Treatment of Polarized Cystic Fibrosis Airway Cells With HGF Prevents VX-661-Rescued F508del-CFTR Destabilization Caused by Prolonged Co-exposure to VX-770

Cystic fibrosis (CF), the most common inherited disease in Caucasians, is caused by mutations in CFTR, the most frequent of which is F508del. F508del causes ER retention and degradation of the mutant CFTR protein, but also defective channel gating and decreased half-life at the plasma membrane. Despite the recent successes with small molecule CFTR modulator drugs, the folding-corrector/gating-potentiator drug combinations approved for CF individuals carrying F508del-CFTR have sometimes produced severe side effects. Previously, we showed that a prolonged, 15-days treatment of polarized bronchial epithelial monolayers with the VX-809+VX-770 combination resulted in epithelial dedifferentiation effects that we found were caused specifically by VX-809. Moreover, prolonged VX-770 exposure also led to the destabilization of VX-809-rescued F508del-CFTR. Notably, co-treatment with the physiological factor HGF prevented VX-809-mediated epithelial differentiation and reverted the destabilizing effect of VX-770 on VX-809-rescued CFTR. Here, we show that prolonged treatment with VX-661, a second-generation corrector developed based on VX-809 structure, does not perturb epithelial integrity of polarized bronchial epithelial monolayers. Yet, its efficacy is still affected by co-exposure to VX-770, the potentiator present in all VX-661-containing combination therapies approved in the United States and Europe for treatment of F508del-CFTR carriers. Importantly, we found that co-treatment with HGF still ameliorated the impact of VX-770 in F508del-CFTR functional rescue by VX-661, without increasing cell proliferation (Ki-67) or altering the overall expression of epithelial markers (ZO-1, E-cadherin, CK8, CK18). Our findings highlight the importance of evaluating the cellular effects of prolonged exposure to CFTR modulators and suggest that the benefits of adding HGF to current combination therapies should be further investigated.This work was supported by the Grant PTDC/BIA-CEL/28408/2017 (to PJ and PM) and Center Grant UID/MULTI/04046/2019 to BioISI, both from the Portuguese Fundação para a Ciência e a Tecnologia.info:eu-repo/semantics/publishedVersio

YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1

(This article belongs to the Section Molecular Biology)Recent developments in CFTR modulator drugs have had a significant transformational effect on the treatment of individuals with Cystic Fibrosis (CF) who carry the most frequent F508del- CFTR mutation in at least one allele. However, the clinical effects of these revolutionary drugs remain limited by their inability to fully restore the plasma membrane (PM) stability of the rescued mutant channels. Here, we shed new light on the molecular mechanisms behind the reduced half-life of rescued F508del-CFTR at the PM of airway cells. We describe that YES1 protein kinase is enriched in F508del-CFTR protein PM complexes, and that its interaction with rescued channels is mediated and dependent on the adaptor protein YAP1. Moreover, we show that interference with this complex, either by depletion of one of these components or inhibiting YES1 activity, is sufficient to significantly improve the abundance and stability of modulator-rescued F508del-CFTR at the surface of airway cells. In addition, we found that this effect was mediated by a decreased phosphorylation of the scaffold protein SHC1, a key regulator of MAPK pathway activity. In fact, we showed that depletion of SHC1 or inhibition of MAPK pathway signaling was sufficient to improve rescued F508del-CFTR surface levels, whereas an ectopic increase in pathway activation downstream of SHC1, through the use of a constitutively active H-RAS protein, abrogated the stabilizing effect of YES1 inhibition on rescued F508del-CFTR. Taken together, our findings not only provide new mechanistic insights into the regulation of modulator-rescued F508del-CFTR membrane stability, but also open exciting new avenues to be further explored in CF research and treatment.Funding: This work was supported by the Grant PTDC/BIA-CEL/28408/2017 (to PJ and PM) and Center Grant UID/MULTI/04046/2019 to BioISI from the Portuguese Fundação para a Ciência e a Tecnologia.info:eu-repo/semantics/publishedVersio

Trends in mortality from pulmonary tuberculosis before and after antibiotics in the Portuguese sanatorium Carlos Vasconcelos Porto (1918-1991): archival evidence and its paleopathological relevance

