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Customization of deuterated biomolecules is vital for many advanced biological experiments including neutron scattering. However, because it is challenging to control the proportion and regiospecificity of deuterium incorporation in live systems, often only two or three synthetic lipids are mixed together to form simplistic model membranes. This limits the applicability and biological accuracy of the results generated with these synthetic membranes. Despite some limited prior examination of deuterating Escherichia coli lipids in vivo, this approach has not been widely implemented. Here, an extensive mass spectrometry-based profiling of E. coli phospholipid deuteration states with several different growth media was performed, and a computational method to describe deuterium distributions with a one-number summary is introduced. The deuteration states of 36 lipid species were quantitatively profiled in 15 different growth conditions, and tandem mass spectrometry was used to reveal deuterium localization. Regressions were employed to enable the prediction of lipid deuteration for untested conditions. Small-angle neutron scattering was performed on select deuterated lipid samples, which validated the deuteration states calculated from the mass spectral data. Based on these experiments, guidelines for the design of specifically deuterated phospholipids are described. This unlocks even greater capabilities from neutron-based techniques, enabling experiments that were formerly impossible.
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Difracción de Neutrones , Fosfolípidos , Deuterio/química , Difracción de Neutrones/métodos , Escherichia coli/metabolismo , Espectrometría de Masas en TándemRESUMEN
The cell-cell adhesion cadherin-catenin complexes recruit vinculin to the adherens junction (AJ) to modulate the mechanical couplings between neighboring cells. However, it is unclear how vinculin influences the AJ structure and function. Here, we identified two patches of salt bridges that lock vinculin in the head-tail autoinhibited conformation and reconstituted the full-length vinculin activation mimetics bound to the cadherin-catenin complex. The cadherin-catenin-vinculin complex contains multiple disordered linkers and is highly dynamic, which poses a challenge for structural studies. We determined the ensemble conformation of this complex using small-angle x-ray and selective deuteration/contrast variation small-angle neutron scattering. In the complex, both α-catenin and vinculin adopt an ensemble of flexible conformations, but vinculin has fully open conformations with the vinculin head and actin-binding tail domains well separated from each other. F-actin binding experiments show that the cadherin-catenin-vinculin complex binds and bundles F-actin. However, when the vinculin actin-binding domain is removed from the complex, only a minor fraction of the complex binds to F-actin. The results show that the dynamic cadherin-catenin-vinculin complex employs vinculin as the primary F-actin binding mode to strengthen AJ-cytoskeleton interactions.
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Actinas , Cadherinas , Cadherinas/metabolismo , Actinas/metabolismo , Vinculina/metabolismo , alfa Catenina/química , Unión Proteica , Citoesqueleto de Actina/metabolismo , Adhesión CelularRESUMEN
Tumor-stroma interactions are critical in pancreatic ductal adenocarcinoma (PDAC) progression and therapeutics. Patient-derived xenograft (PDX) models recapitulate tumor-stroma interactions, but the conventional antibody-based immunoassay is inadequate to discriminate tumor and stromal proteins. Here, we describe a species-deconvolved proteomics approach embedded in IonStar that can unambiguously quantify the tumor (human-derived) and stromal (mouse-derived) proteins in PDX samples, enabling unbiased investigation of tumor and stromal proteomes with excellent quantitative reproducibility. With this strategy, we studied tumor-stroma interactions in PDAC PDXs that responded differently to Gemcitabine combined with nab-Paclitaxel (GEM+PTX) treatment. By analyzing 48 PDX animals 24 h/192 h after treatment with/without GEM+PTX, we quantified 7262 species-specific proteins under stringent cutoff criteria, with high reproducibility. For the PDX sensitive to GEM+PTX, the drug-dysregulated proteins in tumor cells were involved in suppressed oxidative phosphorylation and the TCA cycle, and in the stroma, inhibition of glycolytic activity was predominant, suggesting a relieved reverse Warburg effect by the treatment. In GEM+PTX-resistant PDXs, protein changes suggested extracellular matrix deposition and activation of tumor cell proliferation. Key findings were validated by immunohistochemistry (IHC). Overall, this approach provides a species-deconvolved proteomic platform that could advance cancer therapeutic studies by enabling unbiased exploration of tumor-stroma interactions in the large number of PDX samples required for such investigations.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Carcinoma Ductal Pancreático/tratamiento farmacológico , Línea Celular Tumoral , Gemcitabina , Xenoinjertos , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Neoplasias Pancreáticas/patología , Proteómica , Reproducibilidad de los ResultadosRESUMEN
