RESUMO
The function of integral membrane proteins is critically dependent on their naturally surrounding lipid membrane. Detergent-solubilized and purified membrane proteins are therefore often reconstituted into cell-membrane mimics and analyzed for their function with single-molecule microscopy. Expansion of this approach toward a broad range of pharmaceutically interesting drug targets and biomarkers however remains hampered by the fact that these proteins have low expression levels, and that detergent solubilization and reconstitution often cause protein conformational changes and loss of membrane-specific cofactors, which may impair protein function. To overcome this limitation, we here demonstrate how antibody-modified nanoparticles can be used to achieve affinity purification and enrichment of selected integral membrane proteins directly from cell membrane preparations. Nanoparticles were first bound to the ectodomain of ß-secretase 1 (BACE1) contained in cell-derived membrane vesicles. In a subsequent step, these were merged into a continuous supported membrane in a microfluidic channel. Through the extended nanoparticle tag, a weak (â¼fN) hydrodynamic force could be applied, inducing directed in-membrane movement of targeted BACE1 exclusively. This enabled selective thousand-fold enrichment of the targeted membrane protein while preserving a natural lipid environment. In addition, nanoparticle-targeting also enabled simultaneous tracking analysis of each individual manipulated protein, revealing how their mobility changed when moved from one lipid environment to another. We therefore believe this approach will be particularly useful for separation in-line with single-molecule analysis, eventually opening up for membrane-protein sorting devices analogous to fluorescence-activated cell sorting.
Assuntos
Anticorpos Imobilizados/química , Membrana Celular/química , Proteínas de Membrana/isolamento & purificação , Nanopartículas/química , Secretases da Proteína Precursora do Amiloide/isolamento & purificação , Animais , Ácido Aspártico Endopeptidases/isolamento & purificação , Linhagem Celular , Humanos , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/química , Lipossomos/químicaRESUMO
Antimicrobial peptides (AMPs) can kill bacteria by destabilizing their membranes, yet translating these molecules' properties into a covalently attached antibacterial coating is challenging. Rational design efforts are obstructed by the fact that standard microbiology methods are ill-designed for the evaluation of coatings, disclosing few details about why grafted AMPs function or do not function. It is particularly difficult to distinguish the influence of the AMP's molecular structure from other factors controlling the total exposure, including which type of bonds are formed between bacteria and the coating and how persistent these contacts are. Here, we combine label-free live-cell microscopy, microfluidics, and automated image analysis to study the response of surface-bound Escherichia coli challenged by the same small AMP either in solution or grafted to the surface through click chemistry. Initially after binding, the grafted AMPs inhibited bacterial growth more efficiently than did AMPs in solution. Yet, after 1 h, E. coli on the coated surfaces increased their expression of type-1 fimbriae, leading to a change in their binding mode, which diminished the coating's impact. The wealth of information obtained from continuously monitoring the growth, shape, and movements of single bacterial cells allowed us to elucidate and quantify the different factors determining the antibacterial efficacy of the grafted AMPs. We expect this approach to aid the design of elaborate antibacterial material coatings working by specific and selective actions, not limited to contact-killing. This technology is needed to support health care and food production in the postantibiotic era.
Assuntos
Peptídeos Antimicrobianos , Escherichia coli , Microscopia , Biofilmes , Antibacterianos/farmacologia , Antibacterianos/química , Bactérias , Materiais Revestidos Biocompatíveis/químicaRESUMO
A single-chip electrochemical method based on impedance measurements in resonance mode has been employed to study lipid monolayer and bilayer formation on hydrophobic alkanethiolate and SiO(2) substrates, respectively. The processes were monitored by temporally resolving changes in interfacial capacitance and resistance, revealing information about the rate of formation, coverage, and defect density (quality) of the layers at saturation. The resonance-based impedance measurements were shown to reveal significant differences in the layer formation process of bilayers made from (i) positively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), (ii) neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on SiO(2), and (iii) monolayers made from POEPC on hydrophobic alkanethiolate substrates. The observed responses were represented with an equivalent circuit, suggesting that the differences primarily originate from the presence of a conductive aqueous layer between the lipid bilayers and the SiO(2). In addition, by adding the ion channel gramicidin D to bilayers supported on SiO(2), channel-mediated charge transport could be measured with high sensitivity (resolution around 1 pA).
