| 1. |
- Qian, Xiaoyan, et al.
(författare)
-
Target sequence design of padlock probes based on experimentally determined in situ synthesized cDNA fragments
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Padlock probes are widely used to target a short fragment of DNA. For example, in in situ sequencing (ISS), an image-based technology for highly multiplexed spatial gene expression analysis, cDNA target detection is mediated by padlock probes. Transcript counts from ISS generally has good correlation with next-generation sequencing read counts, but bias between different genes are also observed. Therefore, we developed a new method to isolate and sequence in situ synthesized cDNA and sought to use the read coverage information from it to guide padlock probe design. The results show limited correlation between cDNA library sequencing and ISS counts, but it can still help the probe design process by eliminating target sequences that are very unlikely to be detected. In addition, the method provides a way to systematically characterize in situ reverse transcription.
|
|
| 2. |
- Asp, Michaela, et al.
(författare)
-
A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart
- 2019
-
Ingår i: Cell. - : CELL PRESS. - 0092-8674 .- 1097-4172. ; 179:7, s. 1647-
-
Tidskriftsartikel (refereegranskat)abstract
- The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.
|
|
| 3. |
- de Miranda, Noel F. C. C., et al.
(författare)
-
DNA repair genes are selectively mutated in diffuse large B cell lymphomas
- 2013
-
Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 210:9, s. 1729-1742
-
Tidskriftsartikel (refereegranskat)abstract
- DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis.
|
|
| 4. |
- de Miranda, Noel F. C. C., et al.
(författare)
-
Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
- 2014
-
Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 124:16, s. 2544-2553
-
Tidskriftsartikel (refereegranskat)abstract
- Next-generation sequencing studies on diffuse large B-cell lymphomas (DLBCLs) have revealed novel targets of genetic aberrations but also high intercohort heterogeneity. Previous studies have suggested that the prevalence of disease subgroups and cytogenetic profiles differ between Western and Asian patients. To characterize the coding genome of Chinese DLBCL, we performed whole-exome sequencing of DNA derived from 31 tumors and respective peripheral blood samples. The mutation prevalence of B2M, CD70, DTX1, LYN, TMSB4X, and UBE2A was investigated in an additional 105 tumor samples. We discovered 11 novel targets of recurrent mutations in DLBCL that included functionally relevant genes such as LYN and TMSB4X. Additional genes were found mutated at high frequency (>= 10%) in the Chinese cohort including DTX1, which was the most prevalent mutation target in the Notch pathway. We furthermore demonstrated that mutations in DTX1 impair its function as a negative regulator of Notch. Novel and previous unappreciated targets of somatic mutations in DLBCL identified in this study support the existence of additional/alternative tumorigenic pathways in these tumors. The observed differences with previous reports might be explained by the genetic heterogeneity of DLBCL, the germline genetic makeup of Chinese individuals, and/or exposure to distinct etiological agents.
|
|
| 5. |
- Liu, Haipeng, et al.
(författare)
-
Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?
- 2011
-
Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 35:1, s. 51-61
-
Tidskriftsartikel (refereegranskat)abstract
- Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30 kDa protein from hemocytes of the freshwater crayfish, Pacifastocus leniusculus, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type pepticloglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the Pl-SPH1 and lipopolysaccharide- and beta-1,3-glucan-bincling protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two Pl-SPlis are involved in this activation possibly by forming a complex with LGBP and without a PGRP.
|
|
| 6. |
- Liu, Xin, et al.
