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/

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

Impact of biofilm growth in the microbial community composition of a sequencing batch reactor

Conventional activated sludge wastewater treatment plants (WWTP) for carbon and nutrient removal operate at high sludge retention time and, consequently, at a low food to microorganisms ratio. While the first condition is favourable for nitrifying bacteria growth, an excessive growth of certain species of filamentous bacteria often occurs in the presence of the second one. Interestingly, no problems with excessive growth of filamentous bacteria have been reported for the activated sludge process when combined with biofilm growth more than a decade ago. In this context, the present work aims to compare the microbial community composition of two sequencing batch reactors (SBRs) operating in the presence and in the absence of biofilm growth, and to correlate microbial composition with the performance of the reactors. Two SBRs with a working volume of 1.5 L were operated in parallel with a constant cycle time of 4 h, a volume exchange ratio of 0.5 L/L and a resulting hydraulic retention time of 8 h. The duration of the individual operating phases was: 5 min fill, 225 min aerated, 5 min settle and 5 min draw. During the aerated phase airflow was applied through membrane diffusers, causing the reactor contents including the carrier bed to circulate. The SBRs were operated with synthetic water containing acetate as the only carbon source and ammonium as nitrogen source. One SBR was operated just with suspended biomass while the second one combined suspended biomass with biofilm cultivation. The biofilm was formed on a new type of polyethylene support developed by University of Minho, consisting of hollow, star-shaped, carriers with 17 mm external diameter and a height of 10 mm. The bed formed by these carriers had a specific surface area of 407 m2/m3, an average porosity of 0.74 and occupied 20 % of the reactor volume. Grab samples were taken and analyzed for acetate using a HPLC system. Observation of the filamentous microbial and microfauna communities was accomplished by microscope inspection at 100 to 1000X magnification, in fresh samples and after Gram and Neisser staining. The microbial community of the SBR operating just with suspended growth was dominated by fungi micelar growth, while in the other reactor the communities were clearly more complex. The incorporation of carriers for biofilm growth in the other SBR apparently suppressed the excessive growth of fungi. Differences in the microfaunas community were also observed, the SBR operating just with suspended growth showing clearly a delay in the microfauna succession comparing to the other reactor. Despite this, acetate was completely removed in both SBRs

Troubleshooting of filamentous bulking using biofilm reactors

The present work aims to evaluate filamentous bulking control in systems combining suspended biomass with biofilm growth. For this study, four sequencing batch reactors (SBR)fed with an easy biodegradable substrate (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 a filamentous fungi-like microorganism in the SBR operating just with suspended biomass. The incorporation of an optimized amount of support for biofilm growth (10 % and 20 %) seemed to suppress the overgrowth of this filamentous microorganism probably due to the combined effect of a decreased biomass loading rate and the physical cut or breakdown of filaments induced by particle-to-particle collisions. Besides the observed differences in terms of the filamentous fungi-like microorganism, the incorporation of a support material for biofilm growth was also found to induce increasing differences in the bacterial community structure as the concentration of support increased in the SBR

Influence of carrier concentration on the control of Galactomyces geotrichum bulking and bacterial community of biofilm reactors

The present work aims to evaluate a strategy to solve Galactomyces geotrichum bulking based on the incorporation of a biofilm carrier in activated sludge systems. For this study, four sequencing batch reactors (SBR) were operated without (SBR1) and with a carrier for biofilm growth (5% [SBR2], 10% [SBR3], and 20% [SBR4] of the reactor volume). As expected, G. geotrichum bulking was observed in the SBR operating just with suspended biomass, as ascertained by direct microscopic inspection. The incorporation of an optimized amount of biofilm carrier (10 and 20%) suppressed the overgrowth of the filamentous fungus probably due to the combined effect of a decreased biomass loading rate and an increased shear stress. Polymerase chain reaction—denaturing gradient gel electrophoresis (PCR–DGGE) analysis of the bacterial community suggested that the incorporation of a biofilm carrier induced increasing differences in the bacterial community structure as the concentration of carrier increased in the SBR. However, the observed differences did not seem to affect the activated sludge system since bacterial groups usually present in these systems prevailed.The work described in this paper was financially supported by FCT-Foundation for Science and Technology (Portugal)-by the PhD student grant SFRH/BD/44596/2008