Assuntos
Espectroscopia Dielétrica , Canais Iônicos/metabolismo , Bicamadas Lipídicas/química , Dióxido de Silício/química , Gramicidina/química , Transporte de Íons , Fosfatidilcolinas/químicaRESUMO
We describe a 23-year-old male patient who presented with spontaneous intermittent and increasing attacks of severe, left-sided thunderclap headache combined with rapidly progressive muscle weakness and dysphasia, including gradual loss of consciousness. Subsequent CT, MRI and DSA showed progressive brain ischaemia and oedema within the left cerebral hemisphere with strict ipsilateral segmental arterial vasoconstriction. Despite extensive medical care, including steroids, the patient deteriorated rapidly. However, the clinical course changed dramatically within 15 h after the start of an intravenous infusion of prostacyclin at a dose of 0.9 ng/kg/min, with an almost complete recovery of consciousness and speech. In addition the pathophysiological alterations seen on magnetic resonance (imaging and digital) subtraction angiography including diffusion-weighted imaging and apparent diffusion coefficient maps shortly before prostacyclin treatment were clearly reduced when the patient was examined 3-4 days later and he continued to recover thereafter. Although not fully compatible, our case had several clinical characteristics and radiological findings reminiscent of those of the 'segmental reversible vasoconstriction syndrome', sometimes called the Call-Fleming syndrome.
Assuntos
Anti-Hipertensivos/administração & dosagem , Isquemia Encefálica/tratamento farmacológico , Encéfalo/irrigação sanguínea , Epoprostenol/administração & dosagem , Transtornos da Cefaleia Primários/etiologia , Vasoconstrição/efeitos dos fármacos , Acidentes por Quedas , Adulto , Angiografia Digital , Afasia/etiologia , Isquemia Encefálica/patologia , Circulação Cerebrovascular , Imagem de Difusão por Ressonância Magnética , Transtornos da Cefaleia Primários/tratamento farmacológico , Hematoma Subdural Intracraniano/complicações , Humanos , Infusões Intravenosas , Masculino , Debilidade Muscular/etiologia , Fraturas da Coluna Vertebral/complicações , Inconsciência/etiologiaRESUMO
Quartz crystal microbalance with dissipation monitoring and total internal reflection fluorescence microscopy have been used to investigate binding of norovirus-like particles (noroVLPs) to a supported (phospho)lipid bilayer (SLB) containing a few percent of H or B type 1 glycosphingolipid (GSL) receptors. Although neither of these GSLs spontaneously form domains, noroVLPs were observed to form micron-sized clusters containing typically up to about 30 VLP copies, especially for B type 1, which is a higher-affinity receptor. This novel finding is explained by proposing a model implying that VLP-induced membrane deformation promotes VLP clustering, a hypothesis that was further supported by observing that functionalized gold nanoparticles were able to locally induce SLB deformation. Because similar effects are likely possible also at cellular membranes, our findings are interesting beyond a pure biophysicochemical perspective as they shed new light on what may happen during receptor-mediated uptake of viruses as well as nanocarriers in drug delivery.
Assuntos
Glicoesfingolipídeos/química , Bicamadas Lipídicas/metabolismo , Nanopartículas Metálicas/química , Norovirus/química , Carbocianinas/química , Fluorescência , Corantes Fluorescentes/química , Ouro/química , Humanos , Bicamadas Lipídicas/química , Microscopia de Fluorescência , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismoRESUMO
BACKGROUND: Switching patients from a branded antiepileptic drug (AED) to a generic is often challenging. Several studies have shown that considerable proportions of patients report deteriorated seizure control or increased adverse effects, enforcing a switchback to the original drug. Since tolerability and seizure control usually correlate with AED serum concentrations, we examined the fluctuation of levetiracetam (LEV) serum concentrations in patients with epilepsy before and after generic substitution. METHODS: This was an 18-week, naturalistic, open, prospective, two-center study. After a baseline period of 10 weeks, 33 outpatients on stable treatment with branded LEV (Keppra®) either continued with this product or were switched overnight to a generic LEV preparation (1A Pharma) for an eight-week study period. Throughout the study, patients were monitored with bi-weekly LEV serum concentration measurements and seizure diaries. RESULTS: 16 out of 33 patients were switched to a generic LEV product. No switchbacks were seen. LEV dose, LEV serum concentrations, fluctuation index and concentration/dose-ratio (C/D-ratio) were not significantly different within-group (baseline vs. study period) or between-group. Large within-subject variability in serum concentrations was seen in both groups. None of the patients that were seizure-free before inclusion experienced seizures while on the generic LEV product. CONCLUSIONS: Our results show equal fluctuation of LEV serum concentrations with branded LEV and the generic LEV. Most importantly, within-subject variability was much larger than the small, non-significant differences between brands.
