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The ability to generate mode-engineered single photons to interface with disparate quantum systems is of importance for building a quantum network. Here we report on the generation of a pulsed, heralded single photon source with a sub-GHz spectral bandwidth that couples to indium arsenide quantum dots centered at 942 nm. The source is built with a type-II PPKTP down-conversion crystal embedded in a semi-confocal optical cavity and pumped with a 76 MHz repetition rate pulsed laser to emit collinear, polarization-correlated photon pairs resonant with a single quantum dot. In order to demonstrate direct coupling, we use the mode-engineered cavity-SPDC single-photon source to resonantly excite an isolated single quantum dot.
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The amyloid-ß peptides (Aß40 and Aß42) feature prominently in the synaptic dysfunction and neuronal loss associated with Alzheimer's disease (AD). This has been proposed to be due either to interactions between Aß and cell surface receptors affecting cell signaling, or to the formation of calcium-permeable channels in the membrane that disrupt calcium homeostasis. In both mechanisms the cell membrane is the primary cellular structure with which Aß interacts. Aß concentrations in human bodily fluids are very low (pM-nM) rendering studies of the size, composition, cellular binding sites and mechanism of action of the oligomers formed in vivo very challenging. Most studies, therefore, have utilized Aß oligomers prepared at micromolar peptide concentrations, where Aß forms oligomeric species which possess easily observable cell toxicity. Such toxicity has not been observed when nM concentrations of peptide are used in the experiment highlighting the importance of employing physiologically relevant peptide concentrations for the results to be of biological significance. In this paper single-molecule microscopy was used to monitor Aß oligomer formation and diffusion on a supported lipid bilayer at nanomolar peptide concentrations. Aß monomers, the dominant species in solution, tightly associate with the membrane and are highly mobile whereas trimers and higher-order oligomers are largely immobile. Aß dimers exist in a mixture of mobile and immobile states. Oligomer growth on the membrane is more rapid for Aß40 than for the more amyloidogenic Aß42 but is largely inhibited for a 1:1 Aß40:Aß42 mixture. The mechanism underlying these Aß40-Aß42 interactions may feature in Alzheimer's pathology.
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The collection efficiency of light from a point-like emitter may be extremely poor due to aberrations induced by collection optics and the emission distribution of the source. Analyzing the aberrant wavefront (e.g., with a Shack-Hartmann sensor) and correcting accordingly can be infeasible on the single-photon level. We present a technique that uses a genetic algorithm to control a deformable mirror for correcting wavefront aberrations in single-photon signals from point emitters. We apply our technique to both a simulated point source and a real InAs quantum dot, achieving coupling increases of up to 50% and automatic reduction of system drift.
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We demonstrate the extension of coherence between all four two-electron spin ground states of an InAs quantum dot molecule (QDM) via nonlocal suppression of nuclear spin fluctuations in two vertically stacked quantum dots (QDs), while optically addressing only the top QD transitions. Long coherence times are revealed through dark-state spectroscopy as resulting from nuclear spin locking mediated by the exchange interaction between the QDs. Line shape analysis provides the first measurement of the quieting of the Overhauser field distribution correlating with reduced nuclear spin fluctuations.
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Amyloid-ß peptide (Aß) oligomers may represent the proximal neurotoxin in Alzheimer's disease. Single-molecule microscopy (SMM) techniques have recently emerged as a method for overcoming the innate difficulties of working with amyloid-ß, including the peptide's low endogenous concentrations, the dynamic nature of its oligomeric states, and its heterogeneous and complex membrane interactions. SMM techniques have revealed that small oligomers of the peptide bind to model membranes and cells at low nanomolar-to-picomolar concentrations and diffuse at rates dependent on the membrane characteristics. These methods have also shown that oligomers grow or dissociate based on the presence of specific inhibitors or promoters and on the ratio of Aß40 to Aß42. Here, we discuss several types of single-molecule imaging that have been applied to the study of Aß oligomers and their membrane interactions. We also summarize some of the recent insights SMM has provided into oligomer behavior in solution, on planar lipid membranes, and on living cell membranes. A brief overview of the current limitations of the technique, including the lack of sensitive assays for Aß-induced toxicity, is included in hopes of inspiring future development in this area of research.