(författare)
-
Plasma metabolites mediate the association of coarse grain intake with blood pressure in hypertension-free adults
- 2020
-
Ingår i: Nutrition, Metabolism and Cardiovascular Diseases. - : Elsevier BV. - 0939-4753 .- 1590-3729. ; 30:9, s. 1512-1519
-
Tidskriftsartikel (refereegranskat)abstract
- Increased intake of whole/coarse grains was associated with improved blood pressure control, but concurrent metabolism alterations are less clear. We sought to identify metabolomic profiles of blood pressure, and to explore their mediation effects on the coarse grain intake-blood pressure association among young adults free of hypertension. Methods and results: Plasma metabolome of 86 participants from the Carbohydrate Alternatives and Metabolic Phenotypes study was characterized by untargeted lipidomics and metabolomics using liquid chromatography–high-resolution mass spectrometry. We identified 24 and 117 metabolites associated with systolic blood pressure (SBP) and diastolic blood pressure (DBP), respectively, using random forest modeling and partial correlation analysis. Moreover, metabolite panels for highly specific prediction of blood pressure (8 metabolites for SBP and 11 metabolites for DBP) were determined using ten-fold cross-validated ridge regression (R2 ≥ 0.70). We also observed an inverse association between metabolite panel of SBP (β ± SE = −0.02 ± 0.01, P = 0.04) or DBP (β ± SE = −0.03 ± 0.01, P = 0.02) and coarse grain intake. Furthermore, we observed significant mediating effects of metabolites, in particular, sphingolipid ceramides, on the association between coarse grain exposure and blood pressure using both bias-corrected bootstrap tests and high-dimensional mediation analysis adapted for large-scale and high-throughput omics data. Conclusions: We identified metabolomic profiles specifically associated with blood pressure in young Chinese adults without diagnosed hypertension. The inverse association between coarse grain intake and blood pressure may be mediated by sphingolipid metabolites.
|
|
| 7. |
- Lundin, Elin, 1983-, et al.
(författare)
-
Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. However, the spatiotemporal and diversification properties of RNA editing in the brain are largely unknown. Here, we applied in situ sequencing to distinguish between edited and unedited transcripts in distinct regions of the mouse brain at four developmental stages, and investigate the diversity of the RNA landscape.Results: We analyzed RNA editing at codon-altering sites using in situ sequencing at single-cell resolution, in combination with the detection of individual ADAR enzymes and specific cell type marker transcripts. This approach revealed cell-type specific regulation of RNA editing of a set of transcripts, and developmental and regional variation in editing levels for many of the targeted sites. We found increasing editing diversity throughout development, which arises through regional- and cell type-specific regulation of ADAR enzymes and target transcripts.Conclusions: Our single-cell in situ sequencing method has proved useful to study the complex landscape of RNA editing and our results indicate that this complexity arises due to distinct mechanisms of regulating individual RNA editing sites, acting both regionally and in specific cell types.
|
|
| 8. |
- Lundin, Elin, et al.
(författare)
-
Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation
- 2020
-
Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 18:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. However, the spatiotemporal and diversification properties of RNA editing in the brain are largely unknown. Here, we applied in situ sequencing to distinguish between edited and unedited transcripts in distinct regions of the mouse brain at four developmental stages, and investigate the diversity of the RNA landscape. Results We analyzed RNA editing at codon-altering sites using in situ sequencing at single-cell resolution, in combination with the detection of individual ADAR enzymes and specific cell type marker transcripts. This approach revealed cell-type-specific regulation of RNA editing of a set of transcripts, and developmental and regional variation in editing levels for many of the targeted sites. We found increasing editing diversity throughout development, which arises through regional- and cell type-specific regulation of ADAR enzymes and target transcripts. Conclusions Our single-cell in situ sequencing method has proved useful to study the complex landscape of RNA editing and our results indicate that this complexity arises due to distinct mechanisms of regulating individual RNA editing sites, acting both regionally and in specific cell types.
|
|
| 9. |
- Strell, Carina, et al.
(författare)
-
Placing RNA in context and space - methods for spatially resolved transcriptomics
- 2019
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 286:8, s. 1468-1481
-
Forskningsöversikt (refereegranskat)abstract
- Single-cell transcriptomics provides us with completely new insights into the molecular diversity of different cell types and the different states they can adopt. The technique generates inventories of cells that constitute the building blocks of multicellular organisms. However, since the method requires isolation of discrete cells, information about the original location within tissue is lost. Therefore, it is not possible to draw detailed cellular maps of tissue architecture and their positioning in relation to other cells. In order to better understand the cellular and tissue function of multicellular organisms, we need to map the cells within their physiological, morphological, and anatomical context and space. In this review, we will summarize and compare the different methods of in situ RNA analysis and the most recent developments leading to more comprehensive and highly multiplexed spatially resolved transcriptomic approaches. We will discuss their highlights and advantages as well as their limitations and challenges and give an outlook on promising future applications and directions both within basic research as well as clinical integration.
|
|
| 10. |
- Söderhäll, Irene, et al.
(författare)
-
A novel protein acts as a negative regulator of prophenoloxidase activation and melanization in the freshwater crayfish Pacifastacus leniusculus
- 2009
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:10, s. 6301-6310
-
Tidskriftsartikel (refereegranskat)abstract
- Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response.
|
|