While solution micellization of ionic block copolymers (BCP) with randomly distributed ionization sites along the hydrophilic segments has been extensively studied, the roles of positionally controlled ionization sites along the BCP chains in their micellization and resulting micellar structure remain comparatively less understood. Herein, three amphoteric polypeptoid block copolymers carrying two oppositely charged ionizable sites, with one fixed at the hydrophobic terminus and the other varyingly positioned along the hydrophilic segment, have been synthesized by sequential ring-opening polymerization method. The presence of the ionizable site at the hydrophobic segment terminus is expected to promote polymer association toward equilibrium micellar structures in an aqueous solution. The concurrent presence of oppositely charged ionizable sites on the polymer chains allows the polymer association to be electrostatically modulated in a broad pH range (ca. 2-12). Micellization of the amphoteric polypeptoid BCP in dilute aqueous solution and the resulting micellar structure at different solution pHs was investigated by a combination of scattering and microscopic methods. Negative-stain transmission-electron microscopy (TEM), small-angle neutron scattering (SANS), and small-angle X-ray scattering (SAXS) analyses revealed the dominant presence of core-shell-type spherical micelles and occasional rod-like micelles with liquid crystalline (LC) domains in the micellar core. The micellar structures (e.g., aggregation number, radius of gyration, chain packing in the micelle) were found to be dependent on the solution pH and the position of the ionizable site along the chain. This study has highlighted the potential of controlling the position of ionizable sites along the BCP polymer to modulate the electrostatic and LC interactions, thus tailoring the micellar structure at different solution pH values in water.
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Micelas , Polímeros , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Polímeros/química , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people, with limited treatment options available for most patients. AMD involves the death of retinal pigment epithelium (RPE) and photoreceptor cells, with mitochondria dysfunction being a critical early event. In the current study, we utilized our unique resource of human donor RPE graded for AMD presence and severity to investigate proteome-wide dysregulation involved in early AMD. Organelle-enriched fractions of RPE were isolated from donors with early AMD (n = 45) and healthy age-matched controls (n = 32) and were analyzed by UHR-IonStar, an integrated proteomics platform enabling reliable and in-depth proteomic quantification in large cohorts. A total of 5941 proteins were quantified with excellent analytical reproducibility, and with further informatics analysis, many biological functions and pathways were found to be significantly dysregulated in donor RPE samples with early AMD. Several of these directly pinpointed changes in mitochondrial functions, e.g., translation, ATP metabolic process, lipid homeostasis, and oxidative stress. These novel findings highlighted the value of our proteomics investigation by allowing a better understanding of the molecular mechanisms underlying early AMD onset and facilitating both treatment development and biomarker discovery.
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Degeneración Macular , Epitelio Pigmentado de la Retina , Humanos , Anciano , Epitelio Pigmentado de la Retina/metabolismo , Proteómica , Reproducibilidad de los Resultados , Degeneración Macular/metabolismo , Estrés OxidativoRESUMEN
PURPOSE OF REVIEW: Hyperbilirubinemia is commonly seen in neonates. Though hyperbilirubinemia is typically asymptomatic, severe elevation of bilirubin levels can lead to acute bilirubin encephalopathy and progress to kernicterus spectrum disorder, a chronic condition characterized by hearing loss, extrapyramidal dysfunction, ophthalmoplegia, and enamel hypoplasia. Epidemiological data show that the implementation of universal pre-discharge bilirubin screening programs has reduced the rates of hyperbilirubinemia-associated complications. However, acute bilirubin encephalopathy and kernicterus spectrum disorder are still particularly common in low- and middle-income countries. RECENT FINDINGS: The understanding of the genetic and biochemical processes that increase the susceptibility of defined anatomical areas of the central nervous system to the deleterious effects of bilirubin may facilitate the development of effective treatments for acute bilirubin encephalopathy and kernicterus spectrum disorder. Scoring systems are available for the diagnosis and severity grading of these conditions. The treatment of hyperbilirubinemia in newborns relies on the use of phototherapy and exchange transfusion. However, novel therapeutic options including deep brain stimulation, brain-computer interface, and stem cell transplantation may alleviate the heavy disease burden associated with kernicterus spectrum disorder. Despite improved screening and treatment options, the prevalence of acute bilirubin encephalopathy and kernicterus spectrum disorder remains elevated in low- and middle-income countries. The continued presence and associated long-term disability of these conditions warrant further research to improve their prevention and management.
