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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. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 4. |
- Kristanl, Matej, et al.
(författare)
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The Seventh Visual Object Tracking VOT2019 Challenge Results
- 2019
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Ingår i: 2019 IEEE/CVF INTERNATIONAL CONFERENCE ON COMPUTER VISION WORKSHOPS (ICCVW). - : IEEE COMPUTER SOC. - 9781728150239 ; , s. 2206-2241
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Konferensbidrag (refereegranskat)abstract
- The Visual Object Tracking challenge VOT2019 is the seventh annual tracker benchmarking activity organized by the VOT initiative. Results of 81 trackers are presented; many are state-of-the-art trackers published at major computer vision conferences or in journals in the recent years. The evaluation included the standard VOT and other popular methodologies for short-term tracking analysis as well as the standard VOT methodology for long-term tracking analysis. The VOT2019 challenge was composed of five challenges focusing on different tracking domains: (i) VOT-ST2019 challenge focused on short-term tracking in RGB, (ii) VOT-RT2019 challenge focused on "real-time" short-term tracking in RGB, (iii) VOT-LT2019 focused on long-term tracking namely coping with target disappearance and reappearance. Two new challenges have been introduced: (iv) VOT-RGBT2019 challenge focused on short-term tracking in RGB and thermal imagery and (v) VOT-RGBD2019 challenge focused on long-term tracking in RGB and depth imagery. The VOT-ST2019, VOT-RT2019 and VOT-LT2019 datasets were refreshed while new datasets were introduced for VOT-RGBT2019 and VOT-RGBD2019. The VOT toolkit has been updated to support both standard short-term, long-term tracking and tracking with multi-channel imagery. Performance of the tested trackers typically by far exceeds standard baselines. The source code for most of the trackers is publicly available from the VOT page. The dataset, the evaluation kit and the results are publicly available at the challenge website(1).
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| 5. |
- Daré, Elisabetta, et al.
(författare)
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Methylmercury and H2O2 provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis
- 2001
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Ingår i: Free Radical Biology & Medicine. - Linköping : Elsevier. - 0891-5849 .- 1873-4596. ; 30:12, s. 1347-1356
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Tidskriftsartikel (refereegranskat)abstract
- Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H2O2) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H2O2 preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H2O2 stimulated divergent pathways, with caspases being activated only by H2O2. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H2O2, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H2O2. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.
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| 6. |
- Li, Wei, et al.
(författare)
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Autophagy dysfunction and regulatory cystatin C in macrophage death of atherosclerosis
- 2016
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Ingår i: Journal of Cellular and Molecular Medicine. - : Wiley. - 1582-1838 .- 1582-4934. ; 20:9, s. 1664-1672
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Tidskriftsartikel (refereegranskat)abstract
- Autophagy dysfunction in mouse atherosclerosis models has been associated with increased lipid accumulation, apoptosis and inflammation. Expression of cystatin C (CysC) is decreased in human atheroma, and CysC deficiency enhances atherosclerosis in mice. Here, we first investigated the association of autophagy and CysC expression levels with atheroma plaque severity in human atherosclerotic lesions. We found that autophagy proteins Atg5 and LC3β in advanced human carotid atherosclerotic lesions are decreased, while markers of dysfunctional autophagy p62/SQSTM1 and ubiquitin are increased together with elevated levels of lipid accumulation and apoptosis. The expressions of LC3β and Atg5 were positively associated with CysC expression. Second, we investigated whether CysC expression is involved in autophagy in atherosclerotic apoE-deficient mice, demonstrating that CysC deficiency (CysC-/-) in these mice results in reduction of Atg5 and LC3β levels and induction of apoptosis. Third, macrophages isolated from CysC-/- mice displayed increased levels of p62/SQSTM1 and higher sensitivity to 7-oxysterol-mediated lysosomal membrane destabilization and apoptosis. Finally, CysC treatment minimized oxysterol-mediated cellular lipid accumulation. We conclude that autophagy dysfunction is a characteristic of advanced human atherosclerotic lesions and is associated with reduced levels of CysC. The deficiency of CysC causes autophagy dysfunction and apoptosis in macrophages and apoE-deficient mice. The results indicate that CysC plays an important regulatory role in combating cell death via the autophagic pathway in atherosclerosis. Copyright
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| 7. |
- Mahajan, Anubha, et al.