The comparative study of patients’ profiles and outcomes from pulmonary tuberculosis (TB), before and after the discovery of antibiotic therapy, using sanatoria archives is an unexplored approach in paleopathology. Although higher mortality rates are assumed before chemotherapy, scarce information exists regarding the disease’s duration in institutionalized patients and to what extent tuberculous sufferers lived enough to develop skeletal lesions. To fill this gap, 315 clinical files from the former male Sanatorium Carlos Vasconcelos Porto, located in São Brás de Alportel, Portugal, were studied. Two periods of hospitalization were considered: 1931-1944 (n=128, Group 1) and 1955-1961 (n=187, Group 2). The average duration of hospitalization (350.3 days for Group 1 and 371.8 for Group 2) and the crude mortality (18.2% and 11.2%, respectively in Groups 1 and 2) did not differ significantly between groups. However, Cox’s regression revealed significant differences between survival curves, after adjusting for age at admission (14-74 years old), with pre-chemotherapy patients presenting a higher risk of dying during hospitalization (p=0.37, hazard ratio=1.94, IC95%=1.03-3.63). This study also confirms poorer prognoses for pulmonary tuberculosis sufferers hospitalized in sanatoria before antibiotics and reveals that a significant number of patients survived enough time to develop bone lesions

Implementing RRI in a Research and Innovation Ecosystem

Funding Information: We acknowledge the support from the European Commission through the ETHNA System project (Grant Nº 872360). Partial financial support was also provided by Portuguese FCT program UIDB/00066/2020 (CTS – Center of Technology and Systems). Publisher Copyright: © 2023, The Author(s).New organizational forms are emerging today at all levels of society, and more and more research is conducted in dynamic collaborative networks or ecosystems. Unlike traditional research centers, these new types of organization are very dynamic, with fluid boundaries, and volatile in terms of membership. This characteristic requires that more attention be paid to research ethics and RRI. This work reports on an implementation process carried out in a research and innovation ecosystem according to the principles and guidelines proposed by the ETHNA project. The process, its barriers and drivers are described, and finally, learned lessons and recommendations are presented.publishersversionpublishe

Biofilm reactor technology as an alternative to control fungal filamentous bulking caused by Galactomyces geotrichum

The present work aims to evaluate a strategy for solving fungal filamentous bulking caused by Galactomyces geotrichum. For this study, four sequencing batch reactors (SBR) fed with acetate were operated without (SBR1) and with support for biofilm growth [5 % (SBR2), 10 % (SBR3) and 20 % (SBR4) of the reactor volume]. The results demonstrated an overabundance of G. geotrichum in the SBR operating just with suspended biomass. The incorporation of an optimized amount of support for biofilm growth (10 %) seemed to suppress the overgrowth of the G. geotrichum filaments probably due to the combined effect of a decreased biomass loading and an increased shear force

Nature-based solutions contribution for urban resilience and sustainability

info:eu-repo/semantics/publishedVersio

Comprehensive review on the interaction between natural compounds and brain receptors: Benefits and toxicity

