10 research outputs found

    Brain regions that showed higher functional connectivity with the left SOG in Far blocks versus the Near blocks.

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    <p>Brain regions that showed higher functional connectivity with the left SOG in Far blocks versus the Near blocks.</p

    Psychophysiological interaction (PPI) analysis with the POJ as the source region and with the ‘Near > Far’ contrast as the psychological factor.

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    <p>The source region in the POJ is marked in red. (A) PPI activation in ‘match’ condition. Both the dorsal and ventral streams (blue) showed significant context-dependent co-variations with the neural activity in the POJ. The coupling was stronger in the far space than in the near space. In order to give a clearer view of ventral cortical structures, the cerebellum was removed in the ventral view display. PPI analysis based on the neural activity in the POJ (red) for a representative participants were shown. Mean corrected neural activity in the right inferior occipital gyrus, the left fusiform gyrus, the left inferior temporal gyrus, the left superior parietal gyrus and the right precuneus is displayed as a function of mean corrected activity in the POJ (i.e., first principal component from a sphere of 4 mm radius) in the near space (blue dots and lines) and far space (red dots and lines) blocks. (B) PPI activation in ‘natural’ condition. Both the dorsal and ventral streams (blue) showed significant context-dependent co-variations with the neural activity in the POJ. The coupling was stronger in the far space than in the near space. In order to give a clearer view of ventral cortical structures, the cerebellum was removed in the ventral view display. PPI analysis based on the neural activity in the POJ (red) for a representative participants were shown. Mean corrected neural activity in the left inferior occipital gyrus, the right fusiform gyrus, the right lingual gyrus, the right middle occipital gyrus, the left precuneus is displayed as a function of mean corrected activity in the POJ (i.e., first principal component from a sphere of 4 mm radius) in the near space (blue dots and lines) and far space (red dots and lines) blocks.</p

    Main effect of spatial domain.

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    <p>(A) The bilateral SOG and the POJ showed significantly higher neural activity in the near space processing than in the far space processing (i.e., ‘Near > Far’) in the ‘match’ condition. No significant activation was found for the reverse contrast (i.e., ‘Far > Near’). (B) The primary visual cortex, the POJ and SOG showed significantly higher neural activity in the near space processing than in the far space processing (i.e., ‘Near > Far’) in the ‘natural’ condition (red regions). The bilateral inferior occipital gyrus (IOG) were activated for the reverse contrast (i.e., ‘Far > Near’). (C) Conjunction between the ‘Near > Far’ contrast in the ‘match’ and ‘natural’ conditions revealed the POJ and bilateral SOG.</p

    Development of Copper-Catalyzed Azide–Alkyne Cycloaddition for Increased in Vivo Efficacy of Interferon β‑1b by Site-Specific PEGylation

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    The development of protein conjugate therapeutics requires control over the site of modification to allow for reproducible generation of a product with the desired potency, pharmacokinetic, and safety profile. Placement of a single nonnatural amino acid at the desired modification site of a recombinant protein, followed by a bioorthogonal reaction, can provide complete control. To this end, we describe the development of copper-catalyzed azide–alkyne cycloaddition (CuAAC, a click chemistry reaction) for site-specific PEGylation of interferon β-1b (IFNb) containing azidohomoalanine (Aha) at the N-terminus. Reaction conditions were optimized using various propargyl-activated PEGs, tris­(benzyltriazolylmethyl)­amine (TBTA), copper sulfate, and dithiothreitol (DTT) in the presence of SDS. The requirement for air in order to advance the redox potential of the reaction was investigated. The addition of unreactive PEG diol reduced the required molar ratio to 2:1 PEG–alkyne to IFNb. The resultant method produced high conversion of Aha-containing IFNb to the single desired product. PEG–IFNbs with 10, 20, 30, and 40 kDa linear or 40 kDa branched PEGs were produced with these methods and compared. Increasing PEG size yielded decreasing in vitro antiviral activities along with concomitant increases in elimination half-life, AUC, and bioavailability when administered in rats or monkeys. A Daudi tumor xenograft model provided comparative evaluation of these combined effects, wherein a 40 kDa branched PEG–IFNb was much more effective than conjugates with smaller PEGs or unPEGylated IFNb at preventing tumor growth in spite of dosing with fewer units and lesser frequency. The results demonstrate the capability of site-specific nonnatural amino acid incorporation to generate novel biomolecule conjugates with increased in vivo efficacy

    Cysteine as a Monothiol Reducing Agent to Prevent Copper-Mediated Oxidation of Interferon Beta During PEGylation by CuAAC

