| 1. |
- Asp, Michaela, et al.
(författare)
-
Spatial detection of fetal marker genes expressed at low level in adult human heart tissue
- 2017
-
Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Heart failure is a major health problem linked to poor quality of life and high mortality rates. Hence, novel biomarkers, such as fetal marker genes with low expression levels, could potentially differentiate disease states in order to improve therapy. In many studies on heart failure, cardiac biopsies have been analyzed as uniform pieces of tissue with bulk techniques, but this homogenization approach can mask medically relevant phenotypes occurring only in isolated parts of the tissue. This study examines such spatial variations within and between regions of cardiac biopsies. In contrast to standard RNA sequencing, this approach provides a spatially resolved transcriptome- and tissue-wide perspective of the adult human heart, and enables detection of fetal marker genes expressed by minor subpopulations of cells within the tissue. Analysis of patients with heart failure, with preserved ejection fraction, demonstrated spatially divergent expression of fetal genes in cardiac biopsies.
|
|
| 2. |
- Das, Sarbashis, et al.
(författare)
-
Transcriptomics of cardiac biopsies reveals differences in patients with or without diagnostic parameters for heart failure with preserved ejection fraction
- 2019
-
Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Heart failure affects 2-3% of adult Western population. Prevalence of heart failure with preserved left ventricular (LV) ejection fraction (HFpEF) increases. Studies suggest HFpEF patients to have altered myocardial structure and functional changes such as incomplete relaxation and increased cardiac stiffness. We hypothesised that patients undergoing elective coronary bypass surgery (CABG) with HFpEF characteristics would show distinctive gene expression compared to patients with normal LV physiology. Myocardial biopsies for mRNA expression analysis were obtained from sixteen patients with LV ejection fraction >= 45%. Five out of 16 patients (31%) had echocardiographic characteristics and increased NTproBNP levels indicative of HFpEF and this group was used as HFpEF proxy, while 11 patients had Normal LV physiology. Utilising principal component analysis, the gene expression data clustered into two groups, corresponding to HFpEF proxy and Normal physiology, and 743 differentially expressed genes were identified. The associated top biological functions were cardiac muscle contraction, oxidative phosphorylation, cellular remodelling and matrix organisation. Our results also indicate that upstream regulatory events, including inhibition of transcription factors STAT4, SRF and TP53, and activation of transcription repressors HEY2 and KDM5A, could provide explanatory mechanisms to observed gene expression differences and ultimately cardiac dysfunction in the HFpEF proxy group.
|
|
| 3. |
- Hu, Yue O. O., et al.
(författare)
-
Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systems
- 2018
-
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Urban sewer systems consist of wastewater and stormwater sewers, of which only wastewater is processed before being discharged. Occasionally, misconnections or damages in the network occur, resulting in untreated wastewater entering natural water bodies via the stormwater system. Cultivation of faecal indicator bacteria (e.g. Escherichia coli; E. coli) is the current standard for tracing wastewater contamination. This method is cheap but has limited specificity and mobility. Here, we compared the E. coli culturing approach with two sequencing-based methodologies (Illumina MiSeq 16S rRNA gene amplicon sequencing and Oxford Nanopore MinION shotgun metagenomic sequencing), analysing 73 stormwater samples collected in Stockholm. High correlations were obtained between E. coli culturing counts and frequencies of human gut microbiome amplicon sequences, indicating E. coli is indeed a good indicator of faecal contamination. However, the amplicon data further holds information on contamination source or alternatively how much time has elapsed since the faecal matter has entered the system. Shotgun metagenomic sequencing on a subset of the samples using a portable real-time sequencer, MinION, correlated well with the amplicon sequencing data. This study demonstrates the use of DNA sequencing to detect human faecal contamination in stormwater systems and the potential of tracing faecal contamination directly in the field.
|
|
| 4. |
- Jemt, Anders, 1985-, et al.
(författare)
-
An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries.
- 2016
-
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- Sequencing the nucleic acid content of individual cells or specific biological samples is becoming increasingly common. This drives the need for robust, scalable and automated library preparation protocols. Furthermore, an increased understanding of tissue heterogeneity has lead to the development of several unique sequencing protocols that aim to retain or infer spatial context. In this study, a protocol for retaining spatial information of transcripts has been adapted to run on a robotic workstation. The method spatial transcriptomics is evaluated in terms of robustness and variability through the preparation of reference RNA, as well as through preparation and sequencing of six replicate sections of a gingival tissue biopsy from a patient with periodontitis. The results are reduced technical variability between replicates and a higher throughput, processing four times more samples with less than a third of the hands on time, compared to the standard protocol.
|
|
| 5. |
- Järver, Peter, et al.
