| 1. |
- Qian, Xiaoyan, et al.
(författare)
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Target sequence design of padlock probes based on experimentally determined in situ synthesized cDNA fragments
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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.
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| 2. |
- Asp, Michaela, et al.
(författare)
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A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart
- 2019
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Ingår i: Cell. - : CELL PRESS. - 0092-8674 .- 1097-4172. ; 179:7, s. 1647-
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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.
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| 3. |
- de Miranda, Noel F. C. C., et al.
(författare)
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DNA repair genes are selectively mutated in diffuse large B cell lymphomas
- 2013
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 210:9, s. 1729-1742
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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.
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| 4. |
- de Miranda, Noel F. C. C., et al.
(författare)
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Exome sequencing reveals novel mutation targets in diffuse large B-cell lymphomas derived from Chinese patients
- 2014
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Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 124:16, s. 2544-2553
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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.
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| 5. |
- Li, Wenyun, et al.
(författare)
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Personalized microbial fingerprint associated with differential glycemic effects of a whole grain rye intervention on Chinese adults
- 2024
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Ingår i: Molecular Nutrition & Food Research. - : John Wiley & Sons. - 1613-4125 .- 1613-4133.
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Tidskriftsartikel (refereegranskat)abstract
- Scope: This study aims to identify the gut enterotypes that explain differential responses to intervention with whole grain rye by proposing an “enterotype - metabolic” model.Methods and results: A 12-week randomized controlled trial is conducted in Chinese adults, with 79 subjects consuming whole grain products with fermented rye bran (FRB) and 77 consuming refined wheat products in this exploratory post-hoc analysis. Responders or non-responders are identified according to whether blood glucose decreased by more than 10% after rye intervention. Compared to non-responders, responders in FRB have higher baseline Bacteroides (p < 0.001), associated with reduced blood glucose (p < 0.001), increased Faecalibacterium (p = 0.020) and Erysipelotrichaceae_UCG.003 (p = 0.022), as well as deceased 7β-hydroxysteroid dehydrogenase (p = 0.033) after intervention. The differentiated gut microbiota and metabolites between responders and non-responders after intervention are enriched in aminoacyl-tRNA biosynthesis.Conclusion: The work confirms the previously suggested importance of microbial enterotypes in differential responses to whole grain interventions and supports taking enterotypes into consideration for improved efficacy of whole grain intervention for preventing type 2 diabetes. Altered short-chain fatty acids and bile acid metabolism might be a potential mediator for the beneficial effects of whole grain rye on glucose metabolism.
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| 6. |
- Liu, Haipeng, et al.
(författare)
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Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?
- 2011
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 35:1, s. 51-61
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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.
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| 7. |
- Liu, Xin, et al.
(författare)
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Plasma metabolites mediate the association of coarse grain intake with blood pressure in hypertension-free adults
- 2020
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Ingår i: Nutrition, Metabolism and Cardiovascular Diseases. - : Elsevier BV. - 0939-4753 .- 1590-3729. ; 30:9, s. 1512-1519
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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.
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| 8. |
- Lundin, Elin, 1983-, et al.
(författare)
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Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation
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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.
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| 9. |
- Lundin, Elin, et al.
(författare)
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Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation
- 2020
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Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 18:1
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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.
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| 10. |
- Strell, Carina, et al.
(författare)
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Placing RNA in context and space - methods for spatially resolved transcriptomics
- 2019
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 286:8, s. 1468-1481
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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.
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| 11. |
- Söderhäll, Irene, et al.
(författare)
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A novel protein acts as a negative regulator of prophenoloxidase activation and melanization in the freshwater crayfish Pacifastacus leniusculus
- 2009
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:10, s. 6301-6310
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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.
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| 12. |
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| 13. |
- Wu, Chenglin, et al.
