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- 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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| 2. |
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| 3. |
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| 4. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 5. |
- Cheung, Pierre, et al.
(författare)
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[18F]MK-7246 for Positron Emission Tomography Imaging of the Beta-Cell Surface Marker GPR44
- 2023
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 15:2
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Tidskriftsartikel (refereegranskat)abstract
- The progressive loss of beta-cell mass is a hallmark of diabetes and has been suggested as a complementary approach to studying the progression of diabetes in contrast to the beta-cell function. Non-invasive nuclear medicinal imaging techniques such as Positron Emission Tomography using radiation emitting tracers have thus been suggested as more viable methodologies to visualize and quantify the beta-cell mass with sufficient sensitivity. The transmembrane G protein-coupled receptor GPR44 has been identified as a biomarker for monitoring beta-cell mass. MK-7246 is a GPR44 antagonist that selectively binds to GPR44 with high affinity and good pharmacokinetic properties. Here, we present the synthesis of MK-7246, radiolabeled with the positron emitter fluorine-18 for preclinical evaluation using cell lines, mice, rats and human pancreatic cells. Here, we have described a synthesis and radiolabeling method for producing [18F]MK-7246 and its precursor compound. Preclinical assessments demonstrated the strong affinity and selectivity of [18F]MK-7246 towards GPR44. Additionally, [18F]MK-7246 exhibited excellent metabolic stability, a fast clearance profile from blood and tissues, qualifying it as a promising radioactive probe for GPR44-directed PET imaging.
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| 6. |
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| 7. |
- Cheung, Pierre
(författare)
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PET imaging of beta-cell mass
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A hallmark of diabetes is the progressive loss of the beta-cell mass (BCM) and function. Most of the diagnostic methods for diabetes are mostly reflective of the beta-cell function, whereas current methodologies for studying the beta-cell mass are limited to post-mortem biopsies or highly invasive methods. Positron-emission tomography (PET) is a highly sensitive and quantitative medical imaging technique that was suggested as a tool for non-invasive imaging of the pancreas and quantification of the BCM. GPR44 is a membrane protein recently identified as a promising beta-cell biomarker highly expressed in beta cells, but not in other exocrine or endocrine tissues within the pancreas. Paper I describes the preclinical evaluation of the carbon-11 labeled GPR44 antagonist [11C]MK-7246 using cell lines and pigs. We have demonstrated strong binding of [11C]MK-7246 to the GPR44 receptor as well as its specificity to the pig pancreas. Based on the success of this study, we have initiated the clinical translation of [11C]MK-7246 to first-in-man BCM imaging studies in diabetic patients. As a direct continuation to paper I, paper II focused on the preclinical evaluation of MK-7246 labeled with a different isotope, namely fluorine-18. Additional characterizations were obtained such as the binding affinity and specificity to endocrine pancreatic fractions. Proof of concept in vivo GPR44 directed imaging has been demonstrated as well using an immunodeficient mouse model grafted with GPR44 expressing cells.Paper III is focused on the development and screening of first-in-class affibody molecules targeting DGCR2, another biomarker specific to beta cells. After binding and biodistribution assessments, the affibody molecule ZDGCR2:AM106 has been retained as the lead candidate for DGCR2 targeted imaging. In Paper IV, we have labeled the affibody molecule ZDGCR2:AM106 screened from paper III using fluorine-18, and the resulting [18F]ZDGCR2:AM106 has been assessed for in vivo PET imaging of stem-cells derived pancreatic islets grafts. In conclusion, this thesis describes the preclinical evaluation of several PET imaging radiotracers with the end goal of in vivo imaging of the BCM.
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| 8. |
- Cheung, Pierre, et al.
(författare)
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PET Imaging of GPR44 by Antagonist [C-11]MK-7246 in Pigs
- 2021
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Ingår i: Biomedicines. - : MDPI. - 2227-9059. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- A validated imaging marker for beta-cell mass would improve understanding of diabetes etiology and enable new strategies in therapy development. We previously identified the membrane-spanning protein GPR44 as highly expressed and specific to the beta cells of the pancreas. The selective GPR44 antagonist MK-7246 was radiolabeled with carbon-11 and the resulting positron-emission tomography (PET) tracer [C-11]MK-7246 was evaluated in a pig model and in vitro cell lines. The [C-11]MK-7246 compound demonstrated mainly hepatobiliary excretion with a clearly defined pancreas, no spillover from adjacent tissues, and pancreatic binding similar in magnitude to the previously evaluated GPR44 radioligand [C-11]AZ12204657. The binding could be blocked by preadministration of nonradioactive MK-7246, indicating a receptor-binding mechanism. [C-11]MK-7246 showed strong potential as a PET ligand candidate for visualization of beta-cell mass (BCM) and clinical translation of this methodology is ongoing.
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| 9. |
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| 10. |
- Cheung, Pierre, et al.
(författare)
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Preclinical evaluation of Affibody molecule for PET imaging of human pancreatic islets derived from stem cells
- 2023
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Ingår i: EJNMMI Research. - : Springer Nature. - 2191-219X. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Beta-cell replacement methods such as transplantation of isolated donor islets have been proposed as a curative treatment of type 1 diabetes, but widespread application is challenging due to shortages of donor tissue and the need for continuous immunosuppressive treatments. Stem-cell-derived islets have been suggested as an alternative source of beta cells, but face transplantation protocols optimization difficulties, mainly due to a lack of available methods and markers to directly monitor grafts survival, as well as their localization and function. Molecular imaging techniques and particularly positron emission tomography has been suggested as a tool for monitoring the fate of islets after clinical transplantation. The integral membrane protein DGCR2 has been demonstrated to be a potential pancreatic islet biomarker, with specific expression on insulin-positive human embryonic stem-cell-derived pancreatic progenitor cells. The candidate Affibody molecule ZDGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [18F]ZDGCR2:AM106 was evaluated for binding to recombinant human DGCR2 and cryosections of stem-cell-derived islets, as well as in vivo using an immune-deficient mouse model transplanted with stem-cell-derived islets. Biodistribution of the [18F]ZDGCR2:AM106 was also assessed in healthy rats and pigs. Results: [18F]ZDGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [18F]ZDGCR2:AM106 retained binding to recombinant human DCGR2 as well as to cryosectioned stem-cell-derived islets, but in vivo binding to native pancreatic tissue in both rat and pig was low. However, in vivo uptake of [18F]ZDGCR2:AM106 in stem-cell-derived islets transplanted in the immunodeficient mice was observed, albeit only within the early imaging frames after injection of the radiotracer. Conclusion: Targeting of DGCR2 is a promising approach for in vivo detection of stem-cell-derived islets grafts by molecular imaging. The synthesis of [18F]ZDGCR2:AM106 was successfully performed via a pretargeting method to label a site-specific covalently bonded fluorine-18 to the Affibody molecule. However, the rapid washout of [18F]ZDGCR2:AM106 from the stem-cell-derived islets graft indicates that dissociation kinetics can be improved. Further studies using alternative binders of similar classes with improved binding potential are warranted.
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