Assuntos
Peptídeos beta-Amiloides/química , Microscopia de Fluorescência/métodos , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/metabolismo , Animais , Membrana Celular/metabolismo , Humanos , Fragmentos de Peptídeos/metabolismo , Multimerização ProteicaRESUMO
Two amyloid-ß peptides (Aß40 and Aß42) feature prominently in the extracellular brain deposits associated with Alzheimer's disease. While Aß40 is the prevalent form in the cerebrospinal fluid, the fraction of Aß42 increases in the amyloid deposits over the course of disease development. The low in vivo concentration (pM-nM) and metastable nature of Aß oligomers have made identification of their size, composition, cellular binding sites and mechanism of action challenging and elusive. Furthermore, recent studies have suggested that synergistic effects between Aß40 and Aß42 alter both the formation and stability of various peptide oligomers as well as their cytotoxicity. These studies often utilized Aß oligomers that were prepared in solution and at µM peptide concentrations. The current work was performed using physiological Aß concentrations and single-molecule microscopy to follow peptide binding and association on primary cultured neurons. When the cells were exposed to a 1:1 mixture of nM Aß40:Aß42, significantly larger membrane-bound oligomers developed compared to those formed from either peptide alone. Fluorescence resonance energy transfer experiments at the single molecule level reveal that these larger oligomers contained both Aß40 and Aß42, but that the growth of these oligomers was predominantly by addition of Aß42. Both pure peptides form very few oligomers larger than dimers, but either membrane bound Aß40/42 complex, or Aß40, bind Aß42 to form increasingly larger oligomers. These findings may explain how Aß42-dominant oligomers, suspected of being more cytotoxic, develop on the neuronal membrane under physiological conditions.
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Peptídeos beta-Amiloides/farmacologia , Microscopia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Peptídeos beta-Amiloides/química , Animais , Sinergismo Farmacológico , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/citologia , Fragmentos de Peptídeos/química , Multimerização Proteica , Estrutura Secundária de Proteína , Ratos , Propriedades de Superfície , Fatores de TempoRESUMO
Soluble oligomers of the amyloid-ß peptide have been implicated as proximal neurotoxins in Alzheimer's disease. However, the identity of the neurotoxic aggregate(s) and the mechanisms by which these species induce neuronal dysfunction remain uncertain. Physiologically relevant experimentation is hindered by the low endogenous concentrations of the peptide, the metastability of Aß oligomers, and the wide range of observed interactions between Aß and biological membranes. Single-molecule microscopy represents one avenue for overcoming these challenges. Using this technique, we find that Aß binds to primary rat hippocampal neurons at physiological concentrations. Although amyloid-ß(1-40) as well as amyloid-ß(1-42) initially form larger oligomers on neurites than on glass slides, a 1:1 mix of the two peptides result in smaller neurite-bound oligomers than those detected on-slide or for either peptide alone. With 1 nM peptide in solution, Aß40 oligomers do not grow over the course of 48 h, Aß42 oligomers grow slightly, and oligomers of a 1:1 mix grow substantially. Evidently, small Aß oligomers are capable of binding to neurons at physiological concentrations and grow at rates dependent on local Aß42:Aß40 ratios. These results are intriguing in light of the increased Aß42:Aß40 ratios shown to correlate with familial Alzheimer's disease mutations.
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Peptídeos beta-Amiloides/química , Neuritos/metabolismo , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/metabolismo , Animais , Membrana Celular/metabolismo , Hipocampo/citologia , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Microscopia de Fluorescência , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica , Subunidades Proteicas , RatosRESUMO
Recent evidence supports the hypothesis that the oligomers formed by the ß-amyloid peptide early in its aggregation process are neurotoxic and may feature in Alzheimer's disease. Although the mechanism underlying this neurotoxicity remains unclear, interactions of these oligomers with neuronal membranes are believed to be involved. Identifying the neurotoxic species is challenging because ß-amyloid peptides form oligomers at very low physiological concentrations (nM), and these oligomers are highly heterogeneous and metastable. Here, we report the use of single-molecule imaging techniques to study the interactions between ß-amyloid (1-40) peptides and supported synthetic model anionic lipid membranes. The evolution of the ß-amyloid species on the membranes was monitored for up to several days, and the results indicate an initial tight, uniform, binding of ß-amyloid (1-40) peptides to the lipid membranes, followed by oligomer formation in the membrane. At these low concentrations, the behavior at early times during the formation of small oligomers is interpreted qualitatively in terms of the two-state model proposed by H. W. Huang for the interaction between amphipathic peptides and membranes. However, the rate of oligomer formation in the membrane and their size are highly dependent on the concentrations of ß-amyloid (1-40) peptides in aqueous solution, suggesting two different pathways of oligomer formation, which lead to drastically different species in the membrane and a departure from the two-state model as the concentration increases.
