Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 88
Filtrar
Mais filtros

Bases de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Mol Ther ; 32(1): 103-123, 2024 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-37919899

RESUMO

Targeted delivery and cell-type-specific expression of gene-editing proteins in various cell types in vivo represent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of artificial vectors for intravascular delivery (AVIDs), an engineered adenovirus-based gene delivery platform that allows for highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) in vivo after intravenous vector administration. Due to a set of refined structural modifications, intravenous administration of AVIDs did not trigger cytokine storm, hepatotoxicity, or thrombocytopenia. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34+ cells, led to up to 20% transduction of CD34+CD38-CD45RA- HSPC subsets in the bone marrow. Importantly, targeted in vivo transduction of CD34+CD38-CD45RA-CD90-CD49f+ subsets, highly enriched for human hematopoietic stem cells (HSCs), reached up to 19%, which represented a 1,900-fold selectivity in gene delivery to HSC-enriched over lineage-committed CD34-negative cell populations. Because the AVID platform allows for regulated, cell-type-specific expression of gene-editing technologies as well as expression of immunomodulatory proteins to ensure persistence of corrected HSCs in vivo, the HSC-targeted AVID platform may enable development of curative therapies through in vivo gene correction in human HSCs after a single intravenous administration.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Animais , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Técnicas de Transferência de Genes , Antígenos CD34/metabolismo , Terapia Genética , Adenoviridae/genética , Adenoviridae/metabolismo
2.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32051269

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi's sarcoma (KS), the most common malignancy in people living with human immunodeficiency virus (HIV)/AIDS. The oral cavity is a major route for KSHV infection and transmission. However, how KSHV breaches the oral epithelial barrier for spreading to the body is not clear. Here, we show that exosomes purified from either the saliva of HIV-positive individuals or the culture supernatants of HIV-1-infected T-cell lines promote KSHV infectivity in immortalized and primary human oral epithelial cells. HIV-associated saliva exosomes contain the HIV trans-activation response element (TAR), Tat, and Nef RNAs but do not express Tat and Nef proteins. The TAR RNA in HIV-associated exosomes contributes to enhancing KSHV infectivity through the epidermal growth factor receptor (EGFR). An inhibitory aptamer against TAR RNA reduces KSHV infection facilitated by the synthetic TAR RNA in oral epithelial cells. Cetuximab, a monoclonal neutralizing antibody against EGFR, blocks HIV-associated exosome-enhanced KSHV infection. Our findings reveal that saliva containing HIV-associated exosomes is a risk factor for the enhancement of KSHV infection and that the inhibition of EGFR serves as a novel strategy for preventing KSHV infection and transmission in the oral cavity.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi's sarcoma (KS), the most common malignancy in HIV/AIDS patients. Oral transmission through saliva is considered the most common route for spreading the virus among HIV/AIDS patients. However, the role of HIV-specific components in the cotransfection of KSHV is unclear. We demonstrate that exosomes purified from the saliva of HIV-positive patients and secreted by HIV-infected T-cell lines promote KSHV infectivity in immortalized and primary oral epithelial cells. HIV-associated exosomes promote KSHV infection, which depends on HIV trans-activation response element (TAR) RNA and EGFR of oral epithelial cells, which can be targeted for reducing KSHV infection. These results reveal that HIV-associated exosomes are a risk factor for KSHV infection in the HIV-infected population.


Assuntos
Exossomos/metabolismo , Sarcoma de Kaposi/metabolismo , Adulto , Linhagem Celular , Epitélio/metabolismo , Epitélio/virologia , Receptores ErbB/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , HIV-1/fisiologia , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Humanos , Masculino , Saliva/química , Saliva/virologia , Sarcoma de Kaposi/virologia , Ativação Viral , Replicação Viral
4.
J Paediatr Child Health ; 56(5): 742-745, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31849136

