Biofilm formation on polystyrene in detached vs. planktonic cells of polyhydroxyalkanoate-accumulating Halomonas venusta

Biofilm development is characterized by distinct stages of initial attachment, microcolony formation and maturation (sessile cells), and final detachment (dispersal of new, planktonic cells). In this work we examined the influence of polyhydroxyalkanoate (PHA) accumulation on bacterial surface properties and biofilm formation on polystyrene in detached vs. planktonic cells of an environmental strain isolated from microbial mats, Halomonas venusta MAT28. This strain was cultured either in an artificial biofilm in which the cells were immobilized on alginate beads (sessile) or as free-swimming (planktonic) cells. For the two modes of growth, conditions allowing or preventing PHA accumulation were established. Cells detached from alginate beads and their planktonic counterparts were used to study cell surface properties and cellular adhesion on polystyrene. Detached cells showed a slightly higher affinity than planktonic cells for chloroform (Lewis-acid) and a greater hydrophobicity (affinity for hexadecane and hexane). Those surface characteristics of the detached cells may explain their better adhesion on polystyrene compared to planktonic cells. Adhesion to polystyrene was not significantly different between H. venusta cells that had accumulated PHA vs. those that did not. These observations suggest that the surface properties of detached cells clearly differ from those of planktonic cells and that for at least the first 48 h after detachment from alginate beads H. venusta retained the capacity of sessile cells to adhere to polystyrene and to form a biofilm. [Int Microbiol 2014; 17(4):205-212]Keywords: Halomonas venusta MAT-28 · PHA · alginate beads · cell surface physicochemical characteristics · adhesion on polystyren

Integració de coneixements de diferents matèries a través d’un treball col·laboratiu en xarxa: Experiència pilot en els graus del Campus de l’Alimentació

Projecte: 2015PID-UB/018Els docents que impartim diferents matèries de Grau compartim la sensació que els nostres alumnes assoleix els continguts de cada matèria com habilitats i coneixements estancs i independents, necessaris per aprovar, i no veu la necessitat de fer cap esforç per integrar i relacionar els coneixements adquirits. En aquest projecte d’innovació hem desenvolupat una activitat de treball col·laboratiu en xarxa on l’alumne ha pogut interrelacionar els conceptes treballats en les diferents matèries de primer curs de dos ensenyaments de Grau del Campus de l’Alimentació de Torribera: Nutrició Humana i Dietètica (NHD) i Ciència i Tecnologia dels Aliments (CTA). Es va crear des de la plataforma Moodle una aula invertida on els docents vam ser els guies, facilitadors i activadors de l’aprenentatge. Aquesta modalitat de flipped classroom va permetre una millora del disseny pedagògic de l’activitat d’aprenentatge que consistia en la creació, per part dels alumnes, d’una wiki que integrés els coneixements de les tres assignatures a l’entorn d’un tema determinat. Es van elaborar dues rúbriques que van servir per l’avaluació de l’activitat d’aprenentatge. La rúbrica1 es va utilitzar per dur a terme una autoavaluació, una coavaluació entre companys i l’avaluació per part dels docents. La rúbrica2 es va utilitzar per part dels docents per avaluar ítems referents a la competència transversal Treball en Equip. Els alumnes que han intervingut en l’activitat van mostrar unes habilitats significativament més elevades a l’hora d'interpretar els resultats numèrics de les activitats proposades a les classes de problemes

Enhanced polyhydroxyalkanoates accumulation by Halomonas spp. in artifi cial biofi lms of alginate beads

Microbial mats are complex but stable, multi-layered and multi-functional biofilms, which are the most frequent bacterial formations in nature. The functional strategies and physiological versatility of the bacterial populations growing in microbial mats allow bacteria to resist changing conditions within their environment. One of these strategies is the accumulation of carbon- and energy-rich polymers that permit the recovery of metabolic activities when favorable conditions are restored. In the present study, we systematically screened microbial mats for bacteria able to accumulate large amounts of the ester carbon polymers polyhydroxyalkanoates (PHA). Several of these strains were isolated from Ebro Delta microbial mats and their ability to accumulate PHA up to 40-60 % of their dry weight was confirmed. According to two identification approaches (16S rRNA and ropD genes), these strains were identified as Halomonas alkaliphila (MAT-7, -13, -16), H. neptunia (MAT-17), and H. venusta (MAT-28). To determine the mode of growth yielding maximum PHA accumulation, these three different species were cultured in an artificial biofilm in which the cells were immobilized on alginate beads. PHA accumulation by cells that had detached from the biofilm was compared with that of their planktonic counterparts. Experiments in different culture media showed that PHA accumulation, measured as the relative fluorescence intensity after 48 h of incubation at 30 ºC, was higher in immobilized than in planktonic cells, with the exception of cells growing in 5 % NaCl, in which PHA accumulation was drastically lower in both. Therefore, for obtaining high PHA concentrations, the use of immobilized cells may be a good alternative to the PHA accumulation by bacteria growing in the classical, planktonic mode. From the ecological point of view, increased PHA accumulation in detached cells frombiofi lms would be a natural strategy to improve bacterial dispersion capacity and, consequently, to increase survival in stressed environments. [Int Microbiol 2012; 15(4):191-199

