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Functionalization of carbon nanotubes through protein corona formation with bovine plasma is a novel waste-to-wealth approach in agri-environmental nanoscience towards remediation of pollutants from water.

Four amphiphilic peptides were synthesized, characterized, and evaluated regarding their efficiency in the catalysis of direct aldol reactions in water. The lipopeptides differ by having a double lipid chain and a guanidinium pyrrole group functionalizing one Lys side chain. All the samples are composed of the amino acids L-proline (P), L-arginine (R), or L-lysine (K) functionalized with the cationic guanidiniocarbonyl pyrrole unit (GCP), L-tryptophan (W), and L-glycine (G), covalently linked to one or two long aliphatic chains, leading to surfactant-like designs with controlled proline protonation state and different stereoselectivity. Critical aggregation concentrations (cac) were higher in the presence of the GCP group, suggesting that self-assembly depends on charge distribution along the peptide backbone. Cryogenic Transmission Electron Microscopy (Cryo-TEM) and Small Angle X-ray Scattering (SAXS) showed a rich polymorphism including spherical, cylindrical, and bilayer structures. Molecular dynamics simulations performed to assess the lipopeptide polymorphs revealed an excellent agreement with core–shell arrangements derived from SAXS data and provided an atomistic view of the changes incurred by modifying head groups and lipid chains. The resulting nanostructures behaved as excellent catalysts for aldol condensation reactions, in which superior conversions (>99%), high diastereoselectivities (ds = 94 : 6), and enantioselectivities (ee = 92%) were obtained. Our findings contribute to elucidate the effect of nanoscale organization of lipopeptide assemblies in the catalysis of aldol reactions in an aqueous environment.

Nanoparticle morphology, size and dispersion are key parameters for the application of cellulose nanomaterials in various areas, such as polymer nanocomposites, catalysts, gel and so on. Transmission electron microscopy (TEM) is the most suitable technique for the morphological characterization of these particles. However, nanocellulose low contrast in TEM images is the major drawback for their adequate morphological characterization and size determination. Even though it is widespread knowledge that negative staining using uranyl acetate is the best approach for intensifying cellulose contrast, up to now few have succeeded in achieving high quality images and reliable size measurements of these nanomaterials. This protocol presents an optimization of the standard uranyl acetate protocol commonly used for biological specimens in order to suit cellulose nanomaterials. Drying method and grid conditions were proven to be the most significant variables for effective TEM specimen preparation. These guidelines could also be successfully applied to enhance the cellulose nanomaterial contrast in polymer matrices.

Septins are GTP binding proteins considered to be novel components of the cytoskeleton. They polymerize into filaments based on hexameric or octameric core particles in which two copies of either three or four different septins, respectively, assemble into a specific sequence. Viable combinations of the 13 human septins are believed to obey substitution rules in which the different septins involved must come from distinct subgroups. The hexameric assembly, for example, has been reported to be SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7. Here, we have replaced SEPT2 by SEPT5 according to the substitution rules and used transmission electron microscopy to demonstrate that the resulting recombinant complex assembles into hexameric particles which are inverted with respect that predicted previously. MBP-SEPT5 constructs and immunostaining show that SEPT5 occupies the terminal positions of the hexamer. We further show that this is also true for the assembly including SEPT2, in direct contradiction with that reported previously. Consequently, both complexes expose an NC interface, as reported for yeast, which we show to be more susceptible to high salt concentrations. The correct assembly for the canonical combination of septins 2-6-7 is therefore established to be SEPT2-SEPT6-SEPT7-SEPT7-SEPT6-SEPT2, implying the need for revision of the mechanisms involved in filament assembly.

Made available in DSpace on 2021-06-25T10:30:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-01 Lignin from pre-treated sugarcane bagasse was sulfomethylated to overcome its high hydrophobicity (Lig-S) and tested at different concentrations as an emulsifier for stabilizing oil-in-water nanoemulsions. The average diameter of the oil droplets was higher in the nanoemulsion prepared with 0.1 % (w/w) Lig-S (∼380 nm) than those prepared with 0.5 and 1.0 % (w/w) (∼180 nm and ∼170 nm, respectively). Zeta potential measurements predicted the long-term stability of Lig-S nanoemulsion. GC–MS analysis of the volatile carbon compounds derived from the oxidation of soybean oil indicated the highest oxidation rates were in preparations with the smallest droplet size. However, all the Lig-S nanoemulsions showed oxidation rates below the threshold values described in the literature. Microscopy analysis confirmed that all the preparations nanosized, dispersed spherical droplets. Collectively, this study has demonstrated that modified lignin isolated from sugarcane bagasse is an excellent emulsifier for the production of oil-in-water nanoemulsions that have both high physical and oxidative stability, providing prospects for the development of nanosystems, based on sustainable strategies, that can be explored for applications such as entrapment and delivery of hydrophobic or bioactive molecules. Brazilian Biorenewables National Laboratory (LNBR) Brazilian Center for Research in Energy and Materials (CNPEM) Faculty of Food Engineering University of Campinas (UNICAMP) Brazilian Nanotechnology National Laboratory (LNNano) Brazilian Center for Research in Energy and Materials (CNPEM) Interdisciplinary Center of Energy Planning University of Campinas (UNICAMP) School of Chemical Engineering University of Campinas (UNICAMP) Department of Bioprocess and Biotechnology College of Agricultural Sciences São Paulo State University (UNESP) Programa de Processos Tecnológicos e Ambientais University of Sorocaba Department of Bioprocess and Biotechnology College of Agricultural Sciences São Paulo State University (UNESP)

In order to form functional filaments, human septins must assemble into hetero-oligomeric rod-like particles which polymerize end-to-end. The rules governing the assembly of these particles and the subsequent filaments are incompletely understood. Although crystallographic approaches have been successful in studying the separate components of the system, there has been difficulty in obtaining high resolution structures of the full particle. Here we report a first cryo-EM structure for a hexameric rod composed of human septins 2, 6 and 7 with a global resolution of ~3.6 A and a local resolution of between ~3.0 A and ~5.0 A. By fitting the previously determined high-resolution crystal structures of the component subunits into the cryo-EM map, we are able to provide an essentially complete model for the particle. This exposes SEPT2 NC-interfaces at the termini of the hexamer and leaves internal cavities between the SEPT6-SEPT7 pairs. The floor of the cavity is formed by the two α0 helices including their polybasic regions. These are locked into place between the two subunits by interactions made with the α5 and α6 helices of the neighbouring monomer together with its polyacidic region. The cavity may serve to provide space allowing the subunits to move with respect to one another. The elongated particle shows a tendency to bend at its centre where two copies of SEPT7 form a homodimeric G-interface. Such bending is almost certainly related to the ability of septin filaments to recognize and even induce membrane curvature.