Assuntos
Anticonvulsivantes/sangue , Anticonvulsivantes/uso terapêutico , Substituição de Medicamentos , Epilepsia/tratamento farmacológico , Piracetam/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Relação Dose-Resposta a Droga , Medicamentos Genéricos/uso terapêutico , Epilepsia/sangue , Feminino , Humanos , Levetiracetam , Masculino , Pessoa de Meia-Idade , Pacientes Ambulatoriais , Piracetam/sangue , Piracetam/uso terapêutico , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Biological nanoparticles (BNPs) are of high interest due to their key role in various biological processes and use as biomarkers. BNP size and composition are decisive for their functions, but simultaneous determination of both properties with high accuracy remains challenging. Optical microscopy allows precise determination of fluorescence/scattering intensity, but not the size of individual BNPs. The latter is better determined by tracking their random motion in bulk, but the limited illumination volume for tracking this motion impedes reliable intensity determination. Here, we show that by attaching BNPs to a supported lipid bilayer, subjecting them to hydrodynamic flows and tracking their motion via surface-sensitive optical imaging enable determination of their diffusion coefficients and flow-induced drifts, from which accurate quantification of both BNP size and emission intensity can be made. For vesicles, the accuracy of this approach is demonstrated by resolving the expected radius-squared dependence of their fluorescence intensity for radii down to 15 nm.
RESUMO
Emerging biomedical applications such as molecular imaging and drug delivery often require directed binding of nanoparticles to cell-membrane receptors. The specific apparent affinity of such ligand-functionalized particles is size-dependent, an observation so far solely attributed to multivalent receptor-ligand interaction. We question the universality of this explanation by demonstrating that the binding kinetics also depends on weak, attractive colloidal-type interaction between nanoparticles and a lipid membrane. Applying label-free single-particle imaging, we correlate binding of nanoparticles targeted to a cell-mimetic lipid membrane with the distribution of nontargeted particles freely diffusing close to the membrane interface. This analysis shows that already a weak, kBT-scale attraction present between 50 nm gold nanoparticles and the membrane renders these particles an order of magnitude higher avidity compared to 20 nm particles. A stronger emphasis on nonspecific particle-membrane interaction might thus be required to accurately predict nanoparticle targeting and other similar processes such as cellular uptake of exosomes and viruses.
Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas Metálicas , Membrana Celular , Ouro , Ligantes , Nanopartículas , Tamanho da Partícula , Ligação Proteica , Vírus/efeitos dos fármacosRESUMO
Advancement in the understanding of biomolecular interactions has benefited greatly from the development of surface-sensitive bioanalytical sensors. To further increase their broad impact, significant efforts are presently being made to enable label-free and specific biomolecule detection with high sensitivity, allowing for quantitative interpretation and general applicability at low cost. In this work, we have addressed this challenge by developing a waveguide chip consisting of a flat silica core embedded in a symmetric organic cladding with a refractive index matching that of water. This is shown to reduce stray light (background) scattering and thereby allow for label-free detection of faint objects, such as individual sub-20 nm gold nanoparticles as well as sub-100 nm lipid vesicles. Measurements and theoretical analysis revealed that light-scattering signals originating from single surface-bound lipid vesicles enable characterization of their sizes without employing fluorescent lipids as labels. The concept is also demonstrated for label-free measurements of protein binding to and enzymatic (phospholipase A2) digestion of individual lipid vesicles, enabling an analysis of the influence on the measured kinetics of the dye-labeling of lipids required in previous assays. Further, diffraction-limited imaging of cells (platelets) binding to a silica surface showed that distinct subcellular features could be visualized and temporally resolved during attachment, activation, and spreading. Taken together, these results underscore the versatility and general applicability of the method, which due to its simplicity and compatibility with conventional microscopy setups may reach a widespread in life science and beyond.
