Plasma membrane-specific interactome analysis reveals calpain 1 as a druggable modulator of rescued Phe508del-CFTR cell surface stability

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del–Na+/H+-exchange regulatory factor-1 (NHERF1) complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR– or rPhe508del–containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 downregulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an attractive target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.This work was supported by a center grant UID/MULTI/04046/2019 to BioISI and project PTDC/BIA-CEL/28408/2017 and IF2012 to PM, both from FCT, Portugal. AMM was recipient of fellowship SFRH/BD/52490/2014 from BioSYS PhD programme PD65-2012, and PB of fellowship SFRH/BPD/94322/2013.N/

Pherotypes are driving genetic differentiation within Streptococcus pneumoniae

<p>Abstract</p> <p>Background</p> <p>The boundaries of bacterial species and the mechanisms underlying bacterial speciation are matters of intense debate. Theoretical studies have shown that recombination acts as a strong cohesive force preventing divergence in bacterial populations. <it>Streptococcus pneumoniae </it>populations have the telltale signs of high recombination with competence implicated as the major driving force behind gene exchange. Competence in <it>S. pneumoniae </it>is triggered by a quorum-sensing mechanism controlled by the competence-stimulating peptide pheromone.</p> <p>Results</p> <p>We studied the distribution of the two major pherotypes in the pneumococcal population and their association with serotype, antimicrobial resistance and genetic lineage. Using multilocus sequence data we evaluated pherotype influence on the dynamics of horizontal gene transfer. We show that pherotype is a clonal property of pneumococci. Standard population genetic analysis and multilocus infinite allele model simulations support the hypothesis that two genetically differentiated populations are defined by the major pherotypes.</p> <p>Conclusion</p> <p>Severe limitations to gene flow can therefore occur in bacterial species in the absence of geographical barriers and within highly recombinogenic populations. This departure from panmixia can have important consequences for our understanding of the response of pneumococci to human imposed selective pressures such as vaccination and antibiotic use.</p

The presence of the pilus locus is a clonal property among pneumococcal invasive isolates

<p>Abstract</p> <p>Background</p> <p>Pili were recently recognized in <it>Streptococcus pneumoniae </it>and implicated in the virulence of this bacterium, which led to the proposal of using these antigens in a future pneumococcal vaccine. However, pili were found to be encoded by the <it>rlrA </it>islet that was not universally distributed in the species. We examined the distribution of the pilus islet, using the presence of the <it>rlrA </it>gene as a marker for the locus, among a collection of invasive isolates recovered in Portugal and analyzed its association with capsular serotypes, clusters defined by the pulsed-field gel electrophoretic profiles (PFGE) and multilocus sequence types.</p> <p>Results</p> <p>Only a minority of the isolates were positive for the presence of the <it>rlrA </it>gene (27%). There was a high correspondence between the serotype and the presence or absence of <it>rlrA </it>(Wallace coefficient, W = 0.778). In particular, there was an association between the presence of <it>rlrA </it>and the vaccine serotypes 4, 6B, 9V and 14 whereas the gene was significantly absent from other serotypes, namely 1, 7F, 8, 12B and 23F, a group that included a vaccine serotype (23F) and serotype 1 associated with enhanced invasiveness. Even within serotypes, there was variation in the presence of the pilus islet between PFGE clones and a higher Wallace coefficient (W = 0.939) indicates that carriage of the islet is a clonal property of pneumococci. Analysis of <it>rlrA </it>negative isolates revealed heterogeneity in the genomic region downstream of the <it>rfl </it>gene, the region where the islet is found in other isolates, compatible with recent loss of the islet in some lineages.</p> <p>Conclusion</p> <p>The pilus islet is present in a minority of pneumococcal isolates recovered from human invasive infections and is therefore not an essential virulence factor in these infections. Carriage of the pilus islet is a clonal property of pneumococci that may vary between isolates expressing the same serotype and loss and acquisition of the islet may be ongoing.</p

