| 1. |
- Marco Salas, Sergio, et al.
(författare)
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De novo spatiotemporal modelling of cell-type signatures in the developmental human heart using graph convolutional neural networks
- 2022
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Ingår i: PloS Computational Biology. - : Public Library of Science (PLoS). - 1553-734X .- 1553-7358. ; 18:8
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Tidskriftsartikel (refereegranskat)abstract
- With the emergence of high throughput single cell techniques, the understanding of the molecular and cellular diversity of mammalian organs have rapidly increased. In order to understand the spatial organization of this diversity, single cell data is often integrated with spatial data to create probabilistic cell maps. However, targeted cell typing approaches relying on existing single cell data achieve incomplete and biased maps that could mask the true diversity present in a tissue slide. Here we applied a de novo technique to spatially resolve and characterize cellular diversity of in situ sequencing data during human heart development. We obtained and made accessible well defined spatial cell-type maps of fetal hearts from 4.5 to 9 post conception weeks, not biased by probabilistic cell typing approaches. With our analysis, we could characterize previously unreported molecular diversity within cardiomyocytes and epicardial cells and identified their characteristic expression signatures, comparing them with specific subpopulations found in single cell RNA sequencing datasets. We further characterized the differentiation trajectories of epicardial cells, identifying a clear spatial component on it. All in all, our study provides a novel technique for conducting de novo spatial-temporal analyses in developmental tissue samples and a useful resource for online exploration of cell-type differentiation during heart development at sub-cellular image resolution.
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| 2. |
- Neumann, Felix, 1989-
(författare)
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Advancing isothermal nucleic acid amplification tests : Towards democratization of diagnostics
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Modern healthcare is the result of scientific advancement across disciplines and has enabled us to understand the rationale behind many diseases and how to treat or cure them; but still a myriad of unanswered questions remains. Especially infectious diseases play an important role in healthcare as they pose a constant threat for global health and well-being. This was painfully highlighted in this year's ongoing COVID-19 pandemic with more than 40 million people infected and over 1 million deaths. Pandemics like this have not only devastating effects on global health but also economy.Therefore, scientific research in the field of infectious diseases is paramount to ensure outbreak control and surveillance of emerging threats. Current healthcare relies heavily on the diagnosis of infectious diseases in centralized healthcare centers thereby overlooking the access of molecular diagnostics for other areas such as airports, home-testing and especially the developing world with its limited resources. Towards this, various isothermal nucleic acid amplification technologies have been developed that hold the promise to bring state-of-the-art molecular diagnostics into these areas as they are versatile, sensitive and specific, and cost-effective. One such technique is rolling circle amplification which was used in this thesis.This research work provides an overview of the developments in biochemistry, related disciplines and their combination to design methods for diagnostic platforms tackling infectious diseases. The studies conducted in this work can be considered as individual modules for addressing challenges, like typing of pathogens and disease-related antibodies, and inexpensive bulk as well as digital quantification and simplified assay schemes. These approaches and their combinations aim to bring rolling circle amplification-based assay schemes into the molecular diagnostic field and towards decentralized healthcare.
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| 3. |
- Soares, Ruben R. G., et al.
(författare)
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Circle-to-circle amplification coupled with microfluidic affinity chromatography enrichment for in vitro molecular diagnostics of Zika fever and analysis of anti-flaviviral drug efficacy
- 2021
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Ingår i: Sensors and actuators. B, Chemical. - : Elsevier BV. - 0925-4005 .- 1873-3077. ; 336
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Tidskriftsartikel (refereegranskat)abstract
- Sensitive viral diagnostic methods are increasingly in demand to tackle emerging epidemics. The Zika virus (ZIKV) is particularly relevant in tropical resource limited settings (RLS) and is associated with intermittent epidemics such as the recent 2016 ZIKV outbreak in South America, wherein Zika fever was classified by WHO as a public health emergency of international concern. Thus, there is an urgent need for widespread Zika fever diagnostics and efficient drug therapies. ZIKV diagnostics are typically performed using RT-qPCR in centralized laboratories. While extremely sensitive, RT-qPCR requires rapid heating-cooling cycles, combined with continuous fluorescence measurements to allow quantification, implying high costs and limiting availability of molecular diagnostics in RLS. Here, we report isothermal amplification of ZIKV cDNA using padlock probes followed by two rounds of Rolling Circle Amplification (RCA), termed as circle-to-circle amplification (C2CA), combined with a microfluidic affinity chromatography enrichment (mu ACE) platform. This platform allowed the detection of <17 vRNA copies per reaction mixture, equivalent to similar to 3 aM, showed a positive correlation with RT-qPCR in both average (r = 0.80) and discrete (r = 0.95) signal modes, and was validated for drug efficiency tests using in vitro infected peripheral blood mononuclear cells from 3 healthy donors. This performance shows significant promise towards highly sensitive, albeit simple and cost-effective point-of-care viral diagnostics.
