Microbiota–host communication is primarily achieved by secreted factors that can penetrate the mucosal surface, such as extracellular membrane vesicles (EVs). The EVs released by the gut microbiota have been extensively studied in... more
Microbiota–host communication is primarily achieved by secreted factors that can penetrate the mucosal surface, such as extracellular membrane vesicles (EVs). The EVs released by the gut microbiota have been extensively studied in cellular and experimental models of human diseases. However, little is known about their in vivo effects in early life, specifically regarding immune and intestinal maturation. This study aimed to investigate the effects of daily administration of EVs from probiotic and commensal E. coli strains in healthy suckling rats during the first 16 days of life. On days 8 and 16, we assessed various intestinal and systemic variables in relation to animal growth, humoral and cellular immunity, epithelial barrier maturation, and intestinal architecture. On day 16, animals given probiotic/microbiota EVs exhibited higher levels of plasma IgG, IgA, and IgM and a greater proportion of Tc, NK, and NKT cells in the spleen. In the small intestine, EVs increased the villi ar...
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Research Interests: Chemistry, Inorganic Chemistry, Magnetic Resonance Spectroscopy, Apoptosis, Medicine, and 12 moreMacromolecular X-Ray Crystallography, DNA, Humans, Reactive Oxygen Species, Platinum, Ascorbic Acid, Cell cycle checkpoints, Molecular Conformation, Cell Proliferation, Antineoplastic Agents, Isomerism, and cathepsin B
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Research Interests: Biochemistry, Chemistry, Kinetics, Biological Chemistry, Medicine, and 11 moreBiological Sciences, Escherichia coli, CHEMICAL SCIENCES, Amino Acids, Dehydrogenase, Carbohydrate metabolism, Substrate Specificity, Molecular weight, Metabolic pathway, Species Specificity, and Medical and Health Sciences
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Research Interests: Biochemistry, Bacteriology, Biology, Oxidative Stress, DNA damage, and 14 moreDNA repair, Medicine, Catalytic Mechanism, Biological Sciences, Protein Structure and Function, Escherichia coli, Enzyme, Molecular cloning, Western blot, Bleomycin, Amino Acid Sequence, Carbon Source, Molecular Sequence Data, and Medical and Health Sciences
To examine the substrate specificity of the membrane transport carriers LldP (L-lactate permease) and GlcA (glycolate permease) of Escherichia coli, a mutant strain lacking their structural genes and blocked in the metabolism of the... more
To examine the substrate specificity of the membrane transport carriers LldP (L-lactate permease) and GlcA (glycolate permease) of Escherichia coli, a mutant strain lacking their structural genes and blocked in the metabolism of the tested substrates was constructed and transformed with a plasmid bearing either the lldP or the glcA gene. Each transformant acquired the ability to accumulate L-lactate, D-lactate, and glycolate against a high concentration gradient. Substrate accumulation was inhibited by carbonyl cyanide m-chlorophenylhydrazone, a hydrophobic proton conductor that dissipates proton motive force. Competition of (14)C-L-lactate transport by nonradioactive L-lactate, D-lactate, and glycolate in LldP synthesizing cells and competition of (14)C-glycolate transport by the same three substrates in GlcA synthesizing cells showed that both carriers effectively transported all three substrates with a K(i) value ranging from 10 to 20 microM. D-Lactate does not appear to have a permease of its own. Utilization of the compound depends mainly on LldP.
