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1.
Psychogeriatrics ; 23(3): 466-474, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36959368

RESUMO

BACKGROUND: Semantic dementia (SD), a subtype of frontotemporal dementia, manifests as verbal symptoms, including social and behavioural deficits, associated with focal atrophy of the frontotemporal lobes. This study aimed to clarify the experiences of individuals with early-onset SD receiving speech and language rehabilitation (hereafter referred to as 'rehabilitation'), with the intent of making it routine, as well as the experiences of their families. METHODS: Individual interviews were conducted with nine families with members who had adopted rehabilitation. Verbatim transcripts were used as data, and analyzed inductively according to the content analysis process. RESULTS: The family members realised the changes in the personality and behaviour of the individual with SD early, to the extent that they thought the individual with SD was different from before and were distressed by the loss of verbal communication. Nevertheless, the family members found a way to communicate by maintaining residual functions through rehabilitation and utilising their unique relationship with the individual with SD. CONCLUSIONS: It is important to carefully explain the characteristics of the disease and the long-term significance of rehabilitation to individuals with SD and their families in the early stages of the disease.


Assuntos
Demência Frontotemporal , Humanos , Demência Frontotemporal/diagnóstico , Testes Neuropsicológicos , Idioma , Família , Pesquisa Qualitativa
2.
Anal Chem ; 94(51): 18025-18033, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36511577

RESUMO

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogeneous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein, the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 µm) of SPM compared to a typical agarose gel. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling. This is the first approach to clarify the variation of protein contents in EVs by the difference of surface glycans via lectin immobilized media.


Assuntos
Vesículas Extracelulares , Lectinas , Lectinas/metabolismo , Concanavalina A/química , Cromatografia de Afinidade/métodos , Vesículas Extracelulares/metabolismo , Polissacarídeos/metabolismo
3.
Bioconjug Chem ; 32(4): 680-684, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33719402

RESUMO

PKH dyes, which are currently the most widely used fluorescent probes for extracellular vesicle (EV) labeling, have some limitations. For example, these dyes tend to aggregate, leading to formation of EV-like nanoparticles that can be taken up by cells. Moreover, it has been suggested that PKH dyes trigger an enlargement of EVs because of membrane fusion or intercalation. To overcome these limitations, we developed three novel extracellular vesicular-membrane-binding fluorescent probes-Mem dye-Green, Mem dye-Red, and Mem dye-Deep Red-for monitoring EV uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold and amphiphilic moieties on the cyanine. The three dyes have different photophysical characteristics. To investigate the characteristics of the Mem dyes for EV labeling, we performed nanoparticle tracking, zeta potential measurements, and confocal microscopy. The dyes enable highly sensitive fluorescence imaging of EVs. They can also be used to observe EV dynamics in live cells. The Mem dyes show excellent EV labeling with no aggregation and less particle enlargement.


Assuntos
Vesículas Extracelulares/química , Corantes Fluorescentes/química , Metabolismo dos Lipídeos , Células HeLa , Humanos , Microscopia Confocal
4.
Biomater Sci ; 7(4): 1617-1622, 2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30702722

RESUMO

We report the combined enzymatic synthesis of five glucan dendrimers with control of molecular weight and particle size. Amphiphilic properties were introduced through dodecyl group substitution. A Tokyo-green fluorescence assay showed that amphiphilic glucan dendrimers were able to successfully deliver active ß-galactosidase to cells.


Assuntos
Dendrímeros/química , Glucanos/química , Nanopartículas/química , Tensoativos/química , beta-Galactosidase/química , Dendrímeros/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Endocitose/efeitos dos fármacos , Glucanos/biossíntese , Células HeLa , Humanos , Peso Molecular , Nanopartículas/metabolismo , Tamanho da Partícula , Tensoativos/metabolismo , beta-Galactosidase/metabolismo
5.
Biomacromolecules ; 18(12): 3913-3923, 2017 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-29059529

RESUMO

Nanometer-size gel particles, or nanogels, have potential for delivering therapeutic macromolecules. A cationic surface promotes cellular internalization of nanogels, but undesired electrostatic interactions, such as with blood components, cause instability and toxicities. Poly(ethylene glycol) coating has been used to shield charges, but this decreases delivery efficiency. Technical difficulties in synthesis and controlling molecular weights make it unfeasible to, instead, coat with biodegradable polymers. Our proposed solution is cationized nanogels enzymatically functionalized with branched polysaccharide chains, forming a shell to shield charges and increase stability. Biodegradation of the polysaccharides by an endogenous enzyme would then expose the cationic charges, allowing cellular internalization and cargo delivery. We tested this concept, preparing maltopentaose functionalized cholesteryl poly(l-lysine) nanogel and using tandem enzymatic polymerization with glycogen phosphorylase and glycogen branching enzyme, to add branched amylose moieties, forming a CbAmyPL nanogel. We characterized CbAmyPL nanogels and investigated their suitability as small interfering RNA (siRNA) carriers in murine renal carcinoma (Renca) cells. The nanogels had neutral ζ potential values that became positive after degradation by α-amylase. Foster resonance energy transfer demonstrated that the nanogels formed stable complexes with siRNA, even in the presence of bovine serum albumin and after α-amylase exposure. The nanogels, with or without α-amylase, were not cytotoxic. Complexes of CbAmyPL with siRNA against vascular endothelial growth factor (VEGF), when incubated alone with Renca cells decreased VEGF mRNA levels by only 20%. With α-amylase added, however, VEGF mRNA knockdown by the siRNA/nanogels complexes was 50%. Our findings strongly supported the hypothesis that enzyme-responsive nanogels are promising as a therapeutic siRNA delivery platform.