In Silico Exploration of Mycobacterium tuberculosis Metabolic Networks Shows Host-Associated Convergent Fluxomic Phenotypes

Mycobacterium tuberculosis, the causative agent of tuberculosis, is composed of several lineages characterized by a genome identity higher than 99%. Although the majority of the lineages are associated with humans, at least four lineages are adapted to other mammals, including different M. tuberculosis ecotypes. Host specificity is associated with higher virulence in its preferred host in ecotypes such as M. bovis. Deciphering what determines the preference of the host can reveal host-specific virulence patterns. However, it is not clear which genomic determinants might be influencing host specificity. In this study, we apply a combination of unsupervised and supervised classification methods on genomic data of ~27,000 M. tuberculosis clinical isolates to decipher host-specific genomic determinants. Host-specific genomic signatures are scarce beyond known lineage-specific mutations. Therefore, we integrated lineage-specific mutations into the iEK1011 2.0 genome-scale metabolic model to obtain lineage-specific versions of it. Flux distributions sampled from the solution spaces of these models can be accurately separated according to host association. This separation correlated with differences in cell wall processes, lipid, amino acid and carbon metabolic subsystems. These differences were observable when more than 95% of the samples had a specific growth rate significantly lower than the maximum achievable by the models. This suggests that these differences might manifest at low growth rate settings, such as the restrictive conditions M. tuberculosis suffers during macrophage infection

Group A Streptococci clones associated with invasive infections and pharyngitis in Portugal present differences in emm types, superantigen gene content and antimicrobial resistance

© 2012 Friães et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly citedBackground: A few lineages of Group A streptococci (GAS) have been associated with a reemergence of severe invasive streptococcal disease in developed countries. However, the majority of the comparisons between invasive and non-invasive GAS isolates have been performed for collections of reduced genetic diversity or relied on limited typing information to distinguish clones. We characterized by several typing methods and compared a collection of 160 isolates recovered from normally sterile sites with 320 isolates associated with pharyngitis and recovered in the same time period in Portugal. Results: Although most of the isolates belonged to clones that were equally prevalent in invasive infections and pharyngitis, we identified markers of invasiveness, namely the emm types 1 and 64, and the presence of the speA and speJ genes. In contrast, emm4, emm75, and the ssa and speL/M genes were significantly associated with pharyngitis. There was a strong agreement between the emm type, the superantigen (SAg) genes and the clusters defined by pulsed-field gel electrophoresis (PFGE) profiling. Therefore, combinations of particular emm types and SAg genes frequently co-occurred in the same PFGE cluster, but there was no synergistic or antagonistic interaction between them in determining invasiveness. Only macrolide-susceptible PFGE clones were significantly associated with invasive infections or pharyngitis, while the clones of resistant isolates sharing all other molecular properties analyzed were equally prevalent in the two groups of isolates. Conclusions: This study confirmed the importance of the widely disseminated emm1-T1-ST28 clone in invasive infections but also identified other clones linked to either invasive infections (emm64-ST164) or pharyngitis (emm4-T4-ST39), which may be more limited in their temporal and geographical spread. Clonal properties like some emm types or SAg genes were associated with disease presentation, highlighting the importance of bacterial genetic factors to the outcome of GAS infections, although other, yet unidentified factors may also play an important role.This work was partially supported by Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-ESA/72321/2006), Fundação Calouste Gulbenkian and unrestricted research grant from Glaxo SmithKline.info:eu-repo/semantics/publishedVersio

Metodologia para diagnóstico e intervenção em edifícios correntes: habitação social no Porto e Recife