Research Interests: Biochemistry, Biology, Biophysical Chemistry, Medicine, Escherichia coli, and 12 moreMembrane transport, Biochemical, Membrane transport proteins, Substrate Specificity, Acetic Acid, Proton Motive Force, Lactic Acid, Protons, Biochemistry and cell biology, dctA permease, Medical biochemistry and metabolomics, and stereoisomerism
Research Interests: Biochemistry, Molecular Biology, Bacteriology, Kinetics, Biology, and 12 moreMedicine, Biological Sciences, Escherichia coli, Gene, Membrane Transport Protein, Membrane transport proteins, Substrate Specificity, Base Sequence, Culture Media, dctA permease, Molecular Sequence Data, and Medical and Health Sciences
Extracellular matrix components of bacterial biofilms include biopolymers such as polysaccharides, nucleic acids and proteins. Similar to polysaccharides, the secretion of adhesins and other matrix proteins can be regulated by the second... more
Extracellular matrix components of bacterial biofilms include biopolymers such as polysaccharides, nucleic acids and proteins. Similar to polysaccharides, the secretion of adhesins and other matrix proteins can be regulated by the second messenger cyclic diguanylate (cdG). We have performed quantitative proteomics to determine the extracellular protein contents of a Rhizobium etli strain expressing high cdG intracellular levels. cdG promoted the exportation of proteins that likely participate in adhesion and biofilm formation: the rhizobial adhesion protein RapA and two previously undescribed likely adhesins, along with flagellins. Unexpectedly, cdG also promoted the selective exportation of cytoplasmic proteins. Nearly 50% of these cytoplasmic proteins have been previously described as moonlighting or candidate moonlighting proteins in other organisms, often found extracellularly. Western blot assays confirmed cdG-promoted export of two of these cytoplasmic proteins, the translatio...
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Background Acne is a common skin disorder that involves an infection inside the hair follicle, which is usually treated with antibiotics, resulting in unbalanced skin microbiota and microbial resistance. For this reason, we developed... more
Background Acne is a common skin disorder that involves an infection inside the hair follicle, which is usually treated with antibiotics, resulting in unbalanced skin microbiota and microbial resistance. For this reason, we developed polymeric nanoparticles encapsulating thymol, a natural active compound with antimicrobial and antioxidant properties. In this work, optimization physicochemical characterization, biopharmaceutical behavior and therapeutic efficacy of this novel nanostructured system were assessed. Results Thymol NPs (TH-NP) resulted on suitable average particle size below 200 nm with a surface charge around − 28 mV and high encapsulation efficiency (80%). TH-NP released TH in a sustained manner and provide a slow-rate penetration into the hair follicle, being highly retained inside the skin. TH-NP possess a potent antimicrobial activity against Cutibacterium acnes and minor effect towards Staphylococcus epidermis, the major resident of the healthy skin microbiota. Addi...
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Aim: Development of fluorometholone-loaded PEG-PLGA nanoparticles (NPs) functionalized with cell-penetrating peptides (CPPs) for the treatment of ocular inflammatory disorders. Materials & methods: Synthesized polymers and peptides were... more
Aim: Development of fluorometholone-loaded PEG-PLGA nanoparticles (NPs) functionalized with cell-penetrating peptides (CPPs) for the treatment of ocular inflammatory disorders. Materials & methods: Synthesized polymers and peptides were used for elaboration of functionalized NPs, which were characterized physicochemically. Cytotoxicity and ability to modulate the expression of proinflammatory cytokines were evaluated in vitro using human corneal epithelial cells (HCE-2). NPs uptake was assayed in both in vitro and in vivo models. Results: NPs showed physicochemical characteristics suitable for ocular administration without evidence of cytotoxicity. TAT-NPs and G2-NPs were internalized and displayed anti-inflammatory activity in both HCE-2 cells and mouse eye. Conclusion: TAT-NPs and G2-NPs could be considered a novel strategy for the treatment of ocular inflammatory diseases of the anterior and posterior segment.
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The locus glc (min 64.5), associated with the glycolate utilization trait in Escherichia coli, is known to contain glcB, encoding malate synthase G, and the gene(s) needed for glycolate oxidase activity. Subcloning, sequencing, insertion... more
The locus glc (min 64.5), associated with the glycolate utilization trait in Escherichia coli, is known to contain glcB, encoding malate synthase G, and the gene(s) needed for glycolate oxidase activity. Subcloning, sequencing, insertion mutagenesis, and expression studies showed five additional genes: glcC and in the other direction glcD, glcE, glcF, and glcG followed by glcB. The gene glcC may encode the glc regulator protein. Consistently a chloramphenicol acetyltransferase insertion mutation abolished both glycolate oxidase and malate synthase G activities. The proteins encoded from glcD and glcE displayed similarity to several flavoenzymes, the one from glcF was found to be similar to iron-sulfur proteins, and that from glcG had no significant similarity to any group of proteins. The insertional mutation by a chloramphenicol acetyltransferase cassette in either glcD, glcE, or glcF abolished glycolate oxidase activity, indicating that presumably these proteins are subunits of th...