RESUMO

AIM: To examine the impact of changes to the endocrine/diabetes after-hours service model of care at a major tertiary children's hospital in Australia. The model aimed to enhance the independence of families and reduce dependency on after-hours calls to health professionals. METHODS: The after-hours activity was captured prospectively using an iPad with a customised FileMaker database. Data were collected for 9 months prior to and for 8 months after the implementation of a modified model of service. Questionnaires gathered information from endocrine junior medical officers (JMOs) and other hospital staff. Data on emergency department visits were analysed for presentations before and after the implementation of the service changes. RESULTS: Changes to the after-hours service resulted in a significant reduction in median calls from 9 (range 0-39) to 2 (range 0-7) per shift. The number of shifts with no calls increased from 2 to 24% and the number of shifts with <3 calls increased from 8 to 60%. Disturbed nights (calls between 10 pm and 6 am) decreased from 75 to 29%. Junior medical officer experience was positive and there was no perceivable increase in workload from in-hospital staff. The number of endocrine patients presenting to the emergency department did not change significantly following the implementation of the new after-hours service. CONCLUSION: This is the only Australian study to prospectively gather accurate on-call data in order to elucidate the impact of changing a hospital's after-hours endocrine/diabetes service to a model that enhanced family empowerment and independence. Historical 24-h on-call service models are not indispensable, and changes can improve sustainability without compromising patient care.


Assuntos
Diabetes Mellitus , Serviço Hospitalar de Emergência , Austrália , Criança , Diabetes Mellitus/terapia , Hospitais Pediátricos , Humanos , Centros de Atenção Terciária
5.
Proc Natl Acad Sci U S A ; 114(13): E2608-E2615, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28289214

RESUMO

Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged Gt activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates Gt in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.


Assuntos
Rodopsina/química , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Isomerismo , Processos Fotoquímicos , Rodopsina/fisiologia , Rodopsina/efeitos da radiação
6.
Nano Lett ; 19(3): 2099-2105, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30801195

RESUMO

In situ cancer vaccination that uses immune stimulating agents is revolutionizing the way that cancer is treated. In this realm, viruses and noninfectious virus-like particles have gained significant traction in reprogramming the immune system to recognize and eliminate malignancies. Recently, cowpea mosaic virus-like particles (VLPs) have shown exceptional promise in their ability to fight a variety of cancers. However, the current methods used to produce CPMV VLPs rely on agroinfiltration in plants. These protocols remain complicated and labor intensive and have the potential to introduce unwanted immunostimulatory agents, like lipopolysaccharides. This Letter describes a simple "post-processing" method to remove RNA from wild-type CPMV, while retaining the structure and function of the capsid. Lyophilization was able to eject encapsulated RNA to form lyo-eCPMV and, when purified, eliminated nearly all traces of encapsulated RNA. Lyo-eCPMV was characterized by cryo-electron microscopy single particle reconstruction to confirm the structural integrity of the viral capsid. Finally, lyo-eCPMV showed  equivalent anticancer efficacy as eCPMV, produced by agroinfiltration, when using an invasive melanoma model. These results describe a straightforward method to prepare CPMV VLPs from infectious virions.


Assuntos
Vacinas Anticâncer/química , Comovirus/química , Melanoma/tratamento farmacológico , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Comovirus/genética , Microscopia Crioeletrônica , Liofilização , Humanos , Melanoma/imunologia , Plantas/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vírion/química , Vírion/genética
7.
Langmuir ; 35(39): 12765-12772, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532686

RESUMO

Colloidal oil-in-water nanoemulsions are gaining increasing interest as a nanoparticle delivery system because of their large oil droplet core that can carry a large payload. In order to formulate these particles with long-term stability, an appropriate oil media and block copolymer pair must be selected. The interaction between the nanoemulsion core and the polymer shell is critical to forming stable nanoparticles. Herein, we probed how interactions between various polymers with hydrocarbon and perfluorocarbon oil media influenced nanoemulsion formation, stability, and size. Through a series of nanoemulsions with unique polymer/oil media combinations, we examined the effects of oil core hydrophobicity, fluorophilicity, surface charge, and volume as well as the effects of polymer tail composition. Surprisingly, we found that nanoemulsions formulated with pure perfluorocarbon oil cores versus perfluoro poly(ether) oil cores exhibited very different characteristics. We also found that both hydrocarbon and fluorocarbon polymer tails interacted favorably with perfluoro poly(ethers) as well as hydrocarbon oil cores forming stable nanoemulsions. We believe these results are focused on the unique properties of perfluorocarbons especially their rigidity, low polarizability, and near-zero surface charge. Interestingly, we saw that perfluoro poly(ethers) deviated from these expected properties resulting in an increased versatility when formulating nanoemulsions with perfluoro poly(ether) oil cores compared to pure perfluorocarbon oil cores. Nanoemulsion size, stability, growth rate, and life time were explored to probe these factors. Experimental and computational data are presented as a rationale.