Robustesa de serveis ferroviaris

La robustesa de serveis ferroviaris, també anomenada estabilitat d’horaris, consisteix en l’elaboració d’horaris i itineraris de ferrocarril per a línies en condicions pròximes a saturació de manera que permetin una explotació dels serveis sense experimentar retards. La recerca i innovació en aquest àmbit s’ha centrat principalment en el desenvolupament d’una metodologia de mesura de la robustesa, basada en mètodes de simulació de serveis ferroviaris. La metodologia s’ha aplicat als horaris actuals de la línia Barcelona – Vallès de Ferrocarrils de la Generalitat de Catalunya, així com també en casos pràctics de modificació d’horaris per a escenaris futurs d’obres a la xarxa

Engineering and development of model lipid membranes mimicking the HeLa cell membrane

Cells are complex systems whose interaction with nanocarriers, i.e., liposomes, are continuously under investigation to improve drug uptake. Model membranes can facilitate the understanding of the processes involved in fusion or endocytosis. In this work, we engineered two different lipid model membranes, vesicles and supported lipid bilayers (SLBs), mimicking the lipid composition of the HeLa cell plasma membrane. We characterized the model using atomic force microscopy (AFM) and fluorescence. We found that liposomes formed with four lipid components mimicking the HeLa cell bilayer show a liquid ordered fluid nature between 13 °C and 34 °C and yield featureless SLBs onto mica. We evaluated the fusion between the model and liposomes positively charged with and without cholesterol by AFM-based force spectroscopy and fluorescence techniques, such as Förster resonance energy transfer, fluorescence lifetime decay and fluorescence anisotropy. The results indicated a primary electrostatic interaction between the HeLa bilayer model and the liposomes. It was also confirmed the well-known fact that cholesterol enhances the fusion process with the engineered HeLa bilayer. All results support the usefulness of the engineered model in the rationale design of liposomes for drug deliver

Multidisciplinary approach to the transfection of plasmid DNA by a nonviral nanocarrier based on a Gemini-Bolaamphiphilic hybrid lipid

A multidisciplinary strategy, including bothbiochemical and biophysical studies, was proposed here toevaluate the potential of lipid nanoaggregates consisting of amixture of a gemini−bolaamphiphilic lipid (C6C22C6) and thewell-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidy-lethanolamine (DOPE) to transfect plasmid DNA into livingcells in an efficient and safe way. For that purpose, severalexperimental techniques were employed, such as zeta potential(phase analysis light scattering methodology), agarose gelelectrophoresis (pDNA compaction and pDNA protectionassays), small-angle X-ray scattering, cryo-transmission electronmicroscopy, atomic force microscopy,fluorescence-assisted cellsorting, luminometry, and cytotoxicity assays. The resultsrevealed that the cationic lipid and plasmid offer only 70 and30% of their nominal positive (=++q2.0nom,C C C6226) and negative charges (=−−q2/bpnom,pDNA), respectively. Upon mixing withDOPE, they form lipoplexes that self-aggregate in typical multilamellar Lαlyotropic liquid-crystal nanostructures with sizes in therange of 100−200 nm and low polydispersities, very suitablyfitted to remain in the bloodstream and cross the cell membrane.Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNAplasmids (pEGFP-C3, encoding the greenfluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with anefficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/−charge ratio wasmaintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic becausethe viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosysteman optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experimentsFinancial support from the Ministerio de Economia y Competitividad of Spain (projects CTQ2012-30821, CTQ2015-65972-R, CTQ2015-64425-C2-2-R, and CTQ2014-55208-P), Madrid Regional Government (S2013/MIT-2807), Xunta de Galicia (GR 2007/085; IN607C 2016/03 and Centro Singular de Investigación de Galicia accreditation 2016–2019, ED431G/09), the European Regional Development Fund (ERDF), and Universidad Complutense de Madrid, Spain (project UCMA05-33-010), is gratefully acknowledgedS

Multidisciplinary Approach to the Transfection of Plasmid DNA by a Nonviral Nanocarrier Based on a Gemini-Bolaamphiphilic Hybrid Lipid

A multidisciplinary strategy, including both biochemical and biophysical studies, was proposed here to evaluate the potential of lipid nanoaggregates consisting of a mixture of a gemini-bolaamphiphilic lipid (C6C22C6) and the well-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) to transfect plasmid DNA into living cells in an efficient and safe way. For that purpose, several experimental techniques were employed, such as zeta potential (phase analysis light scattering methodology), agarose gel electrophoresis (pDNA compaction and pDNA protection assays), small-angle X-ray scattering, cryo-transmission electron microscopy, atomic force microscopy, fluorescence-assisted cell sorting, luminometry, and cytotoxicity assays. The results revealed that the cationic lipid and plasmid offer only 70 and 30% of their nominal positive () and negative charges (), respectively. Upon mixing with DOPE, they form lipoplexes that self-aggregate in typical multilamellar Lα lyotropic liquid-crystal nanostructures with sizes in the range of 100-200 nm and low polydispersities, very suitably fitted to remain in the bloodstream and cross the cell membrane. Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNA plasmids (pEGFP-C3, encoding the green fluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with an efficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/- charge ratio was maintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic because the viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosystem an optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments

Baricitinib Liposomes as a New Approach for the Treatment of Sjögren's Syndrome

Sjögren's syndrome is a chronic systemic autoimmune disease affecting from 0.2 to 3% of the general population. The current treatment for Sjögren's syndrome is aimed at controlling symptoms such as dry eyes and xerostomia. Systemic therapy with glucocorticoids or immunosuppressants is also used. Baricitinib is an immunosuppressant drug, specifically a Janus kinases 1 and 2 selective inhibitor. We propose ocular liposomal formulations loaded with baricitinib for the management of Sjögren's syndrome. The novelty of the work relies on the fact that, for the first time, baricitinib is intended to be used for topical delivery. Two liposomal formulations were prepared with different lipids: (i) L-α-phosphatidylcholine (Lα-PC) and (ii) a combination of lipids 1-palmitoyl-2-oleoyl-phosphatidylethanolamine: s1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoglycerol (3:1, mol/mol) (POPE:POPG), and they were physicochemically characterized. The in vitro drug release and the ex vivo permeation through corneal and scleral tissues were also assessed. Finally, the tolerance of the formulations on the ocular tissues was evaluated by the HET-CAM technique, as well as through the histological analysis of the cornea and sclera and the cornea transparency. Both liposomes resulted in small, spherical shapes, with suitable physicochemical properties for the ocular administration. Lα-PC led to higher flux, permeation, and retention in the sclera, whereas POPE:POPG led to higher flux and permeation in the cornea. The formulations showed no irritant effects on the chorioallantoic membrane. Additionally, the liposomes did not affect the cornea transparency when they were applied, and the histological analysis did not reveal any structural alteratio

The lipid-protein interplay in efflux plump

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/103042Reproducció del capítol del llibre publicat a: http://www.trnres.com/ebookcontents.php?id=267In this work lactose permease (LacY) of Escherichia coli has been taken as model efflux pump to investigate the interactions between the protein and the main lipid components (POPE and POPG) of the inner membrane. Two main approaches have been followed: (i) measuring the fluorescence energy transfer between a single tryptophan mutant of the protein (W151/C154G LacY) and pyrene labeled phospholipids (Pyr-PE and Pyr-PG); and (ii) pulling the protein from the supported lipid bilayers where it is embedded by using the tip of the atomic force microscope (AFM). On one hand, fluorescence measurements at different pHs indicate that LacY present selectivity for PE. On the other hand the observations of the reconstituted protein in lipid bilayers by AFM show a preference of LacY for the fluid phase (Lα) rather than for the gel phase (Lβ). To get an estimation of the proportion of each lipid in each phase we have constructed a phase diagram for the system POPE:POPG. The diagram shows that at the temperature of the experiments (24 ºC) there is an almost equimolar proportion of each lipid. The results suggest the existence of a boundary region around LacY formed mainly by POPE laterally segregated from a bulk with a random distribution of POPE and POPG. Force spectroscopy allows to establish the force required and the mechanism to unspecifically unfold the protein

Assessment of Efficacy and Safety Using PPAR-γ Agonist-Loaded Nanocarriers for Inflammatory Eye Diseases

Drug-loaded nanocarriers (NCs) are new systems that can greatly improve the delivery and targeting of drugs to specific tissues and organs. In our work, a PPAR-γ agonist loaded into polymeric NCs was prepared, stabilized by spray-drying, and tested in vitro, ex vivo, and in vivo (animal models) to provide a safe formulation for optical anti-inflammatory treatments. The NCs were shown to be well tolerated, and no signs of irritancy or alterations of the eye properties were detected by the in vitro HET-CAM test and in vivo Draize test. Furthermore, no signs of cytotoxicity were found in the NC formulations on retinoblastoma cells (Y-79) analyzed using the alamarBlue assay, and the transmittance experiments evidenced good corneal transparency with the formulations tested. The ocular anti-inflammatory study confirmed the significant prevention efficacy using the NCs, and these systems did not affect the corneal tissue structure. Moreover, the animal corneal structure treated with the NCs was analyzed using X-ray diffraction using synchrotron light. Small-angle X-ray scattering (SAXS) analysis did not show a significant difference in corneal collagen interfibrillar spacing after the treatment with freshly prepared NCs or NCs after the drying process compared to the corresponding negative control when inflammation was induced. Considering these results, the PPAR-γ agonist NCs could be a safe and effective alternative for the treatment of inflammatory ocular processes