Phagosomal Proteins of \u3ci\u3eDictyostelium discoideum\u3c/i\u3e

In recognizing food particles, Dictyostelium cell-surface molecules initiate cytoskeletal rearrangements that result in phagosome formation. After feeding D. discoideum cells latex beads, early phagosomes were isolated on sucrose step gradients. Protein analyses of these vesicles showed that they contained glycoproteins and surface-labeled species corresponding to integral plasma membrane proteins. Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa-actin bundling protein. As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H+-ATPase that was detected by immunoblotting. Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete. The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others. Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis

Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase

Innovación y Creación De Valor Compartido

Con sus dificultades, la CVC -nueva realidad colectiva y social, superadora de la RSE (Responsabilidad Social Empresaria)- continúa haciendo pie en un mundo altamente enfocado en el corto plazo, lo material y lo próximo, arriesgando el porvenir de las generaciones futuras. Así y a partir de las teorías de innovación disruptiva e inversa, este artículo analiza cómo la implementación de estaciones de servicio con tanques aéreos (MAS, por Módulos de Abastecimiento Social) -destinadas al mercado automotor liviano, en pequeñas localidades del interior de Argentina- cuenta con el potencial de generar beneficios para la comunidad, el medio ambiente y la empresa, condiciones imprescindibles para que la CVC tenga lugar y se desenvuelva. Se concluye que la CVC –en la medida que logre mayor difusión, escala y se la combine con otras fuertes teorías, en este caso de innovación- puede resultar un negocio rentable y transformador para las empresas, ayudando a solucionar o mitigar la crisis ambiental y social que sufre el planeta. Además, que los MAS y productos similares pueden ser implementados en pequeñas localidades y en las grandes ciudades, contando con un gran potencial de negocios que desafía a las estaciones de servicio tradicionales. El objetivo de este estudio se refiere a analizar los MAS, proponiendo ideas que ayuden a potenciarlos como negocio, dentro de los entendimientos que ofrecen la CVC, la innovación disruptiva y la innovación inversa. Finalmente, los beneficios sociales y ambientales de la implementación de este concepto han sido constatados, aunque se enfatiza que sus objetivos no han buscado la generación de un negocio rentable y expansivo, sino –simplemente- mejorar la imagen corporativa de la empresa

Decidua-derived mesenchymal stem cells as carriers of mesoporous silica nanoparticles. In vitro and in vivo evaluation on mammary tumors

The potential use of human Decidua-derived mesenchymal stem cells (DMSCs) as a platform to carry mesoporous silica nanoparticles in cancer therapy has been investigated. Two types of nanoparticles were evaluated. The nanoparticles showed negligible toxicity to the cells, a fast uptake and a long retention inside them. Nanoparticle location in the cell was studied by colocalization with the lysosomes. Moreover, the in vitro and in vivo migration of DMSCs towards tumors was not modified by the evaluated nanoparticles. Finally, DMSCs transporting doxorubicin-loaded nanoparticles were capable of inducing cancer cell death in vitro

Production of aromatics from biomass by computer-aided selection of the zeolite catalyst