Assuntos
Óleos/química , Polímeros/química , Água/química , Emulsões , Éteres/química , Modelos Moleculares , Conformação Molecular , Eletricidade Estática
9.
Mol Pharm ; 15(8): 2900-2909, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29733602

RESUMO

Nanoparticle delivery systems offer advantages over free drugs, in that they increase solubility and biocompatibility. Nanoparticles can deliver a high payload of therapeutic molecules while limiting off-target side effects. Therefore, delivery of an existing drug with a nanoparticle frequently results in an increased therapeutic index. Whether of synthetic or biologic origin, nanoparticle surface coatings are often required to reduce immune clearance and thereby increase circulation times allowing the carriers to reach their target site. To this end, polyethylene glycol (PEG) has long been used, with several PEGylated products reaching clinical use. Unfortunately, the growing use of PEG in consumer products has led to an increasing prevalence of PEG-specific antibodies in the human population, which in turn has fueled the search for alternative coating strategies. This review highlights alternative bioinspired nanoparticle shielding strategies, which may be more beneficial moving forward than PEG and other synthetic polymer coatings.


Assuntos
Materiais Biomiméticos/química , Portadores de Fármacos/química , Nanopartículas/química , Materiais Biomiméticos/efeitos adversos , Engenharia Química/métodos , Química Farmacêutica , Química Click , Ensaios Clínicos como Assunto , Portadores de Fármacos/efeitos adversos , Humanos , Sistema Imunitário/efeitos dos fármacos , Nanopartículas/efeitos adversos , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/química
10.
J Am Chem Soc ; 139(9): 3312-3315, 2017 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-28121424

RESUMO

Covalent conjugation of water-soluble polymers to proteins is critical for evading immune surveillance in the field of biopharmaceuticals. The most common and long-standing polymer modification is the attachment of methoxypoly(ethylene glycol) (mPEG), termed PEGylation, which has led to several clinically approved pharmaceuticals. Recent data indicate that brush-type polymers significantly enhance in vitro and in vivo properties. Herein, the polymer conformation of poly(ethylene glycol) is detailed and compared with those of water-soluble polyacrylate and polynorbornene (PNB) when attached to icosahedral virus-like particles. Small-angle neutron scattering reveals vastly different polymer conformations of the multivalent conjugates. Immune recognition of conjugated particles was evaluated versus PEGylated particles, and PNB conjugation demonstrated the most effective shielding from antibody recognition.


Assuntos
Acrilatos/química , Plásticos/química , Polietilenoglicóis/química , Vacinas de Partículas Semelhantes a Vírus/química , Animais , Camundongos , Modelos Moleculares , Estrutura Molecular , Difração de Nêutrons , Espalhamento a Baixo Ângulo
11.
Mol Pharm ; 14(11): 3815-3823, 2017 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-28881141

RESUMO

Thrombotic cardiovascular disease, including acute myocardial infarction, ischemic stroke, and venous thromboembolic disease, is the leading cause of morbidity and mortality worldwide. While reperfusion therapy with thrombolytic agents reduces mortality from acute myocardial infarction and disability from stroke, thrombolysis is generally less effective than mechanical reperfusion and is associated with fatal intracerebral hemorrhage in up to 2-5% of patients. To address these limitations, we propose the tobacco mosaic virus (TMV)-based platform technology for targeted delivery of thrombolytic therapies. TMV is a plant virus-based nanoparticle with a high aspect ratio shape measuring 300 × 18 nm. These soft matter nanorods have favorable flow and margination properties allowing the targeting of the diseased vessel wall. We have previously shown that TMV homes to thrombi in a photochemical mouse model of arterial thrombosis. Here we report the synthesis of TMV conjugates loaded with streptokinase (STK). Various TMV-STK formulations were produced through bioconjugation of STK to TMV via intervening PEG linkers. TMV-STK was characterized using SDS-PAGE and Western blot, transmission electron microscopy, cryo-electron microscopy, and cryo-electron tomography. We investigated the thrombolytic activity of TMV-STK in vitro using static phantom clots, and in a physiologically relevant hydrodynamic model of shear-induced thrombosis. Our findings demonstrate that conjugation of STK to the TMV surface does not compromise the activity of STK. Moreover, the nanoparticle conjugate significantly enhances thrombolysis under flow conditions, which can likely be attributed to TMV's shape-mediated flow properties resulting in enhanced thrombus accumulation and dissolution. Together, these data suggest TMV to be a promising platform for the delivery of thrombolytics to enhance clot localization and potentially minimize bleeding risk.