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Encefalopatías , Kernicterus , Bilirrubina , Humanos , Recién Nacido , Kernicterus/diagnóstico , Kernicterus/epidemiología , Kernicterus/etiología , Fototerapia/efectos adversosRESUMEN
The cadherin-catenin adhesion complex is the central component of the cell-cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-cateninâ¢ß-cateninâ¢epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and ß-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of ß-catenin forms a flexible "tongue" that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin-catenin adhesion complex in mechanotransduction.
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Cadherinas/química , Cadherinas/metabolismo , Mecanotransducción Celular , alfa Catenina/química , alfa Catenina/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Uniones Adherentes/química , Uniones Adherentes/genética , Uniones Adherentes/metabolismo , Secuencias de Aminoácidos , Cadherinas/genética , Humanos , Conformación Molecular , Unión Proteica , Dominios Proteicos , Dispersión del Ángulo Pequeño , alfa Catenina/genética , beta Catenina/genéticaRESUMEN
Quantitative proteomics in large cohorts is highly valuable for clinical/pharmaceutical investigations but often suffers from severely compromised reliability, accuracy, and reproducibility. Here, we describe an ultra-high-resolution IonStar method achieving reproducible protein measurement in large cohorts while minimizing the ratio compression problem, by taking advantage of the exceptional selectivity of ultra-high-resolution (UHR)-MS1 detection (240k_FWHM@m/z = 200). Using mixed-proteome benchmark sets reflecting large-cohort analysis with technical or biological replicates (N = 56), we comprehensively compared the quantitative performances of UHR-IonStar vs a state-of-the-art SWATH-MS method, each with their own optimal analytical platforms. We confirmed a cutting-edge micro-liquid chromatography (LC)/Triple-TOF with Spectronaut outperforms nano-LC/Orbitrap for SWATH-MS, which was then meticulously developed/optimized to maximize sensitivity, reproducibility, and proteome coverage. While the two methods with distinct principles (i.e., MS1- vs MS2-based) showed similar depth-of-analysis (â¼6700-7000 missing-data-free proteins quantified, 1% protein-false discovery rate (FDR) for entire set, 2 unique peptides/protein) and good accuracy/precision in quantifying high-abundance proteins, UHR-IonStar achieved substantially superior quantitative accuracy, precision, and reproducibility for lower-abundance proteins (a category that includes most regulatory proteins), as well as much-improved sensitivity/selectivity for discovering significantly altered proteins. Furthermore, compared to SWATH-MS, UHR-IonStar showed markedly higher accuracy for a single analysis of each sample across a large set, which is an inadequately investigated albeit critical parameter for large-cohort analysis. Finally, we compared UHR-IonStar vs SWATH-MS in measuring the time courses of altered proteins in paclitaxel-treated cells (N = 36), where dysregulated biological pathways have been very well established. UHR-IonStar discovered substantially more well-recognized biological processes/pathways induced by paclitaxel. Additionally, UHR-IonStar showed markedly superior ability than SWATH-MS in accurately depicting the time courses of well known to be paclitaxel-induced biomarkers. In summary, UHR-IonStar represents a reliable, robust, and cost-effective solution for large-cohort proteomic quantification with excellent accuracy and precision.
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Proteómica , Espectrometría de Masas en Tándem , Estudios de Cohortes , Proteoma , Reproducibilidad de los ResultadosRESUMEN
How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that sphingomyelin and cholesterol-rich (SCOR) lipid mixtures with phosphatidylcholine can be cosolubilized by n-dodecyl-ß-melibioside to form bicelles. Small-angle X-ray and neutron scattering, as well as cryo-electron microscopy, demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios. SCOR bicelles are shown to be compatible with a wide array of experimental techniques, as applied to the transmembrane human amyloid precursor C99 protein in this medium. These studies reveal an equilibrium between low-order oligomer structures that differ significantly from previous experimental structures of C99, providing an example of how ordered membranes alter membrane protein structure.