[EN] Taking into account that the transformation of biomass-derived 2,5-dimethylfuran (DMF) top-xylene involves Diels-Alder (DA) cycloaddition as the limiting step, the use of an ITQ-2 zeolite obtained by direct synthesis (DS-ITQ-2) as a catalyst for this reaction is proposed based on the fact that the organic molecule employed for its synthesis mimics the size and shape of the DA oxanorbornene cycloadduct intermediate. Periodic Density Functional Theory (DFT) calculations reveal a better stabilization of the oxanorbornene intermediate within the external hemicavities or "cups" of the DS-ITQ-2 zeolite (MWW-framework) than in other zeolites employed for this reaction, such as FAU and Beta. Interestingly, experimental results also show improved catalytic conversion values for the DS-ITQ-2 zeolite compared to FAU and Beta, in good agreement with the stabilization energies calculated by DFT. The "ab initio" catalyst design presented here to enhance the catalytic performance for the transformation of biomass-derived products is a valuable example that could be employed for the rationalization of other chemical processes catalyzed by zeolites.This work has been supported by the European Union through ERC-AdG-2014-671093 (SynCatMatch) and by Spanish Government through "Severo Ochoa" (SEV-2016-0683, MINECO), MAT2017-82288-C2-1-P (AEI/FEDER, UE) and RTI2018-101033-B-I00 (MCIU/AEI/FEDER, UE). E. M. G. acknowledges "La Caixa - Severo Ochoa" International PhD Fellowships (call 2015). Elisa Garcia is acknowledged for her technical assistance in this work. The Electron Microscopy Service of the UPV is also acknowledged for their help in sample characterization. We appreciate the support of ExxonMobil Research and Engineering for their help with our efforts in fundamental catalytic research.Margarit Benavent, VJ.; Gallego, EM.; Paris, C.; Boronat Zaragoza, M.; Moliner Marin, M.; Corma Canós, A. (2020). Production of aromatics from biomass by computer-aided selection of the zeolite catalyst. Green Chemistry. 22(15):5123-5131. https://doi.org/10.1039/d0gc01031fS512351312215Owusu, P. A., & Asumadu-Sarkodie, S. (2016). A review of renewable energy sources, sustainability issues and climate change mitigation. Cogent Engineering, 3(1), 1167990. doi:10.1080/23311916.2016.1167990Corma, A., Iborra, S., & Velty, A. (2007). Chemical Routes for the Transformation of Biomass into Chemicals. Chemical Reviews, 107(6), 2411-2502. doi:10.1021/cr050989dSettle, A. E., Berstis, L., Rorrer, N. A., Roman-Leshkóv, Y., Beckham, G. T., Richards, R. M., & Vardon, D. R. (2017). Heterogeneous Diels–Alder catalysis for biomass-derived aromatic compounds. 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Renewablep-Xylene from 2,5-Dimethylfuran and Ethylene Using Phosphorus-Containing Zeolite Catalysts. ChemCatChem, 9(3), 398-402. doi:10.1002/cctc.201601294Zhao, R., Zhao, Z., Li, S., Parvulescu, A.-N., Müller, U., & Zhang, W. (2018). Excellent Performances of Dealuminated H-Beta Zeolites from Organotemplate-Free Synthesis in Conversion of Biomass-derived 2,5-Dimethylfuran to Renewable p -Xylene. ChemSusChem, 11(21), 3803-3811. doi:10.1002/cssc.201801504Chang, C.-C., Green, S. K., Williams, C. L., Dauenhauer, P. J., & Fan, W. (2014). Ultra-selective cycloaddition of dimethylfuran for renewable p-xylene with H-BEA. Green Chem., 16(2), 585-588. doi:10.1039/c3gc40740cLi, Y.-P., Head-Gordon, M., & Bell, A. T. (2014). Computational Study of p-Xylene Synthesis from Ethylene and 2,5-Dimethylfuran Catalyzed by H-BEA. The Journal of Physical Chemistry C, 118(38), 22090-22095. doi:10.1021/jp506664cNikbin, N., Feng, S., Caratzoulas, S., & Vlachos, D. G. 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Exploring endolysin-loaded liposomes for a transtympanic treatment of S. pneumoniae otitis media

Otitis media, the main reason for which antibiotics are prescribed in childhood, is often caused by Streptococcus pneumoniae. The exogenous use of recombinantly produced endolysins, peptidoglycan hydrolases encoded by bacteriophages at the end of their lytic cycle, have been shown to be very effective against this pathogen. To increase bioavailability, and consequently reduce the probability of a recurrent or chronic infection, endolysins could be applied topically in the ear. However, delivery systems with permeation enhancing characteristics are needed to surpass the barrier provided by the tympanic membrane, which separates the ear canal from the middle ear. Therefore, this work aimed to develop a novel delivery system for a transtympanic treatment of pneumococcal otitis media using endolysins. The MSlys endolysin was encapsulated into deformable liposomes composed of L-alpha-lecithin and sodium cholate (L:SC:MSlys) or PEG2000 PE (L:PEG:MSlys) with a efficiency of approximately 35% in average, being released in a controlled manner. Liposomes loaded with MSlys showed no cytotoxicity against keratinocyte and fibroblast cell lines. Moreover, MSlys-loaded liposomes interacted with S. pneumoniae cells, being able to significantly reduce planktonic and biofilm cells. Transtympanic permeation studies demonstrated that PEGylated liposomes significantly enhanced the transport of MSlys through human tympanic membranes in an ex vivo model, showing antipneumococcal effect after 2 hours. Nevertheless, degradation of MSlys occurred during extended incubation at 37 ºC, which affected its effectiveness. In conclusion, endolysin-loaded liposomes are a promising approach for transtympanic treatment of otitis media caused by S. pneumoniae. Nevertheless, further optimization is required in order to increase effectiveness.info:eu-repo/semantics/publishedVersio

Liposomes loaded with the pneumococcal endolysin MSlys: From in vitro characterization to ex vivo permeation across the tympanic membrane