Assuntos
Nanopartículas/química , Vírus de Plantas/química , Terapia Trombolítica/métodos , Western Blotting , Sistemas de Liberação de Medicamentos/métodos , Eletroforese em Gel de Poliacrilamida , Fibrinolíticos/química , Fibrinolíticos/uso terapêutico , Plasminogênio/química , Trombose/tratamento farmacológico , Vírus do Mosaico do Tabaco/química
12.
Anal Biochem ; 499: 63-65, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26851339

RESUMO

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the Kd values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT-PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL L-cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT-PCR machines in a hydrophilic or hydrophobic environment.


Assuntos
Compostos de Boro/química , Cistina/análogos & derivados , Corantes Fluorescentes/química , Fluorometria , Proteínas/análise , Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Compostos de Sulfidrila/química , Cistina/química , Interações Hidrofóbicas e Hidrofílicas , Reação em Cadeia da Polimerase em Tempo Real/normas , Temperatura
13.
Clin Infect Dis ; 60(2): 228-36, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25301212

RESUMO

BACKGROUND: Infections with human parechoviruses (HPeVs) are associated with a wide range of clinical presentations in children, ranging from mild or asymptomatic infections to severe sepsis-like presentations or meningoencephalitis. METHODS: We reviewed medical records of infants admitted to 5 hospitals in New South Wales, Australia, during an outbreak of HPeV-3 infection. Data were collected on clinical presentation, laboratory markers, and outcome of infants with HPeV infection confirmed by reverse transcription polymerase chain reaction. RESULTS: We identified 118 infected infants. Most presented with an acute sepsis-like syndrome with high fever, tachycardia, poor perfusion, and severe irritability. Other common features were erythrodermic rash, abdominal distension, edema, and hepatitis. The age range of infants was 4 days to 9.5 months; 75% were <2 months old, including all but 1 of the 30 infants (25%) admitted to intensive care units (ICUs), who as a group, were significantly younger than infants not admitted to ICUs. Only 4% of evaluable cerebrospinal fluid samples had pleocytosis, but HPeV was detected in 95%. Brain magnetic resonance imaging on a small number of children demonstrated white matter changes and diffusion restriction. Sequencing of the VP1 gene confirmed HPeV-3 in all samples tested. All children recovered without ongoing complications at last follow-up. CONCLUSIONS: We report the largest series of HPeV-3 infection in infants, and the first outbreak in Australia. Infants presented with a severe sepsis-like syndrome with a high rate of ICU admissions, but all recovered from the acute infection without complications. Long-term sequelae are unknown.


Assuntos
Surtos de Doenças , Parechovirus/isolamento & purificação , Infecções por Picornaviridae/complicações , Infecções por Picornaviridae/epidemiologia , Sepse/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , New South Wales/epidemiologia , Parechovirus/classificação , Parechovirus/genética , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/virologia , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/virologia
14.
J Virol ; 88(1): 469-76, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24155406

RESUMO

Previous human antibody studies have shown that the human VH1-46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody-double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação de Anticorpos , Biopolímeros/imunologia , Microscopia Crioeletrônica , Primers do DNA , Epitopos/química , Espectrometria de Massas , Dados de Sequência Molecular
15.
Bioconjug Chem ; 26(2): 262-269, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25611133

