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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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- Lawrenson, Kate, et al.
(författare)
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Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10-20), ER-negative BC (P=1.1 × 10-13), BRCA1-associated BC (P=7.7 × 10-16) and triple negative BC (P-diff=2 × 10-5). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10-3) and ABHD8 (P<2 × 10-3). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3′-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk.
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- Bulten, W, et al.
(författare)
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Artificial intelligence for diagnosis and Gleason grading of prostate cancer: the PANDA challenge
- 2022
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Ingår i: Nature medicine. - : Springer Science and Business Media LLC. - 1546-170X .- 1078-8956. ; 28:21, s. 154-
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Tidskriftsartikel (refereegranskat)abstract
- Artificial intelligence (AI) has shown promise for diagnosing prostate cancer in biopsies. However, results have been limited to individual studies, lacking validation in multinational settings. Competitions have been shown to be accelerators for medical imaging innovations, but their impact is hindered by lack of reproducibility and independent validation. With this in mind, we organized the PANDA challenge—the largest histopathology competition to date, joined by 1,290 developers—to catalyze development of reproducible AI algorithms for Gleason grading using 10,616 digitized prostate biopsies. We validated that a diverse set of submitted algorithms reached pathologist-level performance on independent cross-continental cohorts, fully blinded to the algorithm developers. On United States and European external validation sets, the algorithms achieved agreements of 0.862 (quadratically weighted κ, 95% confidence interval (CI), 0.840–0.884) and 0.868 (95% CI, 0.835–0.900) with expert uropathologists. Successful generalization across different patient populations, laboratories and reference standards, achieved by a variety of algorithmic approaches, warrants evaluating AI-based Gleason grading in prospective clinical trials.
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- Ge, C. R., et al.
(författare)
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Antibodies to Cartilage Oligomeric Matrix Protein Are Pathogenic in Mice and May Be Clinically Relevant in Rheumatoid Arthritis
- 2022
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Ingår i: Arthritis & Rheumatology. - : Wiley. - 2326-5191 .- 2326-5205.
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Tidskriftsartikel (refereegranskat)abstract
- Objective Cartilage oligomeric matrix protein (COMP) is an autoantigen in rheumatoid arthritis (RA) and experimental models of arthritis. This study was undertaken to investigate the structure, function, and relevance of anti-COMP antibodies. Methods We investigated the pathogenicity of monoclonal anti-COMP antibodies in mice using passive transfer experiments, and we explored the interaction of anti-COMP antibodies with cartilage using immunohistochemical staining. The interaction of the monoclonal antibody 15A11 in complex with its specific COMP epitope P6 was determined by x-ray crystallography. An enzyme-linked immunosorbent assay and a surface plasma resonance technique were used to study the modulation of calcium ion binding to 15A11. The clinical relevance and value of serum IgG specific to the COMP P6 epitope and its citrullinated variants were evaluated in a large Swedish cohort of RA patients. Results The murine monoclonal anti-COMP antibody 15A11 induced arthritis in naive mice. The crystal structure of the 15A11-P6 complex explained how the antibody could bind to COMP, which can be modulated by calcium ions. Moreover, serum IgG specific to the COMP P6 peptide and its citrullinated variants was detectable at significantly higher levels in RA patients compared to healthy controls and correlated with a higher disease activity score. Conclusion Our findings provide the structural basis for binding a pathogenic anti-COMP antibody to cartilage. The recognized epitope can be citrullinated, and levels of antibodies to this epitope are elevated in RA patients and correlate with higher disease activity, implicating a pathogenic role of anti-COMP antibodies in a subset of RA patients.
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- Steigner, Michael L, et al.
(författare)
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Iodinated Contrast Opacification Gradients in Normal Coronary Arteries Imaged With Prospectively ECG-Gated Single Heart Beat 320-Detector Row Computed Tomography
- 2010
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Ingår i: CIRCULATION-CARDIOVASCULAR IMAGING. - : Lippincott Williams & Wilkins. - 1941-9651 .- 1942-0080. ; 3:2, s. 179-186
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Tidskriftsartikel (refereegranskat)abstract
- Background-To define and evaluate coronary contrast opacification gradients using prospectively ECG-gated single heart beat 320-detector row coronary angiography (CTA). Methods and Results-Thirty-six patients with normal coronary arteries determined by 320 x 0.5-mm detector row coronary CTA were retrospectively evaluated with customized image postprocessing software to measure Hounsfield Units at 1-mm intervals orthogonal to the artery center line. Linear regression determined correlation between mean Hounsfield Units and distance from the coronary ostium (regression slope defined as the distance gradient G(d)), lumen cross-sectional area (G(a)), and lumen short-axis diameter (G(s)). For each gradient, differences between the 3 coronary arteries were analyzed with ANOVA. Linear regression determined correlations between measured gradients, heart rate, body mass index, and cardiac phase. To determine feasibility in lesions, all 3 gradients were evaluated in 22 consecutive patients with left anterior descending artery lesions andgt;= 50% stenosis. For all 3 coronary arteries in all patients, the gradients G(a) and G(s) were significantly different from zero (P andlt; 0.0001), highly linear (Pearson r values, 0.77 to 0.84), and had no significant difference between the left anterior descending, left circumflex, and right coronary arteries (P andgt; 0.503). The distance gradient G(d) demonstrated nonlinearities in a small number of vessels and was significantly smaller in the right coronary artery when compared with the left coronary system (P andlt; 0.001). Gradient variations between cardiac phases, heart rates, body mass index, and readers were low. Gradients in patients with lesions were significantly different (P andlt; 0.021) than in patients considered normal by CTA. Conclusions-Measurement of contrast opacification gradients from temporally uniform coronary CTA demonstrates feasibility and reproducibility in patients with normal coronary arteries. For all patients, the gradients defined with respect to the coronary lumen cross-sectional area and short-axis diameters are highly linear, not significantly influenced by the coronary artery (left anterior descending artery versus left circumflex versus right coronary artery), and have only small variation with respect to patient parameters. Preliminary evaluation of gradients across coronary artery lesions is promising but requires additional study.
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