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1.
Nat Struct Mol Biol ; 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778821

RESUMO

The formation of amyloid deposits in human tissues is a defining feature of more than 50 medical disorders, including Alzheimer's disease. Strong genetic and histological evidence links these conditions to the process of protein aggregation, yet it has remained challenging to identify a definitive connection between aggregation and pathogenicity. Using time-resolved fluorescence microscopy of individual synthetic vesicles, we show for the Aß42 peptide implicated in Alzheimer's disease that the disruption of lipid bilayers correlates linearly with the time course of the levels of transient oligomers generated through secondary nucleation. These findings indicate a specific role of oligomers generated through the catalytic action of fibrillar species during the protein aggregation process in driving deleterious biological function and establish a direct causative connection between amyloid formation and its pathological effects.

3.
Cell Death Differ ; 2020 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-32341450

RESUMO

Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of ß-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson's disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

4.
Lancet ; 395(10234): 1434-1443, 2020 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-32197107

RESUMO

BACKGROUND: An unmet clinical need remains for an effective tetravalent dengue vaccine suitable for all age groups, regardless of serostatus. We assessed the immunogenicity and safety of three different dose schedules of a tetravalent dengue vaccine (TAK-003) over a 48-month period in children living in dengue-endemic countries. METHODS: We did a large, phase 2, double-blind, placebo-controlled trial at three sites in the Dominican Republic, Panama, and the Philippines. Healthy participants aged 2-17 years were randomly assigned 1:2:5:1 using an interactive web response system with stratification by age to receive either a two-dose primary series (days 1 and 91), one primary dose (day 1), one primary dose plus booster (days 1 and 365), or placebo. Participants and relevant study personnel were masked to the random assignment until completion of the study at month 48. To maintain masking, TAK-003 recipients were administered placebo doses when appropriate. The primary objective was assessment of neutralising geometric mean titres for each serotype to month 48 assessed in the per-protocol immunogenicity subset. Secondary safety endpoints included proportions of participants with serious adverse events and symptomatic virologically confirmed dengue. This study is registered with ClinicalTrials.gov, NCT02302066. FINDINGS: Between Dec 5, 2014, and Feb 13, 2015, 1800 children were randomly assigned to the following groups: two-dose primary series (n=201), one primary dose (n=398), one primary dose plus 1-year booster (n=1002), and placebo (n=199). Of them, 1479 (82%) participants completed the 48-month study. Immunogenicity endpoints were assessed in 562 participants enrolled in the immunogenicity subset, of whom 509 were included in the per-protocol subset. At month 48, antibody titres remained elevated in all TAK-003 groups compared with placebo, irrespective of baseline serostatus. At month 48, geometric mean titres were 378 (95% CI 226-632) in two-dose, 421 (285-622) in one-dose, 719 (538-960) in one-dose plus 1-year booster, and 100 (50-201) in placebo recipients against DENV 1; 1052 (732-1511), 1319 (970-1794), 1200 (927-1553), and 208 (99-437) against DENV 2; 183 (113-298), 201 (135-298), 288 (211-392), and 71 (37-139) against DENV 3; and 152 (97-239), 164 (114-236), 219 (165-290), and 46 (26-82) against DENV 4; and tetravalent seropositivity rate was 89% (79-96), 86% (80-92), 97% (93-99), and 60% (47-72), respectively. Virologically confirmed dengue was recorded in 37 (2%) TAK-003 and 13 (7%) placebo participants, with a relative risk of 0·35 (0·19-0·65). No vaccine-related serious adverse events or severe dengue virus disease were reported. INTERPRETATION: TAK-003 elicited antibody responses against all four serotypes, which persisted to 48 months post-vaccination, regardless of baseline serostatus. No important safety risks were identified. We observed a long-term reduction in risk of symptomatic dengue virus disease in vaccinees. Results from this study provide a long-term safety database and support assessment of the vaccine in the ongoing phase 3 efficacy study. FUNDING: Takeda Vaccines.


