| 1. |
- 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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| 2. |
- Andrusivova, Zaneta
(författare)
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Development and application of spatial transcriptomics methods
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transcriptomics is one of the pivotal fields in molecular biology, enabling comprehensive analysis of gene expression patterns. Recent advancements in the biotechnology field have transformed the transcriptomics research, providing insights into the complexity of cellular processes in a greater detail. However, conventional transcriptomics methods such as bulk RNA sequencing or single-cell RNA sequencing rely on tissue dissociation and therefore lack spatial information, which limits our understanding of gene expression patterns within the tissue structures. The development of spatially resolved transcriptomics methods has revolutionized the study of transcriptomes, enabling analysis of gene expression patterns in the spatial context. The wide range of available transcriptomics technologies offer various levels of resolution and throughput, and combination of multiple techniques can be beneficial for studying biological systems and gain deeper understanding of their molecular processes. In this thesis, particular emphasis is given to the Visium spatial gene expression technology, which has gain widespread popularity in the research community over the recent years. In the article I, we expand the application of the Visium platform to fresh-frozen samples of lower RNA quality or otherwise challenging characteristics. To achieve this, we introduce specific modifications to the commercially available protocol and test its effectiveness across different tissue types of varying RNA quality, including pediatric brain tumors, human small intestine, and mouse bone and cartilage. By conducting comparative analysis, we demonstrate that the new protocol outperforms the standard Visium protocol when working with samples of moderate and lower RNA quality.Article II introduces a novel method that enhances the resolution of the Visium gene expression method through tissue expansion. We showcase the implementation of this new protocol on two regions of mouse brain, olfactory bulb and hippocampus. We demonstrate the ability of this approach to study smaller tissue structures that were previously beyond the resolution capabilities of the Visium platform.In the article III and IV, we demonstrate the practical application of the Visium approach and its combination with other methodologies in the field of developmental biology. We show how utilizing spatial transcriptomics methods help elucidate the spatial organization of cell types and cell states during organogenesis in the developing human spinal cord (article III) and developing lung tissue (article IV). By deploying single-cell RNA sequencing and spatial methods, we described the spatiotemporal gene expression profiles of various cell types as well as shared and unique events occurring during the spinal cord development in humans and rodents (article III). Applying this multimodal approach to lung tissue (article IV) allowed us to characterize novel cell states emerging during lung development and provided valuable insights into the structural organization of developing lungs. These studies highlight the findings and observations that can be gained by combining spatially resolved transcriptomics with other laboratory techniques to shed light on the spatial dynamics of cellular processes during organ development.
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| 3. |
- Bergenstråhle, Joseph
(författare)
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Exploring the transcriptional space
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transcriptomics promises biological insight into gene regulation, cell diversity, and mechanistic understanding of dysfunction. Driven by technological advancements in sequencing technologies, the field has witnessed an exponential growth in data output. Not only has the amount of raw data increased tremendously but it’s granularity as well. From only being able to obtain aggregated transcript information from large tissue samples, we can now pinpoint the precise origin of transcripts within the tissue, sometimes even within the confines of individual cells. This thesis focuses on the different aspects of how to use these emergent technologies to obtain a greater understanding of biological mechanisms. The work conducted here spans only a few years of the much longer history of spatially resolved transcriptomics, which started with the early in situ hybridization techniques and will continue to a potential future with complete molecular profiling ofevery cell in their natural, active state. Thus, at the same time the workpresented here introduces and demonstrates the use of the latest techniques within spatial transcriptomics, it also deals with the shortcomings of the current state of the field, which undoubtedly will see extensive improvements in the not too distant future. Article I is part of a series of articles where we mechanistically examine the biological underpinnings of a serendipitous finding that single-stranded nucleic acids have immunomodulatory effects. In particular, we look at influenza-infected innate immune cells and the ability of the oligonucleotide to inhibit viral entry. The oligonucleotides prevent the cells from responding to certain types of pattern recognitionand cause a decrease in viral load. Our hypothesis is that the administration of oligonucleotides blocks certain endocytic routes. While the invivo experiments suggest that the influenza virus is still able to infect and promote disease in the host, changes in signaling response due to the inhibition of the endocytotic routes could represent an avenue for future therapeutics. The conclusions were drawn by combining protein labeling and conventional methods for RNA profiling in the form of quantitative realtime PCR and bulk RNA sequencing. As a transition into the concept of spatial RNA profiling, the thesis includes an Additional material review article on spatial transcriptomics, where we give an overview of the current state of the field, as it looked like in the beginning of 2020. In Article II, we report on the development of an R package for analyzing spatial transcriptomics datasets. The package offers visualization features and an automated pipeline for masking tissue images and aligning serially sectioned experiments. The tool is extensively used throughout the rest of the articles where spatial transcript information is analyzed and is available for all scientists that use the supported spatial transcriptomics platforms in their research. In Article III, we propose a method to spatially map long-read sequencing data. While previously described methods for high-throughput spatial transcriptomics produce short-read data, full-length transcript information allows us to spatially profile alternatively spliced transcripts. Using the proposed method, we find alternatively spliced transcripts and find isoforms of the same gene to be differentially expressed in different regions of the mouse brain. Furthermore, we profile RNA editing across the full-length transcripts and find certain parts of the mouse left hemisphere to display a substantially higher degree of editing events compared to the rest of the brain. The proposed method is based on readily available reagents and does not require advanced instrumentation. We believe full-length transcript information obtained in this manner could help scientists obtain a deeper understanding from transcriptome data. Finally, in Article IV, we explore how the latest technologies for spatial transcriptomics can be used to characterize the expression landscape of respiratory syncytial virus infections by comparing infected and non-infected mouse lungs. By integration of annotated single-cell data and spatially resolved transcriptomics, we map the location of the single cells onto the spatial grid to localize immune cell populations across the tissue sections. By correlating the locations to gene expression, we profile locally confined cellular processes and immune responses. We believe that high-throughput spatial information obtained without predefined targets will become an important tool for exploratory analysis and hypothesis generation, which in turn could unlock mechanistic knowledge of the differences between experimental models that are important for translational research.