Troubleshooting of filamentous bulking using hybdrid systems

The present work aims to evaluate filamentous bulking control in hybrid systems. 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 a filamentous fungi-like microorganism in the SBR operating just with suspended biomass. The incorporation of an optimized amount of support for biofilm growth (10 % and 20 %) seemed to suppress the overgrowth of this filamentous microorganism probably due to the combined effect of a decreased biomass loading rate and the physical cut or breakdown of filaments induced by support-to-support collisions

Growth of Galactomyces geotrichum in sequencing batch reactors under different organic loading conditions

The present work aims to study the effect of the organic loading rate on the growth of filamentous fungi. For this purpose, three sequencing batch reactors (SBR), fed with an easy biodegradable substrate (acetate), were operated at different organic loading rates: 4.3 g COD L -1 day-1 (SBR1), 1.0 g COD L-1 day-1 (SBR2) and 0.5 g COD L-1 day-1 (SBR3). High amounts of fungal filaments were observed in the SBR operating at higher organic loading rate, as ascertained by direct microscopic inspection, while, at lower organic loading rates, overabundance of fungal filaments was not observed. Sequence retrieved from the isolated fungal filaments presented high similarity (99 %) to Galactomyces geotrichum

Synthesis of tricyanovinyl-substituted thienylpyrroles and characterization of the solvatochromic, electrochemical and non-linear optical properties

Tricyanovinyl-substituted 1-(alkyl)aryl-2-(2´-thienyl)pyrroles 1 have been synthesized by direct tricyanovinylation reaction of 1-(alkyl)aryl-2-(2´-thienyl)pyrroles 2 using TCNE. The tricyanovinyl- derivatives 1 display dramatic reductions in both their optical and electrochemical band gaps relative to thienylpyrrole precursors 2. The solvatochromic behavior of compounds 1 was investigated in a variety of solvents. Hyper-Rayleigh scattering was used to measure the first hyperpolarizabilities β of the mentioned compounds. The β values show that the new compounds prepared could be used on the manufacture of materials with good non-linear (NLO) properties.Fundação para a Ciência e Tecnologia (FCT) - POCTI, FEDER (ref.POCTI/QUI/37816/2001)

Using zeta-potential measurements to quantify peptide partition to lipid membranes

© The Author(s) 2011. This article is published with open access at Springerlink.com.Open Access: This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.Many cellular phenomena occur on the biomembranes. There are plenty of molecules (natural or xenobiotics) that interact directly or partially with the cell membrane. Biomolecules, such as several peptides (e.g., antimicrobial peptides) and proteins, exert their effects at the cell membrane level. This feature makes necessary investigating their interactions with lipids to clarify their mechanisms of action and side effects necessary. The determination of molecular lipid/water partition constants (Kp) is frequently used to quantify the extension of the interaction. The determination of this parameter has been achieved by using different methodologies, such as UV-Vis absorption spectrophotometry, fluorescence spectroscopy and ζ-potential measurements. In this work, we derived and tested a mathematical model to determine the Kp from ζ-potential data. The values obtained with this method were compared with those obtained by fluorescence spectroscopy, which is a regular technique used to quantify the interaction of intrinsically fluorescent peptides with selected biomembrane model systems. Two antimicrobial peptides (BP100 and pepR) were evaluated by this new method. The results obtained by this new methodology show that ζ-potential is a powerful technique to quantify peptide/lipid interactions of a wide variety of charged molecules, overcoming some of the limitations inherent to other techniques, such as the need for fluorescent labeling.This work was partially supported by project PTDC/QUI/ 69937/2006 from Fundação para a Ciência e Tecnologia-Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), and by Fundação Calouste Gulbenkian (Portugal). JMF and MMD also thank FCT-MCTES for grants IMM/BT/37-2010 and SFRH/BD/41750/2007, respectively

Synthesis of polymer-based triglycine sulfate nanofibres by electrospinning

In this work we present the synthesis and characterization of polyethylene oxide (PEO) based triglycine sulfate (NH2(CH2OOH)3H2S04, TGS) nanofibres obtained by electrospinning. The fibres, with typical diameters of about 190–750 nm and above several hundred micrometres in length, present the nanocrystals of TGS embedded in a polymer matrix. The obtained nanofibres were characterized by FT-IR spectroscopy and the domain structure was examined by piezoforce microscopy. Dielectric permittivity measurements on the TGS–PEO nanofibres exhibit the characteristic ferroelectric–paraelectric phase transition at around 50 ºC.This work was financially supported by Fundacao para a Ciencia e Tecnologia (reference CIENCIA-2007 UMINHOCF-06). The authors would like to acknowledge Luis Vieira for help in FT-IR measurements