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Peptídeos beta-Amiloides/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Imagem Molecular , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
We demonstrate the suppression of nuclear-spin fluctuations in an InAs quantum dot and measure the timescales of the spin narrowing effect. By initializing for tens of milliseconds with two continuous wave diode lasers, fluctuations of the nuclear spins are suppressed via the hole-assisted dynamic nuclear polarization feedback mechanism. The fluctuation narrowed state persists in the dark (absent light illumination) for well over 1 s even in the presence of a varying electron charge and spin polarization. Enhancement of the electron spin coherence time (T2*) is directly measured using coherent dark state spectroscopy. By separating the calming of the nuclear spins in time from the spin qubit operations, this method is much simpler than the spin echo coherence recovery or dynamic decoupling schemes.
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Understanding how amyloid-ß peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric Aß interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high Aß concentrations required for most biochemical analyses, the metastable nature of Aß aggregates, and the complex variety of Aß species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small Aß(1-40) oligomers bind to living neuroblastoma cells at physiological Aß concentrations. Single particle fluorescence intensity measurements indicate that cell-bound Aß species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound Aß oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary Aß species that interact with neurons at physiological concentrations.
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Peptídeos beta-Amiloides/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Neuroblastoma/metabolismo , Multimerização ProteicaRESUMO
Type II diabetes, in its late stages, is often associated with the formation of extracellular islet amyloid deposits composed of islet amyloid polypeptide (IAPP or amylin). IAPP is stored before secretion at millimolar concentrations within secretory granules inside the ß-cells. Of interest, at these same concentrations in vitro, IAPP rapidly aggregates and forms fibrils, yet within secretory granules of healthy individuals, IAPP does not fibrillize. Insulin is also stored within the secretory granules before secretion, and has been shown in vitro to inhibit IAPP fibril formation. Because of insulin's inhibitory effect on IAPP fibrillization, it has been suggested that insulin may also inhibit IAPP-mediated permeabilization of the ß-cell plasma membrane in vivo. We show that although insulin is effective at preventing fiber-dependent membrane disruption, it is not effective at stopping the initial phase of membrane disruption before fibrillogenesis, and does not prevent the formation of small IAPP oligomers on the membrane. These results suggest that insulin has a more complicated role in inhibiting IAPP fibrillogenesis, and that other factors, such as the low pH of the secretory granule, may also play a role.
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Membrana Celular/efeitos dos fármacos , Insulina/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Cinética , Estrutura Quaternária de Proteína , Soluções , Fatores de Tempo , Lipossomas Unilamelares/metabolismoRESUMO
OBJECTIVE: We sought to identify immunologic and virologic correlates of spontaneous viral control among long-term survivors of perinatal HIV infection expressing the protective human leukocyte antigen (HLA)-B57 allele. DESIGN: The frequency, epitope specificity, and functional attributes of HIV-specific T cells and sequence variation within B57-restricted epitopes were compared between 'spontaneous controllers' who maintained normal CD4 percentages and viral loads less than 3000 copies/ml without antiretroviral therapy, and 'treated progressors' who had initiated HAART. METHODS: Recognition of HIV optimal epitopes was assessed by interferon gamma (IFNgamma) enzyme-linked immunosorbent spot. Functional characterization of CD8 cells targeting B57 epitopes was performed by staining for cytokine production (intracellular IFNgamma, interleukin-2, tumor necrosis factor alpha) and degranulation. Sequencing of autologous RNA was performed to determine the prevalence of viral escape mutations within B57-restricted epitopes and associated compensatory mutations. RESULTS: HLA-B57 remained immunodominant during chronic infection in both controllers and progressors, but controllers recognized fewer epitopes and targeted epitopes within Gag and reverse transcriptase only, whereas progressors demonstrated a broader response targeting additional proteins. No individual epitope was targeted more frequently by spontaneous controllers. CD8 cytokine production patterns were heterogeneous among individuals and even among different epitopes in the same individual and did not correlate with spontaneous viral control. Extensive sequence variation within B57 epitopes was observed in both groups, but only progressors displayed additional capsid mutations that are known to offset the viral fitness cost of B57-driven immune escape. CONCLUSION: Among HLA-B57-positive long-term survivors, spontaneous control of viremia is not associated with a qualitatively or quantitatively superior T-cell response, but with uncompensated fitness-attenuating mutations in the viral capsid.