Given their therapeutic activity, natural products have been used in traditional medicines throughout the centuries. The growing interest of the scientific community in phytopharmaceuticals, and more recently in marine products, has resulted in a significant number of research efforts towards understanding their effect in the treatment of neurodegenerative diseases, such as Alzheimer's (AD), Parkinson (PD) and Huntington (HD). Several studies have shown that many of the primary and secondary metabolites of plants, marine organisms and others, have high affinities for various brain receptors and may play a crucial role in the treatment of diseases affecting the central nervous system (CNS) in mammalians. Actually, such compounds may act on the brain receptors either by agonism, antagonism, allosteric modulation or other type of activity aimed at enhancing a certain effect. The current manuscript comprehensively reviews the state of the art on the interactions between natural compounds and brain receptors. This information is of foremost importance when it is intended to investigate and develop cutting-edge drugs, more effective and with alternative mechanisms of action to the conventional drugs presently used for the treatment of neurodegenerative diseases. Thus, we reviewed the effect of 173 natural products on neurotransmitter receptors, diabetes related receptors, neurotrophic factor related receptors, immune system related receptors, oxidative stress related receptors, transcription factors regulating gene expression related receptors and blood-brain barrier receptors.The author A.R.S. is grateful to the authors C.G. and J.M.R for the scientific assistance and suggestions shared throughout the supervision of her B.Sc. final project. The author A.R.S. also acknowledges the Department of Biology (DB) and the Centro de Biologia Molecular e Ambiental [Centre of Molecular and Environmental Biology] (CBMA) from School of Sciences (EC), University of Minho (UM), Braga, Portugal, by providing all the conditions leading to the B.Sc. in Biochemistry. The corresponding author C.G. is grateful to Fundação para a Ciência e a Tecnologia [Foundation for Science and Technology, FCT I.P.] (FCT I.P.) for the FCT Investigator (IF/01332/2014/CP1255/CT0001). The author J.M.R. acknowledges the CBMA and the Instituto de Ciência e Inovação para a Biosustentabilidade [Institute of Science and Innovation for Biosustainability] (IBS) from University of Minho (UM), Braga, Portugal, where he recently carried out his activities as invited assistant researcher and professor. The author J.M.R. is also grateful to the Laboratório Associado para a Química Verde (LAQV) [Green Chemistry Laboratory] from REQUIMTE – Rede de Química e Tecnologia [REQUIMTE – Chemistry and Technology Network], as well as to the Department of Chemistry and Biochemistry (DCB) from the Faculty of Sciences from University of Porto (FCUP), Porto, Portugal, where he is currently researcher. Regarding to the author J.M.R., this work was supported by the strategic programmes UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) (Research project entitled “EcoAgriFood: Innovative green products and processes to promote Agri-Food BioEconomy”) and PTDC/SAUNUT/30448/2017 (POCI-01-0145-FEDER-030448) (Research project entitled “Poly4CD: Action of Dietary Polyphenols in Preventing Celiac Disease”) funded by Portuguese national funds through Fundação para a Ciência e Tecnologia [Foundation for Science and Technology] (FCT-I.P.)/Ministério da Ciência, Tecnologia e Ensino Superior [Ministry of Science, Technology and Higher Education] (MCTES), and Fundo Europeu de Desenvolvimento Regional [European Regional Development Fund] (FEDER), under the scope of the COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) [COMPETE2020 – Competitiveness and Internationalization Operational Program] and by COST Action 18101 “SOURDOMICS”, supported by COST (European Cooperation in Science and Technology). C. G. and C. D.-M. are grateful for the financial support from FCT/MCTES through national funds (UID/QUI/50006/2019). C. G. and C. D.-M. would also like to thank the EU and FCT for funding through the projects: DESignBIOtecHealth - New Technologies for three Health Challenges of Modern Societies: Diabetes, Drug Abuse and Kidney Diseases (Portugal2020, Norte-01-0145-FEDER-000024) and project PTDC/OCE-ETA/30240/2017- SilverBrain - From sea to brain: Green neuroprotective extracts for nanoencapsulation and functional food production (POCI-01-0145-FEDER-030240); and to the REQUIMTE for the project “Sea_4_Brain_Food”. All the authors acknowledge the CBMA and IB-S by the financial support provided specifically for this open access publication.info:eu-repo/semantics/publishedVersio

Ciência, Tecnologia e Desenvolvimento Industrial no Portugal Oitocentista. O caso dos lanifícios do Alentejo

Na primeira parte deste trabalho pretendemos analisar até que ponto Portugal estava desfasado em termos científicos e tecnológicos daquilo que era feito nos outros países da Europa, procurando verificaras possibilidades de circulação e transmissão do conhecimentos e a aproximação que, ao longo do período em análise, se verificou entre o mundo científico e a actividade económica e política. Análise que passa necessariamente pela acção das instituições, como as academias, as sociedades e as associações, de estruturas administrativas, como a Junta do Comércio ou o Ministério das Obras, Públicas Comércio e Industria, e pelo papel desempenhado pelos homens ligados a essas instituições ou às estruturas administrativas- Na segunda parte do trabalho analisamos a indústria dos lanifícios em dois distritos do Alentejo, Évora e Portalegre, procurando verificar quer a forma como tradicionalmente se organizava esta actividade, quer a forma como os discursos sobre o progresso e as vantagens de aplicação de maquinismos ou de princípios científicos e técnicos foram entendidos e aplicados pelos fabricantes e pelos industriais que na região alentejana se dedicavam a esta actividade