(författare)
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Multi-ancestry genetic study of type 2 diabetes highlights the power of diverse populations for discovery and translation
- 2022
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Ingår i: Nature Genetics. - : Springer Nature. - 1061-4036 .- 1546-1718. ; 54:5, s. 560-572
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Tidskriftsartikel (refereegranskat)abstract
- We assembled an ancestrally diverse collection of genome-wide association studies (GWAS) of type 2 diabetes (T2D) in 180,834 affected individuals and 1,159,055 controls (48.9% non-European descent) through the Diabetes Meta-Analysis of Trans-Ethnic association studies (DIAMANTE) Consortium. Multi-ancestry GWAS meta-analysis identified 237 loci attaining stringent genome-wide significance (P < 5 x 10(-9)), which were delineated to 338 distinct association signals. Fine-mapping of these signals was enhanced by the increased sample size and expanded population diversity of the multi-ancestry meta-analysis, which localized 54.4% of T2D associations to a single variant with >50% posterior probability. This improved fine-mapping enabled systematic assessment of candidate causal genes and molecular mechanisms through which T2D associations are mediated, laying the foundations for functional investigations. Multi-ancestry genetic risk scores enhanced transferability of T2D prediction across diverse populations. Our study provides a step toward more effective clinical translation of T2D GWAS to improve global health for all, irrespective of genetic background. Genome-wide association and fine-mapping analyses in ancestrally diverse populations implicate candidate causal genes and mechanisms underlying type 2 diabetes. Trans-ancestry genetic risk scores enhance transferability across populations.
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| 8. |
- Han, Yang, et al.
(författare)
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X-radiation inhibits histone deacetylase 1 and 2, upregulates Axin expression and induces apoptosis in non-small cell lung cancer
- 2012
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Ingår i: Radiation Oncology. - : BioMed Central. - 1748-717X .- 1748-717X. ; 7:183
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundHistone deacetylase (HDAC) plays an important role in the deacetylation of histone, which can alter gene expression patterns and affect cell behavior associated with malignant transformation. The aims of this study were to investigate the relationships between HDAC1, HDAC2, clinicopathologic characteristics, patient prognosis and apoptosis, to clarify the mechanism of upregulation of the Axis inhibitor Axin (an important regulator of the Wnt pathway) by X-radiation and to elucidate the effect of siRNA on radiation therapy of non-small cell lung cancer (NSCLC).MethodsHDAC1 and HDAC2 expression levels were measured by immunohistochemistry and reverse transcription PCR. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling and fluorescence activated cell sorting. BE1 cells expressing Axin were exposed to 2 Gy of X-radiation.ResultsExpression of HDAC1 and that of HDAC2 were correlated, and significantly higher in NSCLC tissues than in normal lung tissues (P < 0.05). HDAC1 and HDAC2 expression was correlated with pTNM stage and negatively correlated with differentiation of NSCLC and apoptotic index (P < 0.05). The prognosis of patients with low expression of HDAC1 and HDAC2 was better than that of those with high expression. X-radiation and siRNA inhibited HDAC1 and HDAC2 expression in NSCLC cells and Axin levels were significantly higher in BE1 cells.ConclusionsX-radiation and siRNA inhibit expression of HDAC1 and HDAC2, weaken the inhibitory effect of HDAC on Axin, upregulate Axin expression and induce apoptosis of lung cancer cells. Inhibition of HDAC1 and HDAC2 is a means of enhancing the radiosensitivity of NSCLC.
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| 9. |
- Jacobsson, Leif, 1945-, et al.
