-hour-post-load hyperglycemia is associated with morpho-functional subcortical brain alterations and poor memory performance tests