Assuntos
Vacinas contra Dengue/efeitos adversos , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Imunogenicidade da Vacina/imunologia , Adolescente , Criança , Pré-Escolar , Dengue/imunologia , Dengue/virologia , Vacinas contra Dengue/administração & dosagem , Vírus da Dengue/genética , República Dominicana/epidemiologia , Método Duplo-Cego , Esquema de Medicação , Feminino , Humanos , Imunização Secundária/métodos , Masculino , Panamá/epidemiologia , Filipinas/epidemiologia , Placebos/administração & dosagem , Segurança , Sorogrupo , Vacinação/métodos
5.
Acta Neuropathol Commun ; 7(1): 120, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31349874

RESUMO

Soluble aggregates of amyloid-ß (Aß) have been associated with neuronal and synaptic loss in Alzheimer's disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we characterised soluble aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls using a range of sensitive biophysical methods. We used super-resolution imaging and atomic force microscopy to characterise the size and structure of the aggregates present in CSF and correlate this with their ability to permeabilise lipid membranes and induce an inflammatory response. We found that these aggregates are extremely heterogeneous and exist in a range of sizes, varying both structurally and in their mechanisms of toxicity during the disease progression. A higher proportion of small aggregates of Aß that can cause membrane permeabilization are found in MCI CSF; in established AD, a higher proportion of the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells, while there was no detectable change in aggregate concentration. These results show that large aggregates, some longer than 100 nm, are present in the CSF of AD patients and suggest that different neurotoxic mechanisms are prevalent at different stages of AD.

6.
Chem Sci ; 10(17): 4588-4597, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31123569

RESUMO

The aggregation of the prion protein (PrP) plays a key role in the development of prion diseases. In the past decade, a similar process has been associated with other proteins, such as Aß, tau, and α-synuclein, which participate in other neurodegenerative diseases. It is increasingly recognized that the small oligomeric species of aggregates can play an important role in the development of prion diseases. However, determining the nature of the oligomers formed during the aggregation process has been experimentally difficult due to the lack of suitable methods capable of the detection and characterization of the low level of oligomers that may form. To address this problem, we have utilized single-aggregate methods to study the early events associated with aggregation of recombinant murine PrP in vitro to approach the bona fide process in vivo. PrP aggregation resulted in the formation of thioflavin T (ThT)-inactive and ThT-active species of oligomers. The ThT-active oligomers undergo conversion from a Proteinase K (PK)-sensitive to PK-resistant conformer, from which mature fibrils can eventually emerge. Overall, our results show that single-aggregate methods can provide structural and mechanistic insights into PrP aggregation, identify the potential species that mediates cytotoxicity, and reveal that a range of distinct oligomeric species with different properties is formed during prion protein aggregation.

7.
Nat Commun ; 10(1): 1541, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948723

RESUMO

Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer's disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aß42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aß42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aß42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aß42 may contribute to the onset and progression of Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Bicamadas Lipídicas/metabolismo , Agregação Patológica de Proteínas , Peptídeos beta-Amiloides/metabolismo , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Ultracentrifugação
8.
ACS Chem Neurosci ; 10(5): 2374-2384, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30793584

RESUMO

Aggregation of the amyloid-ß (Aß) peptide into plaques is believed to play a crucial role in Alzheimer's disease. Amyloid plaques consist of fibrils of full length Aß peptides as well as N-terminally truncated species. ß-Site amyloid precursor protein-cleaving enzyme (BACE1) cleaves amyloid precursor protein in the first step in Aß peptide production and is an attractive therapeutic target to limit Aß generation. Inhibition of BACE1, however, induces a unique pattern of Aß peptides with increased levels of N-terminally truncated Aß peptides starting at position 5 (Aß5-X), indicating that these peptides are generated through a BACE1-independent pathway. Here we elucidate the aggregation mechanism of Aß5-42 and its influence on full-length Aß42. We find that, compared to Aß42, Aß5-42 is more aggregation prone and displays enhanced nucleation rates. Aß5-42 oligomers cause nonspecific membrane disruption to similar extent as Aß42 but appear at earlier time points in the aggregation reaction. Noteworthy, this implies similar toxicity of Aß42 and Aß5-42 and the toxic species are generated faster by Aß5-42. The increased rate of secondary nucleation on the surface of existing fibrils originates from a higher affinity of Aß5-42 monomers for fibrils, as compared to Aß42: an effect that may be related to the reduced net charge of Aß5-42. Moreover, Aß5-42 and Aß42 peptides coaggregate into heteromolecular fibrils and either species can elongate existing Aß42 or Aß5-42 fibrils but Aß42 fibrils are more catalytic than Aß5-42 fibrils. Our findings highlight the importance of the N-terminus for surface-catalyzed nucleation and thus the production of toxic oligomers.