(författare)
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An insect TEP in a crustacean is specific for cuticular tissues and involved in intestinal defense
- 2012
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Ingår i: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 0965-1748 .- 1879-0240. ; 42:2, s. 71-80
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Tidskriftsartikel (refereegranskat)abstract
- In an attempt to identify genes encoding thioester-containing proteins in the freshwater crayfish, Pacifastacus leniusculus, three different cDNAs were found. A phylogenetic analysis of these proteins indicates that they can be classified into two subfamilies: two alpha-2-macroglobulins (Pl-A2M1, Pl-A2M2) showing a close similarity to shrimp A2M, and one insect TEP-like protein (Pl-TEP). This is the first report of an insect TEP-like protein in a crustacean. Crayfish Pl-A2M1, Pl-A2M2 and Pl-TEP cDNAs encode proteins with 1480, 1586 or 1507 amino acids, respectively. Pl-A2M1, Pl-A2M2 and Pl-TEP have the basic domain structure and functionally important residues for each molecule, and their mRNA was detected in different parts of the body, suggesting that they may have different functions. Pl-A2M1 was mainly expressed in hemocytes and Pl-A2M2 was highly expressed in heart and nerve, while Pl-TEP was exclusively expressed in cuticular tissues such as gill and intestine. RNA interference of Pl-TEP in vivo resulted in that these animals were slightly less resistant when fed with the bacterium, Pseudomonas libanensis/gessardii. Furthermore, when TEP activity was blocked using methylamine followed by bacterial feeding, the animals were killed to a higher extent compared to a control group. Taken together, this indicates that Pl-TEP and/or Pl-A2M1, Pl-A2M2 may be important for the immune defense in crayfish intestine and function as a pattern recognition protein in crayfish cuticular tissues.
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| 14. |
- Wu, Chenglin, et al.
(författare)
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An MBL-like protein may interfere with the activation of the proPO-system, an important innate immune reaction in invertebrates
- 2013
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Ingår i: Immunobiology. - : Elsevier BV. - 0171-2985 .- 1878-3279. ; 218:2, s. 159-168
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Tidskriftsartikel (refereegranskat)abstract
- An important characteristic of the innate immune systems of crayfish and other arthropods is the activation of a serine proteinase cascade in the hemolymph, which results in the activation of prophenoloxidase and subsequently leading to the formation of toxic quinones and melanin. Although no true complement homologues have been detected in crayfish or crustaceans, several proteins with similarities to vertebrate pattern recognition receptors (PRRs), which are involved in the lectin pathway of complement activation in vertebrates, are present. One is a C-type lectin, a mannose-binding lectin (Pl-MBL), which is secreted from granular hemocytes. Here we report that Pl-MBL has LPS-binding capacity and is dependent upon high Ca(2+) for its solubility and Pl-MBL interferes with proPO activation in vitro when HLS is prepared at high Ca(2+). The proPO-activating system is efficiently activated by microbial polysaccharides and it has to be neatly regulated to avoid activation in places where it is inappropriate and the active enzyme PO should be prevented from spreading throughout the body of the animal. This may be particularly important during molting when proPO is involved in hardening of a new cuticle and the animal is vulnerable to microbes. The presence of high amount of Pl-MBL in the granular hemocytes may play a role in this process. Since a hemocyte lysate supernatant (HLS) prepared at 100mM Ca(2+) could become activated when the concentration of LPS was increased up to 3mg/ml, this may indicate that Pl-MBL acts as a scavenger for LPS to prevent spreading of LPS in the hemolymph to avoid further activation of the proPO-system.
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| 15. |
- Wu, Chenglin, et al.
(författare)
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Genetic heterogeneity in primary and relapsed mantle cell lymphomas : Impact of recurrent CARD11 mutations
- 2016
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 7:25, s. 38180-38190
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Tidskriftsartikel (refereegranskat)abstract
- The genetic mechanisms underlying disease progression, relapse and therapy resistance in mantle cell lymphoma (MCL) remain largely unknown. Whole-exome sequencing was performed in 27 MCL samples from 13 patients, representing the largest analyzed series of consecutive biopsies obtained at diagnosis and/or relapse for this type of lymphoma. Eighteen genes were found to be recurrently mutated in these samples, including known (ATM, MEF2B and MLL2) and novel mutation targets (S1PR1 and CARD11). CARD11, a scaffold protein required for B-cell receptor (BCR)-induced NF-kappa B activation, was subsequently screened in an additional 173 MCL samples and mutations were observed in 5.5% of cases. Based on in vitro cell line-based experiments, overexpression of CARD11 mutants were demonstrated to confer resistance to the BCR-inhibitor ibrutinib and NF-kappa B-inhibitor lenalidomide. Genetic alterations acquired in the relapse samples were found to be largely non-recurrent, in line with the branched evolutionary pattern of clonal evolution observed in most cases. In summary, this study highlights the genetic heterogeneity in MCL, in particular at relapse, and provides for the first time genetic evidence of BCR/NF-kappa B activation in a subset of MCL.