Mayaro virus (MAYV) is an emerging arbovirus of the Americas that may cause a debilitating arthritogenic disease. The biology of MAYV is not fully understood and largely inferred from related arthritogenic alphaviruses. Here, we present the structure of MAYV at 4.4 Å resolution, obtained from a preparation of mature, infective virions. MAYV presents typical alphavirus features and organization. Interactions between viral proteins that lead to particle formation are described together with a hydrophobic pocket formed between E1 and E2 spike proteins and conformational epitopes specific of MAYV. We also describe MAYV glycosylation residues in E1 and E2 that may affect MXRA8 host receptor binding, and a molecular “handshake” between MAYV spikes formed by N262 glycosylation in adjacent E2 proteins. The structure of MAYV is suggestive of structural and functional complexity among alphaviruses, which may be targeted for specificity or antiviral activity., Mayaro virus (MAYV) is an emerging arbovirus in Central and South America that is transmitted by mosquitoes and causes arthritogenic disease. Here, the authors present the 4.4 Å resolution cryo-EM structure of MAYV and describe specific features of the virus, which could be exploited for the design of MAYV-specific diagnostics and therapeutics.

Mayaro virus (MAYV) is an emerging arbovirus of the Americas that may cause a debilitating arthritogenic disease. The biology of MAYV is not fully understood and largely inferred from related arthritogenic alphaviruses. Here we present the structure of MAYV at 4.4 Å resolution, obtained from a preparation of mature, infective virions. MAYV presents typical alphavirus features and organization. Interactions between viral proteins that lead to particle formation are described together with a hydrophobic pocket formed between E1 and E2 spike proteins and conformational epitopes specific of MAYV. We also describe MAYV glycosylation residues in E1 and E2 that may affect MXRA8 host receptor binding, and a molecular “handshake” between MAYV spikes formed by N262 glycosylation in adjacent E2 proteins. The structure of MAYV is suggestive of structural and functional complexity among alphaviruses, which may be targeted for specificity or antiviral activity.

Oral administration of antioxidant and anti-inflammatory drugs have the potential to improve the current therapy of inflammatory bowel disease. Success of oral treatments, however, depends on the capacity of drugs to remain structurally stable along the gastrointestinal tract, and on the feasibility of accessing the target cells. Delivering anti-inflammatory and antioxidant drugs to macrophages using targeted nanoparticles, could make treatments more efficient. In this work structural features and in vitro activity of macrophage-targeted nanostructured archaeolipid carriers (NAC) containing the high antioxidant dipolar C50 carotenoid bacterioruberin (BR) plus dexamethasone (Dex): NAC-Dex, are described. Ultra-small (66 nm), -32 mV  potential, 1200 g Dex /ml NAC-Dex, consisted of a compritol and BR core, covered by a shell of sn 2,3 ether linked archaeolipids and Tween 80 (2: 2: 1.2: 3 % w/w) were obtained. NAC-Dex were extensively captured by macrophages and Caco-2 cells and displayed high anti-inflammatory and antioxidant activities on a gut inflammation model made of Caco-2 cells and lipopolysaccharide stimulated THP-1 derived macrophages reducing 65 % and 55 % TNF- and IL-8 release, respectively and 60 % reactive oxygen species production. NAC-Dex also reversed the morphological changes induced by inflammation and increased the transepithelial electrical resistance, partly reconstituting the barrier function. Activity of BR and Dex in NAC-Dex was partially protected after simulated gastrointestinal digestion, improving the chances of BR-Dex joint activity. Results suggest that oral NAC-Dex deserve further exploration as intestinal barrier repairing agent. Fil: Higa, Leticia Herminia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schilrreff, Priscila. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina Fil: Briski, Andrés Martín. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina Fil: Jerez, Horacio Emanuel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: de Farias, Marcelo Alexandre. Brazilian Nanotechnology National Laboratory; Brasil Fil: Villares Portugal, Rodrigo. Brazilian Nanotechnology National Laboratory; Brasil Fil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Morilla, María José. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

During transcription initiation, the RNA polymerase holoenzyme (RNAP) and the promoter form an open complex. For many promoters, this interaction involves upstream DNA wrapping, downstream promoter bending, and associated large-scale protein rearrangements. Although these processes have been reported across the life kingdom, their structure, energetics, and role in transcription remain an area of active research. Using optical tweezers, we find that these processes become energetically and reversibly coupled after the formation of the open promoter complex, providing the main contribution to their stability. Using electron microscopy and single particle analysis, we find that the interaction encompasses from positions −76 to +18 along the template, that it involves an overall DNA bent angle of ~245°, and that the upstream wrapping is enabled by interactions between the C-terminal domains of RNAP’s alpha subunits and proximal and middle upstream promoter regions. The energy associated with upstream wrapping, downstream bending and its coupling to downstream rearrangements does not require specific upstream promoter sequence, and correlate positively with the rate of transcription DNA bubble formation as reported by a real-time fluorescence assay. Our results suggest that the coupling between upstream and downstream events are part of a cis-regulatory network established after the opening of the DNA bubble, that could furnish a control mechanism of gene expression by protein factors and regulatory metabolites.SummaryThe first step of gene expression involves transcription of DNA into RNA by RNA polymerase (RNAP). RNAP recognizes a promoter sequence forming the transcriptionally active open complex. For several promoters, DNA wraps around the RNAP. We find that upstream wrapping contacts are energetically coupled and occur cooperatively with downstream rearrangements in the open complexes, providing the largest contribution to their stability. We also determined that upstream wrapping is enabled by interactions between non-specific upstream promoter regions and RNAP α subunit C-terminal domains. Significantly, the strength of these contacts correlates with the rate of DNA bubble opening, and is regulated by factors such as the transcriptional regulator ppGpp. We suggest that any modulator altering upstream wrapping and downstream rearrangements could finely tune gene expression in response to the needs of the cell

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

In recent years, cryogenic electron microscopy (Cryo-EM) has revolutionized the structure determination of wet samples and especially that of biological macromolecules. The glassy-water medium in which the molecules are embedded is considered an almost

Lysosomal Storage Disorders (LSDs) are characterized by an abnormal accumulation of substrates within the lysosome and comprise more than 50 genetic disorders with a frequency of 1:5000 live births. Nanotechnology may be a promising way to circumvent the drawbacks of the current therapies for lysosomal diseases. The blood circulation time and bioavailability of the enzymes or drugs could be improved by inserting them in nanocarriers, which could decrease and/or avoid the need of frequent intravenous infusions along with the minimization or elimination of associated immunogenic responses. Considering the exposed, we aimed to build monoolein-based nanoparticles stabilized by polysorbate 80 as a smart platform able to reach the central nervous system (CNS) to deliver drugs or enzymes inside lysosomes. We developed and characterized the nanoparticles by dynamic light scattering (DLS), small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (Cryo-TEM). The nanoparticles showed a diameter of 115 nm, which is compatible with in vivo application. The SAXS patterns of the formulations displayed a single broad correlation peak that was fitted to the Teubner-Strey model confirming that disordered bicontinuous structures were obtained. Cryo-TEM images corroborated this finding and showed nanoparticles with size values that are similar to those determined by DLS. Furthermore, the nanoparticles did not present cytotoxicity when they were incubated with human fibroblasts, and demonstrated hemolytic activity proportional to the negative control, proving to be safe for parenteral administration. Through the use of a fluorescent dye to track the nanoparticles inside the cell, we demonstrated that they reached lysosomes after 1 h of treatment. More interestingly, the fluorescent dye was detected in the CNS of mice just after 3 h of treatment. The nanoparticles show great potential to improve the treatment of LSDs with brain impairment, acting as a smart platform to targeted delivery of drugs or enzymes.