9.
Biochim Biophys Acta Proteins Proteom ; 1867(10): 870-878, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30611780

RESUMO

Protein aggregates play a key role in the initiation and spreading of neurodegenerative disease but have been difficult to study due to their low abundance and heterogeneity, in both size and structure. Fluorescence based methods capable of detecting and characterising single aggregates have recently been developed and can be used to measure many important aggregate properties, and can be combined with sensitive assays to measure aggregate toxicity. Here we review these methods and discuss recent examples of their application to determine the molecular mechanism of aggregation and the detection of aggregates in cells and cerebrospinal fluid. The further development of these methods and their application to the aggregates present in humans has the potential to solve a major problem in the field and allow the identification of the key toxic species that should be targeted in therapies.


Assuntos
Imagem Molecular , Doenças Neurodegenerativas , Imagem Óptica , Agregados Proteicos , Animais , Humanos , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/metabolismo
10.
Nano Lett ; 18(12): 7494-7501, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30380895

RESUMO

Proteins fold into a single structural ensemble but can also misfold into many diverse structures including small aggregates and fibrils, which differ in their toxicity. The aggregate surface properties play an important role in how they interact with the plasma membrane and cellular organelles, potentially inducing cellular toxicity, however, these properties have not been measured to date due to the lack of suitable methods. Here, we used a spectrally resolved, super-resolution imaging method combined with an environmentally sensitive fluorescent dye to measure the surface hydrophobicity of individual aggregates formed by the protein α-synuclein (αS), whose aggregation is associated with Parkinson's disease. We show that the surface of soluble oligomers is more hydrophobic than fibrils and populates a diverse range of coexisting states. Overall, our data show that the conversion of oligomers to fibril-like aggregates and ultimately to fibrils results in a reduction in both hydrophobicity and the variation in hydrophobicity. This funneling characteristic of the energy landscape explains many of the observed properties of αS aggregates and may be a common feature of aggregating proteins.


Assuntos
Agregados Proteicos , alfa-Sinucleína/química , Corantes Fluorescentes/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imagem Óptica , Doença de Parkinson/metabolismo , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , Solubilidade , alfa-Sinucleína/metabolismo
11.
ACS Nano ; 12(11): 10855-10866, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30371053

RESUMO

Small oligomers of the protein α-synuclein (αS) are highly cytotoxic species associated with Parkinson's disease (PD). In addition, αS can form co-aggregates with its mutational variants and with other proteins such as amyloid-ß (Aß) and tau, which are implicated in Alzheimer's disease. The processes of self-oligomerization and co-oligomerization of αS are, however, challenging to study quantitatively. Here, we have utilized single-molecule techniques to measure the equilibrium populations of oligomers formed in vitro by mixtures of wild-type αS with its mutational variants and with Aß40, Aß42, and a fragment of tau. Using a statistical mechanical model, we find that co-oligomer formation is generally more favorable than self-oligomer formation at equilibrium. Furthermore, self-oligomers more potently disrupt lipid membranes than do co-oligomers. However, this difference is sometimes outweighed by the greater formation propensity of co-oligomers when multiple proteins coexist. Our results suggest that co-oligomer formation may be important in PD and related neurodegenerative diseases.