RESUMO

Molecular imaging approaches and targeted drug delivery hold promise for earlier detection of diseases and treatment with higher efficacy while reducing side effects, therefore increasing survival rates and quality of life. Virus-based nanoparticles are a promising platform because their scaffold can be manipulated both genetically and chemically to simultaneously display targeting ligands while carrying payloads for diagnosis or therapeutic intervention. Here, we displayed a 12-amino-acid peptide ligand, GE11 (YHWYGYTPQNVI), on nanoscale filaments formed by the plant virus potato virus X (PVX). Bioconjugation was used to produce fluorescently labeled PVX-GE11 filaments targeted toward the epidermal growth factor receptor (EGFR). Cell detection and imaging was demonstrated using human skin epidermoid carcinoma, colorectal adenocarcinoma, and triple negative breast cancer cell lines (A-431, HT-29, MDA-MB-231), all of which upregulate EGFR to various degrees. Nonspecific uptake in ductal breast carcinoma (BT-474) cells was not observed. Furthermore, co-culture experiments with EGFR(+) cancer cells and macrophages indicate successful targeting and partitioning toward the cancer cells. This study lays a foundation for the development of EGFR-targeted filaments delivering contrast agents for imaging and diagnosis, and/or toxic payloads for targeted drug delivery.


Assuntos
Receptores ErbB/metabolismo , Nanopartículas , Neoplasias/diagnóstico , Peptídeos , Vírus de Plantas/química , Sequência de Aminoácidos , Linhagem Celular Tumoral , Diagnóstico por Imagem , Humanos , Nanopartículas/química , Nanopartículas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Peptídeos/química , Peptídeos/metabolismo , Vírus de Plantas/metabolismo
16.
Langmuir ; 31(31): 8680-8, 2015 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-26174179

RESUMO

We present an in situ cryo-electron microscopy (cryoEM) study of mixed poly(acrylic acid) (PAA)/polystyrene (PS) brush-grafted 67 nm silica nanoparticles in organic and aqueous solvents. These organic-inorganic nanoparticles are predicted to be environmentally responsive and adopt distinct brush layer morphologies in different solvent environments. Although the self-assembled morphology of mixed PAA/PS brush-grafted particles has been studied previously in a dried state, no direct visualization of microphase separation was achieved in the solvent environment. CryoEM allows the sample to be imaged in situ, that is, in a frozen solvated state, at the resolution of a transmission electron microscope. Cryo-electron tomograms (cryoET) were generated for mixed PAA/PS brush-grafted nanoparticles in both N,N-dimethylformamide (DMF, a nonselective good solvent) and water (a selective solvent for PAA). Different nanostructures for the mixed brushes were observed in these two solvents. Overall, the brush layer is more compact in water, with a thickness of 18 nm, as compared with an extended layer of 27 nm in DMF. In DMF, mixed PAA/PS brushes are observed to form laterally separated microdomains with a ripple wavelength of 13.8 nm. Because of its lower grafting density than that of PAA, PS domains form more or less cylindrical or truncated cone-shaped domains in the PAA matrix. In water, PAA chains are found to form a more complete shell around the nanoparticle to maximize their interaction with water, whereas PS chains collapse into the core of surface-tethered micelles near the silica core. The cryoET results presented here confirm the predicted environmentally responsive nature of PAA/PS mixed brush-grafted nanoparticles. This experimental approach may be useful for the design of future mixed brush-grafted nanoparticles for nano- and biotechnology applications.


Assuntos
Resinas Acrílicas/química , Microscopia Crioeletrônica , Dimetilformamida/química , Nanopartículas/química , Poliestirenos/química , Dióxido de Silício/química , Tamanho da Partícula , Solventes/química , Propriedades de Superfície , Água/química
17.
Soft Matter ; 11(27): 5501-12, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26061172

RESUMO

Environmentally responsive self-assembly of nearly symmetric mixed poly(tert-butyl acrylate) (PtBA, 22.2 kDa)/polystyrene (PS, 23.4 kDa) brushes grafted onto 67 nm silica nanoparticles in selective homopolymer matrices [PtBA for the grafted PtBA chains and poly(cyclohexyl methacrylate) (PCHMA) for the grafted PS chains] was investigated using both conventional transmission electron microscopy (TEM) and electron tomography (i.e., 3D TEM). A variety of self-assembled phase morphologies were observed for the mixed brushes in selective polymer matrices with different molecular weights, and these can be explained by entropy-driven wet- and dry-brush theories. In a low molecular weight selective matrix, the wet-brush regime was formed with the miscible chains stretching out and the immiscible chains collapsing into isolated domains. In contrast, when the molecular weight of the selective matrix was higher than that of the compatible grafted polymer chains, the dry-brush regime was formed with the mixed brushes exhibiting the unperturbed morphology. In addition to the molecular weight, the size of nanoparticles (or the substrate curvature) was also observed to play an important role. For small particles (core size less than 50 nm), the wet brush-like morphology with a surface-tethered micellar structure was observed. Finally, the wet- and dry-brush regimes also significantly affected the dispersion of mixed brush particles in selective polymer matrices.