Controlled release of genetic material such as small interfering RNA (siRNA) using lipid-based non-viral vectors has gained substantial importance in gene therapy applications. Therefore, the interaction between siRNA and these vectors must be well understood. This study aims to investigate the effect of different molar charge ratios (R+/-) between positive charges from microfluidics-produced cationic liposomes (CL) (egg phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and negative charges from siRNA and on physico-chemical and morphological properties of the lipoplexes (CL/siRNA) as well as their in vitro luciferase silencing effect in HeLa cells. R+/- 3.27 was found to be the optimum point for complexation. This finding was confirmed by gel retardation and siRNA accessibility assays. According to Cryo-TEM analysis, the lipoplexes had multi-lamellarity. In vitro transfection efficiency of lipoplexes in HeLa cells was tested at three different siRNA concentrations (10, 25, and 35 nM). Significant knockdown of luciferase by siRNA lipoplexes was observed based on reduced luciferase activity of transfected HeLa cells. Our findings were comparable with the silencing effect of siRNA complexed with Lipofectamine®. No cytotoxicity of lipoplexes was detected at the tested concentrations. This study was essential for further complexation studies which will be performed using microfluidic systems to formulate next-generation lipid-based controlled release systems.

In this work, we developed a coarse-grained model of sumatriptan suitable for extensive molecular dynamics simulations. First, we confirmed the interfacial distribution of this drug in bilayers through cryogenic transmission electron microscopy and small-angle X-ray scattering techniques, as was predicted by our previous atomistic simulations. Based on these simulations, we developed a coarse-grained model for sumatriptan able to reproduce its overall molecular behavior, captured by atomistic simulations and experiments. We then tested the sumatriptan model in a micellar environment along with experimental characterization of sumatriptan-loaded micelles. The simulation results showed good agreement with photon correlation spectroscopy and electrophoretic mobility experiments performed in this work. The particle size of the obtained micelles was comparable with the simulated ones; meanwhile, zeta-potential results suggest adsorption of the drug on the micellar surface. This model is a step forward in the search for a suitable drug-delivery system for sumatriptan. Fil: Wood, Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Albano, Juan Manuel Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Filho, Pedro L. O.. Universidade de Sao Paulo; Brasil Fil: Couto, Veronica Muniz. Universidade Estadual de Campinas; Brasil Fil: Farias, Marcelo A. de. Ministério da Ciência, Tecnologia, Inovações e Comunicações. Centro Nacional de Pesquisa em Energia e Materiais; Brasil Fil: Portugal, Rodrigo V.. Ministério da Ciência, Tecnologia, Inovações e Comunicações. Centro Nacional de Pesquisa em Energia e Materiais; Brasil Fil: de Paula, Eneida. Universidade Estadual de Campinas; Brasil Fil: Oliveira, Cristiano L. P.. Universidade de Sao Paulo; Brasil Fil: Pickholz, Mónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina

An efficient and fast pipeline is presented for obtaining near-atomic resolution structures from large single-particle cryo-EM data sets. The approach is virtually reference-free and is therefore less prone to the perils of reference bias., Single-particle cryogenic electron microscopy (cryo-EM) can now yield near-atomic resolution structures of biological complexes. However, the reference-based alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the ‘Einstein from random noise’ problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous (‘four-dimensional’) cryo-EM data sets. The methodologies integrated in this pipeline include a posteriori camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, ‘random-startup’ three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external ‘starting models’. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive ‘ABC-4D’ pipeline is based on the two-dimensional reference-free ‘alignment by classification’ (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography versus single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from Lumbricus terrestris at a global resolution of ∼3.8 Å is presented as an example of the use of the ABC-4D procedure.

Aim: Develop nanoparticulate agents for oral targeted delivery of dexamethasone (Dex) to macrophages of inflamed mucosa. Materials & methods: Solid archaeolipid nanoparticles (SAN-Dex) (compritol/Halorubrum tebenquichense polar archaeolipids/soybean phosphatidylcholine/Tween-80 4; 0.9; 0.3; 3% w/w) loaded with Dex were prepared. Their mucopenetration, stability under digestion and in vitro anti-inflammatory activity, were determined. Results: Ultra-small SAN-Dex strongly reduced the levels of TNF-α, IL-6 and IL-12 on J774A1 cells stimulated with lipopolysaccharides as compared with free Dex or loaded in ordinary solid lipid nanoparticles-Dex. After in vitro digestion, the anti-inflammatory activity of SAN-Dex was retained, while that of solid lipid nanoparticles-Dex was lost. Conclusion: Because of their structural and pharmacodynamic features, SAN-Dex may be suitable for oral targeted delivery to inflamed mucosa. Fil: Higa, Leticia Herminia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jerez, Horacio Emanuel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: de Farias, Marcelo Alexandre. Brazilian Nanotechnology National Laboratory; Brasil Fil: Portugal, Rodrigo Villares. Brazilian Nanotechnology National Laboratory; Brasil Fil: Romero, Eder Lilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina Fil: Morilla, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina

β-Cyclodextrin (β-CD) is being considered a promising therapy for Niemann-Pick C (NPC) disease because of its ability to mobilise the entrapped cholesterol from lysosomes, however, a major limitation is its inability to cross the blood-brain barrier (BBB) and address the central nervous system (CNS) manifestations of the disease. Considering this, we aimed to design nanoparticles able to cross the BBB and deliver β-CD into the CNS lysosomes. The physicochemical characteristics of β-CD-loaded nanoparticles were evaluated by dynamic light scattering, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The in vitro analyses were performed with NPC dermal fibroblasts and the β-CD-loaded nanoparticles were tracked in vivo. The nanoparticles showed a mean diameter around 120 nm with a disordered bicontinuous inner structure. The nanoparticles did not cause decrease in cell viability, impairment in the antioxidant enzymes activity, damage to biomolecules or release of reactive species in NPC dermal fibroblasts; also, they did not induce genotoxicity or alter the mitochondrial function in healthy fibroblasts. The β-CD-loaded nanoparticles were taken up by lysosomes reducing the cholesterol accumulated in NPC fibroblasts and reached the CNS of mice more intensely than other organs, demonstrating advantages compared to the free β-CD. The results demonstrated the potential of the β-CD-loaded nanoparticles in reducing the brain impairment of NPC.