De acordo com o INE (2010), o parque de habitação social distribui-se por 246 Municípios, sendo constituído por cerca de 97 mil fogos e 22 mil edifícios. O município do Porto detém 12 682 fogos, correspondendo a 13% do total. Durante o ano de 2009 os municípios portugueses executaram obras de conservação em 2 252 edifícios (10,2% do total) e procederam à reabilitação de 6 636 fogos (6,8% do total). Este património municipal gerou, em 2009, uma receita média por fogo de 706 euros, entre rendas cobradas e fogos vendidos. Por seu lado a despesa média (incluindo os encargos fixos) ficou-se pelos 676 euros por fogo. Atendendo apenas a estas rubricas de receitas e despesas relacionadas com o parque de habitação social dos municípios, verifica-se a existência de um “défice” nas regiões do Norte de 12,7 milhões, o que demonstra o investimento em curso actualmente. O Município do Porto registou um défice superior a 1 000 euros por fogo (receita de 604 euros e despesa de 1 718 euros). A Habitação Social resulta de uma necessidade passada e presente de pensar nas pessoas, isto é, pensar numa política de valorização da qualidade de vida da população que passando muito pela habitação, não acaba nela. A política social da habitação dá início a um processo global de melhoria da qualidade de vida das pessoas, sendo necessário fazer coincidir a melhoria das condições de alojamento, com a melhoria das condições envolventes aos conjuntos habitacionais. Só com uma participação activa dos moradores é possível a sua identificação com o conjunto habitacional onde residem. A missão descrita é incompatível com habitação social em deficiente estado de conservação ou mesmo muito degradada, tal como se verifica em inúmeros países. Importa desta forma assegurar a reabilitação deste património construído e a sua posterior conservação. Nos últimos anos ocorreram desenvolvimentos muito significativos no que diz respeito à capacidade de utilizar técnicas experimentais (in situ ou em laboratório) e simulações em computador. Um aspecto relevante é que a engenharia “de conservação” deve ter uma abordagem e capacidade diferentes das usadas em construções novas. Frequentemente, os materiais e técnicas tradicionais são desconhecidos para os envolvidos. Também se verifica que a tendência das entidades reguladoras e dos projectistas para que os regulamentos actuais sejam cumpridos. Isto é muitas vezes inaceitável, visto que os regulamentos foram escritos tendo em mente outras formas de construção, pelo que a sua aplicação em materiais, tecnologias e formas tradicionais é excessivamente conservadora ou penalizadora. A necessidade de reconhecer a diferença entre o projecto moderno e a conservação também é relevante no contexto dos custos associados à contribuição da engenharia. O procedimento habitual de cálculo de honorários de engenharia, como uma percentagem do trabalho realizado, está claramente em oposição com as melhores práticas de conservação. Ser capaz de recomendar não tomar qualquer medida pode, na realidade, implicar mais estudos e mais custos reais do que uma recomendação para grandes intervenções. Os procedimentos das intervenções modernas exigem um levantamento cuidado da construção, bem como a compreensão da sua história, tendo em vista obter um diagnóstico claro, que requer muitas vezes técnicas de inspecção adequadas e experiência adquirida relevante, num processo muito semelhante à medicina Após reconstituir o historial do paciente (o edifício) e requerer exames (técnicas de inspecção e ensaios), é possível um diagnóstico. Este diagnóstico permite, se necessário, uma terapia adequada (projecto de intervenção) e o respectivo controlo de resultados (monitorização dos resultados). Tendo em vista demonstrar a abordagem metodológica necessária, no presente artigo serão apresentados casos de estudo em Portugal e Brasil, onde os autores estiveram envolvidos

Analyzing the Implementation of Lean Methodologies and Practices in the Portuguese Industry: A Survey