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| 4. |
- Lundin, Elin, 1983-
(författare)
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RNA-based spatial characterization of cell and tissue heterogeneity
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Technical advances in cell biology have revolutionized the field of cell biology. With new technology it is now possible to address scientific questions in cell biology at the molecular level. Single-cell RNA-sequencing can reveal transcriptomic information for single cells and spatially resolved transcriptomic technology can visualize thousands or millions of cells and transcripts for spatial molecular profiling. The work in this thesis describes the technological development from traditional in situ hybridization to the current state-of-the-art technology for spatial multiplexed gene expression analysis. This development has enabled RNA-based molecular characterization of cells and tissues with the spatial dimension maintained. The work included in the thesis highlights the potential and the advantages of padlock-probe-based technology for spatial RNA-based profiling of cells and tissues. Furthermore, it demonstrates the possibilities arising from the inherent ability of padlock probes to distinguish between transcripts based on differences in single nucleotides.The study in paper I investigates the prevalence of Enterovirus species B in patients with Crohn’s disease by a chromogenic in situ hybridization assay combined with immunohistochemistry to detect viral RNA and proteins directly in tissue samples.In paper II, padlock probes were used to study the spatial gene expression of gene homologs from the X and Y chromosome in human embryonic nervous tissue. Furthermore, a strategy was devised to visualize and evaluate spatial expression patterns.The padlock probe-based approach for multiplexed spatial transcriptional profiling, in situ sequencing, was applied in paper III to study the regional and cell-type-specific dynamics of A-to-I RNA editing in the developing mouse brain.In paper IV, a technical characterization of padlock probes was performed with the aim of determining how to design a padlock probe to obtain optimal detection efficiency.The work in this thesis demonstrates the dramatic shift in how biological questions in cell and tissue biology can be addressed, enabled by the technological evolution of traditional in situ hybridization assays into high-throughput, multiplexed spatial transcription profiling.
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| 5. |
- Qian, Xiaoyan, et al.
(författare)
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Probabilistic cell typing enables fine mapping of closely related cell types in situ
- 2020
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 17:1, s. 101-106
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the function of a tissue requires knowing the spatial organization of its constituent cell types. In the cerebral cortex, single-cell RNA sequencing (scRNA-seq) has revealed the genome-wide expression patterns that define its many, closely related neuronal types, but cannot reveal their spatial arrangement. Here we introduce probabilistic cell typing by in situ sequencing (pciSeq), an approach that leverages prior scRNA-seq classification to identify cell types using multiplexed in situ RNA detection. We applied this method by mapping the inhibitory neurons of mouse hippocampal area CA1, for which ground truth is available from extensive prior work identifying their laminar organization. Our method identified these neuronal classes in a spatial arrangement matching ground truth, and further identified multiple classes of isocortical pyramidal cell in a pattern matching their known organization. This method will allow identifying the spatial organization of closely related cell types across the brain and other tissues.
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| 6. |
- Soares, Ruben R. G., et al.
(författare)
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Sub-attomole detection of HIV-1 using padlock probes and rolling circle amplification combined with microfluidic affinity chromatography
- 2020
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 166
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Tidskriftsartikel (refereegranskat)abstract
- Despite significant progress in diagnostics and disease management during the past decades, human immunodeficiency virus (HIV) infections are still responsible for nearly 1 million deaths every year, mostly in resource-limited settings. Thus, novel, accurate and cost-effective tools for viral load monitoring become crucial to allow specific diagnostics and the effective monitoring of the associated antiviral therapies. Herein, we report an effective combination of a (1) padlock probe (PLP)-mediated rolling circle amplification (RCA) bioassay and an (2) agarose bead-based microfluidic device for the affinity chromatography-based capture and detection of RCA products (RCPs) pre-labelled simultaneously with biotin and an organic fluorophore. This method allowed the efficient capture of similar to 1 mu m-sized RCPs followed by their quantification either as discrete signals or an average fluorescence signal, thus being compatible with both high-resolution imaging for maximum sensitivity as well as simpler optical detection setups. A limit of detection < 30 fM was obtained for HIV-1 synthetic target with just a single round of RCA, comparable to recently reported procedures requiring technically complex amplification strategies such as hyperbranching and/or enzymatic digestion/amplification. Furthermore, targeting a set of five conserved regions in the HIV-1 gag gene, the method could specifically detect HIV-1 in 293T cell culture supernatants, as well as a set of 11 HIV-1 NIH reference samples with four different subtypes. The reported method provides simplicity of operation, unique versatility of signal transduction (i.e. average or discrete signals), and potential coupling with previously reported miniaturized photodetectors. These combined features hold promise for bringing RCA-based molecular diagnostics closer to the point-of-care.
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| 7. |
- Serk, Henrik, 1980-, et al.