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Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1 , Antígenos HLA-B/imunologia , Adolescente , Criança , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Infecções por HIV/genética , Infecções por HIV/virologia , Sobreviventes de Longo Prazo ao HIV , HIV-1/genética , HIV-1/imunologia , Antígenos HLA-B/metabolismo , Humanos , Tolerância Imunológica , Interferon gama/imunologia , Interferon gama/metabolismo , Masculino , ViremiaRESUMO
The extracellular senile plaques prevalent in brain tissue in Alzheimer's disease (AD) are composed of amyloid fibrils formed by the Abeta peptide. These fibrils have been traditionally believed to be featured in neurotoxicity; however, numerous recent studies provide evidence that cytotoxicity in AD may be associated with low-molecular weight oligomers of Abeta that associate with neuronal membranes and may lead to membrane permeabilization and disruption of the ion balance in the cell. The underlying mechanism leading to disruption of the membrane is the subject of many recent studies. Here we report the application of single-molecule optical detection, using fluorescently labeled human Alphabeta40, combined with membrane conductivity measurements, to monitor the interaction of single-oligomeric peptide structures with model planar black lipid membranes (BLMs). In a qualitative study, we show that the binding of Alphabeta to the membrane can be described by three distinctly different behaviors, depending on the Alphabeta monomer concentration. For concentrations much below 10 nM, there is uniform binding of monomers over the surface of the membrane with no evidence of oligomer formation or membrane permeabilization. Between 10 nM and a few hundred nanomolar, the uniform monomer binding is accompanied by the presence of peptide species ranging from dimers to small oligomers. The dimers are not found to permeabilize the membrane, but the larger oligomers lead to permeabilization with individual oligomers producing ion conductances of <10 pS/pore. At higher concentrations, perhaps beyond physiologically relevant concentrations, larger extended and dynamic structures are found with large conductances (hundreds of picosiemens), suggesting a major disruption of the membrane.
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Peptídeos beta-Amiloides/química , Bicamadas Lipídicas/química , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Amiloide/química , Condutividade Elétrica , Corantes Fluorescentes , Humanos , Permeabilidade , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
Amyloid diseases are traditionally characterized by the appearance of inter- and intracellular fibrillar protein deposits, termed amyloid. Historically, these deposits have been thought to be the etiology of the disease. However, recent evidence suggests that small oligomers of the amyloidogenic protein/peptide are the origin of neurotoxicity. Although the importance of identifying the toxic oligomeric species is widely recognized, such identification is challenging because these oligomers are metastable, occur at low concentration, and are characterized by a high degree of heterogeneity. In this work, a fluorescently labeled beta-amyloid(1-40) is used as a model amyloidogenic peptide to test the effectiveness of what we believe is a novel approach based on single-molecule spectroscopy. We find that by directly counting the photobleaching steps in the fluorescence, we can determine the number of subunits in individual beta-amyloid(1-40) oligomers, which allows us to easily distinguish among different species in the mixtures. The results are further analyzed by comparison with Monte Carlo simulations to show that the variability seen in the size of photobleaching steps can be explained by assuming random dipole orientations for the chromophores in a given oligomer. In addition, by accounting for bias in the oligomer size distribution due to the need to subtract background noise, we can make the results more quantitative. Although the oligomer size determined in this work is limited to only small species, our single-molecule results are in good quantitative agreement with high-performance liquid chromatography gel filtration data and demonstrate that single-molecule spectroscopy can provide useful insights into the issues of heterogeneity and ultimately cellular toxicity in the study of amyloid diseases.