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Colesterol/química , Proteínas de la Membrana/química , Esfingolípidos/química , Microscopía por Crioelectrón , HumanosRESUMEN
Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium Bacillus subtilis to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron scattering spectra, we confirmed that the B. subtilis cell membrane is lamellar and determined that its average hydrophobic thickness is 24.3 ± 0.9 Ångstroms (Å). Furthermore, by creating neutron contrast within the plane of the membrane using a mixture of H- and D-fatty acids, we detected lateral features smaller than 40 nm that are consistent with the notion of lipid rafts. These experiments-performed under biologically relevant conditions-answer long-standing questions in membrane biology and illustrate a fundamentally new approach for systematic in vivo investigations of cell membrane structure.
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Bacillus subtilis/metabolismo , Membrana Celular/metabolismo , Ácidos Grasos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Microdominios de Membrana/metabolismo , Modelos Biológicos , Algoritmos , Bacillus subtilis/química , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Cerulenina/farmacología , Deuterio , Enoil-CoA Hidratasa/genética , Enoil-CoA Hidratasa/metabolismo , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Ácidos Grasos/química , Eliminación de Gen , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Microdominios de Membrana/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Difracción de Neutrones , Ácidos Palmíticos/química , Ácidos Palmíticos/metabolismo , Dispersión del Ángulo Pequeño , EstereoisomerismoRESUMEN
The membrane is one of the key structural materials of biology at the cellular level. Composed predominantly of a bilayer of lipids with embedded and bound proteins, it defines the boundaries of the cell and many organelles essential to life and therefore is involved in almost all biological processes. Membrane-specific interactions, such as drug binding to a membrane receptor or the interactions of an antimicrobial compound with the lipid matrix of a pathogen membrane, are of interest across the scientific disciplines. Herein we present a review, aimed at nonexperts, of the major neutron scattering techniques used in membrane studies: small-angle neutron scattering, neutron membrane diffraction, neutron reflectometry, quasielastic neutron scattering, and neutron spin echo. Neutron scattering techniques are well suited to studying biological membranes. The nondestructive nature of cold neutrons means that samples can be measured for long periods without fear of beam damage from ultraviolet, electron, or X-ray radiation, and neutron beams are highly penetrating, thus offering flexibility in samples and sample environments. Most important is the strong difference in neutron scattering lengths between the two most abundant forms of hydrogen, protium and deuterium. Changing the relative amounts of protium/deuterium in a sample allows the production of a series of neutron scattering data sets, enabling the observation of differing components within complex membrane architectures. This approach can be as simple as using the naturally occurring neutron contrast between different biomolecules to study components in a complex by changing the solution H2O/D2O ratio or as complex as selectively labeling individual components with hydrogen isotopes. This review presents an overview of each experimental technique with the neutron instrument configuration, related sample preparation and sample environment, and data analysis, highlighted by a special emphasis on using prominent neutron contrast to understand structure and dynamics. This review gives researchers a practical introduction to the often enigmatic suite of neutron beamlines, thereby lowering the barrier to taking advantage of these large-facility techniques to achieve new understandings of membranes and their interactions with other molecules.
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The structure and dynamics of lipid membranes in the presence of extracellular macromolecules are critical for cell membrane functions and many pharmaceutical applications. The pathogen virulence-suppressing end-phosphorylated polyethylene glycol (PEG) triblock copolymer (Pi-ABAPEG) markedly changes the interactions with lipid vesicle membranes and prevents PEG-induced vesicle phase separation in contrast to the unphosphorylated copolymer (ABAPEG). Pi-ABAPEG weakly absorbs on the surface of lipid vesicle membranes and slightly changes the structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) unilamellar vesicles at 37 °C, as evidenced by small angle neutron scattering. X-ray reflectivity measurements confirm the weak adsorption of Pi-ABAPEG on DMPC monolayer, resulting in a more compact DMPC monolayer structure. Neutron spin-echo results show that the adsorption of Pi-ABAPEG on DMPC vesicle membranes increases the membrane bending modulus κ.