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    Bioconjugation by copper-catalyzed azide–alkyne cycloaddition (CuAAC) provides a powerful means to produce site-specifically modified proteins. However, the use of a copper catalyst brings about the possible generation of reactive oxygen species that could cause degradation of vulnerable amino acid residues. We investigated whether PEGylation by CuAAC caused any modifications to the therapeutic protein interferon beta-1b, which was produced via global amino acid substitution with azidohomo-alanine at the N-terminus and contains no methionine residues. Using previously reported reaction conditions, LC-MS peptide mapping detected +32 Da and +48 Da oxidation modifications of tryptic peptides 28–33 (LEYCLK) and 137–147 (EYSHCAWTIVR) in the protein post-PEGylation. The oxidative degradation increased with reaction time, whereas reducing the copper concentration slowed the PEGylation rate as well as the oxidation rate. Replacing dithiothreitol (DTT) with any of five different monothiol reducing agents in anaerobic conditions allowed efficient PEGylation in 2–4 h and abrogated oxidative degradation. Free cysteine provided reproducible reaction results as a reducing agent in this system and has been successfully applied to other protein conjugations. Monothiol reducing agents, such as cysteine, may be useful tools as protective reducing agents for CuAAC in some bioconjugation systems

    Development and Characterization of Bioinspired Lipid Raft Nanovesicles for Therapeutic Applications

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    Lipid rafts are highly ordered regions of the plasma membrane enriched in signaling proteins and lipids. Their biological potential is realized in exosomes, a subclass of extracellular vesicles (EVs) that originate from the lipid raft domains. Previous studies have shown that EVs derived from human placental mesenchymal stromal cells (PMSCs) possess strong neuroprotective and angiogenic properties. However, clinical translation of EVs is challenged by very low, impure, and heterogeneous yields. Therefore, in this study, lipid rafts are validated as a functional biomaterial that can recapitulate the exosomal membrane and then be synthesized into biomimetic nanovesicles. Lipidomic and proteomic analyses show that lipid raft isolates retain functional lipids and proteins comparable to PMSC-EV membranes. PMSC-derived lipid raft nanovesicles (LRNVs) are then synthesized at high yields using a facile, extrusion-based methodology. Evaluation of biological properties reveals that LRNVs can promote neurogenesis and angiogenesis through modulation of lipid raft-dependent signaling pathways. A proof-of-concept methodology further shows that LRNVs could be loaded with proteins or other bioactive cargo for greater disease-specific functionalities, thus presenting a novel type of biomimetic nanovesicles that can be leveraged as targeted therapeutics for regenerative medicine

    Development and Characterization of Bioinspired Lipid Raft Nanovesicles for Therapeutic Applications

    No full text
    Lipid rafts are highly ordered regions of the plasma membrane enriched in signaling proteins and lipids. Their biological potential is realized in exosomes, a subclass of extracellular vesicles (EVs) that originate from the lipid raft domains. Previous studies have shown that EVs derived from human placental mesenchymal stromal cells (PMSCs) possess strong neuroprotective and angiogenic properties. However, clinical translation of EVs is challenged by very low, impure, and heterogeneous yields. Therefore, in this study, lipid rafts are validated as a functional biomaterial that can recapitulate the exosomal membrane and then be synthesized into biomimetic nanovesicles. Lipidomic and proteomic analyses show that lipid raft isolates retain functional lipids and proteins comparable to PMSC-EV membranes. PMSC-derived lipid raft nanovesicles (LRNVs) are then synthesized at high yields using a facile, extrusion-based methodology. Evaluation of biological properties reveals that LRNVs can promote neurogenesis and angiogenesis through modulation of lipid raft-dependent signaling pathways. A proof-of-concept methodology further shows that LRNVs could be loaded with proteins or other bioactive cargo for greater disease-specific functionalities, thus presenting a novel type of biomimetic nanovesicles that can be leveraged as targeted therapeutics for regenerative medicine

    Development and Characterization of Bioinspired Lipid Raft Nanovesicles for Therapeutic Applications

    No full text
    Lipid rafts are highly ordered regions of the plasma membrane enriched in signaling proteins and lipids. Their biological potential is realized in exosomes, a subclass of extracellular vesicles (EVs) that originate from the lipid raft domains. Previous studies have shown that EVs derived from human placental mesenchymal stromal cells (PMSCs) possess strong neuroprotective and angiogenic properties. However, clinical translation of EVs is challenged by very low, impure, and heterogeneous yields. Therefore, in this study, lipid rafts are validated as a functional biomaterial that can recapitulate the exosomal membrane and then be synthesized into biomimetic nanovesicles. Lipidomic and proteomic analyses show that lipid raft isolates retain functional lipids and proteins comparable to PMSC-EV membranes. PMSC-derived lipid raft nanovesicles (LRNVs) are then synthesized at high yields using a facile, extrusion-based methodology. Evaluation of biological properties reveals that LRNVs can promote neurogenesis and angiogenesis through modulation of lipid raft-dependent signaling pathways. A proof-of-concept methodology further shows that LRNVs could be loaded with proteins or other bioactive cargo for greater disease-specific functionalities, thus presenting a novel type of biomimetic nanovesicles that can be leveraged as targeted therapeutics for regenerative medicine
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