Assuntos
Acrilatos/química , Nanocompostos/química , Nanopartículas/química , Poliestirenos/síntese química , Entropia , Peso Molecular , Nanocompostos/ultraestrutura , Nanopartículas/ultraestrutura , Tamanho da Partícula , Polimerização , Ácidos Polimetacrílicos/química , Compostos de Rutênio/química
18.
J Paediatr Child Health ; 51(4): 458-60, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25266956

RESUMO

A 24-day-old boy presented with fever, irritability and poor feeding. Blood culture grew methicillin-resistant Staphylococcus aureus. Cerebrospinal fluid analysis showed pleocytosis, and methicillin-resistant Staphylococcus aureus grew from enrichment broth. Magnetic resonance imaging revealed an epidural abscess extending from C2-3 to T8-9. Staphylococcal infections of the central nervous system are uncommon in neonates. This case demonstrates the importance of performing a lumbar puncture in isolated staphylococcal bacteraemia. The case also highlights that cerebrospinal fluid pleocytosis may indicate a parameningeal focus of infection.


Assuntos
Bacteriemia/diagnóstico , Infecções Bacterianas do Sistema Nervoso Central/diagnóstico , Abscesso Epidural/diagnóstico , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/diagnóstico , Vértebras Cervicais , Abscesso Epidural/microbiologia , Humanos , Recém-Nascido , Masculino
19.
J Struct Biol ; 187(1): 76-83, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24333094

RESUMO

Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder structural analyses. CryoEM and single particle image processing techniques offer the possibility of imaging samples with significant flexibility. Division of particle images into more homogenous subsets after data acquisition can help compensate for heterogeneity within the sample. We present the utility of an eigenimage sorting analysis for examining two protein/DNA complexes with significant conformational flexibility and heterogeneity. These complexes are integral to the non-homologous end joining pathway, and are involved in the repair of double strand breaks of DNA. Both complexes include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and biotinylated DNA with bound streptavidin, with one complex containing the Ku heterodimer. Initial 3D reconstructions of the two DNA-PKcs complexes resembled a cryoEM structure of uncomplexed DNA-PKcs without additional density clearly attributable to the remaining components. Application of eigenimage sorting allowed division of the DNA-PKcs complex datasets into more homogeneous subsets. This led to visualization of density near the base of the DNA-PKcs that can be attributed to DNA, streptavidin, and Ku. However, comparison of projections of the subset structures with 2D class averages indicated that a significant level of heterogeneity remained within each subset. In summary, image sorting methods allowed visualization of extra density near the base of DNA-PKcs, suggesting that DNA binds in the vicinity of the base of the molecule and potentially to a flexible region of DNA-PKcs.


Assuntos
Antígenos Nucleares/química , Reparo do DNA/genética , Proteínas de Ligação a DNA/química , DNA/química , Proteínas Serina-Treonina Quinases/química , Antígenos Nucleares/genética , Biotina/química , Microscopia Crioeletrônica , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Expressão Gênica , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Imageamento Tridimensional/estatística & dados numéricos , Autoantígeno Ku , Modelos Moleculares , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Estreptavidina/química
20.
J Biol Chem ; 288(7): 4819-30, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23277356

RESUMO

Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used cryoelectron microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the α-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human αB-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition.


Assuntos
Proteínas Arqueais/metabolismo , Bacteriófago T4/enzimologia , Microscopia Crioeletrônica/métodos , Proteínas de Choque Térmico/metabolismo , Muramidase/química , Clonagem Molecular , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Conformação Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Temperatura , alfa-Cristalinas/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA