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| 2. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 3. |
- Simonsen, Anja H., et al.
(författare)
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Pre-analytical factors influencing the stability of cerebrospinal fluid proteins
- 2013
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Ingår i: Journal of Neuroscience Methods. - : Elsevier BV. - 1872-678X .- 0165-0270. ; 215:2, s. 234-240
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Tidskriftsartikel (refereegranskat)abstract
- Cerebrospinal fluid (CSF) is a potential source for new biomarkers due to its proximity to the brain. This study aimed to clarify the stability of the CSF proteome when undergoing pre-analytical factors. We investigated the effects of repeated freeze/thaw cycles, protease inhibitors and delayed storage for 4 h, 24 h or 14 days at -20 degrees C, 4 degrees C and room temperature (RT) after centrifugation compared with our standard practice of two hours at RT before placing the samples in an -80 degrees C environment. The results were obtained using immunoassays for amyloid-beta 1-42 (A beta 42), tau, phosphorylated tau (P-tau) and cystatin C and using surface-enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) mass spectrometry for proteomic profiling. Tau and P-tau were susceptible to repeated freeze/thaw cycles while SELDI-TOF analysis produced eight significant peaks and additional artefact peaks from samples with added protease inhibitors. Delayed storage for different durations and in different temperatures produced six significant SELDI-TOF peaks. A beta 42 and tau were susceptible to increased temperatures and the duration before storage, whereas P-tau and cystatin C were not. Transthyretin and several of its isoforms were found using SELDI-TOF and were susceptible to freeze/thaw cycles and to increased temperature and length of time prior to storage. We recommend that CSF should be collected and centrifuged immediately after sampling and prior to storage at -80 degrees C without the addition of protease inhibitors. Freeze/thawing should be avoided because of the instability of tau, P-tau and transthyretin. Standardised CSF sampling, handling and storage for biomarker research are essential for accurately comparing the results obtained by different studies and institutions. (c) 2013 Elsevier B.V. All rights reserved.
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| 4. |
- Simonsen, A H, et al.
(författare)
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Amyloid beta1-40 quantification in CSF: comparison between chromatographic and immunochemical methods.
- 2007
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Ingår i: Dementia and geriatric cognitive disorders. - : S. Karger AG. - 1420-8008 .- 1421-9824. ; 23:4, s. 246-50
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND/AIMS: Amyloid beta (Abeta) is the principal component of senile plaques, one of the hallmarks of Alzheimer's disease (AD). Evidence is accumulating that soluble aggregates (oligomers) of Abeta are important in the pathogenesis of AD. METHODS: We compared three different methods for quantification of the 40 amino acid form of Abeta (Abeta40) in CSF, two based on antibodies [ELISA and surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) with antibody-coated arrays] and one based on direct binding of proteins to a protein array [SELDI-TOF and immobilized metal affinity [copper] (IMAC30)]. RESULTS: CSF Abeta40 concentration was only found to be significantly elevated in AD (127% of control levels; p=0.0095) using SELDI-TOF with IMAC30 arrays. CONCLUSIONS: These data suggest that the measured Abeta level in CSF may differ depending on whether antibody-based methods are used or not, possibly caused by epitope masking due to Abeta oligomerization or to binding of Abeta to carrier proteins.
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| 5. |
- Simonsen, A H, et al.
(författare)
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Identification of a novel panel of cerebrospinal fluid biomarkers for Alzheimer's disease.
- 2008
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Ingår i: Neurobiology of aging. - : Elsevier BV. - 1558-1497 .- 0197-4580. ; 29:7, s. 961-8
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Tidskriftsartikel (refereegranskat)abstract
- An early and accurate diagnosis of Alzheimer's disease (AD) is required to initiate symptomatic treatment with currently approved drugs and will be of even greater importance if disease modifying compounds in development display a clinical effect. Protein profiles of human cerebrospinal fluid samples from AD patients (n=95) and population-based healthy controls (n=72) were analyzed by SELDI-TOF-MS in order to discover and characterize novel candidate biomarker combinations that differentiate AD patients from normal aging in this explorative study. Thirty candidate biomarkers (ROC AUC>0.7) were discovered that could differentiate patients with AD from healthy controls. Protein sequence determination and positive identification of 15 biomarkers revealed potential associations between the identified markers and AD pathogenesis. A multi-marker combination of five peaks could distinguish AD from healthy control individuals with high sensitivity (97%) and specificity (98%). The panel of five markers was tested on a blinded independent data set of 30 AD samples and 28 controls giving 100% sensitivity and 97% specificity. This novel panel of biomarkers could potentially be used to improve the accuracy of diagnosis of AD.
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| 6. |
- Jing, Xiaona, et al.
(författare)
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Interaction of Peptidomimetics with Bilayer Membranes : Biophysical Characterization and Cellular Uptake
- 2012
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 28:11, s. 5167-5175
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Tidskriftsartikel (refereegranskat)abstract
- Enzymatically stable cell-penetrating alpha-peptide/beta-peptoid peptidomimetics constitute promising drug delivery vehicles for the transport of therapeutic biomacromolecules across membrane barriers. The aim of the present study was to elucidate the mechanism of peptidomimetic-lipid bilayer interactions. A series of peptidomimetics consisting of alternating cationic and hydrophobic residues displaying variation in length and N-terminal end group were applied to fluid-phase, anionic lipid bilayers, and their interaction was investigated using isothermal titration calorimetry (ITC) and ellipsometry. Titration of lipid vesicles into solutions of peptidomimetics resulted in exothermic adsorption processes, and the interaction of all studied peptidomimetics with anionic lipid membranes was found to be enthalpy-driven. The enthalpy and Gibbs free energy (Delta G) proved more favorable with increasing chain length. However, not all charges contribute equally to the interaction, as evidenced by the charge-normalized Delta G being inversely correlated to the sequence length. Ellipsometry data suggested that the hydrophobic residues also played an important role in the interaction process. Furthermore, Delta G extracted from ellipsometry data showed good agreement with that obtained with ITC. To further elucidate their interaction with biological membranes, quantitative uptake and cellular distribution were studied in proliferating HeLa cells by flow cytometry and confocal microscopy. The cellular uptake of carboxyfluorescein-labeled peptidomimetics showed a similar ranking as that obtained from the adsorbed amount, and binding energy to model membranes demonstrated that the initial interaction with the membrane is of key importance for the cellular uptake.
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