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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)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. |
- Bahr, Carsten, et al.
(author)
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A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies
- 2018
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In: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 553:7689, s. 515-520
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Journal article (peer-reviewed)abstract
- The transcription factor Myc is essential for the regulation of haematopoietic stem cells and progenitors and has a critical function in haematopoietic malignancies1. Here we show that an evolutionarily conserved region located 1.7 megabases downstream of the Myc gene that has previously been labelled as a ‘super-enhancer’2 is essential for the regulation of Myc expression levels in both normal haematopoietic and leukaemic stem cell hierarchies in mice and humans. Deletion of this region in mice leads to a complete loss of Myc expression in haematopoietic stem cells and progenitors. This caused an accumulation of differentiation-arrested multipotent progenitors and loss of myeloid and B cells, mimicking the phenotype caused by Mx1-Cre-mediated conditional deletion of the Myc gene in haematopoietic stem cells3. This super-enhancer comprises multiple enhancer modules with selective activity that recruits a compendium of transcription factors, including GFI1b, RUNX1 and MYB. Analysis of mice carrying deletions of individual enhancer modules suggests that specific Myc expression levels throughout most of the haematopoietic hierarchy are controlled by the combinatorial and additive activity of individual enhancer modules, which collectively function as a ‘blood enhancer cluster’ (BENC). We show that BENC is also essential for the maintenance of MLL–AF9-driven leukaemia in mice. Furthermore, a BENC module, which controls Myc expression in mouse haematopoietic stem cells and progenitors, shows increased chromatin accessibility in human acute myeloid leukaemia stem cells compared to blasts. This difference correlates with MYC expression and patient outcome. We propose that clusters of enhancers, such as BENC, form highly combinatorial systems that allow precise control of gene expression across normal cellular hierarchies and which also can be hijacked in malignancies.
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| 6. |
- Millar, JE, et al.
(author)
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Administration of mesenchymal stem cells during ECMO results in a rapid decline in oxygenator performance
- 2019
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In: Thorax. - : BMJ. - 1468-3296 .- 0040-6376. ; 74:2, s. 194-196
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Journal article (peer-reviewed)abstract
- Mesenchymal stem cells (MSCs) have attracted attention as a potential therapy for Acute Respiratory Distress Syndrome (ARDS). At the same time, the use of extracorporeal membrane oxygenation (ECMO) has increased among patients with severe ARDS. To date, early clinical trials of MSCs in ARDS have excluded patients supported by ECMO. Here we provide evidence from an ex-vivo model of ECMO to suggest that the intravascular administration of MSCs during ECMO may adversely impact the function of a membrane oxygenator. The addition of clinical grade MSCs resulted in a reduction of flow through the circuit in comparison to controls (0.6 ±0.35 L min-1vs 4.12 ± 0.03 L min-1, at 240 minutes) and an increase in the transoygenator pressure gradient (101±9 mmHg vs 21±4 mmHg, at 240 minutes). Subsequent immunohistochemistry analysis demonstrated quantities of MSCs highly adherent to membrane oxygenator fibres. This study highlights the potential harm associated with MSC therapy during ECMO and suggests further areas of research required to advance the translation of cell therapy in this population.
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| 9. |
- von Bahr, M, et al.
(author)
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Spreading dynamics of liquids and surfactant solutions on partially wettable hydrophobic substrates
- 2001
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In: Colloids and Surfaces A. - 0927-7757 .- 1873-4359. ; 193, s. 85-96
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Journal article (peer-reviewed)abstract
- The drop spreading of water and aqueous solutions of ethanol and nonionic surfactant on hydrophobic substrates (alkylsilane treated glass) have been investigated. For the low viscous liquids and solutions, the spreading on the surface of hydrophobic glass rod was also studied and compared to the drop spreading experiment. In both experiments, care was taken to ensure a minimum impact of inertial forces. The results for the aqueous systems show rapid initial spreading processes that abruptly halts after less than 30 ms, as the interfacial tension forces are balanced. In the case of surfactants solutions, this is followed by slower adsorption driven drift towards equilibrium conditions. During the initial spreading phase, the wetting front exhibits ~t1/2 spreading law. Two more viscous liquids, ethylene glycol and glycerol, were also examined and found to show a weaker time-dependence in the whole spreading regime. An ~t1/10 scaling of the drop radius versus time was for these liquids observed in the asymptotic long-time regime. For the surfactant solution, a slow relaxation towards equilibrium was observed following the initial fast spreading phase. The rate-limiting process in this regimes was in the drop spreading experiment found to be surfactant adsorption from the bulk to the expanding liquid¯vapour interface, whereas surface diffusion at the liquid¯vapour interface appeared rate-determining in the rod experiment. The reason for this is the differences in aspect ratio between relative expansion of the liquid¯vapour and solid¯liquid interfaces during spreading in the two experiments. In the study of surfactant solution spreading, the importance of surface relaxation prior to contact of the solution and the solid was also pointed out.
