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Ultrasensitive protein quantification using Rolling Circle Amplification
Title
Ultrasensitive protein quantification using Rolling Circle Amplification
Creator
Hetzel, Laura Ann
Date
2019
Description
There are several protein biomarkers that can aid in diagnosing and evaluating the progression of Alzheimer’s Disease (AD), including Amyloid...
Show moreThere are several protein biomarkers that can aid in diagnosing and evaluating the progression of Alzheimer’s Disease (AD), including Amyloid Beta-42 (Aβ42), Amyloid Beta-40 (Aβ40), and Tau proteins. The proteins are most prevalent in the brain and cerebral spinal fluid, becoming more diluted in the bloodstream. Since diagnosis and progression would require evaluating and comparing protein levels over time and identifying miniscule changes, an assay with high sensitivity is paramount. Similarly, evaluating how a drug treatment affects the levels of protein requires a highly sensitive assay. Currently, enzyme-linked immunosorbent assay (ELISA) is accepted as the most sensitive assay for protein detection and quantification. However, in the case of Aβ40 and Aβ42 proteins, oftentimes the levels of the proteins in patients are very close to the sensitivity of the commercial ELISA. The uncertainty in these measurements is very high, which results in reporting of conflicting outcomes. One of the challenges of quantifying proteins is that proteins, unlike nucleic acids, cannot be amplified. To overcome this limitation, we have cleverly pseudo-amplified proteins using rolling circle amplification (RCA). By doing so, we have demonstrated a ten to forty times improvement in sensitivity over ELISA and radioimmunoassays. In previous experiments, C-peptide has been used as the protein of interest, and ELISA reports the smallest detectable quantity is 0.01 ng in buffer. Using RCA, we have found that as little as 0.00075 ng C-peptide in buffer could be quantified, and 0.004 ng in 10% serum could be quantified. The same process can be applied to other proteins such as Aβ40 and Aβ42, and the results are expected to be similar. In fact, we have measured Type I Diabetes autoantibodies with approximately forty times improvement over the gold standard radioimmunoassay. With excellent results in buffer and 10% serum, expansion to human samples holds great potential. If the human experiments are as successful as anticipated, RCA could be used to precisely evaluate the effect of a drug on protein levels, contributing to the overall evaluation of the success of the drug.
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Cardiolipin Modulates the Insertion of Adsorbed Helical Amyloid Beta Peptide Into Model Mitochondrial Membranes
Title
Cardiolipin Modulates the Insertion of Adsorbed Helical Amyloid Beta Peptide Into Model Mitochondrial Membranes
Creator
Kaczmarek, Julia A.
Date
2023
Description
The loss of mitochondrial phospholipid cardiolipin (CL) may play a role in both the pathogenesis of Alzheimer's Disease (AD) and its treatment...
Show moreThe loss of mitochondrial phospholipid cardiolipin (CL) may play a role in both the pathogenesis of Alzheimer's Disease (AD) and its treatment. An effector molecule of the disease, amyloid-beta (Aβ), has been observed to interact with lipid membranes, but its relevance to mitochondrial membranes containing CL remained elusive. The present study investigated if the presence of CL modulated the insertion of adsorbed helical amyloid beta (Aβ14-40) into model mitochondrial membranes, and if this effect was more pronounced for its N-terminus or C-terminus. I conducted a coarse-grained computer simulation using well-tempered metadynamics to traverse the free energy landscape that maps the translocation of Aβ14-40. Insertion into CL-containing bilayers created larger local membrane deformations and modulated the location of the transition path but had an inconclusive impact on the free energy cost of translocation. Since the generation of toxic calcium-permeable pores depends on the insertion of Aβ into the bilayer, the loss of CL seen in AD may prime the inner mitochondrial membrane for pore formation, but more research is needed to pursue this hypothesis.
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Cardiolipin Modulates the Insertion of Adsorbed Helical Amyloid Beta Peptide Into Model Mitochondrial Membranes
Title
Cardiolipin Modulates the Insertion of Adsorbed Helical Amyloid Beta Peptide Into Model Mitochondrial Membranes
Creator
Kaczmarek, Julia A.
Date
2023
Description
The loss of mitochondrial phospholipid cardiolipin (CL) may play a role in both the pathogenesis of Alzheimer's Disease (AD) and its treatment...
Show moreThe loss of mitochondrial phospholipid cardiolipin (CL) may play a role in both the pathogenesis of Alzheimer's Disease (AD) and its treatment. An effector molecule of the disease, amyloid-beta (Aβ), has been observed to interact with lipid membranes, but its relevance to mitochondrial membranes containing CL remained elusive. The present study investigated if the presence of CL modulated the insertion of adsorbed helical amyloid beta (Aβ14-40) into model mitochondrial membranes, and if this effect was more pronounced for its N-terminus or C-terminus. I conducted a coarse-grained computer simulation using well-tempered metadynamics to traverse the free energy landscape that maps the translocation of Aβ14-40. Insertion into CL-containing bilayers created larger local membrane deformations and modulated the location of the transition path but had an inconclusive impact on the free energy cost of translocation. Since the generation of toxic calcium-permeable pores depends on the insertion of Aβ into the bilayer, the loss of CL seen in AD may prime the inner mitochondrial membrane for pore formation, but more research is needed to pursue this hypothesis.
Show less