Research Interests: Biochemistry, Molecular Biology, Bacteriology, Biology, Medicine, and 13 moreBiological Sciences, Mutation, Escherichia coli, Gene, Enzyme, Metabolic pathway, Amino Acid Sequence, Base Sequence, Branch point, Molecular Sequence Data, acetyltransferase, insertional mutagenesis, and Medical and Health Sciences
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Essential oils are a complex mixture of aromatic substances whose pharmacological actions, including antimicrobial, antioxidant, anticancer, and anti-inflammatory activities, have been widely reported. This study aimed to evaluate the... more
Essential oils are a complex mixture of aromatic substances whose pharmacological actions, including antimicrobial, antioxidant, anticancer, and anti-inflammatory activities, have been widely reported. This study aimed to evaluate the anti-Candida and dermal anti-inflammatory activity of essential oils from native and cultivated Ecuadorian plants. Essential oils from Bursera graveolens, Dacryodes peruviana, Mespilodaphne quixos, and Melaleuca armillaris were isolated by hydrodistillation and were characterized physically and chemically. Its tolerance was analyzed by in vitro and in vivo studies. The antifungal activity was studied against Candida albicans, Candida glabrata, and Candida parapsilosis, whereas the anti-inflammatory effect was evaluated by a mouse ear edema model. The main compounds were limonene, α-phellandrene, (E)-methyl cinnamate, and 1,8-cineole, respectively. All essential oils showed high tolerability for skin application, antifungal activity against the three Ca...
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ABSTRACT Trans- and cis-isomers of [Pt(L)Cl2(DMSO)] complexes with a new 3-methoxyimino-2-phenyl-3H-indole ligand have been prepared and characterized by X-ray diffraction.Conclusive proofs of the key role of the DMSO ligand and its... more
ABSTRACT Trans- and cis-isomers of [Pt(L)Cl2(DMSO)] complexes with a new 3-methoxyimino-2-phenyl-3H-indole ligand have been prepared and characterized by X-ray diffraction.Conclusive proofs of the key role of the DMSO ligand and its location in the coordination sphere of the Pt(II) and on the crystal architecture are presented.The study of the antitumoral activity of cis- and trans- [Pt(L)Cl2(DMSO)] shows that most of them are potent cytotoxic agents.The influence of the relative disposition of the Cl- ligands in the cis- and trans-isomers on their cytotoxic activities is discussed.Compounds 4b and 5a are more cytotoxic than cisplatin in MDA-MB231 and MCF-7 breast cancer lines.
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Trefoil factor 3 (TFF3) plays a key role in the maintenance and repair of intestinal mucosa. TFF3 expression is upregulated by the microbiota through TLR2. At the posttranscriptional level, TFF3 is downregulated by miR-7-5p. Reduced TFF3... more
Trefoil factor 3 (TFF3) plays a key role in the maintenance and repair of intestinal mucosa. TFF3 expression is upregulated by the microbiota through TLR2. At the posttranscriptional level, TFF3 is downregulated by miR-7-5p. Reduced TFF3 levels have been detected in the damaged tissue of IBD patients. Here, we investigate the regulation of TFF3 expression by microbiota extracellular vesicles (EVs) in LS174T goblet cells using RT-qPCR and inhibitors of the TLR2 or PI3K pathways. To evaluate the subsequent impact on epithelial barrier function, conditioned media from control and vesicle-stimulated LS174T cells were used to treat Caco-2 monolayers. The barrier-strengthening effects were evaluated by analysing the expression and subcellular distribution of tight junction proteins, and the repairing effects were assessed using wound-healing assays. The results showed a differential regulation of TFF3 in LS174T via EVs from the probiotic EcN and the commensal ECOR12. EcN EVs activated the...