Assuntos
Peptídeos beta-Amiloides/biossíntese , alfa-Sinucleína/metabolismo , Proteínas tau/biossíntese , Peptídeos beta-Amiloides/química , Humanos , Modelos Moleculares , Termodinâmica , alfa-Sinucleína/química , Proteínas tau/química
12.
Chembiochem ; 19(19): 2033-2038, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30051958

RESUMO

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-ß. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Neurônios/patologia , Doença de Parkinson/patologia , Agregados Proteicos , alfa-Sinucleína/química , Peptídeos beta-Amiloides/metabolismo , Aptâmeros de Peptídeos/química , Humanos , Agregação Patológica de Proteínas , alfa-Sinucleína/metabolismo
13.
Cell Rep ; 23(12): 3492-3500, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29924993

RESUMO

The aberrant aggregation of α-synuclein is associated with several human diseases, collectively termed the α-synucleinopathies, which includes Parkinson's disease. The progression of these diseases is, in part, mediated by extracellular α-synuclein oligomers that may exert effects through several mechanisms, including prion-like transfer, direct cytotoxicity, and pro-inflammatory actions. In this study, we show that two abundant extracellular chaperones, clusterin and α2-macroglobulin, directly bind to exposed hydrophobic regions on the surface of α-synuclein oligomers. Using single-molecule fluorescence techniques, we found that clusterin, unlike α2-macroglobulin, exhibits differential binding to α-synuclein oligomers that may be related to structural differences between two previously described forms of αS oligomers. The binding of both chaperones reduces the ability of the oligomers to permeabilize lipid membranes and prevents an oligomer-induced increase in ROS production in cultured neuronal cells. Taken together, these data suggest a neuroprotective role for extracellular chaperones in suppressing the toxicity associated with α-synuclein oligomers.


Assuntos
Espaço Extracelular/metabolismo , Multimerização Proteica , alfa-Sinucleína/química , alfa-Sinucleína/toxicidade , Interações Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/metabolismo , Ligação Proteica
14.
ACS Chem Biol ; 13(3): 636-646, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29300447

RESUMO

As a key player of the protein quality control network of the cell, the molecular chaperone Hsp70 inhibits the aggregation of the amyloid protein tau. To date, the mechanism of this inhibition and the tau species targeted by Hsp70 remain unknown. This is partly due to the inherent difficulty of studying amyloid aggregates because of their heterogeneous and transient nature. Here, we used ensemble and single-molecule fluorescence measurements to dissect how Hsp70 counteracts the self-assembly process of the K18 ΔK280 tau variant. We found that Hsp70 blocks the early stages of tau aggregation by suppressing the formation of tau nuclei. Additionally, Hsp70 sequesters oligomers and mature tau fibrils with nanomolar affinity into a protective complex, efficiently neutralizing their ability to damage membranes and seed further tau aggregation. Our results provide novel insights into the molecular mechanisms by which the chaperone Hsp70 counteracts the formation, propagation, and toxicity of tau aggregates.


Assuntos
Proteínas de Choque Térmico HSP70/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , Proteínas tau/antagonistas & inibidores , Amiloide/efeitos dos fármacos , Fluorescência , Humanos , Imagem Individual de Molécula
15.
Angew Chem Int Ed Engl ; 57(18): 4886-4890, 2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29342318

RESUMO

Small aggregates of misfolded proteins play a key role in neurodegenerative disorders. Such species have proved difficult to study due to the lack of suitable methods capable of resolving these heterogeneous aggregates, which are smaller than the optical diffraction limit. We demonstrate here an all-optical fluorescence microscopy method to characterise the structure of individual protein aggregates based on the fluorescence anisotropy of dyes such as thioflavin-T, and show that this technology is capable of studying oligomers in human biofluids such as cerebrospinal fluid. We first investigated in vitro the structural changes in individual oligomers formed during the aggregation of recombinant α-synuclein. By studying the diffraction-limited aggregates we directly evaluated their structural conversion and correlated this with the potential of aggregates to disrupt lipid bilayers. We finally characterised the structural features of aggregates present in cerebrospinal fluid of Parkinson's disease patients and age-matched healthy controls.