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Peptídeos beta-Amiloides/química , Amiloide/química , Fragmentos de Peptídeos/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Simulação por Computador , Corantes Fluorescentes , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Modelos Químicos , Método de Monte Carlo , Fotodegradação , Análise EspectralRESUMO
A single electron or hole spin trapped inside a semiconductor quantum dot forms the foundation for many proposed quantum logic devices. In group III-V materials, the resonance and coherence between two ground states of the single spin are inevitably affected by the lattice nuclear spins through the hyperfine interaction, while the dynamics of the single spin also influence the nuclear environment. Recent efforts have been made to protect the coherence of spins in quantum dots by suppressing the nuclear spin fluctuations. However, coherent control of a single spin in a single dot with simultaneous suppression of the nuclear fluctuations has yet to be achieved. Here we report the suppression of nuclear field fluctuations in a singly charged quantum dot to well below the thermal value, as shown by an enhancement of the single electron spin dephasing time T(2)*, which we measure using coherent dark-state spectroscopy. The suppression of nuclear fluctuations is found to result from a hole-spin assisted dynamic nuclear spin polarization feedback process, where the stable value of the nuclear field is determined only by the laser frequencies at fixed laser powers. This nuclear field locking is further demonstrated in a three-laser measurement, indicating a possible enhancement of the electron spin T(2)* by a factor of several hundred. This is a simple and powerful method of enhancing the electron spin coherence time without use of 'spin echo'-type techniques. We expect that our results will enable the reproducible preparation of the nuclear spin environment for repetitive control and measurement of a single spin with minimal statistical broadening.
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Expression of HLA-B57 is associated with restricted replication of human immunodeficiency virus (HIV), but the mechanism for its protective effect remains unknown. If this advantage depends upon CD8 T-cell recognition of B57-restricted epitopes, mother-to-child transmission of escape mutations within these epitopes could nullify its protective effect. However, if the B57 advantage is largely mediated by selection for fitness-attenuating viral mutations within B57-restricted epitopes, such as T242N in TW10-Gag, then the transmission of such mutations could facilitate viral control in the haploidentical infant. We assessed the consequences of B57-associated mutations on replication capacity, viral control, and clinical outcome after vertical transmission in 13 mother-child pairs. We found that expression of HLA-B57 was associated with exceptional control of HIV during infancy, even when mutations within TW10 and most other B57-restricted epitopes were transmitted, subverting the natural immunodominance of HLA-B57. In contrast, most B57-negative infants born to B57-positive mothers progressed rapidly to AIDS. The presence of T242N led to a reproducible reduction in viral fitness, as demonstrated by in vitro assays using NL4-3 constructs encoding p24 sequences from individual mothers and infants. Associated compensatory mutations within p24-Gag were observed to reverse this impairment and to influence the propensity of T242N to revert after transmission to B57-negative hosts. Moreover, primary failure to control viremia was observed in one infant to whom multiple compensatory mutations were transmitted along with T242N. These parallel in vivo and in vitro data suggest that HLA-B57 confers its advantage primarily by driving and maintaining a fitness-attenuating mutation in p24-Gag.
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Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/imunologia , HIV/crescimento & desenvolvimento , HIV/imunologia , Antígenos HLA-B/imunologia , Transmissão Vertical de Doenças Infecciosas , Mutação de Sentido Incorreto/imunologia , Sequência de Aminoácidos , Animais , Pré-Escolar , Progressão da Doença , Feminino , HIV/genética , Proteína do Núcleo p24 do HIV/genética , Infecções por HIV/transmissão , Infecções por HIV/virologia , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The 40 and 42 residue amyloid-beta (Abeta) peptides are major components of the proteinaceous plaques prevalent in the Alzheimer's disease-afflicted brain and have been shown to have an important role in instigating neuronal degeneration. Whereas it was previously thought that Abeta becomes cytotoxic upon forming large fibrillar aggregates, recent studies suggest that soluble intermediate-sized oligomeric species cause cell death through membrane permeabilization. The present study examines the interactions between Abeta40 and lipid membranes using liposomes as a model system to determine how changes in membrane composition influence the conversion of Abeta into these toxic species. Abeta40 membrane binding was monitored using fluorescence-based assays with a tryptophan-substituted peptide (Abeta40 [Y10W]). We extend previous observations that Abeta40 interacts preferentially with negatively charged membranes, and show that binding of nonfibrillar, low molecular mass oligomers of Abeta40 to anionic, but not neutral, membranes involves insertion of the peptide into the bilayer, as well as sequential conformational changes corresponding to the degree of oligomerization induced. Significantly, while anionic membranes in the gel, liquid crystalline, and liquid ordered phases induce these conformational changes equally, membrane permeabilization is reduced dramatically as the fluidity of the membrane is decreased. These findings demonstrate that binding alone is not sufficient for membrane permeabilization, and that the latter is also highly dependent on the fluidity and phase of the membrane. We conclude that binding and pore formation are two distinct steps. The differences in Abeta behavior induced by membrane composition may have significant implications on the development and progression of AD as neuronal membrane composition is altered with age.