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| 16. |
- Wu, Chenglin, et al.
(författare)
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Hemocyte lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus
- 2008
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 8:20, s. 4226-4235
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Tidskriftsartikel (refereegranskat)abstract
- To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells.
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| 17. |
- Wu, Chenglin
(författare)
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Innate Immune Proteins in a Crustacean Pacifastacus leniusculus
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hemocytes (blood cells) are important in the immune defense against pathogens in invertebrates. In crusteacean, the hemocytes and plasma components mount a strong innate immune response against different pathogens including bacteria and virus. This thesis is aimed to identify marker proteins associated with development of different hemocyte types, and to find a protein involved in the phenoloxidase-induced melanization and other innate immune reactions in freshwater crayfish Pacifastacus leniusculus. In crustaceans, the hemocytes are produced and partly differentiated in the hematopoietic tissue (Hpt) before they are released into the hemolymph circulation. To investigate the connection between semigranular cells, granular cells and precursor cells in Hpt of P. leniusculus and possibly also in other crustaceans, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis was used to identify specific proteins expressed in different hemocytes. The specific expression was analyzed by RT-PCR and western blot. Moreover, RNA interference was used to study the hemocyte differentiation in vivo and in vitro. Melanin formation is essential for host defence in arthopods, and it needs to be tightly regulated since unwanted production of quinone intermediates or melanization is also dangerous to the animal. By using western blot, 2-DE and MS, a melanization inhibiting protein (MIP) was found to have similar function as mealworm Tenebrio molitor MIP. Both of them interfere with the melanization reaction, but do not affect phenoloxidase activity. In order to reveal the mechanism by which peptidoglycan (PGN) induces activation of the prophenoloxidase activating system in P. leniusculus, different forms of Lys-type PGN were used to pull down PGN recognition proteins (PGRPs) from plasma or hemocyte lysate supernatant of crayfish. The binding proteins were separated and then analyzed with MS. Results showed that two serine protease homologues are involved in this activation possibly by forming a complex with lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and without a PGRP. Besides, two ficolin-like proteins (FLPs) have been found from crayfish plasma by using different bacteria including Staphylocuccus aureus as an affinity matrix to pull down bacterial binding proteins, followed by the analysis with 2-DE and MS. Two FLPs can bind to bacteria, and may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria injected into the crayfish hemolymph, which suggests that FLPs may function as pattern recognition receptors in the immune response of crayfish.
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| 18. |
- Wu, Chenglin, et al.
(författare)
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Profiling and genotyping individual mRNA molecules through in situ sequencing of super rolling circle amplification products
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- We have recently developed a technology for localized sequence library preparation with rolling-circle amplification (RCA) as an approach for in situ sequencing. This method involves generation of clonally amplified and specially confined substrates for next-generation sequencing within the preserved context of cells and tissues. Our approach combines padlock probing, RCA, and sequencing-by-ligation chemistry that can resolve expression profiles of sets of genes and mutations in tissues without loss of histological context. Like other fluorescence-based assays, it can be hindered by high level of background fluorescence. To achieve high signal-to-noise ratios, we now describe a method to boost the amplification generated by RCA of padlock probes in situ by super RCA (sRCA). In this technique, a second padlock probe is hybridized, ligated and amplified on the first RCA product for enhanced, localized amplification. We describe and compare different sRCA strategies where gap-fill ligation was showed to be most efficient. The sRCA products co-localize and have comparable sizes as RCA products but they display at least two fold higher signal intensity. This increase in signal to noise also proved to result in two folds increase in the number of sRCA products detected. By combining sRCA with in situ sequencing for highly multiplex detection in tissue a four-time increase was seen. In summary, we demonstrate that sRCA can significantly increase the performance of padlock-based in situ sequencing for gene expression profiling of tissue sections, enabling detection of low abundant transcripts and the analysis of also highly auto-fluorescent samples.