The mass production paradigm on which much of the industry was based has changed. The market is increasingly demanding, requesting diversity and products that are more and more adapted to personal wishes and requirements. This implies producing a greater diversity of products in smaller quantities. Competitiveness is enormous, which forces most companies to be truly effective and efficient, taking care of product quality, delivery time, and final cost. Lean methodologies have been a valuable aid in this field. The diversity of Lean tools has been shown to have answers to the most diverse challenges, and companies are aware of this, increasingly adopting methodologies and processes that aim to progressively reduce waste and adapting their production paradigm to what the market requires. This work intends to provide a vision, as global as possible, of the pathway of Lean implementation in the Portuguese industry. For this purpose, a survey was carried out with a significant sample of Portuguese industrial companies from a wide range of activity sectors. The data collected through the survey were treated statistically, and then a SWOT analysis of the results was performed, which provided a collection of precious information on the evolution of industrial companies in Portugal.The author CP was partially supported by CMUP, which is financed by national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P., under the project with reference UIDB/00144/2020. F.J.G.S. would like to thank INEGI Research Center due to its continuous support, namely to Jorge Seabra, as well as all remaining CETRIB/INEGI/LAETA teal.info:eu-repo/semantics/publishedVersio

Reducing scrap and improving an air conditioning pipe production line

30th International Conference on Flexible Automation and Intelligent Manufacturing (FAIM2021) -15-18 June 2021, Athens, GreeceThe automotive industry is considered one of the most demanding and competitive sectors in the global market. This increasingly implies having a stable and optimized production process, always with a view to continuous improvement. Therefore, it is very important to be aware of all the waste that is generated in all production and logistics operations and take action to reduce them. In this regard and considering the process of producing air conditioning pipes for the automotive industry, a high scrap value was detected mainly due to soldering process. Therefore, the entire production process is analyzed in order to identify the main causes behind the high scrap value. Several Lean and quality tools are used to reduce not only the amount of scrap but also to increase the line productivity. In order to face this challenge, after elaborating the action plan and corresponding implementation, the scrap value is reduced by 12% in general, and productivity increased by 29%, 55% and 22.5% in three different references produced by the same machine. Although this solution is a bit expensive, the corresponding payback is reduced, so it can easily be applied transversally to other similar machines allowing extremely interesting gains in the short term.Andresa Baptista acknowledges the financial support of CIDEM- Research Center of Mechanical Engineering, FCT –Portuguese Foundation for the Development of Science and Technology, Ministry of Science, Technology and Higher ducation, under the Project UID/EMS/0615/2019.info:eu-repo/semantics/publishedVersio

Tree type and forest management effects on the structure of strem wood following wilfires

Wildfires are an increasingly common disturbance influencing wood recruitment to streams, and thereby affecting their physical and biological condition. Mediterranean countries such as Portugal, where more than 25% of the land area has burned since 1990, are ideal areas to study impacts of wildfire effects on streams. We evaluated the physical structure of 2206 downed wood pieces (DWP) across 27 first- to third-order streams in central Portugal, all of which had experienced recent wildfires. The streams flowed through monospecific upland forests of Eucalyptus, Maritime pines, or Cork oaks and were fringed by a mixture of riparian tree species. DWP structure differed between tree types and between burned and unburned pieces. Post-fire timber-production forests (Maritime pines and Eucalyptus) contributed a higher quantity of thinner, longer and straighter DWP to streams than Cork oak stands. Pieces from Maritime pines had more rootwads and branches than DWP from the other tree types. Pieces from Cork oak and riparian species generally had a bent form, were shorter and had no rootwads. Burned DWP in streams were often from riparian trees. Relative to unburned DWP, the burned DWP occurred more frequently, were larger and straighter, had branches less often, and were more decayed. With more complex branches, rootwads, and a larger diameter, inputs from burned Maritime pine forests are more likely to change stream hydraulics and habitat complexity, relative to inputs from Eucalyptus forests with their simpler structure. This study shows that, less than a decade after wildfires, structure of downed wood in and near streams is strongly influenced by wildfire, but also still reflects intrinsic species characteristics and respective silviculture practices, even after the effects of fire have been accounted for. Under an anticipated shift in landscape cover with higher shrubland proportions and more mixing of Maritime pine and Eucalyptus forests, our results suggest that instream large wood will become scarcer and more structurally homogeneous