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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. |
- Ai, Yue-Jie, et al.
(författare)
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Role of non-Condon vibronic coupling and conformation change on two-photon absorption spectra of green fluorescent protein
- 2013
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Ingår i: Molecular Physics. - : Informa UK Limited. - 0026-8976 .- 1362-3028. ; 111:9-11, s. 1316-1321
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Tidskriftsartikel (refereegranskat)abstract
- Two-photon absorption spectra of green fluorescent proteins (GFPs) often show a blue-shift band compared to their conventional one-photon absorption spectra, which is an intriguing feature that has not been well understood. We present here a systematic study on one- and two-photon spectra of GFP chromophore by means of the density functional response theory and complete active space self-consistent field (CASSCF) methods. It shows that the popular density functional fails to provide correct vibrational progression for the spectra. The non-Condon vibronic coupling, through the localised intrinsic vibrational modes of the chromophore, is responsible for the blue-shift in the TPA spectra. The cis to trans isomerisation can be identified in high-resolution TPA spectra. Our calculations demonstrate that the high level ab initio multiconfigurational CASSCF method, rather than the conventional density functional theory is required for investigating the essential excited-state properties of the GFP chromophore.
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| 4. |
- Du, Yaoyao, et al.
(författare)
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Cartilage Oligomeric Matrix Protein Inhibits Vascular Smooth Muscle Calcification by Interacting With Bone Morphogenetic Protein-2.
- 2011
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Ingår i: Circulation Research. - 1524-4571. ; 108, s. 79-917
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Tidskriftsartikel (refereegranskat)abstract
- Rationale: Vascular calcification is a significant contributor to cardiovascular morbidity and mortality. We recently reported that cartilage oligomeric matrix protein (COMP) is pivotal for maintaining the homeostasis of vascular smooth muscle cells (VSMCs). Whether COMP affects the process of vascular calcification is unknown. Objective: We aimed to test whether COMP modulates vascular calcification. Methods and Results: VSMC calcification in vitro was induced by calcifying media containing high inorganic phosphate or calcium. In vivo medial vessel calcification was induced in rats by 5/6 nephrectomy with a high-phosphate diet or by periadventitial application of CaCl(2) to the abdominal aorta. COMP protein level was markedly reduced in both calcified VSMCs and arteries. COMP deficiency remarkably exacerbated VSMC calcification, whereas ectopic expression of COMP greatly reduced calcification. Furthermore, COMP knockdown facilitated osteogenic markers expression by VSMCs even in the absence of calcifying media. By contrast, COMP overexpression significantly inhibited high phosphate- or high calcium-induced VSMC osteochondrogenic transition. Induction of osteogenic marker expression by COMP silencing was reversed by a soluble form of bone morphogenetic protein (BMP)-2 receptor IA, which suggests a BMP-2-dependent mechanism. Our data revealed that COMP bound directly to BMP-2 through the C terminus, inhibited BMP-2 receptor binding, and blocked BMP-2 osteogenic signaling, indicating COMP inhibits osteochondrogenic transition of VSMCs at least partially through inhibiting BMP-2. Conclusions: Our data strongly suggest that COMP is a novel inhibitor of vascular calcification. The imbalance between the effects of COMP and BMP-2 may provide new insights into the pathophysiology of vascular calcification.
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| 5. |
- Zhao, Zeng-Ren, et al.
(författare)
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Nup88 mRNA overexpression in colorectal cancers and relationship with p53
- 2010
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Ingår i: CANCER BIOMARKERS. - : IOS Press. - 1574-0153. ; 8:2, s. 73-80
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: We measured nucleoporin 88 (Nup88) mRNA expression in primary colorectal cancers to investigate its relationship with clinicopathological features and p53. Methods: The primary cancer tissues, adjacent noncancerous tissues and the proximal and distant margins of normal mucosa were collected from 73 colorectal cancer patients during surgery. Nup88 mRNA expression was measured on these fresh specimens and on colon cell lines HCT-116 (P53+/+) and HCT-116 (P53-/-) by RT-PCR while p53 mRNA and beta-actin as controls. Nup88 and p53 protein expression were then immunohistochemistrically examined in other 25 colorectal cancers specimens paraffin embedded and formalin fixed. Results: Nup88 expression was higher in primary cancer tissues than in adjacent noncancerous tissues, and in the proximal and distant margins of normal mucosa. Overexpression of Nup88 mRNA was statistically associated with TNM stage (P = 0.044), lymphatic metastasis (P = 0.022), and cancer location (P = 0.036), while not related to gender, age of patients and histological type, infiltration depth, and differentiation of cancers. The expression of Nup88 mRNA in the HCT-116 (P53-/-) cell line was not significantly different from expression in the HCT-116 (P53+/+) cell line. And there was no correlation between Nup88 and p53 protein expression (r = 0.632, P = 0.368). Conclusions: Nup88 mRNA was overexpressed in colorectal cancers and the overexpression was associated with cancer development and aggressiveness. Nup88 might be regard as essential contributor to nodal metastagenicity of colorectal cancer.
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