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In recent years the role of extracellular vesicles (EVs) of Gram-positive bacteria in host-microbe cross-talk has become increasingly appreciated, although the knowledge of their biogenesis, release and host-uptake is still limited. The... more
In recent years the role of extracellular vesicles (EVs) of Gram-positive bacteria in host-microbe cross-talk has become increasingly appreciated, although the knowledge of their biogenesis, release and host-uptake is still limited. The aim of this study was to characterize the EVs released by the dairy isolate Lactiplantibacillus plantarum BGAN8 and to gain an insight into the putative mechanism of EVs uptake by intestinal epithelial cells. The cryo-TEM observation undoubtedly demonstrated the release of EVs (20 to 140 nm) from the surface of BGAN8, with exopolysaccharides seems to be part of EVs surface. The proteomic analysis revealed that the EVs are enriched in enzymes involved in central metabolic pathways, such as glycolysis, and in membrane components with the most abundant proteins belonging to amino acid/peptide ABC transporters. Putative internalization pathways were evaluated in time-course internalization experiments with non-polarized HT29 cells in the presence of inhi...
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Research Interests: Genetics, Bacteriology, Biology, Medicine, Biological Sciences, and 15 moreDNA, Escherichia coli, Iron, Gene, Genetic linkage analysis, Enzyme, Molecular cloning, Acinetobacter, Enzyme activity, Glyoxylate Cycle, Carbon Source, Allantoin, Molecular Sequence Data, DNA mutational analysis, and Medical and Health Sciences
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L-Lactaldehyde is a branching point in the metabolic pathway of L-fucose and L-rhamnose utilization. Under aerobic conditions, L-lactaldehyde is oxidized to L-lactate by the enzyme lactaldehyde dehydrogenase, while under anaerobic... more
L-Lactaldehyde is a branching point in the metabolic pathway of L-fucose and L-rhamnose utilization. Under aerobic conditions, L-lactaldehyde is oxidized to L-lactate by the enzyme lactaldehyde dehydrogenase, while under anaerobic conditions, L-lactaldehyde is reduced to L-1,2-propanediol by the enzyme propanediol oxidoreductase. Aerobic growth on either of the methyl pentoses induces a lactaldehyde dehydrogenase enzyme which is inhibited by NADH and is very stable under anaerobic conditions. In the absence of oxygen, the cell shifts from the oxidation of L-lactaldehyde to its reduction, owing to both the induction of propanediol oxidoreductase activity and the decrease in the NAD/NADH ratio. The oxidation of L-lactaldehyde to L-lactate is again restored upon a change to aerobic conditions. In this case, only the NAD/NADH ratio may be invoked as a regulatory mechanism, since both enzymes remain active after this change. Experimental evidence in the presence of rhamnose with mutants ...
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<p>HT-29 cells were incubated with rhodamine B-R18-labeled OMVs (2 μg) for the indicated times and analyzed using laser scanning confocal spectral microscope. Scale bar: 20 μm. Clathrin was stained using anti-clathrin mouse... more
<p>HT-29 cells were incubated with rhodamine B-R18-labeled OMVs (2 μg) for the indicated times and analyzed using laser scanning confocal spectral microscope. Scale bar: 20 μm. Clathrin was stained using anti-clathrin mouse monoclonal antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (green). Endosomes were labeled with a rabbit polyclonal antibody against the endosome-associated protein EEA1 and Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Lysosomes were detected using LysoTracker Green DND-26 at 300 nM (green). Images are from a single representative experiment (n = 4). Colocalization of the green (clathrin, EEA1 or the LysoTracker probe)) and red (vesicles) signals was confirmed by histogram analysis of the fluorescence intensities along the yellow lines. Analysis by laser scanning confocal spectral microscope was performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160374#pone.0160374.g001" target="_blank">Fig 1</a>.</p
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The poor water solubility of apremilast (APR) is the main impediment to the penetration of the drug through the skin barrier. The objective of this study was to evaluate the permeability of APR in different solutions enriched with... more
The poor water solubility of apremilast (APR) is the main impediment to the penetration of the drug through the skin barrier. The objective of this study was to evaluate the permeability of APR in different solutions enriched with penetration promoters in ex vivo samples of human skin, and additionally assess its tolerance in vivo. To this end, APR solutions with 5% promoter were developed, and the drug’s ability to penetrate human abdominal skin samples was evaluated; the coefficients of permeability, cumulated amounts permeated, and flow were some of the parameters evaluated; likewise, the in vitro and in vivo tolerance of the solutions was evaluated. The results obtained showed that the solutions containing squalene as a promoter improved the penetration of APR compared to the other promoters evaluated; in the same way, on an in vitro scale in HaCaT cells, the promoters were not toxic, finding a cell viability greater than 80% at the different dilutions evaluated. In the in vivo ...