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| 12. |
- von Bahr, V, et al.
(author)
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Long-term pulmonary function and quality of life in adults after extracorporeal membrane oxygenation for respiratory failure
- 2019
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In: Perfusion. - : SAGE Publications. - 1477-111X .- 0267-6591. ; 34:1_suppl, s. 49-57
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Journal article (peer-reviewed)abstract
- There is a significant long-term burden on survivors after acute respiratory distress syndrome, even 5 years after discharge. This is not well investigated in patients treated with extracorporeal membrane oxygenation. The objective of this study was to describe very-long-term (⩾3 years) disability in lung function and morphology, quality of life, mood disorders, walking capacity, and return to work status in extracorporeal membrane oxygenation survivors. Methods: Single-center retrospective cohort study on long-term survivors treated with extracorporeal membrane oxygenation for respiratory failure between 1995 and 2010 at a tertiary referral center in Sweden. Eligible patients were approached, and those who consented were interviewed and investigated during a day at the hospital. Results: A total of 38 patients were investigated with a median follow-up time of 9.0 years. Quality of life was reduced in several Short form 36 (SF-36) subscales and all domains of the St George’s Respiratory Questionnaire, similar to previous studies in conventionally managed acute respiratory distress syndrome survivors. A reduced diffusion capacity of carbon monoxide was seen in 47% of patients, and some degree of residual lung parenchymal pathology was seen in 82%. Parenchymal pathology correlated with reductions in quality of life and diffusion capacity. Symptoms of anxiety and depression were seen in 22% and 14%, respectively. Conclusion: A significant long-term burden remains even 3–17 years after extracorporeal membrane oxygenation treatment, similar to conventionally managed acute respiratory distress syndrome survivors. Future prospective studies are needed to elucidate risk factors for these sequelae.
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| 15. |
- von Bahr, V, et al.
(author)
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Mesenchymal stem cells may ameliorate inflammation in an ex vivo model of extracorporeal membrane oxygenation
- 2019
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In: Perfusion. - : SAGE Publications. - 1477-111X .- 0267-6591. ; 34:1_suppl, s. 15-21
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Journal article (peer-reviewed)abstract
- Mesenchymal stem cells exhibit immunomodulatory properties which are currently being investigated as a novel treatment option for Acute Respiratory Distress Syndrome. However, the feasibility and efficacy of mesenchymal stem cell therapy in the setting of extracorporeal membrane oxygenation is poorly understood. This study aimed to characterise markers of innate immune activation in response to mesenchymal stem cells during an ex vivo simulation of extracorporeal membrane oxygenation. Methods: Ex vivo extracorporeal membrane oxygenation simulations (n = 10) were conducted using a commercial extracorporeal circuit with a CO2-enhanced fresh gas supply and donor human whole blood. Heparinised circuits (n = 4) were injected with 40 × 106-induced pluripotent stem cell–derived human mesenchymal stem cells, while the remainder (n = 6) acted as controls. Simulations were maintained, under physiological conditions, for 240 minutes. Circuits were sampled at 15, 30, 60, 120 and 240 minutes and assessed for levels of interleukin-1β, interleukin-6, interleukin-8, interleukin-10, tumour necrosis factor-α, transforming growth factor-β1, myeloperoxidase and α-Defensin-1. In addition, haemoglobin, platelet and leukocyte counts were performed. Results: There was a trend towards reduced levels of pro-inflammatory cytokines in mesenchymal stem cell–treated circuits and a significant increase in transforming growth factor-β1. Blood cells and markers of neutrophil activation were reduced in mesenchymal stem cell circuits during the length of the simulation. As previously reported, the addition of mesenchymal stem cells resulted in a reduction of flow and increased trans-oxygenator pressures in comparison to controls. Conclusions: The addition of mesenchymal stem cells during extracorporeal membrane oxygenation may cause an increase in transforming growth factor-β1. This is despite their ability to adhere to the membrane oxygenator. Further studies are required to confirm these findings.
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| 16. |
- von Bahr, V, et al.
(author)
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The authors reply
- 2018
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In: Critical care medicine. - 1530-0293. ; 46:10, s. E1014-E1015
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Journal article (peer-reviewed)
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| 17. |
- von Bahr, V, et al.
(author)
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The authors reply
- 2018
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In: Critical care medicine. - 1530-0293. ; 46:4, s. E349-E349
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Journal article (other academic/artistic)
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