Assuntos
Técnicas Biossensoriais/métodos , Microscopia/métodos , Análise de Célula Única/métodos , Plaquetas/citologia , Células Cultivadas , Ouro/química , Humanos , Luz , Lipossomos/química , Lipossomos/metabolismo , Nanopartículas Metálicas/química , Espalhamento de RadiaçãoAssuntos
Imunoglobulinas Intravenosas/uso terapêutico , Doenças do Sistema Nervoso/tratamento farmacológico , Atitude do Pessoal de Saúde , Doenças Autoimunes do Sistema Nervoso/tratamento farmacológico , Análise Custo-Benefício , Seguimentos , Humanos , Imunoglobulinas Intravenosas/efeitos adversos , Imunoglobulinas Intravenosas/economia , Satisfação do Paciente , Fatores de Risco , Inquéritos e Questionários , Resultado do TratamentoRESUMO
PURPOSE: The objective was to estimate health care costs and productivity losses due to epilepsy in Sweden and to compare these estimates to previously published estimates. METHODS: Register data on health care utilisation, pharmaceutical sales, permanent disability and mortality were used to calculate health care costs and costs that accrue due to productivity losses. By linkage of register information, we were able to distinguish pharmaceuticals prescribed against epilepsy from prescriptions that were prompted by other indications. RESULTS: The estimated total cost of epilepsy in Sweden in 2009 was
Assuntos
Epilepsia/economia , Custos de Cuidados de Saúde , Sistema de Registros , Trabalho/economia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Custos de Medicamentos/estatística & dados numéricos , Epilepsia/tratamento farmacológico , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Distribuição por Sexo , Suécia , Adulto JovemRESUMO
The immune complement (IC) is a cell-free protein cascade system, and the first part of the innate immune system to recognize foreign objects that enter the body. Elevated activation of the system from, for example, biomaterials or medical devices can result in both local and systemic adverse effects and eventually loss of function or rejection of the biomaterial. Here, the researchers have studied the effect of surface nanotopography on the activation of the IC system. By a simple nonlithographic process, gold nanoparticles with an average size of 58 nm were immobilized on a smooth gold substrate, creating surfaces where a nanostructure is introduced without changing the surface chemistry. The activation of the IC on smooth and nanostructured surfaces was viewed with fluorescence microscopy and quantified with quartz crystal microbalance with dissipation monitoring in human serum. Additionally, the ability of pre-adsorbed human immunoglobulin G (IgG) (a potent activator of the IC) to activate the IC after a change in surface hydrophobicity was studied. It was found that the activation of the IC was significantly attenuated on nanostructured surfaces with nearly a 50% reduction, even after pre-adsorption with IgG. An increase in surface hydrophobicity blunted this effect. The possible role of the curvature of the nanoparticles for the orientation of adsorbed IgG molecules, and how this can affect the subsequent activation of the IC, are discussed. The present findings are important for further understanding of how surface nanotopography affects complex protein adsorption, and for the future development of biomaterials and blood-contacting devices.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Ouro/química , Nanopartículas Metálicas/ultraestrutura , Adsorção , Ativação do Complemento/imunologia , Ouro/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/metabolismo , Nanopartículas Metálicas/química , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Tamanho da Partícula , Espectroscopia Fotoeletrônica , Técnicas de Microbalança de Cristal de Quartzo , Propriedades de SuperfícieRESUMO
Gold surfaces and structures modified with octanedithiol were reacted with dithiothreitol prior to immersion in buffered solutions of charge stabilized gold nanoparticles. The procedure gives a dithiol layer with adequate properties for a homogeneous octanedithiol monolayer and uniform and reproducible gold nanoparticle binding. The distance between the adsorbing particles is controlled by the particle electrostatic interactions and can be carefully tuned by variation of ionic strength. To some extent, long-range ordering occurs among the adsorbed particles. This behavior is facilitated by the particles' small size compared to the Debye screening but also by the homogeneity of the surface modification. The simple character of the system makes it attractive for fabrication of controlled nanoparticle arrays where further chemical and biological modifications are required.