Oxidative stress plays an essential role in the pathogenesis and progression of inflammatory bowel disease. Co-administration of antioxidants and anti-inflammatory drugs has shown clinical benefits. Due to its significant reactive oxygen species (ROS) scavenging ability, great interest has been focused on superoxide dismutase (SOD) for therapeutic use. However, oral SOD is exposed to biochemical degradation along gastrointestinal transit. Furthermore, the antioxidant activity of SOD must be achieved intracellularly, therefore its cell entry requires endocytic mediating mechanisms. In this work, SOD was loaded into nanoarchaeosomes (ARC-SOD), nanovesicles fully made of sn 2,3 ether linked phytanyl saturated archaeolipids to protect and target SOD to inflammatory macrophages upon oral administration. Antioxidant and anti-inflammatory activities of ARC-SOD, non-digested and digested in simulated gastrointestinal fluids, on macrophages stimulated with H 2 O 2 and lipopolysaccharide were determined and compared with those of free SOD and SOD encapsulated into highly stable liposomes (LIPO-SOD). Compared to SOD and LIPO-SOD, ARC-SOD (170 ± 14 nm, -30 ± 4 mV zeta potential, 122 mg protein/g phospholipids) showed the highest antioxidant and anti-inflammatory activity: it reversed the cytotoxic effect of H 2 O 2 , decreased intracellular ROS and completely suppressed the production of IL-6 and TNF-α on stimulated J774 A.1 cells. Moreover, while the activity of LIPO-SOD was lost upon preparation, gastrointestinal digestion and storage, ARC-SOD was easy to prepare and retained its antioxidant capacity upon digestion in simulated gastrointestinal fluids and after 5 months of storage. Because of their structural and pharmacodynamic features, ARC-SOD may be suitable for oral targeted delivery of SOD to inflamed mucosa. Fil: Schilrreff, Priscila. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Simioni, Yamila Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina Fil: Jerez, Horacio Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina Fil: Caimi, Ayelen Tatiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina Fil: de Farias, Marcelo Alexandre. Brazilian Nanotechnology National Laboratory; Brasil Fil: Villares Portugal, Rodrigo. Brazilian Nanotechnology National Laboratory; Brasil Fil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Morilla, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina

Lauric acid (LAH) strongly inhibits the growth of acne-causing bacteria. LAH is essentially water-insoluble and the solubility of laurate (LA) salts are medium and temperature dependent. Hence, LAH/LA preparations are difficult to formulate. Here we fully characterized phospholipid vesicles containing up to 50 mol% LAH. Vesicles of dipalmitoylphosphatidylcholine (DPPC) containing LAH, at pHs 7.4 and 5.0, were characterized measuring size, charge, bilayer phase transition temperature (Tm) and permeability of water-soluble probes. Small angle X-ray scattering and cryotransmission electron microscopy showed multilamellar vesicles at low LAH %. Increasing LAH % had a negligible effect on particle size. An internal aqueous compartment in all vesicle's preparations, even at equimolar DPPC: LAH fractions, was demonstrated using water-soluble probes. At pH 5.0, the interaction between DPPC and LAH increased the Tm and phase transition cooperativity showing a single lipid phase formed by hydrogen-bonded DPPC: LAH complexes. At pH 7.4, vesicles containing 50 mol% LAH exhibited distinct phases, ascribed to complex formation between LAH and LA or LAH and DPPC. LAH incorporated in the vesicles minimally permeated a skin preparation at both pHs, indicating that the primary sites of LAH solubilization were the skin layers. These results provide the foundations for developing processes and products containing DPPC: LAH., Biotechnology; Pharmaceutical Science; Cryo-TEM; Lauric acid; DSC; Lipid Vesicles; DLS; Ionizable solute; SAXS

Septins are GTP binding proteins considered to be a novel component of the cytoskeleton. They polymerize into filaments based on hetero-oligomeric core particles which, in humans, are either hexamers or octamers composed of two copies each of either three or four different septins from the 13 available. Not all combinations are possible as it is believed that these must obey substitution rules which determine that different septins must be derived from four distinct and well-established sub-groups. Here, we have purified and characterized one such combinations, SEPT5-SEPT6-SEPT7, and used TEM to derive the first structural information concerning its assembly. The full complex was purified using an affinity tag attached to only one of its components (SEPT7) and was able to bind to and perturb lipid bilayers. Although the complex assembled into elongated hexameric particles, the position of SEPT5 was incompatible with that predicted by the reported structure of SEPT2-SEPT6-SEPT7 based on the substitution rules. MBP-fusion constructs for SEPT5 and SEPT2 and immuno-staining clearly show that these septins occupy the terminal positions of the SEPT5-SEPT6-SEPT7 and SEPT2-SEPT6-SEPT7 hexamers, respectively. In so doing they expose a so-called NC interface which we show to be more susceptible to perturbation at high salt concentrations. Our results show that the true structure of the hexamer is inverted with respect to that described previously and, as such, is more compatible with that reported for yeast. Taken together, our results suggest that the mechanisms involved in spontaneous self-assembly of septin core particles and their filaments deserve further reflection.

The structural determination of biological molecules has recently undergone a revolution with the Cryogenic-Electron Microscopy (cryo-EM) technique advances. However, a poor partitioning of macromolecules into the holes of holey carbon support grids frequently limits 3D structural determination by Single Particles Analysis through cryo-EM. This work present a method to deposit, on gold-coated carbon grids, a self-assembled monolayer whose surface properties can be controlled by chemical modification. There is a demonstration of this approach utility to maximize the grid wettability while drive partitioning lipossomes into the holes, thereby enabling better results using cryo-EM. Gold-coated grids with 2-mercaptoethanol yields a hydrophilic surface to holey carbon cryo-EM supports. The method is simple and accessible requiring commercially available items at relatively low costs. The acquired cryo-micrographs for the liposome sample prepared on the SAM grids confirm the success on the drive partitioning of the species into the holes. This approach may enable structure determination of macromolecular targets that suffer from high affinity for holey carbon support.

Development of needle and pain free noninvasive immunization procedures is a top priority for public health agencies. In this work the topical adjuvant activity of the immunomodulator imiquimod (IMQ) carried by ultradeformable archaeosomes (UDA 2 ) (nanovesicles containing sn-2,3 ether linked phytanyl saturated archaeolipids) was surveyed and compared with that of ultradeformable liposomes lacking archaeolipids (UDL 2 ) and free IMQ, using the model antigen ovalbumin and a seasonal influenza vaccine in Balb/c mice. UDA 2 (250 ± 94 nm, -26 ± 4 mV Z potential) induced higher IMQ accumulation in human skin and higher production of TNF-α and IL-6 by macrophages and keratinocytes than free IMQ and UDL 2 . Mixed with ovalbumin, UDA 2 was more efficient at generating cellular response, as measured by an increase in serum IgG2a and INF-γ production by splenocytes, compared with free IMQ and UDL 2 . Moreover, mixed with a seasonal influenza vaccine UDA 2 produced same IgG titers and IgG2a/IgG1 isotypes ratio (≈1) than the subcutaneously administered influenza vaccine. Topical UDA 2 however, induced highest stimulation index and INF-γ levels by splenocytes. UDA 2 might be a promising adjuvant for topical immunization, since it produced cell-biased systemic response with ≈ 13-fold lower IMQ dose than the delivered as the commercial IMQ cream, Aldara. Fil: Caimi, Ayelen Tatiana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Altube, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: de Farias, Marcelo Alexandre. Centro Nacional de Pesquisa Em Energia E Materiais; Brasil Fil: Portugal, Rodrigo Villares. Centro Nacional de Pesquisa Em Energia E Materiais; Brasil Fil: Perez, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Morilla, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina

Biology is a challenging and complicated mess. Understanding this challenging complexity is the realm of the biological sciences: Trying to make sense of the massive, messy data in terms of discovering patterns and revealing its underlying general rules. Among the most powerful mathematical tools for organizing and helping to structure complex, heterogeneous and noisy data are the tools provided by multivariate statistical analysis (MSA) approaches. These eigenvector/eigenvalue data-compression approaches were first introduced to electron microscopy (EM) in 1980 to help sort out different views of macromolecules in a micrograph. After 35 years of continuous use and developments, new MSA applications are still being proposed regularly. The speed of computing has increased dramatically in the decades since their first use in electron microscopy. However, we have also seen a possibly even more rapid increase in the size and complexity of the EM data sets to be studied. MSA computations had thus become a very serious bottleneck limiting its general use. The parallelization of our programs—speeding up the process by orders of magnitude—has opened whole new avenues of research. The speed of the automatic classification in the compressed eigenvector space had also become a bottleneck which needed to be removed. In this paper we explain the basic principles of multivariate statistical eigenvector-eigenvalue data compression; we provide practical tips and application examples for those working in structural biology, and we provide the more experienced researcher in this and other fields with the formulas associated with these powerful MSA approaches.

Background: Thymus vulgaris essential oil (T) could be an alternative to classical antibiotics against bacterial biofilms, which show increased tolerance to antibiotics and host defence systems and contribute to the persistence of chronic bacterial infections. Hypothesis: A nanovesicular formulation of T may chemically protect the structure and relative composition of its multiple components, potentially improving its antibacterial and antibiofilm activity. Study design: We prepared and structurally characterized T in two types of nanovesicles: nanoliposomes (L80-T) made of Soybean phosphatidylcholine (SPC) and Polysorbate 80 (P80) [SPC:P80:T 1:0.75:0.3 w:w], and nanoarchaeosomes (A80-T) made of SPC, P80 and total polar archaeolipids (TPA) extracted from archaebacteria Halorubrum tebenquichense [SPC:TPA:P80:T 0.5:0.50.75:0.7 w:w]. We determined the macrophage cytotoxicity and the antibacterial activity against Staphylococcus aureus ATCC 25,923 and four MRSA clinical strains. Results: L80-T (Z potential −4.1 ± 0.6 mV, ∼ 115 nm, ∼ 22 mg/ml T) and A80-T (Z potential −6.6 ± 1.5 mV, ∼ 130 nm, ∼ 42 mg/ml T) were colloidally and chemically stable, maintaining size, PDI, Z potential and T concentration for at least 90 days. While MIC 90 of L80-T was > 4 mg/ml T, MIC 90 of A80-T was 2 mg/ml T for all S. aureus strains. The antibiofilm formation activity was maximal for A80-T, while L80-T did not inhibit biofilm formation compared to untreated control. A80-T significantly decreased the biomass of preformed biofilms of S. aureus ATCC 25,923 strain and of 3 of the 4 clinical MRSA isolates at 4 mg/ml T. It was found that the viability of J774A.1 macrophages was decreased significantly upon 24 h incubation with A80-T, L80-T and T emulsion at 0.4 mg/ml T. These results show that from 0.4 mg/ml T, a value lower than MIC 90 and the one displaying antibiofilm activity, with independence of its formulation, T significantly decreased the macrophages viability. Conclusion: Overall, because of its lower MIC 90 against planktonic bacteria, higher antibiofilm formation capacity and stability during storage, A80-T resulted better antibacterial agent than T emulsion and L80-T. These results open new avenues to explode the A80-T antimicrobial intracellular activity. Fil: Perez, Ana Paula. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina Fil: Perez, Noelia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina Fil: Suligoy Lozano, Carlos Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Altube, María Julia. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: de Farias, Marcelo Alexandre. No especifíca; Fil: Portugal, Rodrigo Villares. No especifíca; Fil: Buzzola, Fernanda Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Morilla, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina Fil: Romero, Eder Lilia. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Hyperhalophilic archaebacteria exclusively produce sn2,3 diphytanylglycerol diether archaeolipids, unique structures absent in bacteria and eukaryotes. Nanovesicles made of archaeolipids known as nanoarchaeosomes (nanoARC), possess highly stable bilayers, some of them displaying specific targeting ability. Here we hypothesize that nanoARC made from Halorubrum tebenquichense archaebacteria, may constitute efficient carriers for the TLR7 agonist imiquimod (IMQ). NanoARC-IMQ takes advantage of the intense interaction between IMQ and the highly disordered, poorly fluid branched archaeolipid bilayers, rich in archaeol analog of methyl ester of phosphatidylglycerophosphate (PGP-Me), a natural ligand of scavenger receptor A1 (SR-A1). This approach lacks complex manufacture steps required for bilayers labeling, enabling future analytical characterization, batch reproducibility, and adaptation to higher scale production. SR-A1 mediated internalization of particulate material is mostly targeted to macrophages and is extensive because it is not submitted to a negative feedback. A massive and selective intracellular delivery of IMQ may concentrate its effect specifically into the endosomes, where the TLR7 is expressed, magnifying its immunogenicity, at the same time reducing its systemic bioavailability, and therefore it's in vivo adverse effects. NanoARC-IMQ (600-900 nm diameter oligolamellar vesicles of ~-43 mV Z potential) were heavily loaded with IMQ at ~44 μg IMQ/mg phospholipids [~20 folds higher than the non-SR-A1 ligand soyPC liposomes loaded with IMQ (LIPO-IMQ)]. In vitro, nanoARC-IMQ induced higher TNF-a and IL-6 secretion by J774A1 macrophages compared to same dose of IMQ and same lipid dose of LIPO-IMQ. In vivo, 3 subcutaneous doses of nanoARC-IMQ+ 10 μg total leishmania antigens (TLA) at 50 μg IMQ per Balb/C mice, induced more pronounced DTH response, accompanied by a nearly 2 orders higher antigen-specific systemic IgG titers than IMQ+TLA and LIPO-IMQ. The isotype ratio of nanoARC-IMQ+TLA remained ~0.5 indicating, the same as IMQ+TLA, a Th2 biased response distinguished by a pronounced increase in antibody titers, without negative effects on splenocytes lymphoproliferation, with a potential CD8+LT induction 10 days after the last dose. Overall, this first approach showed that highly SR-A1 mediated internalization of heavily loaded nanoARC-IMQ, magnified the effect of IMQ on TLR7 expressing macrophages, leading to a more intense in vivo immune response. Fil: Parra, Federico Leonel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Caimi, Ayelen Tatiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Altube, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Cargnelutti, Diego Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Vermeulen, Elba Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Farias, Marcelo Alexandre de. Brazilian Nanotechnology National Laboratory; Brasil Fil: Portugal, Rodrigo Villares. Brazilian Nanotechnology National Laboratory; Brasil Fil: Morilla, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Romero, Eder Lilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina

Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 A-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein–protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution. A combination of cryo-electron microscopy (cryo-EM) and targeted mutagenesis studies elucidates the structure and organization of the core complex of the type IV secretion system used by Xanthomonas citri to kill competing bacteria.