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Membrana Celular/química , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Fosfatidilcolinas/química , Membrana Celular/ultraestructura , Dimiristoilfosfatidilcolina/química , Glicerilfosforilcolina/química , Humanos , Membrana Dobles de Lípidos/metabolismo , Polietilenglicoles/química , Polímeros/química , Dispersión del Ángulo Pequeño , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
Flexible tactile sensor array has drawn great attention due to its ability to mimic human skin for sensing weak pressure and distinguishing pressure distribution, but the deficiency of sensitivity, the low resolution, and the complex and costly fabrication process seriously limit its development. Hence, it is urgent to explore a fully flexible sensor array with high sensitivity and high resolution as an electronic-skin. Here, the flexible piezoelectric tactile sensor array based on the composite film of PZT nanowires and polydimethylsiloxane (PDMS) was fabricated by the simple fabrication process (electrospinning process and mixture process). The electrospun PZT nanofibers have high aspect ratio and could enhance the generation and accumulation of the piezoelectric charges in the two electrodes of the composite film. By virtue of the inherently high piezoelectric coefficient of PZT material and high aspect ratio of PZT nanofibers, the composite film (75 wt% PZT nanofibers) presents high force-electric conversion capability and high sensitivity. Owing to the bottom electrode sheet shared by all sensor units and the supporting layer with relatively high elastic modulus, the sensor array shows high resolution to qualitatively sense the distribution and size of the impact in real time. Moreover, the sensor array also shows great durability, repeatability, and large working range. Based on these excellent characteristics, the sensor array has wide potential applications in the field of bionics science, robotics science and human-machine interaction.
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Antimicrobial peptides are one of the most promising classes of antibiotic agents for drug-resistant bacteria. Although the mechanisms of their action are not fully understood, many of them are found to interact with the target bacterial membrane, causing different degrees of perturbations. In this work, we directly observed that a short peptide disturbs membranes by inducing lateral segregation of lipids without forming pores or destroying membranes. Aurein 1.2 (aurein) is a 13-amino acid antimicrobial peptide discovered in the frog Litoria genus that exhibits high antibiotic efficacy. Being cationic and amphiphilic, it binds spontaneously to a membrane surface with or without charged lipids. With a small-angle neutron scattering contrast matching technique that is sensitive to lateral heterogeneity in membrane, we found that aurein induces significant lateral segregation in an initially uniform lipid bilayer composed of zwitterionic lipid and anionic lipid. More intriguingly, the lateral segregation was similar to the domain formed below the order-disorder phase-transition temperature. To our knowledge, this is the first direct observation of lateral segregation caused by a peptide. With quasi-elastic neutron scattering, we indeed found that the lipid lateral motion in the fluid phase was reduced even at low aurein concentrations. The reduced lateral mobility makes the membrane prone to additional stresses and defects that change membrane properties and impede membrane-related biological processes. Our results provide insights into how a short peptide kills bacteria at low concentrations without forming pores or destroying membranes. With a better understanding of the interaction, more effective and economically antimicrobial peptides may be designed.
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Péptidos Catiónicos Antimicrobianos/química , Lípidos/química , Simulación de Dinámica Molecular , Animales , Anuros , Estructura Molecular , Difracción de Neutrones , Dispersión del Ángulo PequeñoRESUMEN
Despite being one of the most potent chemotherapeutics, doxorubicin (DOX) facilitates cardiac toxicity by irreversibly damaging the cardiac muscle as well as severely dysregulating the immune system and impairing the resolution of cardiac inflammation. Herein, we report synthesis and aqueous self-assembly of nanosized polymersomes from temperature-responsive poly(3-methyl-N-vinylcaprolactam)-block-poly(N-vinylpyrrolidone) (PMVC-PVPON) diblock copolymers and demonstrate their potential to minimize DOX cardiotoxicity compared to liposomal DOX. RAFT polymerization of vinylpyrrolidone and 3-methyl-N-vinylcaprolactam, which are structurally similar monomers but have drastically different hydrophobicity, allows decreasing the cloud point of PMVCm-PVPONn copolymers below 20 °C. The lower critical solution temperature (LCST) of the PMVC58-PVPONn copolymer varied from 19.2 to 18.6 and to 15.2 °C by decreasing the length of the hydrophilic PVPONn block from n = 98 to n = 65 and to n = 20, respectively. The copolymers assembled into stable vesicles at room temperature when PVPON polymerization degrees were 65 and 98. Anticancer drug DOX was entrapped with high efficiency into the aqueous PMVC58-PVPON65 polymersomal core surrounded by the hydrophobic temperature-sensitive PMVC shell and the hydrophilic PVPON corona. Unlike many liposomal, micellar, or synthetic drug delivery systems, these polymersomes exhibit an exceptionally high loading capacity of DOX (49%) and encapsulation efficiency (95%) due to spontaneous loading of the drug at room temperature from aqueous DOX solution. We also show that C57BL/6J mice injected with the lethal dose of DOX at 15 mg kg-1 did not survive the 14 day treatment, resulting in 100% mortality. The DOX-loaded PMVC58-PVPON65 polymersomes did not cause any mortality in mice indicating that they can be used for successful DOX encapsulation. The gravimetric analyses of the animal organs from mice treated with liposome-encapsulated DOX (Lipo-DOX) and PMVC58-PVPON65 polymersomes (Poly-DOX) revealed that the Lipo-DOX injection caused some toxicity manifesting as decreased body weight compared to Poly-DOX and saline control. Masses of the left ventricle of the heart, lung, and spleen reduced in the Lipo-DOX-treated mice compared to the nontoxic saline control, while no significant decrease of those masses was observed for the Poly-DOX-treated mice. Our results provide evidence for superior stability of synthetic polymersomes in vivo and show promise for the development of next-generation drug carriers with minimal side effects.