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Peptídeos beta-Amiloides/química , Permeabilidade da Membrana Celular/fisiologia , Bicamadas Lipídicas/química , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/metabolismo , Sítios de Ligação , Dicroísmo Circular , Transferência Ressonante de Energia de Fluorescência , Humanos , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Triptofano/químicaRESUMO
Perinatal HIV infection is characterized by a sustained high-level viremia and a high risk of rapid progression to AIDS, indicating a failure of immunologic containment of the virus. We hypothesized that age-related differences in the specificity or function of HIV-specific T cells may influence HIV RNA levels and clinical outcome following perinatal infection. In this study, we defined the HIV epitopes targeted by 76 pediatric subjects (47 HIV infected and 29 HIV exposed, but uninfected), and assessed the ability of HIV-specific CD8 and CD4 T cells to degranulate and produce IFN-gamma, TNF-alpha, and IL-2. No responses were detected among HIV-uninfected infants, whereas responses among infected subjects increased in magnitude and breadth with age. Gag-specific responses were uncommon during early infancy, and their frequency was significantly lower among children younger than 24 mo old (p = 0.014). Importantly, Gag responders exhibited significantly lower HIV RNA levels than nonresponders (log viral load 5.8 vs 5.0; p = 0.005). Both the total and Gag-specific T cell frequency correlated inversely with viral load after correction for age, whereas no relationship with targeting of other viral proteins was observed. Functional assessment of HIV-specific T cells by multiparameter flow cytometry revealed that polyfunctional CD8 cells were less prevalent in children before 24 mo of age, and that HIV-specific CD4 cell responses were of universally low frequency among antiretroviral-naive children and absent in young infants. These cross-sectional data suggest that qualitative differences in the CD8 response, combined with a deficiency of HIV-specific CD4 cells, may contribute to the inability of young infants to limit replication of HIV.
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Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Carga Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Adolescente , Fatores Etários , Degranulação Celular , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Infecções por HIV/sangue , HIV-1/metabolismo , Humanos , Lactente , Interferon gama/sangue , Interferon gama/imunologia , Interleucina-2/sangue , Interleucina-2/imunologia , Masculino , RNA Viral/sangue , RNA Viral/imunologia , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/imunologia , Replicação Viral/imunologiaRESUMO
BACKGROUND: Paediatric and Perinatal HIV/AIDS remain significant health challenges in the Caribbean where the HIV seroprevalence is second only to Sub-Saharan Africa. METHOD: We describe a collaborative approach to the prevention, treatment and care of HIV in pregnant women, infants and children in Jamaica. A team of academic and government healthcare personnel collaborated to address the paediatric and perinatal HIV epidemic in Greater Kingston as a model for Jamaica (population 2.6 million, HIV seroprevalence 1.5%). A five-point plan was utilized and included leadership and training, preventing mother-to-child transmission (pMTCT), treatment and care of women, infants and children, outcomes-based research and local, regional and international outreach. RESULTS: A core group of paediatric/perinatal HIV professionals were trained, including paediatricians, obstetricians, public health practitioners, nurses, microbiologists, data managers, information technology personnel and students to serve Greater Kingston (birth cohort 20 000). During September 2002 to August 2007, over 69 793 pregnant women presented for antenatal care. During these five years, significant improvements occurred in uptake of voluntary counselling (40% to 91%) and HIV-testing (53% to 102%). Eight hundred and eighty-three women tested HIV-positive with seroprevalence rates of 1-2% each year. The use of modified short course zidovudine or nevirapine in the first three years significantly reduced mother-to-child transmission (MTCT) of HIV from 29% to 6% (RR 0.27; 95% CI - 0.10, 0.68). During 2005 to 2007 using maternal highly active antiretroviral therapy (HAART) with zidovudine and lamivudine with either nevirapine, nelfinavir or lopinavir/ritonavir and infant zidovudine and nevirapine, MTCT was further reduced to an estimated 1.6% in Greater Kingston and 4.75% islandwide. In five years, we evaluated 1570 children in four-weekly paediatric infectious diseases clinics in Kingston, St Andrew and St Catherine and in six rural outreach sites throughout Jamaica; 24% (377) had HIV/AIDS and 76% (1193) were HIV-exposed. Among the infected children, 79% (299 of 377) initiated HAART, resulting in reduced HIV-attributable childhood morbidity and mortality islandwide. An outcomes-based research programme was successfully implemented. CONCLUSION: Working collaboratively, our mission of pMTCT of HIV and improving the quality of life for families living and affected by HIV/AIDS in Jamaica is being achieved.