The interaction of single-layer graphene oxide (SLGO) and multi-layered graphene oxide (MLGO) with a cell culture medium (i.e. DMEM) was studied by evaluating fetal bovine serum (FBS) protein corona formation towards in vitro nanotoxicity assessment and nanobiointeractions. SLGO and MLGO exhibited different colloidal behavior in the culture medium, which was visualized by cryogenic transmission electron microscopy in situ analysis. Exploring proteomics and bioinformatics tools, 394 and 290 proteins were identified on the SLGO and MLGO hard corona compositions, respectively. From this amount, 115 proteins were exclusively detected on the SLGO and merely 11 on MLGO. SLGO enriched FBS proteins involved in metabolic processes and signal transduction, while MLGO enriched proteins involved in cellular development/structure, and lipid transport/metabolic processes. Such a distinct corona profile is due to differences on surface chemistry, aggregation behavior and the surface area of GO materials. Hydrophilic interactions were found to play a greater role in protein adsorption by MLGO than SLGO. Our results point out implications for in vitro studies of graphene oxide materials concerning the effective dose delivered to cells and corona bioactivity. Finally, we demonstrated the importance of integrating conventional and modern techniques thoroughly to understand the GO-FBS complexes towards more precise, reliable and advanced in vitro nanotoxicity assessment.

A new nanohydrogel drug delivery platform based on Laponite nanodiscs, polyacrylate, and sodium phosphate salts is described. The hybrid nanohydrogel is tailored to obtain soft and flexible nanohydrogels with G′ around 3 kPa, which has been proposed as the ideal stiffness for drug delivery applications. In vitro studies demonstrate that the new nanohydrogels are biocompatible, biodegradable, nonswellable, pH-responsive, and noncytotoxic and are able to deliver antineoplastic drugs into cancer cells. The IC50 of nanohydrogels containing cisplatin, 4-fluorouracil, and cyclophosphamide is significantly lower than the IC50 of the free drugs. In vivo experiments suggest that the new nanomaterials are biocompatible and do not accumulate in crucial organs. The simple formulation procedure enables encapsulation of virtually any water-soluble molecule, without the need for chemical modification of the guests. These nanohydrogels are a versatile platform that enables the simultaneous encapsulation of several cancer...

Septinas são GTPases consideradas como um novo componente do citoesqueleto. Essas proteínas interagem entre si para formar heterocomplexos filamentosos e estruturas de alta ordem que são importantes para a citocinese e uma variedade de outros processos celulares. Existem muitos aspectos mecânicos dessas proteínas que não são totalmente compreendidos, incluindo a forma como os heterocomplexos se agrupam corretamente. Em humanos, há 13 genes que codificam septinas, classificadas em quatro grupos quanto à similaridade em relação à estrutura primária. O complexo hexamérico SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 foi o melhor caracterizado, com uma estrutura cristalina resolvida à 4 Å. Segundo às Regras de Kinoshita, as septinas desse complexo podem ser substituídas nesse arranjo por outras pertencentes ao mesmo grupo. Neste trabalho utilizamos a técnica de microscopia eletrônica de transmissão com análise de partícula única para estudar dois complexos de septinas. Um dos complexos estudados neste projeto é formado por septinas humanas, para as quais atualmente não há informações estruturais disponíveis. O complexo SEPT5-SEPT6-SEPT7 foi expresso heterólogamente em E. coli e purificado em alta concentração salina para evitar a polimerização. A análise de partículas únicas de imagens por contrastação negativa mostrou a presença de partículas alongadas de aproximadamente 25 nm de comprimento, compostos por seis monômeros, como esperado. Com o objetivo de localizar a posição da SEPT5 no complexo, foi realizada uma fusão com MBP (Maltose Binding Protein) e imunomarcação com anticorpo monoclonal anti-SEPT5, concluindo que a SEPT5 está localizada na extremidade do complexo hexamérico. Porém, a SEPT5 pertence ao mesmo grupo da SEPT2, que foi relatada estar localizada no centro do hexâmero. Este resultado possibilitou uma nova discussão sobre a maneira que as septinas formam os complexos e, como a sensibilidade à concentração salina está relacionada com a fragilidade da interface NC, análogo ao observado em complexos de Saccharomyces cerevisiae. Um complexo de Ciona intestinalis incluindo a SEPT2, SEPT6, SEPT7 e SEPT9, também expresso heterólogamente em E. coli, foi preparado por contrastação negativa. A análise de partícula única das imagens coletadas mostrou um heterocomplexo aparentemente hexamérico, embora fosse esperado um octâmero devido à presença das quatro septinas diferentes, sendo uma pertencente à cada um dos quatro grupos. Os resultados deste trabalho proporcionaram um avanço na compreensão da formação de heterocomplexos de septinas e como essas proteínas interagem umas com as outras nesta montagem. Septins are GTPases that appear to be a novel component of the cytoskeleton. These proteins interact with each other to form filamentous heterocomplexes and high order structures which are important for cytokinesis and a variety of other cellular processes. There are many mechanistic aspects of these proteins that are not fully understood, including how the heterocomplexes correctly assemble. In humans, there are 13 genes encoding septins, classified in four groups based on primary structure. The SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 hexameric complex was the best characterized, with a crystalline structure solved at 4 Å. According to Kinoshita´s Rules, the septins of this complex can be replaced in this arrangement by others belonging to the same group. In this work, we used transmission electron microscopy with single particle analysis to study two septin complexes. One of the complexes studied in this project is composed of three human septins, for which there is currently no structural information available. The SEPT5-SEPT6-SEPT7 complex was heterologously expressed in E. coli and purified at high salt concentration to avoid polymerization. Single particle analysis of negatively stained samples showed the presence of elongated particles of approximately 25 nm in length. To locate SEPT5 in the complex, a fusion with MBP (Maltose Binding Protein) and immunoblotting with anti-SEPT5 monoclonal antibody was performed, concluding that SEPT5 is located at the end of the hexameric complex. However, SEPT5 belongs to the same group as SEPT2, which was reported to be located in the center of the hexamer. This result allowed for a new discussion on the way that septins form heterocomplexes and also, on how the sensitivity of the NC interface in related to salt concentration, analogous to that observed in the heterocomplex of Saccharomyces cerevisiae. A Ciona intestinalis complex including SEPT2, SEPT6, SEPT7 and SEPT9, also expressed heterologously in E. coli, was prepared by negative staining. The single particle analysis of the collected images showed an apparently hexameric heterocomplex, although an octamer was expected due to the presence of the four different septins, one belonging to each of the four groups. The results of this work represent advances in the understanding of the formation of septin heterocomplexes and how these proteins interact with each other during assembly.

This paper describes the development of a facile and environmentally friendly strategy for supporting crotamine on gold nanoparticles (GNPs). Our approach was based on the covalent binding interaction between the cell penetrating peptide crotamine, which is a snake venom polypeptide with preference to penetrate dividing cells, and a polyethylene glycol (PEG) ligand, which is a nontoxic, water-soluble and easily obtainable commercial polymer. Crotamine was derivatized with ortho-pyridyldisulfide-polyethyleneglycol-N-hydroxysuccinimide (OPSS-PEG-SVA) cross-linker to produce OPSS-PEG-crotamine as the surface modifier of GNP. OPSS-PEG-SVA can serve not only as a surface modifier, but also as a stabilizing agent for GNPs. The successful PEGylation of the nanoparticles was demonstrated using different physicochemical techniques, while the grafting densities of the PEG ligands and crotamine on the surface of the nanoparticles were estimated using a combination of electron microscopy and mass spectrometry analysis. In vitro assays confirmed the internalization of these GNPs, into living HeLa cells. The results described herein suggest that our approach may serve as a simple platform for the synthesis of GNPs decorated with crotamine with well-defined morphologies and uniform dispersion, opening new roads for crotamine biomedical applications.

