| 1. |
- Allalou, Amin, et al.
(author)
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Image Based Measurements of Single Cell mtDNA Mutation Load
- 2007
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In: Image Analysis, Proceedings. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 9783540730392 ; , s. 631-640
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Conference paper (peer-reviewed)abstract
- Cell cultures as well as cells in tissue always display a certain degree of variability, and measurements based on cell averages will miss important information contained in a heterogeneous population. This paper presents automated methods for image based measurements of mitochondiral DNA (mtDNA) mutations in individual cells. The mitochondria are present in the cell’s cytoplasm, and each cytoplasm has to be delineated. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%. The final image based measurements of mitochondrial mutation load are also compared to, and show high agreement with, measurements made using biochemical techniques.
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| 2. |
- Allalou, Amin, et al.
(author)
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Image based measurements of single cell mtDNA mutation load MTD 2007
- 2007
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In: Medicinteknikdagarna 2007.
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Conference paper (pop. science, debate, etc.)abstract
- Cell cultures as well as cells in tissue always display a certain degree of variability,and measurements based on cell averages will miss important information contained in a heterogeneous population. These differences among cells in a population may be essential to quantify when looking at, e.g., protein expression and mutations in tumor cells which often show high degree of heterogeneity.Single nucleotide mutations in the mithochondrial DNA (mtDNA) can accumulate and later be present in large proportions of the mithocondria causing devastating diseases. To study mtDNA accumulation and segregation one needs to measure the amount of mtDNA mutations in each cell in multiple serial cell culture passages. The different degrees of mutation in a cell culture can be quantified by making measurements on individual cells as an alternative to looking at an average of a population. Fluorescence microscopy in combination with automated digital image analysis provides an efficient approach to this type of single cell analysis.Image analysis software for these types of applications are often complicated and not easy to use for persons lacking extensive knowledge in image analysis, e.g., laboratory personnel. This paper presents a user friendly implementation of an automated method for image based measurements of mtDNA mutations in individual cells detected with padlock probes and rolling-circle amplification (RCA). The mitochondria are present in the cell’s cytoplasm, and here each cytoplasm has to be delineated without the presence of a cytoplasmic stain. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%. The final image based measurements of mitochondrial mutation load are also compared to, and show high agreement with, measurements made using biochemical techniques.
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| 3. |
- Allalou, Amin, et al.
(author)
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Segmentation of Cytoplasms of Cultured Cells
- 2007
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In: In Proceedings SSBA 2007, Symposium on image analysis, Linköping.
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Conference paper (other academic/artistic)abstract
- Cell cultures as well as cells in tissue always display a certain degree of variability, and measurements based on cell averages will miss important information contained in a heterogeneous population. This paper presents automated methods for segmentation of cells and cytoplasms. The segmentation results are applied to image based measurements of mitochondiral DNA (mtDNA) mutations in individual cells. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%, compared to an inter observer variability of 79% at manual delineation.
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| 4. |
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| 5. |
- De Prins, L, et al.
(author)
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White Paper on European Patient Needs and Suggestions on Chronic Type 2 Inflammation of Airways and Skin by EUFOREA
- 2022
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In: Frontiers in allergy. - : Frontiers Media SA. - 2673-6101. ; 3, s. 889221-
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Journal article (peer-reviewed)abstract
- Type 2 inflammation underlies the chronicity of disease in subgroups of patients with asthma, chronic rhinosinusitis with nasal polyps (CRSwNP) and atopic dermatitis (AD), that often co-exist. Although several studies have investigated the unmet needs of asthma, AD and CRSwNP as such, little is known about the similarities and differences in experiences and perspectives of the current management of patients with comorbid Type 2 inflammatory diseases.AimsTo improve insight into the common and organ-specific needs of patients with Type 2 inflammation and comorbidities, allowing the formulation of recommendations to better address these needs in the future.MethodologyThis qualitative study was conducted between July 2021 and December 2021 using semi-structured face-to-face or telephone interviews with patients suffering from year-long severe chronic Type 2 inflammation and at least one co-morbid inflammatory condition. Seven participating academic centers in Europe interviewed asthma (Copenhagen and Leuven), CRSwNP (London, Amsterdam and Crete) and/or AD (Oldenburg and Zurich) patients on patient characteristics, disease severity, shortcomings of current care pathways and suggestions for improvement of care. Transcripts were analyzed using an inductive thematic analysis approach.ResultsEighty-one patients with severe Type 2 inflammation and comorbidities were interviewed. Similar needs were recognized by patients with Type 2 inflammation, with both a lack of coordination in care and a lack of a real cure reported as being most frustrating. However, several needs are specific to asthma, CRSwNP and AD. Suggestions for improvement of care were generic across diseases, such as the implementation of a multidisciplinary approach, the improved facilitation of access to better treatments, the increase of general awareness on disease burden, and better educational programs for healthcare providers and patients. Of note, patients with CRSwNP also stated the need for alternatives to sinus surgery, whereas patients with asthma requested better medical care to prevent exacerbations and patients with AD would warmly welcome the reimbursement of emollients.ConclusionPatients with asthma, CRSwNP and AD have shared unmet needs that need to be addressed by physicians, the academic community and health policy makers. This survey provides unique recommendations made by patients for the implementation of better care.
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| 6. |
- Efanov, AM, et al.
(author)
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The novel imidazoline compound BL11282 potentiates glucose-induced insulin secretion in pancreatic beta-cells in the absence of modulation of K(ATP) channel activity
- 2001
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In: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 50:4, s. 797-802
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Journal article (peer-reviewed)abstract
- The insulinotropic activity of the novel imidazoline compound BL11282 was investigated. Intravenous administration of BL11282 (0.3 mg · kg–1 · min–1) to anesthetized rats did not change blood glucose and insulin levels under basal conditions, but produced a higher increase in blood insulin levels and a faster glucose removal from the blood after glucose infusion. Similarly, in isolated Wistar rat pancreatic islets, 0.1–100 μmol/l BL11282 potently stimulated glucose-induced insulin secretion but did not modulate basal insulin secretion. Unlike previously described imidazolines, BL11282 did not block ATP-dependent K+ channels. Furthermore, the compound stimulated insulin secretion in islets depolarized with high concentrations of KCl or permeabilized with electric shock. Insulinotropic activity of BL11282 was dependent on activity of protein kinases A and C. In pancreatic islets from spontaneously diabetic GK rats, the imidazoline compound restored the impaired insulin response to glucose. In conclusion, the imidazoline BL11282 constitutes a new class of insulinotropic compounds that exerts an exclusive glucose-dependent insulinotropic activity in pancreatic islets by stimulating insulin exocytosis.
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| 7. |
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| 8. |
- Jahangir Tafrechi, Roshan S., et al.
(author)
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Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis
- 2007
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In: Journal of Histochemistry and Cytochemistry. - 0022-1554 .- 1551-5044. ; 55:11, s. 1159-1166
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Journal article (peer-reviewed)abstract
- Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification–based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.
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| 9. |
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| 10. |
- Nilsson, Mats, et al.
(author)
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Real-time monitoring of rolling-circle amplification using a modified molecular beacon design
- 2002
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 30:14, s. e66-
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Journal article (peer-reviewed)abstract
- We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2'-O-Me-RNA to prevent 3' exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.
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