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Antineoplásicos/química , Cardiotoxicidad/prevención & control , Doxorrubicina/química , Polímeros/química , Pirrolidinonas/química , Animales , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones , Ratones Endogámicos C57BL , Polimerizacion , TemperaturaRESUMEN
Some synthetic polymers can block cell death when applied following an injury that would otherwise kill the cell. This cellular rescue occurs through interactions of the polymers with cell membranes. However, general principles for designing synthetic polymers to ensure strong, but nondisruptive, cell membrane targeting are not fully elucidated. Here, we tailored biomimetic phosphorylcholine-containing block copolymers to interact with cell membranes and determined their efficacy in blocking neuronal death following oxygen-glucose deprivation. By adjusting the hydrophilicity and membrane affinity of poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC)-based triblock copolymers, the surface active regime in which the copolymers function effectively as membrane-targeting cellular rescue agents was determined. We identified nonintrusive interactions between the polymer and the cell membrane that alter the collective dynamics of the membrane by inducing rigidification without disrupting lipid packing or membrane thickness. In general, our results open new avenues for biological applications of polyMPC-based polymers and provide an approach to designing membrane-targeting agents to block cell death after injury.
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Materiales Biocompatibles/farmacología , Metacrilatos/química , Fosforilcolina/análogos & derivados , Polímeros/química , Materiales Biocompatibles/química , Biomimética/métodos , Muerte Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Metacrilatos/farmacología , Fosforilcolina/química , Fosforilcolina/farmacología , Polímeros/farmacologíaRESUMEN
As a promising functional material, ferroelectric Pb(ZrxTi1-x)O3 (PZT) are widely used in many optical and electronic devices. Remarkably, as the film thickness decreases, the materials' properties deviate gradually from those of solid materials. In this work, multilayered PZT thin films with different thicknesses are fabricated by Sol-Gel technique. The thickness effect on its microstructure, ferroelectric, and optical properties has been studied. It is found that the surface quality and the crystalline structure vary with the film thickness. Moreover, the increasing film thickness results in a significant increase in remnant polarization, due to the interfacial layer effect. Meanwhile, the dielectric loss and tunability are strongly dependent on thickness. In terms of optical properties, the refractive index of PZT films increase with the increasing thickness, and the photorefractive effect are also influenced by the thickness, which could all be related to the film density and photovoltaic effect. Besides, the band gap decreases as the film thickness increases. This work is significant for the application of PZT thin film in optical and optoelectronic devices.