Poly(A)-binding proteins (PABPs) play crucial roles in mRNA biogenesis, stability, transport and translational control in most eukaryotic cells. Although animal PABPs are well-studied proteins, the biological role, three-dimensional structure and RNA-binding mode of plant PABPs remain largely uncharacterized. Here, we report the structural features and RNA-binding mode of a Citrus sinensis PABP (CsPABPN1). CsPABPN1 has a domain architecture of nuclear PABPs (PABPNs) with a single RNA recognition motif (RRM) flanked by an acidic N-terminus and a GRPF-rich C-terminus. The RRM domain of CsPABPN1 displays virtually the same three-dimensional structure and poly(A)-binding mode of animal PABPNs. However, while the CsPABPN1 RRM domain specifically binds poly(A), the full-length protein also binds poly(U). CsPABPN1 localizes to the nucleus of plant cells and undergoes a dimer–monomer transition upon poly(A) interaction. We show that poly(A) binding by CsPABPN1 begins with the recognition of the RNA-binding sites RNP1 and RNP2, followed by interactions with residues of the β2 strands, which stabilize the dimer, thus leading to dimer dissociation. Like human PABPN1, CsPABPN1 also seems to form filaments in the presence of poly(A). Based on these data, we propose a structural model in which contiguous CsPABPN1 RRM monomers wrap around the RNA molecule creating a superhelical structure that could not only shield the poly(A) tail but also serve as a scaffold for the assembly of additional mRNA processing factors.

This work presents a study of the association between low molecular weight hyaluronic acid (16 kDa HA) and cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). The cationic liposome/HA complexes were evaluated to determine their mesoscopic structure, average size, zeta potential, and morphology as a function of the amount of HA in the system. Small angle X-ray scattering results revealed that neighboring cationic liposomes either stick together after a partial coating of low concentration HA or disperse completely in excess of HA, but they never assemble as multilamellar vesicles. Cryo-transmission electron microscopy images confirm the existence of unilamellar vesicles and large aggregates of unilamellar vesicles for HA fractions up to 80% (w/w). High concentrations of HA (> 20% w/w) proved to be efficient for coating extruded liposomes, leading to particle complexes with sizes in the nanoscale range and a negative zeta potential.

On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. However, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We propose an evolutionary model wherein the appearance of the most active enzyme isoform, glutaminase C (GAC), which is expressed in many cancers, was a late retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. To obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C-termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors.

Ultradeformable archaeosomes (UDA) are nanovesicles made of total polar archaeolipids (TPA) from the archaea Halorubrum tebenquichense, soybean phosphatidylcholine and sodium cholate (3:3:1 w/w). Fresh dispersions of UDA including different type of antigens are acknowledged as efficient topical vaccination agents. UDA dispersions however, if manufactured for pharmaceutical use, have to maintain colloidal stability upon liposomicidal processes such as sterilization and lyophilization (SLRUDA), needed to extend shelf life during storage. The remaining capacity of SLRUDA to act as adjuvants was therefore tested here for the first time. Another unexplored issue addressed here, is the outcome of replacing classical antigen inclusion into nanovesicles by their physical mixture. Our results showed that UDA behaved as super-stable nanovesicles because of its high endurance during heat sterilization and storage for 5 months at 40 °C. The archaeolipid content of UDA however, was insufficient to protect it against lyophilization, which demanded the addition of 2.5% v/v glycerol plus 0.07% w/v glucose. No significant differences were found between serum anti-ovalbumin (OVA) IgG titers induced by fresh or SLRUDA upon topical application of 4 weekly doses at 600 μg lipids/75 μg OVA to Balb/c mice. Finally, SLRUDA mixed with OVA elicited the same Th2 biased plus a non-specific cell mediated response than OVA encapsulated within UDA. Concluding, we showed that TPA is key component of super-stable nanovesicles that confers resistance to heat sterilization and to storage under cold-free conditions. The finding of SLRUDA as ready-to-use topical adjuvant would lead to simpler manufacture processing and cheaper products.. Fil: Caimi, Ayelen Tatiana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Parra, Federico Leonel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: de Farias, Marcelo Alexander. Centro Nacional de Pesquisa Em Energia E Materiais; Brasil Fil: Portugal, Rodrigo Villares. Centro Nacional de Pesquisa Em Energia E Materiais; Brasil Fil: Perez, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Romero, Eder Lilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Morilla, María José. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Solution studies permit a direct investigation of the particles on a well-defined environment. A number of thermodynamic and spectroscopic methods can be used directly in solution providing important structural information. In this work, hydrogen deuterium exchange with mass spectrometry, useful for studying protein interactions, dynamics, and conformations, is explained with data analysis approaches having practical implications. Scattering methods, in particular small-angle X-ray scattering (SAXS), are described, and several applications are presented. As it will be shown, directly from the scattering data, it is possible to obtain size, shape, oligomerization state, and aggregation dynamics among several other parameters. We have further provided the details of cryogenic electron microscopy (“cryo-EM”) where vitreous ice-embedded macromolecules, liposomes, nanoparticles, and three-dimensional structures are determined using several 2D projection images obtained from cryogenic TEM.

Aim: To increase the subcellular delivery of dexamethasone phosphate (DP) and stability to nebulization stress, pH-sensitive nanoliposomes (LpH) exhibiting archaeolipids, acting as ligands for scavenger receptors (pH-sensitive archaeosomes [ApH]), were prepared. Materials & methods: The anti-inflammatory effect of 0.18 mg DP/mg total lipid, 100–150 nm DP-containing ApH (dioleylphosphatidylethanolamine: Halorubrum tebenquichense total polar archaeolipids:cholesteryl hemisuccinate 4.2:2.8:3 w:w) was tested on different cell lines. Size and HPTS retention of ApH and conventional LpH (dioleylphosphatidylethanolamine:cholesteryl hemisuccinate 7:3 w:w) before and after nebulization were determined. Results & conclusion: DP-ApH suppressed IL-6 and TNF-α on phagocytic cells. Nebulized after 6-month storage, LpH increased size and completely lost its HPTS while ApH3 conserved size and polydispersity, fully retaining its original HPTS content. Fil: Altube, María Julia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Selzer, Solange Mailen. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: de Farias, Marcelo Alexandre. Brazilian Nanotechnology National Laboratory; Brasil Fil: Portugal, Rodrigo Villares. Brazilian Nanotechnology National Laboratory; Brasil Fil: Morilla, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina Fil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina

The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen–deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B–dependent transcription exercised by GR in cells.