Introduction: The increasing antibiotic resistance triggered interest in novel antimicrobials as well as delivery systems that allow a topical and targeted delivery. However, biological barriers, such as the skin or the tympanic membrane (TM), may hinder the success of the therapy. Drug permeation has been extensively studied in the context of transdermal delivery, but only recently started to be explored for transtympanic applications. Ex vivo Franz diffusion cell permeation tests have been used and validated for the permeation of compounds through the skin prior to in vivo studies, but their exploitation for transtympanic delivery is limited. Endolysins, peptidoglycan hydrolases derived from bacterial viruses, are attractive antimicrobials, with promising action against the otitis media pathogen Streptococcus pneumoniae. Liposomal systems, such as transfersomes or PEGylated liposomes, have been shown to enhance drug permeation across the TM. Here, we describe the in vitro characterization of the endolysin-loaded liposomal carriers as well as their ex vivo permeation through TMs. Objectives: The main objective was to develop a delivery system containing an endolysin for a targeted transtympanic treatment of otitis media. To achieve this, it was necessary: i) to encapsulate the endolysin into liposomes for a controlled delivery; and ii) to evaluate the transtympanic permeation ability of the formulations. Materials and Methods: Liposomes composed of 4 mM of L-alpha-lecithin and sodium cholate (5:1) (L:SC) or L-alpha-lecithin and PEG2000 PE (10:1) (L:PEG) loaded with the MSlys endolysin were prepared. The size, polydispersity index (PDI), zeta potential, stability, deformability, encapsulation efficiency, and in vitro MSlys release were determined. The cytotoxicity against fibroblasts and keratinocytes and the efficacy against pneumococcal planktonic and biofilm cells were also evaluated in vitro. Permeation studies were performed in Franz diffusion cells using porcine skin, sheep TMs, and cadaveric human TMs. The amount of MSlys permeated and its antipneumococcal activity were evaluated, and the protein integrity was analyzed by SDS-PAGE. Results and Discussion: The MSlys endolysin was encapsulated into liposomes, with an average efficiency of about 35%. Liposomes with ca. 100 nm and relatively low PDI were produced, with L:PEG formulations being smaller and less polydisperse than L:SC. Both characteristics remained stable for one year at 4 C. Liposomes were shown to be deformable and to provide a controlled release of MSlys over time following a first-order kinetics. No cytotoxicity was observed. Endolysin-loaded liposomes interacted with S. pneumoniae cells, reducing both planktonic and biofilm cultures. The potential of L:PEG over L:SC formulations to transport MSlys was demonstrated in preliminary transdermal assays. The permeation of MSlys across the TMs was enhanced when loaded in PEGylated liposomes. Samples were shown to significantly reduce pneumococcal cells after 2 h of permeation through the human TM. Nonetheless, loss of antipneumococcal activity after 4 h of permeation and protein hydrolysis at 48 and 72 h were observed. Conclusions: This work reports the delivery of an endolysin through an intact TM using liposomes. However, further optimization is needed to expand the overall therapeutic efficacy of this strategy for use in otitis media.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, the Wyss Institute for Biologically Inspired Engineering and from NIH NIDCD K08-DC018575 grant. MDS acknowledges the FCT grants SFRH/BD/128825/2017 and COVID/BD/152363/2022, and SS acknowledges funding by FCT through the individual scientific employment program contract (2020.03171.CEECIND).info:eu-repo/semantics/publishedVersio

A versatile nanocarrierCubosomes, characterization, and applications

The impact of nanotechnology on the exponential growth of several research areas, particularly nanomedicine, is undeniable. The ability to deliver active molecules to the desired site could significantly improve the efficiency of medical treatments. One of the nanocarriers developed which has drawn researchers’ attention are cubosomes, which are nanosized dispersions of lipid bicontinuous cubic phases in water, consisting of a lipidic interior and aqueous domains folded in a cubic lattice. They stand out due to their ability to incorporate hydrophobic, hydrophilic, and amphiphilic compounds, their tortuous internal configuration that provides a sustained release, and the capacity to protect and safely deliver molecules. Several approaches can be taken to prepare this structure, as well as different lipids like monoolein or phytantriol. This review paper describes the different methods to prepare nanocarriers. As it is known, the physicochemical properties of nanocarriers are very important, as they influence their pharmacokinetics and their ability to incorporate and deliver active molecules. Therefore, an extensive characterization is essential to obtain the desired effect. As a result, we have extensively described the most common techniques to characterize cubosomes, particularly nanocarriers. The exceptional properties of the cubosomes make them suitable to be used in several applications in the biomedical field, from cancer therapeutics to imaging, which will be described. Taking in consideration the outstanding properties of cubosomes, their application in several research fields is envisaged.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, by Marie Skłodowska Curie grant (MSCA-RISE; FODIAC; 778388) and by European Regional Development Fund (ERDF) through the Competitiveness factors Operational program—Norte 2020, COMPETE and National Funds through the FCT—under the project AgriFood XXI (NORTE- 01-0145-FEDER-000041). J.L.P. acknowledge the Instituto de Salud Carlos III for a “Sara Borrell” grant (CD19/00250), cofounded by European Social Fund (“El FSE invierte en futuro”). C.J.O.F. acknowledge the FCT for the grant SFRH/149/BD/14199/2019.info:eu-repo/semantics/publishedVersio