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| 4. |
- Boele, Joost, et al.
(författare)
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PAPD5-mediated 3' adenylation and subsequent degradation of miR-21 is disrupted in proliferative disease.
- 2014
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 111:31, s. 11467-11472
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Tidskriftsartikel (refereegranskat)abstract
- Next-generation sequencing experiments have shown that microRNAs (miRNAs) are expressed in many different isoforms (isomiRs), whose biological relevance is often unclear. We found that mature miR-21, the most widely researched miRNA because of its importance in human disease, is produced in two prevalent isomiR forms that differ by 1 nt at their 3' end, and moreover that the 3' end of miR-21 is posttranscriptionally adenylated by the noncanonical poly(A) polymerase PAPD5. PAPD5 knockdown caused an increase in the miR-21 expression level, suggesting that PAPD5-mediated adenylation of miR-21 leads to its degradation. Exoribonuclease knockdown experiments followed by small-RNA sequencing suggested that PARN degrades miR-21 in the 3'-to-5' direction. In accordance with this model, microarray expression profiling demonstrated that PAPD5 knockdown results in a down-regulation of miR-21 target mRNAs. We found that disruption of the miR-21 adenylation and degradation pathway is a general feature in tumors across a wide range of tissues, as evidenced by data from The Cancer Genome Atlas, as well as in the noncancerous proliferative disease psoriasis. We conclude that PAPD5 and PARN mediate degradation of oncogenic miRNA miR-21 through a tailing and trimming process, and that this pathway is disrupted in cancer and other proliferative diseases.
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| 5. |
- Miliara, Sophia, et al.
(författare)
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The biological and prognostic role of long non-coding RNA NEAT1 in acute myeloid leukemia
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA localized in the cell nucleus that has been associated to promote several malignant solid tumors. Its role in acute myeloid leukemia (AML) remains largely elusive. Therefore, the aim of this study was to define the role of NEAT1 in AML compared to normal hematopoiesis. During normal hematopoiesis, it was identified that NEAT1 expression was low in early progenitors but increased in more differentiated cells, especially in monocytes. NEAT1 expression was increased in AML as a whole compared to normal bone marrow (NBM). It was specifically high in AML with inv(16) and t(8;21), while it was lower in patients with t(15;17). Further, NEAT1 expression correlated positively with ASXL1, KRAS and NRAS mutations and negatively with TP53 mutant AML. Higher NEAT expression was associated to better overall survival in AML, independent of other known risk factors. Antisense oligo-mediated knockdown of NEAT1 in AML cells significantly increased expression of the monocytic marker CD14 while granulocytic markers did not change. Genes affected by NEAT1-knockdown using CAGE-sequencing were significantly enriched for genes involved in glucose metabolism. By investigating genome-wide RNA and DNA interactions using RADICL-sequencing, it was revealed that NEAT1 binds to the loci of key hematopoietic regulator RUNX2 as well as the chromatin regulators KMT2A, KMT5B and CHD7. The results suggest that lncRNA NEAT1 has a potential role in hematopoietic and AML cell differentiation and could be a potential new biomarker in AML.
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| 6. |
- Sampson, Joshua N., et al.
(författare)
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Analysis of Heritability and Shared Heritability Based on Genome-Wide Association Studies for 13 Cancer Types
- 2015
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Ingår i: Journal of the National Cancer Institute. - : Oxford University Press (OUP). - 0027-8874 .- 1460-2105. ; 107:12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Studies of related individuals have consistently demonstrated notable familial aggregation of cancer. We aim to estimate the heritability and genetic correlation attributable to the additive effects of common single-nucleotide polymorphisms (SNPs) for cancer at 13 anatomical sites. Methods: Between 2007 and 2014, the US National Cancer Institute has generated data from genome-wide association studies (GWAS) for 49 492 cancer case patients and 34 131 control patients. We apply novel mixed model methodology (GCTA) to this GWAS data to estimate the heritability of individual cancers, as well as the proportion of heritability attributable to cigarette smoking in smoking-related cancers, and the genetic correlation between pairs of cancers. Results: GWAS heritability was statistically significant at nearly all sites, with the estimates of array-based heritability, h(l)(2), on the liability threshold (LT) scale ranging from 0.05 to 0.38. Estimating the combined heritability of multiple smoking characteristics, we calculate that at least 24% (95% confidence interval [CI] = 14% to 37%) and 7% (95% CI = 4% to 11%) of the heritability for lung and bladder cancer, respectively, can be attributed to genetic determinants of smoking. Most pairs of cancers studied did not show evidence of strong genetic correlation. We found only four pairs of cancers with marginally statistically significant correlations, specifically kidney and testes (rho = 0.73, SE = 0.28), diffuse large B-cell lymphoma (DLBCL) and pediatric osteosarcoma (rho = 0.53, SE = 0.21), DLBCL and chronic lymphocytic leukemia (CLL) (rho = 0.51, SE = 0.18), and bladder and lung (rho = 0.35, SE = 0.14). Correlation analysis also indicates that the genetic architecture of lung cancer differs between a smoking population of European ancestry and a nonsmoking Asian population, allowing for the possibility that the genetic etiology for the same disease can vary by population and environmental exposures. Conclusion: Our results provide important insights into the genetic architecture of cancers and suggest new avenues for investigation.
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