Este trabalho apresenta o estudo e caracterização de dois complexos moleculares, hRXRálfadeltaAB e hemocianina de Acanthoscurria gomesiana, através de técnicas estruturais e biofísicas. O uso da técnica de crio-microscopia eletrônica para o estudo da hemocianina de Acanthoscurria gomesiana, resultou em um modelo estrutural com resolução de 14 angstron- pelo métodode Fourier Shell Correlation com critério de 1/2 bit. Neste limite de resolução, já é possível observar detalhes estruturais que o mostram como sendo comptível com outros modelos de hemocianinas. Com relação ao estudo de hRXRalfadeltaAB, mostrou-se, através das técnicas de cromatografia analítica de exclusão por tamanho, eletroforese de gel de poliacrilamida e SAXS, que a proteína pode se apresentar no estado dimérico em solução, mesmo na ausência do seu ligante, 9-cis-RA. Também foi estudado a associação de hRXRalfadeltaAB a elementos responsivos: DR1, DR4, F2 e PAL. Suas constantes de dissociação foram calculadas através da técnica de espectroscopia por anisotropia de fluorescência. Os resultados obtidos mostram maior afinidade por DR1 e DR2 e indicam uma origem entrópica para o processo de associação This work describes characterization of two biomolecular complexes: hRXR deltaAB and a hemocyanin from Acanthoscurria gomesiana using structural and biophysical techniques. Application of cryo-electron microscopy to studies of a hemocyanin from Acanthoscurria gomesiana resulted in its structural model to 14Å resolution, which was calculated by Fourier Shell Correlation with cut-off of 1/2 bit. At this resolution limit one can observe structural details of the complex which are compatible with other hemocyanin models. With respect to hRXR deltaAB, we showed using analytic size exclusion chromatography, SDS PAGE and SAXS, that the protein is dimeric in solution even at the absence of its ligand, 9-cis-RA. hRXR deltaAB binding to the responsive elements of DNA, DR1, DR4, F2 and PAL was investigated and the binding constants to these responsive elements have been determined using fluorescence anisotropy technique. Our results show higher affinity of the receptor to DR1 and DR4 and indicate entropic mechanism of DNA binding

Large datasets are emerging in many fields of image processing including: electron microscopy, light microscopy, medical X-ray imaging, astronomy, etc. Novel computer-controlled instrumentation facilitates the collection of very large datasets containing thousands of individual digital images. In single-particle cryogenic electron microscopy (“cryo-EM”), for example, large datasets are required for achieving quasi-atomic resolution structures of biological complexes. Based on the collected data alone, large datasets allow us to precisely determine the statistical properties of the imaging sensor on a pixel-by-pixel basis, independent of any “a priori” normalization routinely applied to the raw image data during collection (“flat field correction”). Our straightforward “a posteriori” correction yields clean linear images as can be verified by Fourier Ring Correlation (FRC), illustrating the statistical independence of the corrected images over all spatial frequencies. The image sensor characteristics can also be measured continuously and used for correcting upcoming images.

The Echinococcus granulosus actin filament-fragmenting protein (EgAFFP) is a three domain member of the gelsolin family of proteins, which is antigenic to human hosts. These proteins, formed by three or six conserved domains, are involved in the dynamic rearrangements of the cytoskeleton, being responsible for severing and capping actin filaments and promoting nucleation of actin monomers. Various structures of six domain gelsolin-related proteins have been investigated, but little information on the structure of three domain members is available. In this work, the solution structure of the three domain EgAFFP has been investigated through small-angle x-ray scattering (SAXS) studies. EgAFFP exhibits an elongated molecular shape. The radius of gyration and the maximum dimension obtained by SAXS were, respectively, 2.52 +/- 0.01 nm and 8.00 +/- 1.00 nm, both in the absence and presence of Ca2+. Two different molecular homology models were built for EgAFFP, but only one was validated through SAXS studies. The predicted structure for EgAFFP consists of three repeats of a central beta-sheet sandwiched between one short and one long alpha-helix. Possible implications of the structure of EgAFFP upon actin binding are discussed.

Receptor-like protein tyrosine phosphatases generally contain one or two conserved intracellular catalytic domains with a conserved sequence motif ([I/V]HCXAGXXR[S/T]G), a single transmembrane domain, and an external highly variable part. Here, we describe cloning of the intracellular catalytic domain of the rat protein tyrosine phosphatase eta (rPTPetaCD) into pET28a(+) vector, its expression in Escherichia coli, purification and initial characterization. The purification of His6-tagged rPTPetaCD to near homogeneity was achieved by a combination of affinity and size exclusion chromatography. The His-tag was subsequently removed by thrombin digestion. PhastGel IEF electrophoresis demonstrated that the isoelectric point of this 41 kDa His6-tag free recombinant protein was 7.3, which is just slightly higher than the theoretically predicted value of 7.2. To assess the functionality of the rPTPetaCD we used the pNPP hydrolysis assay and observed that the enzyme has a specific activity of 9 nmol/min/mug. The secondary structure and stability of the recombinant protein was also analyzed by circular dichroism and fluorescence spectroscopy. In summary, the rPTPetaCD is stable at 18 degrees C, properly folded, and fully active, which makes it a suitable candidate for structural and functional studies.

NOR-1 is an orphan member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, fatty acids, retinoic acids, and other lipophilic molecules. The NOR-1 subfamily (NR4), composed also of Nurr1 and Nurr77, has been implicated in cell proliferation, differentiation, apoptosis, chondrosarcomas, inflammation, and atherogenesis. The NOR-1 receptor is an orphan ligand receptor which acts over gene transactivation. No ligands, if such in fact exist, are known for this receptor. Recently, the three-dimensional structure of the homolog receptor Nurr1 has been solved using protein crystallography techniques. Surprisingly, the structure does not present either a typical cavity for ligand binding or a classical co-factor binding site in the ligand binding domain (LBD). To allow for structural studies of other members of NR4 subfamily, we have subcloned, overexpressed in Escherichia coli cells, purified, and characterized the rat orphan nuclear receptor NOR-1 LBD domain. We obtained NOR-1 LBD at a high degree of purity and with an overall yield of 3 mg/L of culture media. CD spectroscopic analysis shows a high alpha-helical secondary structure content (52%), similar to that of Nurr 1 LBD three-dimensional structure. Thermal denaturation monitored by UV absorption and CD spectroscopy suggests proper folding of recombinant NOR-1 LBD.

The phosphate-dependent transition between enzymatically inert dimers into catalytically capable tetramers has long been the accepted mechanism for the glutaminase activation. Here, we demonstrate that activated glutaminase C (GAC) self-assembles into a helical, fiber-like double-stranded oligomer and propose a molecular model consisting of seven tetramer copies per turn per strand interacting via the N-terminal domains. The loop 321LRFNKL326 is projected as the major regulating element for self-assembly and enzyme activation. Furthermore, the previously identified in vivo lysine acetylation (Lys311 in humans, Lys316 in mouse) is here proposed as an important down-regulator of superoligomer assembly and protein activation. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a known glutaminase inhibitor, completely disrupted the higher order oligomer, explaining its allosteric mechanism of inhibition via tetramer stabilization. A direct correlation between the tendency to self-assemble and the activity levels of the three mammalian glutaminase isozymes was established, with GAC being the most active enzyme while forming the longest structures. Lastly, the ectopic expression of a fiber-prone superactive GAC mutant in MDA-MB 231 cancer cells provided considerable proliferative advantages to transformed cells. These findings yield unique implications for the development of GAC-oriented therapeutics targeting tumor metabolism. Background: GAC supplies for increased metabolic needs of tumors because of exclusive localization and kinetic properties. Results: Higher than tetramer oligomers are the active form in in vitro and in cellular assays. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide disrupts oligomers. Conclusion: A novel molecular mechanism for GAC activation is proposed. Significance: The data affect the development of therapies targeting GAC in tumors, with emphasis on allosteric inhibitors.

Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species.