scholarly journals pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers

2015 ◽  
Vol 112 (18) ◽  
pp. 5691-5696 ◽  
Author(s):  
Kevin W. Tipping ◽  
Theodoros K. Karamanos ◽  
Toral Jakhria ◽  
Matthew G. Iadanza ◽  
Sophia C. Goodchild ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Correlation Spectroscopy ◽  
Synthetic Membranes ◽  
Fluorescence Correlation ◽  
Amyloid Toxicity ◽  
Tht Fluorescence ◽  
Amyloid Aggregates ◽  
Key Factor ◽  
Cellular Dysfunction ◽  
Acidic Compartments

Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using β2-microglobulin (β2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.

scholarly journals Single-molecule detection on a portable 3D-printed microscope

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
James W. P. Brown ◽  
Arnaud Bauer ◽  
Mark E Polinkovsky ◽  
Akshay Bhumkar ◽  
Dominic J. B. Hunter ◽  
...  
Keyword(s):  
Single Molecule ◽  
Amyloid Fibrils ◽  
Single Photon ◽  
Photon Counting ◽  
Correlation Spectroscopy ◽  
Single Molecule Detection ◽  
Single Photon Counting ◽  
Fluorescence Correlation ◽  
3D Printed ◽  
Simple Format

AbstractSingle-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson’s disease, with an improved sensitivity of >100,000-fold over bulk measurements.

scholarly journals Comparison of Cerebrospinal Fluid Amyloidogenic Nanoplaques With Core Biomarkers of Alzheimer’s Disease

2021 ◽  
Vol 12 ◽  
Author(s):  
Mari Aksnes ◽  
Ann Tiiman ◽  
Trine Holt Edwin ◽  
Lars Terenius ◽  
Nenad Bogdanović ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cerebrospinal Fluid ◽  
Amyloid Β ◽  
Diagnostic Value ◽  
Correlation Spectroscopy ◽  
Phosphorylated Tau ◽  
Total Tau ◽  
Tht Fluorescence ◽  
Amyloid Aggregates

Accurate biomarkers of Alzheimer’s disease (AD) are essential for early diagnosis and intervention. Available biomarkers are not sufficient to permit the monitoring of AD progression over time, and additional biomarkers are required. Measures of aggregated amyloid-β (Aβ) could be useful biomarkers for AD. Here, we investigate whether levels of Thioflavin-T (ThT) positive amyloid aggregates, i.e., nanoplaques, in cerebrospinal fluid (CSF) could serve as useful biomarkers for AD. One-hundred and eighteen memory clinic patients were AT(N) classified, and CSF nanoplaque concentrations were compared between patients on the “Alzheimer’s continuum” (A+ patients) and patients with “Normal AD biomarkers” or “Non-AD pathologic change” (A− patients). CSF nanoplaque concentrations and sizes were quantified using the novel ThT-Fluorescence Correlation Spectroscopy (ThT-FCS) assay, and core biomarkers (Aβ42, total tau and phosphorylated tau) were determined by enzyme-linked immunosorbent assays. We investigated the association between nanoplaque concentrations and core biomarkers, and the diagnostic value of nanoplaque levels. Nanoplaque levels were increased in A+ patients compared to A− patients. Nanoplaque concentrations were negatively associated with Aβ42, but not related to total tau or phosphorylated tau measures. Quantification of nanoplaques did not improve the classification of patients on the Alzheimer’s continuum compared to the core biomarkers alone. Dynamic changes in nanoplaques concentration and size throughout AD stages should be explored in longitudinal studies.

scholarly journals Variably Sized and Multi-Colored Silica-Nanoparticles Characterized by Fluorescence Correlation Methods for Cellular Dynamics

Materials ◽  
2020 ◽  
Vol 14 (1) ◽  
pp. 19
Author(s):  
Chan-Gi Pack ◽  
Bjorn Paulson ◽  
Yeonhee Shin ◽  
Min Kyo Jung ◽  
Jun Sung Kim ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Correlation Spectroscopy ◽  
Cell Uptake ◽  
Core Shell ◽  
Therapeutic Effects ◽  
Ferrite Core ◽  
Shell Nanoparticles ◽  
Fluorescence Correlation ◽  
Correlation Methods

Controlling the uptake of nanoparticles into cells so as to balance therapeutic effects with toxicity is an essential unsolved problem in the development of nanomedicine technologies. From this point of view, it is useful to use standard nanoparticles to quantitatively evaluate the physical properties of the nanoparticles in solution and in cells, and to analyze the intracellular dynamic motion and distribution of these nanoparticles at a single-particle level. In this study, standard nanoparticles are developed based on a variant silica-based nanoparticle incorporating fluorescein isothiocyanate (FITC) or/and rhodamine B isothiocyanate (RITC) with a variety of accessible diameters and a matching fluorescent cobalt ferrite core-shell structure (Fe2O4/SiO2). The physical and optical properties of the nanoparticles in vitro are fully evaluated with the complementary methods of dynamic light scattering, electron microscopy, and two fluorescence correlation methods. In addition, cell uptake of dual-colored and core/shell nanoparticles via endocytosis in live HeLa cells is detected by fluorescence correlation spectroscopy and electron microscopy, indicating the suitability of the nanoparticles as standards for further studies of intracellular dynamics with multi-modal methods.

scholarly journals Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells

2021 ◽  
Vol 22 (14) ◽  
pp. 7300
Author(s):  
Laura M. Nederveen-Schippers ◽  
Pragya Pathak ◽  
Ineke Keizer-Gunnink ◽  
Adrie H. Westphal ◽  
Peter J. M. van Haastert ◽  
...  
Keyword(s):  
Global Analysis ◽  
Photon Counting ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Protein Dimerization ◽  
Fluorescence Correlation ◽  
Fk506 Binding Protein ◽  
Average Brightness ◽  
Cellular Environment ◽  
Protein Dimers

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.

scholarly journals Nano-antenna enhanced two-focus fluorescence correlation spectroscopy

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Lutz Langguth ◽  
Agata Szuba ◽  
Sander A. Mann ◽  
Erik C. Garnett ◽  
Gijsje H. Koenderink ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

scholarly journals Amyloids: The History of Toxicity and Functionality

Biology ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 394
Author(s):  
Elmira I. Yakupova ◽  
Liya G. Bobyleva ◽  
Sergey A. Shumeyko ◽  
Ivan M. Vikhlyantsev ◽  
Alexander G. Bobylev
Keyword(s):  
Molecular Structure ◽  
Polypeptide Chain ◽  
Amyloid Plaques ◽  
Protein Aggregates ◽  
Specific Function ◽  
Main Hypothesis ◽  
Amyloid Toxicity ◽  
Amyloid Aggregates ◽  
Aβ Amyloid ◽  
History Of

Proteins can perform their specific function due to their molecular structure. Partial or complete unfolding of the polypeptide chain may lead to the misfolding and aggregation of proteins in turn, resulting in the formation of different structures such as amyloid aggregates. Amyloids are rigid protein aggregates with the cross-β structure, resistant to most solvents and proteases. Because of their resistance to proteolysis, amyloid aggregates formed in the organism accumulate in tissues, promoting the development of various diseases called amyloidosis, for instance Alzheimer’s diseases (AD). According to the main hypothesis, it is considered that the cause of AD is the formation and accumulation of amyloid plaques of Aβ. That is why Aβ-amyloid is the most studied representative of amyloids. Therefore, in this review, special attention is paid to the history of Aβ-amyloid toxicity. We note the main problems with anti-amyloid therapy and write about new views on amyloids that can play positive roles in the different organisms including humans.

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