Assuntos
Nanopartículas/química , Peptídeos/química , Polietilenoglicóis/química , Polietilenoimina/química , Polímeros/química , RNA Interferente Pequeno/química , Animais , Cátions/química , Linhagem Celular Tumoral , Lisina/química , Camundongos , Peso Molecular , Nanogéis , Polissacarídeos/química , Fator A de Crescimento do Endotélio Vascular/química , alfa-Amilases/química
6.
Sci Rep ; 6: 21933, 2016 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-26911358

RESUMO

Exosomes are a valuable biomaterial for the development of novel nanocarriers as functionally advanced drug delivery systems. To control and modify the performance of exosomal nanocarriers, we developed hybrid exosomes by fusing their membranes with liposomes using the freeze-thaw method. Exosomes embedded with a specific membrane protein isolated from genetically modified cells were fused with various liposomes, confirming that membrane engineering methods can be combined with genetic modification techniques. Cellular uptake studies performed using the hybrid exosomes revealed that the interactions between the developed exosomes and cells could be modified by changing the lipid composition or the properties of the exogenous lipids. These results suggest that the membrane-engineering approach reported here offers a new strategy for developing rationally designed exosomes as hybrid nanocarriers for use in advanced drug delivery systems.


Assuntos
Exossomos/metabolismo , Lipossomos/metabolismo , Animais , Western Blotting , Exossomos/química , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Congelamento , Células HeLa , Humanos , Lipossomos/química , Fusão de Membrana , Camundongos , Microscopia Confocal , Nanopartículas/química , Nanopartículas/metabolismo , Células RAW 264.7 , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo
7.
Biomed Res Int ; 2015: 962941, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26539548

RESUMO

A new siRNA delivery system using a cationic glyco-star polymer is described. Spermine-modified 8-arm amylose star polymer (with a degree of polymerization of approximately 60 per arm) was synthesized by chemoenzymatic methods. The cationic star polymer effectively bound to siRNA and formed spherical complexes with an average hydrodynamic diameter of 230 nm. The cationic 8-arm star polymer complexes showed superior cellular uptake characteristics and higher gene silencing effects than a cationic 1-arm polymer. These results suggest that amylose-based star polymers are a promising nanoplatform for glycobiomaterials.


Assuntos
Amilose/química , Portadores de Fármacos/química , Polímeros/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética , Animais , Cátions/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia
8.
Int J Nanomedicine ; 7: 4353-62, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22927755

RESUMO

Protein nanocages are self-organized complexes of oligomers whose three-dimensional architecture can been determined in detail. These structures possess nanoscale inner cavities into which a variety of molecules, including therapeutic or diagnostic agents, can be encapsulated. These properties yield these particles suitable for a new class of drug delivery carrier, or as a bioimaging reagent that might respond to biochemical signals in many different cellular processes. We report here the design, synthesis, and biological characterization of a hepatocyte-specific nanocage carrying small heat-shock protein. These nanoscale protein cages, with a targeting peptide composed of a preS1 derivative from the hepatitis B virus on their surfaces, were prepared by genetic engineering techniques. PreS1-carrying nanocages showed lower cytotoxicity and significantly higher specificity for human hepatocyte cell lines than other cell lines in vitro. These results suggested that small heat-shock protein-based nanocages present great potential for the development of effective targeted delivery of various agents to specific cells.


Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Hepatócitos/metabolismo , Nanocápsulas/química , Precursores de Proteínas/metabolismo , Sobrevivência Celular/fisiologia , Células HeLa , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Hepatócitos/citologia , Humanos , Microscopia Confocal , Nanomedicina , Tamanho da Partícula , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
9.
Biochem Biophys Res Commun ; 383(3): 293-7, 2009 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-19341711

RESUMO

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.


Assuntos
Chaperonas Moleculares/metabolismo , Nefelometria e Turbidimetria/métodos , Peptídeo Hidrolases/análise , Fator Xa/genética , Fator Xa/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico Pequenas/metabolismo , Chaperonas Moleculares/genética , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Bioorg Med Chem ; 17(1): 85-93, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19041251

RESUMO

Hsp16.5, a small heat-shock protein (sHSP) from hyperthermophilic archaeon, forms a homogeneous complex comprised of 24 subunits with a molecular mass of 400 kDa. This complex self-organizes under physiological conditions, and the structure of the complex is a nanoscale spherical capsule with small pores. Furthermore, this natural nanocapsule exhibits very high thermal stability. In this paper, we functionalized the nanocapsule to control the structure in response to external stimuli such as a protease signal and temperature. For this purpose, several mutations (Mut1-10) to create a cleavage site for a specific protease, Factor Xa, were introduced on the outer surface of the nanocapsule using a genetic engineering strategy. The resulting mutants were expressed to high levels in Escherichia coli. One of these mutants, Mut6, which has the most accessible cleavage site located at the triangular pore on the surface of the capsule, formed a spherical assembly similar to that observed for the wild-type protein. Mut6 showed the highest sensitivity to Factor Xa, and the structure of the protease digested Mut6 disassembled irreversibly after heating. In contrast, the nanocapsule comprising the wild-type Hsp16.5 was not influenced by the dual stimuli. These results suggest that Mut6 acts as a stimulus-responsive nanocapsule. Such a characteristic of the protein-based nanocapsule has attractive potential as a versatile intelligent system.


Assuntos
Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Nanocápsulas/química , Engenharia de Proteínas , Proteínas Arqueais , Sítios de Ligação/genética , Clonagem Molecular , Fator Xa/metabolismo , Proteínas de Choque Térmico/farmacocinética , Proteínas de Choque Térmico/uso terapêutico , Humanos , Mutagênese Sítio-Dirigida , Nanocápsulas/ultraestrutura , Relação Estrutura-Atividade
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