Assuntos
Imagem Óptica , alfa-Sinucleína/análise , alfa-Sinucleína/química , Humanos , Agregados Proteicos , Conformação Proteica
16.
Cell Rep ; 21(11): 3310-3316, 2017 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-29241555

RESUMO

One potential therapeutic strategy for Alzheimer's disease (AD) is to use antibodies that bind to small soluble protein aggregates to reduce their toxic effects. However, these therapies are rarely tested in human CSF before clinical trials because of the lack of sensitive methods that enable the measurement of aggregate-induced toxicity at low concentrations. We have developed highly sensitive single vesicle and single-cell-based assays that detect the Ca2+ influx caused by the CSF of individuals affected with AD and healthy controls, and we have found comparable effects for both types of samples. We also show that an extracellular chaperone clusterin; a nanobody specific to the amyloid-ß peptide (Aß); and bapineuzumab, a humanized monoclonal antibody raised against Aß, could all reduce the Ca2+ influx caused by synthetic Aß oligomers but are less effective in CSF. These assays could be used to characterize potential therapeutic agents in CSF before clinical trials.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Bioensaio , Cálcio/metabolismo , Líquido Cefalorraquidiano/química , Vesículas Citoplasmáticas/efeitos dos fármacos , Fragmentos de Peptídeos/antagonistas & inibidores , Agregados Proteicos/efeitos dos fármacos , Idoso , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Clusterina/farmacologia , Meios de Cultura/farmacologia , Vesículas Citoplasmáticas/metabolismo , Feminino , Humanos , Transporte de Íons/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Cultura Primária de Células , Ratos , Anticorpos de Domínio Único/farmacologia
17.
Sci Rep ; 7(1): 9996, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28855639

RESUMO

The mitotic spindle, essential for segregating the sister chromatids into the two evolving daughter cells, is composed of highly dynamic cytoskeletal filaments, the microtubules. The dynamics of microtubules are regulated by numerous microtubule associated proteins. We identify here Developmentally regulated GTP binding protein 1 (DRG1) as a microtubule binding protein with diverse microtubule-associated functions. In vitro, DRG1 can diffuse on microtubules, promote their polymerization, drive microtubule formation into bundles, and stabilize microtubules. HeLa cells with reduced DRG1 levels show delayed progression from prophase to anaphase because spindle formation is slowed down. To perform its microtubule-associated functions, DRG1, although being a GTPase, does not require GTP hydrolysis. However, all domains are required as truncated versions show none of the mentioned activities besides microtubule binding.


Assuntos
Divisão Celular , Células Epiteliais/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Microtúbulos/metabolismo , Multimerização Proteica , Células HeLa , Humanos , Ligação Proteica
18.
Angew Chem Int Ed Engl ; 56(27): 7750-7754, 2017 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-28474754

RESUMO

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aß42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aß peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.


Assuntos
Cálcio/metabolismo , Bicamadas Lipídicas/metabolismo , Agregados Proteicos/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Cálcio/química , Clusterina/química , Clusterina/metabolismo , Corantes Fluorescentes/química , Humanos , Cinética , Bicamadas Lipídicas/química , Imagem Óptica , Ligação Proteica , Anticorpos de Domínio Único/imunologia
19.
Methods Appl Fluoresc ; 5(1): 014003, 2017 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-28099171

RESUMO

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in cellular media due to strong cross-talk between energetically separated detection channels.


Assuntos
Mercúrio/análise , Compostos de Boro , Linhagem Celular Tumoral , Corantes Fluorescentes , Humanos , Microscopia de Fluorescência
20.
Methods Mol Biol ; 1486: 137-155, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27844428

RESUMO

Due to their high position and force sensitivity and the ability to remotely apply forces and torques, optical tweezers are widely used in diverse fields, such as biology, material science, and physics. Often, small dielectric particles are trapped and used as probes, which for experimental convenience are mostly spherical and composed of silica or polystyrene. The optical properties of these materials together with the microsphere size determine the trapping efficiency, and the position and force resolution. However, using only a single, homogeneous, isotropic, and unstructured material limits the range of trapping properties and thereby the applications of optical tweezers. Here, we show how custom-made microspheres composed of coated high-refractive-index materials-titania and nanodiamonds-and birefringent, liquid crystals extend the range and combination of desired trapping properties. These custom-made microspheres either enable the generation of high forces, a high force or time resolution, or the applications of torques. Custom-made probes expand the range of possible experiments and approaches broadening the scope and applicability of optical tweezers.


Assuntos
Microesferas , Pinças Ópticas , Óptica e Fotônica , Nanopartículas/química , Óptica e Fotônica/métodos , Dióxido de Silício/química , Titânio/química
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