Microwave- and Ultrasound-Assisted Extraction of Cucurbita pepo Seeds: A Comparison Study of Antioxidant Activity, Phenolic Profile, and In-Vitro Cells Effects

Nowadays there is a growing demand for nutraceuticals to prevent diseases related to redox imbalances, such as atherosclerosis and diabetes, being crucial to search for new matrixes rich in bioactive compounds. This work aims to characterize the value-added compounds extracted from Curcubita pepo seeds using green methodologies, namely microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE), employing water as an extracting solvent for two ratios (condition 1: 1 mg/20 mL; condition 2: 2.5 mg/20 mL). The extract with the best antioxidant/antiradical activity in FRAP (71.09 μmol FSE/g DW) and DPPH (5.08 mg TE/g DW) assays was MAE condition 1, while MAE condition 2 exhibited the highest activity in the ABTS assay (13.29 mg AAE/g DW) and TPC (16.89 mg GAE/g DW). A remarkable scavenging capacity was observed, particularly for HOCl, with IC50 values ranging from 1.88–13.50 μg/mL. A total of 21 phenolic compounds were identified, being catechin (4.567–7.354 mg/g DW), caffeine (1.147–2.401 mg/g DW) and gallic acid (0.945–1.337 mg/g DW) predominant. No adverse effects were observed on Caco-2 viability after exposure to MAE extracts, while the other conditions led to a slight viability decrease in NSC-34. These results highlighted that the extract from MAE condition 2 is the most promising as a potential nutraceutical ingredient.This research was funded by UIDB/50006/2020 and UIDP/50006/2020 by the Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through national funds. Ana Margarida Silva (SFRH/BD/144994/2019) and Ana Sofia Ferreira (2020.07519.BD) are thankful for the Ph.D. grants financed by POPH‐QREN and subsidized by the European Science Foundation and Ministério da Ciência, Tecnologia e Ensino Superior. Manuela M. Moreira (CEECIND/02702/2017) and Francisca Rodrigues (CEECIND/01886/2020) are thankful for their contracts financed by FCT/MCTES—CEEC Individual Program Contract.info:eu-repo/semantics/publishedVersio

Network Biology Identifies Novel Regulators of CFTR Trafficking and Membrane Stability

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31231217/In cystic fibrosis, the most common disease-causing mutation is F508del, which causes not only intracellular retention and degradation of CFTR, but also defective channel gating and decreased membrane stability of the small amount that reaches the plasma membrane (PM). Thus, pharmacological correction of mutant CFTR requires targeting of multiple cellular defects in order to achieve clinical benefit. Although small-molecule compounds have been identified and commercialized that can correct its folding or gating, an efficient retention of F508del CFTR at the PM has not yet been explored pharmacologically despite being recognized as a crucial factor for improving functional rescue of chloride transport. In ongoing efforts to determine the CFTR interactome at the PM, we used three complementary approaches: targeting proteins binding to tyrosine-phosphorylated CFTR, protein complexes involved in cAMP-mediated CFTR stabilization at the PM, and proteins selectively interacting at the PM with rescued F508del-CFTR but not wt-CFTR. Using co-immunoprecipitation or peptide-pull down strategies, we identified around 400 candidate proteins through sequencing of complex protein mixtures using the nano-LC Triple TOF MS technique. Key candidate proteins were validated for their robust interaction with CFTR-containing protein complexes and for their ability to modulate the amount of CFTR expressed at the cell surface of bronchial epithelial cells. Here, we describe how we explored the abovementioned experimental datasets to build a protein interaction network with the aim of identifying novel pharmacological targets to rescue CFTR function in cystic fibrosis (CF) patients. We identified and validated novel candidate proteins that were essential components of the network but not detected in previous proteomic analyses.This work was supported by FCT, Portugal, through center grant UID/MULTI/04046/2019 to BioISI and the BioSys PhD program PD65-2012 (fellowships SFRH/BD/52488/2014, SFRH/ BD/106084/2015, and SFRH/BD/52490/2014 to CL, JS, and AM, respectively).info:eu-repo/semantics/publishedVersio