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BACKGROUND: Cellobiose dehydrogenases have gained interest due to their potential applications in sectors from biofuel production to biomedical devices. The CDHIIA variant is comprised of a cytochrome domain (CYT), a dehydrogenase domain (DH), and a carbohydrate-binding module (CBM) that are connected by two flexible linkers. Upon cellobiose oxidation at the DH, intramolecular electron transfer (IaET) occurs from the DH to the CYT. In vivo, CDHIIA CYT subsequently performs intermolecular electron transfer (IeET) to a lytic polysaccharide monooxygenase (LPMO). The relevant solution-state CDH domain conformations for IaET and IeET have not been fully characterized. METHODS: Small-angle X-ray and neutron scattering measurements of oxidized CDHIIA from Myriococcum thermophilum and Neurospora crassa were performed to investigate the structural landscape explored in solution by MtCDHIIA and NcCDHIIA in response to cations, pH, and the presence of an electron acceptor, LPMO9D from N. crassa. RESULTS: The scattering data complemented by modeling show that, under oxidizing conditions, MtCDHIIA undergoes global conformational rearrangement in the presence of Ca2+. Oxidized NcCDHIIA exhibits conformational changes upon pH variation and, in the presence of NcLPMO9D, primarily adopts a compact conformation. CONCLUSIONS: These results demonstrate different conformational responses of oxidized MtCDHIIA and NcCDHIIA to changes in environment. The results also reveal a shift in the oxidized NcCDHIIA conformational landscape toward interdomain compaction upon co-incubation with NcLPMO9D. GENERAL SIGNIFICANCE: The present study is the first report on the structural landscapes explored in solution by oxidized cellobiose dehydrogenases under various cation concentrations, pH conditions and in the presence of an electron-accepting LPMO.
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Deshidrogenasas de Carbohidratos/química , Proteínas Fúngicas/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Ascomicetos/enzimología , Ascomicetos/genética , Calcio/química , Calcio/metabolismo , Deshidrogenasas de Carbohidratos/genética , Deshidrogenasas de Carbohidratos/metabolismo , Celulosa/química , Celulosa/metabolismo , Transporte de Electrón , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Neurospora crassa/enzimología , Neurospora crassa/genética , Oxidación-Reducción , Unión Proteica , Conformación ProteicaRESUMEN
Supramolecular gel phase crystallization offers a new strategy for drug polymorph screening and discovery. In this method, the crystallization outcome depends on the interaction between solute and gel fibre. While supramolecular gels have shown success in producing new polymorphs and crystals with novel morphologies, role of the gel and nature of gel-solute interaction remains largely unexplored. The present study aims to provide a comprehensive picture of the structural evolution of a supramolecular gel produced from a bis(urea) based gelator (G) in the presence of a polymorphic drug carbamazepine (CBZ). The structural aspects of the gel have been assessed by single crystal X-ray analysis, X-ray powder diffraction (XRPD) and solid state NMR spectroscopy. Small Angle Neutron Scattering (SANS) has been used to follow the changes in gel structure in the presence of CBZ. Visual evidence from morphological study and structural evolution observed at a macroscopic level from rheological measurements, shows good agreement with the SANS results. The concentration of the gelator and the relative proportion of G to CBZ were found to be crucial factors in determining the competitive nucleation events involving gelation and crystallization. At a critical G to CBZ ratio the effect of CBZ on gel structure was maximum and fiber bundling in the gel was found to be critically affected. This study offers important information about how the interplay of gelator assembly and gel-solute interactions can fine-tune the nucleation events in a supramolecular gel phase crystallization.
RESUMEN
Membrane-active peptides (MAPs), which interact directly with the lipid bilayer of a cell and include toxins and host defense peptides, display lipid composition-dependent activity. Phosphatidylserine (PS) lipids are anionic lipids that are found throughout the cellular membranes of most eukaryotic organisms where they serve as both a functional component and as a precursor to phosphatidylethanolamine lipids. The inner leaflet of the plasma membrane contains more PS than the outer one, and the asymmetry is actively maintained. Here, the impact of the MAP melittin on the structure of lipid bilayer vesicles made of a mixture of phosphatidylcholine and phosphatidylserine was studied. Small-angle neutron scattering of the MAP associated with selectively deuterium-labeled lipid bilayer vesicles revealed how the thickness and lipid composition of phosphatidylserine-containing vesicles change in response to melittin. The peptide thickens the lipid bilayer for concentrations up to P/L=1/500, but membrane thinning results when P/L=1/200. The thickness transition is accompanied by a large change in the distribution of DMPS between the leaflets of the bilayer. The change in composition is driven by electrostatic interactions, while the change in bilayer thickness is driven by changes in the interaction of the peptide with the headgroup region of the lipid bilayer. The results provide new information about lipid-specific interactions that take place in mixed composition lipid bilayer membranes.