(författare)
-
Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis
- 2018
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream ofTLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses.
|
|
| 6. |
- Kvastad, Linda, et al.
(författare)
-
Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring
- 2015
-
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Single cell analysis techniques have great potential in the cancer genomics feld. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.
|
|
| 7. |
- Lundin, Sverker, et al.
(författare)
-
Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing
- 2013
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 3, s. 1186-
-
Tidskriftsartikel (refereegranskat)abstract
- Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.
|
|
| 8. |
- Lundmark, A., et al.
(författare)
-
Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics
- 2018
-
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- Periodontitis is a highly prevalent chronic inflammatory disease of the periodontium, leading ultimately to tooth loss. In order to characterize the gene expression of periodontitis-affected gingival tissue, we have here simultaneously quantified and localized gene expression in periodontal tissue using spatial transcriptomics, combining RNA sequencing with histological analysis. Our analyses revealed distinct clusters of gene expression, which were identified to correspond to epithelium, inflamed areas of connective tissue, and non-inflamed areas of connective tissue. Moreover, 92 genes were identified as significantly up-regulated in inflamed areas of the gingival connective tissue compared to non-inflamed tissue. Among these, immunoglobulin lambda-like polypeptide 5 (IGLL5), signal sequence receptor subunit 4 (SSR4), marginal zone B and B1 cell specific protein (MZB1), and X-box binding protein 1 (XBP1) were the four most highly up-regulated genes. These genes were also verified as significantly higher expressed in gingival tissue of patients with periodontitis compared to healthy controls, using reverse transcription quantitative polymerase chain reaction. Moreover, the protein expressions of up-regulated genes were verified in gingival biopsies by immunohistochemistry. In summary, in this study, we report distinct gene expression signatures within periodontitis-affected gingival tissue, as well as specific genes that are up-regulated in inflamed areas compared to non-inflamed areas of gingival tissue. The results obtained from this study may add novel information on the genes and cell types contributing to pathogenesis of the chronic inflammatory disease periodontitis.
|
|
| 9. |
- Lundmark, Anna, et al.
(författare)
-
Transcriptome analysis reveals mucin 4 to be highly associated with periodontitis and identifies pleckstrin as a link to systemic diseases
- 2015
-
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- The multifactorial chronic inflammatory disease periodontitis, which is characterized by destruction of tooth-supporting tissues, has also been implicated as a risk factor for various systemic diseases. Although periodontitis has been studied extensively, neither disease-specific biomarkers nor therapeutic targets have been identified, nor its link with systemic diseases. Here, we analyzed the global transcriptome of periodontitis and compared its gene expression profile with those of other inflammatory conditions, including cardiovascular disease (CVD), rheumatoid arthritis (RA), and ulcerative colitis (UC). Gingival biopsies from 62 patients with periodontitis and 62 healthy subjects were subjected to RNA sequencing. The up-regulated genes in periodontitis were related to inflammation, wounding and defense response, and apoptosis, whereas down-regulated genes were related to extracellular matrix organization and structural support. The most highly up-regulated gene was mucin 4 (MUC4), and its protein product was confirmed to be over-expressed in periodontitis. When comparing the expression profile of periodontitis with other inflammatory diseases, several gene ontology categories, including inflammatory response, cell death, cell motion, and homeostatic processes, were identified as common to all diseases. Only one gene, pleckstrin (PLEK), was significantly overexpressed in periodontitis, CVD, RA, and UC, implicating this gene as an important networking link between these chronic inflammatory diseases.
|
|
| 10. |
- Sandberg, Julia, et al.
(författare)
-
Rapid flow-sorting to simultaneously resolve multiplex massively parallel sequencing products
- 2011
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 1:108
-
Tidskriftsartikel (refereegranskat)abstract
- Sample preparation for Roche/454, ABI/SOLiD and Life Technologies/Ion Torrent sequencing are based on amplification of library fragments on the surface of beads prior to sequencing. Commonly, libraries are barcoded and pooled, to maximise the sequence output of each sequence run. Here, we describe a novel approach for normalization of multiplex next generation sequencing libraries after emulsion PCR. Briefly, amplified libraries carrying unique barcodes are prepared by fluorescent tagging of complementary sequences and then resolved by high-speed flow cytometric sorting of labeled emulsion PCR beads. The protocol is simple and provides an even sequence distribution of multiplex libraries when sequencing the flow-sorted beads. Moreover, since many empty and mixed emulsion PCR beads are removed, the approach gives rise to a substantial increase in sequence quality and mean read length, as compared to that obtained by standard enrichment protocols.
|
|