(författare)
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Effects of α-tocopherol and astaxanthin on LDL oxidation and atherosclerosis in WHHL rabbits
- 2004
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Ingår i: Atherosclerosis. - : Elsevier BV. - 0021-9150 .- 1879-1484. ; 173:2, s. 231-237
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to evaluate the influence of α-tocopherol and astaxanthin on low-density lipoprotein (LDL) oxidation lag time and atherosclerotic lesion formation in Watanabe heritable hyperlipidemic (WHHL) rabbits. Thirty-one, 3-month-old WHHL rabbits were divided into three experimental groups. One group (n=10) was fed standard rabbit feed alone and served as a control, a second group (n=11) was supplied with the same feed containing 500mg α-tocopherol/kg and a third group (n=10) was given a feed containing 100mg astaxanthin/kg. Plasma lipids, lipoproteins and LDL oxidation lag time were followed for 24 weeks. At the end of the treatment period, the animals were killed and the thoracic aorta was used for evaluation of the degree of atherosclerosis. Colour photographs of the intimal surface of the vessel were taken for determination of the atherosclerotic area. Cross-sections of the thoracic aorta were used for histological examination and for determination of intimal thickening. Specimens of the vessel were used for determination of the tissue cholesterol content. Plasma cholesterol remained at a high level during the time of the experiment and there were no differences between the experimental groups. After 24 weeks, the LDL oxidation lag time was 53.7±1.7min, 109±4min (P<0.001) and 56.4±3.4min (P=0.47) in the control, α-tocopherol and astaxanthin groups, respectively. In the thoracic aorta, the atherosclerotic area was 80.7±5.1%, 67.1±6.7% (P=0.13) and 75.2±5.7% (P=0.49) in the control, α-tocopherol and astaxanthin groups, respectively. The intimal thickening was 45.6±3.2%, 44.0±4.1% (P=0.89) and 40.0±4.5% (P=0.33) in the control, α-tocopherol and astaxanthin groups, respectively. Finally, the cholesterol content was 107±9μmol/g, 95.7±11. 5μmol/g (P=0.31) and 101±5μmol/g (P=0.33) in the control, α-tocopherol and astaxanthin groups, respectively. It can be concluded that α-tocopherol but not astaxanthin prolonged the LDL oxidation lag time. The two antioxidative substances did not prevent atherogenesis in WHHL rabbits in this setting.
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| 10. |
- Larsson, David A, et al.
(författare)
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Oxysterol mixtures, in atheroma-relevant proportions, display synergistic and proapoptotic effects
- 2006
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Ingår i: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849 .- 1873-4596. ; 41:6, s. 902-910
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Tidskriftsartikel (refereegranskat)abstract
- Apoptotic cells in atheroma lesions may contribute to plaque development and instability. Oxysterols constitute the major toxic component in oxLDL and are present in mixed forms in human atheroma lesions. However, the cellular effects of oxysterols have been mostly studied individually. In the present study, we investigated the cytotoxic effects of 7β-hydroxycholesterol (7βOH), 7-ketocholesterol (7keto), 25-hydroxycholesterol (25OH), and 27-hydroxycholesterol (27OH) on U937 monocytic cells, both individually and in atheroma-relevant mixtures mimicking the oxysterol composition reported in human atheroma lesions. Apoptosis and necrosis were studied by examining cell morphology, phosphatidylserine exposure, caspase activation, and the terminal dUTP nick end-labeling technique. Cellular reactive oxygen species and total amount of reduced thiols were measured by using fluorescence probes and 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. We found that 7βOH and 7keto induced caspase activation, ROS production, cellular thiol depletion, permeabilization of lysosomal and mitochondrial membranes, and cell death. 25OH and 27OH did not cause any of the above alterations, whereas 7βOH and 7keto exerted synergistic toxic effects. Although single 25OH or 27OH exhibited quenching effects on both 7βOH- and 7keto-induced cell death, the combination of all four oxysterols in atheroma-relevant proportions was proapoptotic. Our findings indicate that the major oxysterols accumulated in human atheroma are proapoptotic and may contribute to atherosclerotic lesion development. © 2006 Elsevier Inc. All rights reserved.
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