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<p><b>(A)</b> Comparison of the protein profile of MVs from EcN and EcN <i>tolR</i>. Isolated vesicles (10 μg protein) were separated in a 10%-SDS-PAGE gel and stained with Sypro<sup>®</sup> Ruby... more
<p><b>(A)</b> Comparison of the protein profile of MVs from EcN and EcN <i>tolR</i>. Isolated vesicles (10 μg protein) were separated in a 10%-SDS-PAGE gel and stained with Sypro<sup>®</sup> Ruby Protein Gel Stain. Molecular size markers are indicated. Seven protein bands (labelled by numbers) were excised from the gel and analyzed by LC-MS/MS (data from these analyses are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169186#pone.0169186.s005" target="_blank">S1 Table</a>). <b>(B)</b> The name of the protein with the highest score is indicated for each band. <b>(C)</b> Western blot analysis of LPS in MVs isolated from EcN and EcN <i>tolR</i> strains. MV samples (0.1μg protein) were separated in a 15%-SDS-PAGE gel and analysed with specific anti-<i>E</i>. <i>coli</i> LPS antibodies. Representative SDS-PAGE and blots from three independent experiments are shown.</p
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An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely... more
An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely... more
An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Effect of EcN Sat on cell viability. (PDF 197 kb)
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An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely... more
An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Ocular inflammation is one of the most common comorbidities associated to ophthalmic surgeries and disorders. Since conventional topical ophthalmic treatments present disadvantages such as low bioavailability and relevant side effects,... more
Ocular inflammation is one of the most common comorbidities associated to ophthalmic surgeries and disorders. Since conventional topical ophthalmic treatments present disadvantages such as low bioavailability and relevant side effects, natural alternatives constitute an unmet medical need. In this sense, lactoferrin, a high molecular weight protein, is a promising alternative against inflammation. However, lactoferrin aqueous instability and high nasolacrimal duct drainage compromises its potential effectiveness. Moreover, nanotechnology has led to an improvement in the administration of active compounds with compromised biopharmaceutical profiles. Here, we incorporate lactoferrin into biodegradable polymeric nanoparticles and optimized the formulation using the design of experiments approach. A monodisperse nanoparticles population was obtained with an average size around 130 nm and positive surface charge. Pharmacokinetic and pharmacodynamic behaviour were improved by the nanoparticles showing a prolonged lactoferrin release profile. Lactoferrin nanoparticles were non-cytotoxic and non-irritant neither in vitro nor in vivo. Moreover, nanoparticles exhibited significantly increased anti-inflammatory efficacy in cell culture and preclinical assays. In conclusion, lactoferrin loaded nanoparticles constitute a safe and novel nanotechnological tool suitable for the treatment of ocular inflammation.
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Research Interests: Biochemistry, Chemistry, Kinetics, Biological Chemistry, Medicine, and 11 moreBiological Sciences, Escherichia coli, CHEMICAL SCIENCES, Amino Acids, Dehydrogenase, Carbohydrate metabolism, Substrate Specificity, Molecular weight, Metabolic pathway, Species Specificity, and Medical and Health Sciences
When grown anaerobically on L-rhamnose, Salmonella typhimurium excreted 1,2-propanediol as a fermentation product. Upon exhaustion of the methyl pentose, 1,2-propanediol was recaptured and further metabolized, provided the culture was... more
When grown anaerobically on L-rhamnose, Salmonella typhimurium excreted 1,2-propanediol as a fermentation product. Upon exhaustion of the methyl pentose, 1,2-propanediol was recaptured and further metabolized, provided the culture was kept under anaerobic conditions. n-Propanol and propionate were found in the medium as end products of this process at concentrations one-half that of 1,2-propanediol. As in Klebsiella pneumoniae (T. Toraya, S. Honda, and S. Fukui, J. Bacteriol. 139:39-47, 1979), a diol dehydratase which transforms 1,2-propanediol to propionaldehyde and the enzymes involved in a dismutation that converts propionaldehyde to n-propanol and propionate were induced in S. typhimurium cultures able to transform 1,2-propanediol anaerobically.