(författare)
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CO2 fertilization of Sphagnum peat mosses is modulated by water table level and other environmental factors
- 2021
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Ingår i: Plant, Cell and Environment. - : John Wiley & Sons. - 0140-7791 .- 1365-3040. ; 44:6, s. 1756-1768
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Tidskriftsartikel (refereegranskat)abstract
- Sphagnum mosses account for most accumulated dead organic matter in peatlands. Therefore, understanding their responses to increasing atmospheric CO2 is needed for estimating peatland C balances under climate change. A key process is photorespiration: a major determinant of net photosynthetic C assimilation that depends on the CO2 to O2 ratio. We used climate chambers to investigate photorespiratory responses of Sphagnum fuscum hummocks to recent increases in atmospheric CO2 (from 280 to 400 ppm) under different water table, temperature, and light intensity levels. We tested the photorespiratory variability using a novel method based on deuterium isotopomers (D6S/D6R ratio) of photosynthetic glucose. The effect of elevated CO2 on photorespiration was highly dependent on water table. At low water table (−20 cm), elevated CO2 suppressed photorespiration relative to C assimilation, thus substantially increasing the net primary production potential. In contrast, a high water table (~0 cm) favored photorespiration and abolished this CO2 effect. The response was further tested for Sphagnum majus lawns at typical water table levels (~0 and −7 cm), revealing no effect of CO2 under those conditions. Our results indicate that hummocks, which typically experience low water table levels, benefit from the 20th century's increase in atmospheric CO2.
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| 8. |
- Soares, Ruben R. G., et al.
(författare)
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Rolling Circle Amplification in Integrated Microsystems : An Uncut Gem toward Massively Multiplexed Pathogen Diagnostics and Genotyping
- 2021
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Ingår i: Accounts of Chemical Research. - : American Chemical Society (ACS). - 0001-4842 .- 1520-4898. ; 54:21, s. 3979-3990
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Forskningsöversikt (refereegranskat)abstract
- The development of robust methods allowing the precise detection of specific nucleic acid sequences is of major societal relevance, paving the way for significant advances in biotechnology and biomedical engineering. These range from a better understanding of human disease at a molecular level, allowing the discovery and development of novel biopharmaceuticals and vaccines, to the improvement of biotechnological processes providing improved food quality and safety, efficient green fuels, and smart textiles. Among these applications, the significance of pathogen diagnostics as the main focus of this Account has become particularly clear during the recent SARS-CoV-2 pandemic. In this context, while RT-PCR is the gold standard method for unambiguous detection of genetic material from pathogens, other isothermal amplification alternatives circumventing rapid heating-cooling cycles up to similar to 95 degrees C are appealing to facilitate the translation of the assay into point-of-care (PoC) analytical platforms. Furthermore, the possibility of routinely multiplexing the detection of tens to hundreds of target sequences with single base pair specificity, currently not met by state-of-the-art methods available in clinical laboratories, would be instrumental along the path to tackle emergent viral variants and antimicrobial resistance genes. Here, we advocate that padlock probes (PLPs), first reported by Nilsson et al. in 1994, coupled with rolling circle amplification (RCA), termed here as PLP-RCA, is an underexploited technology in current arena of isothermal nucleic acid amplification tests (NAATs) providing an unprecedented degree of multiplexing, specificity, versatility, and amenability to integration in miniaturized PoC platforms. Furthermore, the intrinsically digital amplification of PLP-RCA retains spatial information and opens new avenues in the exploration of pathogenesis with spatial multiomics analysis of infected cells and tissue. The Account starts by introducing PLP-RCA in a nutshell focusing individually on the three main assay steps, namely, (1) PLP design and ligation mechanism, (2) RCA after probe ligation, and (3) detection of the RCA products. Each subject is touched upon succinctly but with sufficient detail for the reader to appreciate some assay intricacies and degree of versatility depending on the analytical challenge at hand. After familiarizing the reader with the method, we discuss specific examples of research in our group and others using PLP-RCA for viral, bacterial, and fungal diagnostics in a variety of clinical contexts, including the genotyping of antibiotic resistance genes and viral subtyping. Then, we dissect key developments in the miniaturization and integration of PLPRCA to minimize user input, maximize analysis throughput, and expedite the time to results, ultimately aiming at PoC applications. These developments include molecular enrichment for maximum sensitivity, spatial arrays to maximize analytical throughput, automation of liquid handling to streamline the analytical workflow in miniaturized devices, and seamless integration of signal transduction to translate RCA product titers (and ideally spatial information) into a readable output. Finally, we position PLP-RCA in the current landscape of NAATs and furnish a systematic Strengths, Weaknesses, Opportunities and Threats analysis to shine light upon unpolished edges to uncover the gem with potential for ubiquitous, precise, and unbiased pathogen diagnostics.
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