| 1. |
- Dolinska, Monika, et al.
(författare)
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Leukotriene signaling via ALOX5 and cysteinyl leukotriene receptor 1 is dispensable for in vitro growth of CD34+CD38- stem and progenitor cells in chronic myeloid leukemia
- 2017
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 490:2, s. 378-384
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Tidskriftsartikel (refereegranskat)abstract
- Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL+CD34+CD38- cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34+CD38- cells from 7 CML patients. The majority of the single leukemic BCR-ABL +CD34+CD38- cells expressed cysteinyl leukotriene receptors CYSLT1 and CYSLT2. However, montelukast, an inhibitor of CYSLT1, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLT1 signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients.
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| 2. |
- Gelali, Eleni
(författare)
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Developing and democratizing tools for spatially resolved profiling of genome and transcriptome
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The common thread of the work presented in this thesis is the development and democratization of tools that facilitate the spatial profiling of genome and transcriptome. Paper I describes iFISH, a resource that facilitates the large-scale design and costeffective, flexible production of probes for DNA FISH. More specifically, we created a database of oligos targeting the human genome and a probe design tool which we made publicly available through a user-friendly web interface, which we have named iFISH4U. We used iFISH4U to design hundreds of small probes (<10kb span), targeting equally spaced landmark loci, evenly distributed along all chromosomes. We validated a large fraction of those probes individually and then combined them to create “spotting probes” in order to profile chromosomal territories (CTs) in an unbiased way in 3D fixed cells. We were able to visualize up to 6 different chromosomes in thousands of terminally differentiated fibroblasts and embryonic stem cells and to compare their CTs. In ESCs we observed less distinct CTs, and, in a subset of cells, a remarkable lack of chromosome territoriality. We also assessed whether the degree of chromosomal intermingling differs between pluripotent and differentiated cells. All the tools and probes used in paper I were made available for the community, making iFISH a resource that can greatly facilitate genome architecture studies. In paper II, we developed genomic loci positioning by sequencing (GPSeq), a next generation sequencing based method for profiling of the radial arrangement of chromatin in fixed cells. In GPSeq, we control the incubation time of a restriction enzyme in order to digest chromatin in a gradual manner, starting from the periphery and moving towards the center. By ligating adapters and sequencing the DNA fragments around the cut sites, we can generate high-resolution, genome-wide radiality maps upon computing of a radiality score. In order to define the best radiality score we compared the different estimates with 3D DNA FISH measurements taking advantage of the tools we developed in iFISH. We used GPSeq maps to profile the radiality of various levels of chromatin organization such as chromosomes, A and B compartments and their subcompartments. Moreover, we integrated those maps with different annotation tracks in order to study the radial localization of features and marks of active and inactive chromatin. We also used GPSeq to assess the radial arrangement of SNVs, SNPs and DNA breaks. Finally, we developed a 3D genome reconstruction algorithm that combines GPSeq and Hi-C data, to predict the chromatin structure in single cells. Altogether, GPSeq is a versatile assay that can be used for radial profiling of the 3D genome. Paper III introduces Deconwolf (DW), a publicly available and easy-to-use software for image deconvolution. Even though DW can be used for different type of images, we tested its performance on data generated by imaging-based spatially resolved omics, technologies that (depending on the method) suffer from 2 different limitations, the requirement for imaging with high magnification objectives and the optical crowding problem, or in other words the inability to resolve densely packed targets. As a proof of concept, we applied DW to images from smFISH experiments and we were able to resolve crowded signal as well as to quantify smFISH signals in tissue sections imaged with 20x magnification. We then applied DW to In Situ Sequencing (ISS) and OligoFISSEQ images and we found that DW greatly increases the sensitivity of transcript calling in hybISS and the barcode detection efficiency in OligoFISSEQ. Our results highlight the potential of DW to improve image deconvolution and consequently the amount of information yielded upon imaging-based spatially-resolved molecular profiling
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| 3. |
- Wernersson, Erik, et al.
(författare)
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Deconwolf enables high-performance deconvolution of widefield fluorescence microscopy images
- 2024
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Ingår i: Nature Methods. - 1548-7091 .- 1548-7105.
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Tidskriftsartikel (refereegranskat)abstract
- Microscopy-based spatially resolved omic methods are transforming the life sciences. However, these methods rely on high numerical aperture objectives and cannot resolve crowded molecular targets, limiting the amount of extractable biological information. To overcome these limitations, here we develop Deconwolf, an open-source, user-friendly software for high-performance deconvolution of widefield fluorescence microscopy images, which efficiently runs on laptop computers. Deconwolf enables accurate quantification of crowded diffraction limited fluorescence dots in DNA and RNA fluorescence in situ hybridization images and allows robust detection of individual transcripts in tissue sections imaged with x20 air objectives. Deconvolution of in situ spatial transcriptomics images with Deconwolf increased the number of transcripts identified more than threefold, while the application of Deconwolf to images obtained by fluorescence in situ sequencing of barcoded Oligopaint probes drastically improved chromosome tracing. Deconwolf greatly facilitates the use of deconvolution in many bioimaging applications. Deconwolf is a computationally efficient and user-friendly software tool for fluorescence microscopy image deconvolution that improves the analysis of diverse fluorescence in situ hybridization methods and can handle large datasets.
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