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| 19. |
- Wu, Chenglin, et al.
(författare)
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RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples
- 2018
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Ingår i: Communications biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 1
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Tidskriftsartikel (refereegranskat)abstract
- Single-molecule RNA fluorescence in situ hybridization (smFISH) represents a promising approach to quantify the expression of clinically useful biomarkers in tumor samples. However, routine application of smFISH to formalin-fixed, paraffin-embedded (FFPE) samples is challenging due to the low signal intensity and high background noise. Here we present RollFISH, a method combining the specificity of smFISH with the signal boosting of rolling circle amplification. We apply RollFISH to quantify widely used breast cancer biomarkers in cell lines and FFPE samples. Thanks to the high signal-to-noise ratio, we can visualize selected biomarkers at low magnification (20 x) across entire tissue sections, and thus assess their spatial heterogeneity. Lastly, we apply RollFISH to quantify HER2 mRNA in 150 samples on a single tissue microarray, achieving a sensitivity and specificity of detection of HER2-positive samples of similar to 90%. RollFISH is a robust method for quantifying the expression and intratumor heterogeneity of biomarkers in FFPE tissues.
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| 20. |
- Wu, Chenglin, et al.
(författare)
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Two novel ficolin-like proteins act as pattern recognition receptors for invading pathogens in the freshwater crayfish Pacifastacus leniusculus
- 2011
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 11:11, s. 2249-2264
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Tidskriftsartikel (refereegranskat)abstract
- AbstractTo isolate pathogen-associated molecular patterns (PAMPs)-binding molecules, the bacterium, Staphylococcus aureus was used as an affinity matrix to find bacteria binding proteins in the plasma of the freshwater crayfish, Pacifastacus leniusculus. Two new bacteria binding ficolin-like proteins (FLPs) were identified by 2-DE and MS analysis. The FLPs have a fibrinogen-related domain (FReD) in their C-terminal and a repeat region in their N-terminal region with putative structural similarities to the collagen-like domain of vertebrate ficolins and mannose binding lectins (MBLs). Phylogenetic analysis shows that the newly isolated crayfish FLP1 and FLP2 cluster separately from other FReD containing proteins. A tissue distribution study showed that the mRNA expression of FLP occurred mainly in the hematopoietic tissue (Hpt) and in the hepatopancreas. Recombinant FLPs exhibited agglutination activity of Gram-negative bacteria Escherichia coli and Aeromonas hydrophila in the presence of Ca2+. FLPs were able to bind to A. hydrophila, E.coli and S.aureus as judged by bacteria adsorption. Moreover, the FLPs may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria which had been injected into the hemolymph. When Gram-negative bacteria coated with FLPs were incubated with Hpt cells, a lower death rate of the cells was found compared to control treatment. Our results suggest that FLPs function as pattern recognition receptors in the immune response of crayfish.
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| 21. |
- Zaghlool, Ammar, 1980-, et al.
(författare)
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Expression profiling and in situ screening of circular RNAs in human tissues
- 2018
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Circular RNAs (circRNAs) were recently discovered as a class of widely expressed noncoding RNA and have been implicated in regulation of gene expression. However, the function of the majority of circRNAs remains unknown. Studies of circRNAs have been hampered by a lack of essential approaches for detection, quantification and visualization. We therefore developed a target-enrichment sequencing method suitable for screening of circRNAs and their linear counterparts in large number of samples. We also applied padlock probes and in situ sequencing to visualize and determine circRNA localization in human brain tissue at subcellular levels. We measured circRNA abundance across different human samples and tissues. Our results highlight the potential of this RNA class to act as a specific diagnostic marker in blood and serum, by detection of circRNAs from genes exclusively expressed in the brain. The powerful and scalable tools we present will enable studies of circRNA function and facilitate screening of circRNA as diagnostic biomarkers.
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