ANTECEDENTES: El VIH/SIDA pediátrico y el perinatal continúan siendo retos significativos para la salud en el Caribe, donde la seroprevalencia de VIH ocupa el segundo lugar tras el África Subsahariana. MÉTODO: Se describe un enfoque colaborativo para tratamiento, prevención y cuidado de embarazadas, bebés y niños en Jamaica. Un equipo de personal académico y gubernamental vinculados a la salud, colaboraron para abordar la epidemia de VIH pediátrico y perinatal en Greater Kingston, como modelo para Jamaica (población de 2.6 millones, 1.5% seroprevalencia VIH). Se utilizó un plan de cinco puntos que incluyó liderazgo y entrenamiento, prevención de la transmisión madre a hijo (PTMAH), tratamiento y cuidado de mujeres, bebés y niños, investigaciones basadas en resultados, y outreach local, regional e internacional. RESULTADOS: Un grupo básico de profesionales del VIH pediátrico/perinatal, que incluía pediatras, obstetras, trabajadores de la salud, enfermeras, microbiólogos, administradores de datos, así como personal y estudiantes de la tecnología de la información, fue entrenado para servir en Greater Kingston (cohorte de nacimiento 20 000). De septiembre de 2002 hasta Agosto de 2007, más de 69 793 embarazadas se presentaron para recibir atención prenatal. Durante estos cinco años, tuvieron lugar mejoras significativos en cuanto a la recepción de asesoramiento (40% to 91%) y pruebas (53% to 102%) de VIH voluntarios. Ochocientos ochenta y tres mujeres resultaron VIH positivas en las pruebas, con tasas de seroprevalencia de 1-2% cada año. El uso de un ciclo corto modificado de zidovudina o nevirapina en los primeros tres años, redujo la transmisión madre a hijo (TMAH) de VIH significativamente de 29% a 6% (RR 0.27; 95% CI - 0.10, 0.68). Durante el 2005 hasta 2007, usando terapia antiretroviral altamente activa (TARAA) materna, con zidovudina y lamivudina con nevirapina, nelfinavir o lopinavir/ritonavir y nevirapina y zidovudina para niños, la TMAH se redujo a un estimado de 1.6 % en Greater Kingston y a .75% a lo largo de la isla. En cinco años, evaluamos 1570 niños en cuatro clínicas infecciosas pediátricas semanales en Kingston, Saint Andrew y Saint Catherine, así como en seis otros lugares destinados al servicio comunitario (outreach) por toda Jamaica; 24% (377) tenían VIH/SIDA y 76% (1193) estaba expuestos al VIH. Entre los niños infectados, 79% (299 de 377) iniciaron el TARAA, lo que trajo como resultado una reducción de la mortalidad y la morbilidad infantil atribuible al VIH, en todo el país. Se implementó exitosamente un programa de investigación basado en resultados. CONCLUSIÓN: Trabajando en colaboración, estamos logrando nuestra misión de prevenir la TMAH del VIH, y mejorar la calidad de vida de las familias que viven afectadas por el VIH/SIDA en Jamaica.