| 1. |
- Alekseenko, Zhanna, et al.
(författare)
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Robust derivation of transplantable dopamine neurons from human pluripotent stem cells by timed retinoic acid delivery
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Stem cell therapies for Parkinson’s disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered.
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| 2. |
- Haider, Zahra, et al.
(författare)
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Whole-genome informed circulating tumor DNA analysis by multiplex digital PCR for disease monitoring in B-cell lymphomas : a proof-of-concept study
- 2023
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Ingår i: Frontiers in Oncology. - : Frontiers Media SA. - 2234-943X. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- IntroductionAnalyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA). MethodsIn 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up. ResultsA total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value <0.01). While clearance of ctDNA levels after primary treatment cycle 1 was observed in 3/6 patients, all patients analyzed at final evaluation of primary treatment showed negative ctDNA, hence correlating with PET-CT imaging. One patient with positive ctDNA at interim also displayed detectable ctDNA (average variant allele frequency (VAF) 6.9%) in the follow-up plasma sample collected 2 years after final evaluation of primary treatment and 25 weeks before clinical manifestation of relapse. ConclusionIn summary, we demonstrate that multi-targeted cfDNA analysis, using a combination of SNVs/indels and SVs candidates identified by WGS analysis, provides a sensitive tool for MRD monitoring and can detect lymphoma relapse earlier than clinical manifestation.
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| 3. |
- Jeggari, Ashwini, et al.
(författare)
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miRcode: a map of putative microRNA target sites in the long non-coding transcriptome
- 2012
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Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1367-4811. ; 28:15, s. 2062-2063
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Tidskriftsartikel (refereegranskat)abstract
- Although small non-coding RNAs, such as microRNAs, have well-established functions in the cell, long non-coding RNAs (lncRNAs) have only recently started to emerge as abundant regulators of cell physiology, and their functions may be diverse. A small number of studies describe interactions between small and lncRNAs, with lncRNAs acting either as inhibitory decoys or as regulatory targets of microRNAs, but such interactions are still poorly explored. To facilitate the study of microRNA-lncRNA interactions, we implemented miRcode: a comprehensive searchable map of putative microRNA target sites across the complete GENCODE annotated transcriptome, including 10 419 lncRNA genes in the current version.
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| 4. |
- Jeggari, Ashwini Priya
(författare)
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Exploration of large molecular datasets using global gene networks : computational methods and tools
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Defining gene expression profiles and mapping complex interactions between molecular regulators and proteins is a key for understanding biological processes and the functional properties of cells, which is therefore, the focus on numerous experimental studies. Small-scale biochemical analyses deliver high-quality data, but lack coverage, whereas high throughput sequencing reveals thousands of interactions which can be error-prone and require proper computational methods to discover true relations. Furthermore, all these approaches usually focus on one type of interaction at a time. This makes experimental mapping of the genome-wide network a cost and time-intensive procedure. In the first part of the thesis, I present the developed network analysis tools for exploring large- scale datasets in the context of a global network of functional coupling. Paper I introduces NEArender, a method for performing pathway analysis and determines the relations between gene sets using a global network. Traditionally, pathway analysis did not consider network relations, thereby covering a minor part of the whole picture. Placing the gene sets in the context of a network provides additional information for pathway analysis, which reveals a more comprehensive picture. Paper II presents EviNet, a user-friendly web interface for using NEArender algorithm. The user can either input gene lists or manage and integrate highly complex experimental designs via the interactive Venn diagram-based interface. The web resource provides access to biological networks and pathways from multiple public or users’ own resources. The analysis typically takes seconds or minutes, and the results are presented in a graphic and tabular format. Paper III describes NEAmarker, a method to predict anti-cancer drug targets from enrichment scores calculated by NEArender, thus presenting a practical usage of network enrichment tool. The method can integrate data from multiple omics platforms to model drug sensitivity with enrichment variables. In parallel, alternative methods for pathway enrichment analysis were benchmarked in the paper. The second part of the thesis is focused on identifying spatial and temporal mechanisms that govern the formation of neural cell diversity in the developing brain. High-throughput platforms for RNA- and ChIP-sequencing were applied to provide data for studying the underlying biological hypothesis at the genome-wide scale. In Paper IV, I defined the role of the transcription factor Foxa2 during the specification and differentiation of floor plate cells of the ventral neural tube. By RNA-seq analyses of Foxa2-/- cells, a large set of candidate genes involved in floor plate differentiation were identified. Analysis of Foxa2 ChIP-seq dataset suggested that Foxa2 directly regulated more than 250 genes expressed by the floor plate and identified Rfx4 and Ascl1 as co-regulators of many floor plate genes. Experimental studies suggested a cooperative activator function for Foxa2 and Rfx4 and a suppressive role for Ascl1 in spatially constraining floor plate induction. Paper V addresses how time is measured during sequential specification of neurons from multipotent progenitor cells during the development of ventral hindbrain. An underlying timer circuitry which leads to the sequential generation of motor neurons and serotonergic neurons has been identified by integrating experimental and computational data modeling.
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| 5. |
- Wallander, Karin, et al.
(författare)
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Sensitive Detection of Cell-Free Tumour DNA Using Optimised Targeted Sequencing Can Predict Prognosis in Gastro-Oesophageal Cancer
- 2023
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 15:4, s. 1160-
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Cancer in the stomach and oesophagus is deadly when discovered at a late stage. There are no good biomarkers for its detection or for making a prognostic prediction. In this study, we evaluate the analysis of cell-free DNA as a prognostic cancer biomarker. Cell-free DNA is DNA released from any tissue to a body fluid. When there is a tumour in the body, some of the cell-free DNA will come from that tumour, and it can be detected in a blood sample. We show that the detection of cell-free DNA from the cancer correlates to a worse prognosis than when no tumour DNA is detected. We also show that the method of analysis is important. Either a tissue biopsy must be included as a validation of the genetic variants detected or analysis of the blood cells or another blood sample after tumour resection needs to be analysed to improve detection. In this longitudinal study, cell-free tumour DNA (a liquid biopsy) from plasma was explored as a prognostic biomarker for gastro-oesophageal cancer. Both tumour-informed and tumour-agnostic approaches for plasma variant filtering were evaluated in 47 participants. This was possible through sequencing of DNA from tissue biopsies from all participants and cell-free DNA from plasma sampled before and after surgery (n = 42), as well as DNA from white blood cells (n = 21) using a custom gene panel with and without unique molecular identifiers (UMIs). A subset of the plasma samples (n = 12) was also assayed with targeted droplet digital PCR (ddPCR). In 17/31 (55%) diagnostic plasma samples, tissue-verified cancer-associated variants could be detected by the gene panel. In the tumour-agnostic approach, 26 participants (59%) had cancer-associated variants, and UMIs were necessary to filter the true variants from the technical artefacts. Additionally, clonal haematopoietic variants could be excluded using the matched white blood cells or follow-up plasma samples. ddPCR detected its targets in 10/12 (83%) and provided an ultra-sensitive method for follow-up. Detectable cancer-associated variants in plasma correlated to a shorter overall survival and shorter time to progression, with a significant correlation for the tumour-informed approaches. In summary, liquid biopsy gene panel sequencing using a tumour-agnostic approach can be applied to all patients regardless of the presence of a tissue biopsy, although this requires UMIs and the exclusion of clonal haematopoietic variants. However, if sequencing data from tumour biopsies are available, a tumour-informed approach improves the value of cell-free tumour DNA as a negative prognostic biomarker in gastro-oesophageal cancer patients.
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