| 1. |
- Ciftci, Sibel, et al.
(författare)
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A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The establishment of a robust detection platform for RNA viruses still remains a challenge in molecular diagnostics due to their high mutation rates. Newcastle disease virus (NDV) is one such RNA avian virus with a hypervariable genome and multiple genotypes. Classical approaches like virus isolation, serology, immunoassays and RT-PCR are cumbersome, and limited in terms of specificity and sensitivity. Padlock probes (PLPs) are known for allowing the detection of multiple nucleic acid targets with high specificity, and in combination with Rolling circle amplification (RCA) have permitted the development of versatile pathogen detection assays. In this work, we aimed to detect hypervariable viruses by developing a novel PLP design strategy capable of tolerating mutations while preserving high specificity by targeting several moderately conserved regions and using degenerate bases. For this, we designed nine padlock probes based on the alignment of 335 sequences covering both Class I and II NDV. Our PLP design showed high coverage and specificity for the detection of eight out of ten reported genotypes of Class II NDV field isolated strains, yielding a detection limit of less than ten copies of viral RNA. Further taking advantage of the multiplex capability of PLPs, we successfully extended the assay for the simultaneous detection of three poultry RNA viruses (NDV, IBV and AIV) and combined it with a paper based microfluidic enrichment read-out for digital quantification. In summary, our novel PLP design addresses the current issue of tolerating mutations of highly emerging virus strains with high sensitivity and specificity.
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| 2. |
- Clausson, Carl-Magnus, 1985-, et al.
(författare)
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Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
- 2015
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5, s. 12317:1-10
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Tidskriftsartikel (refereegranskat)abstract
- Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.
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| 3. |
- Lee, Hower, et al.
(författare)
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Direct RNA targeted in situ sequencing for transcriptomic profiling in tissue
- 2022
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Highly multiplexed spatial mapping of transcripts within tissues allows for investigation of the transcriptomic and cellular diversity of mammalian organs previously unseen. Here we explore a direct RNA (dRNA) detection approach incorporating the use of padlock probes and rolling circle amplification in combination with hybridization-based in situ sequencing chemistry. We benchmark a High Sensitivity Library Preparation Kit from CARTANA that circumvents the reverse transcription needed for cDNA-based in situ sequencing (ISS) via direct RNA detection. We found a fivefold increase in transcript detection efficiency when compared to cDNA-based ISS and also validated its multiplexing capability by targeting a curated panel of 50 genes from previous publications on mouse brain sections, leading to additional data interpretation such as de novo cell clustering. With this increased efficiency, we also found to maintain specificity, multiplexing capabilities and ease of implementation. Overall, the dRNA chemistry shows significant improvements in target detection efficiency, closing the gap to other fluorescent in situ hybridization-based technologies and opens up possibilities to explore new biological questions previously not possible with cDNA-based ISS.
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| 4. |
- Maïno, Nicolas, et al.
(författare)
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A microfluidic platform towards automated multiplexed in situ sequencing
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Advancements in multiplexed in situ RNA profiling techniques have given unprecedented insight into spatial organization of tissues by enabling single-molecule quantification and sub-micron localization of dozens to thousands of RNA species simultaneously in cells and entire tissue sections. However, the lack of automation of the associated complex experimental procedures represents a potential hurdle towards their routine use in laboratories. Here, we demonstrate an approach towards automated generation and sequencing of barcoded mRNA amplicons in situ, directly in fixed cells. This is achieved through adaptation of a microfluidic tool compatible with standard microscope slides and cover glasses. The adapted tool combines a programmable reagent delivery system with temperature controller and flow cell to perform established in situ sequencing protocols, comprising hybridization and ligation of gene-specific padlock probes, rolling circle amplification of the probes yielding barcoded amplicons and identification of amplicons through barcode sequencing. By adapting assay parameters (e.g. enzyme concentration and temperature), we achieve a near-identical performance in identifying mouse beta-actin transcripts, in comparison with the conventional manual protocol. The technically adapted assay features i) higher detection efficiency, ii) shorter protocol time, iii) lower consumption of oligonucleotide reagents but slightly more enzyme. Such an automated microfluidic tissue processor for in situ sequencing studies would greatly enhance its research potentials especially for cancer diagnostics, thus paving way to rapid and effective therapies.
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| 5. |
- Osterwalder, Stefan, et al.
(författare)
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Mercury evasion from a boreal peatland shortens the timeline for recovery from legacy pollution
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Peatlands are a major source of methylmercury that contaminates downstream aquatic food webs. The large store of mercury (Hg) in peatlands could be a source of Hg for over a century even if deposition is dramatically reduced. However, the reliability of Hg mass balances can be questioned due to missing long-term land-atmosphere flux measurements. We used a novel micrometeorological system for continuous measurement of Hg peatland-atmosphere exchange to derive the first annual Hg budget for a peatland. The evasion of Hg (9.4 mu g m(-2) yr(-1)) over the course of a year was seven times greater than stream Hg export, and over two times greater than wet bulk deposition to the boreal peatland. Measurements of dissolved gaseous Hg in the peat pore water also indicate Hg evasion. The net efflux may result from recent declines in atmospheric Hg concentrations that have turned the peatland from a net sink into a source of atmospheric Hg. This net Hg loss suggests that open boreal peatlands and downstream ecosystems can recover more rapidly from past atmospheric Hg deposition than previously assumed. This has important implications for future levels of methylmercury in boreal freshwater fish and the estimation of historical Hg accumulation rates from peat profiles.
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| 6. |
- Peichl, Matthias, et al.
(författare)
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Peatland vegetation composition and phenology drive the seasonal trajectory of maximum gross primary production
- 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
- Gross primary production (GPP) is a key driver of the peatland carbon cycle. Although many studies have explored the apparent GPP under natural light conditions, knowledge of the maximum GPP at lightsaturation (GPP(max)) and its spatio-temporal variation is limited. This information, however, is crucial since GPP(max) essentially constrains the upper boundary for apparent GPP. Using chamber measurements combined with an external light source across experimental plots where vegetation composition was altered through long-term (20-year) nitrogen addition and artificial warming, we could quantify GPP(max) in-situ and disentangle its biotic and abiotic controls in a boreal peatland. We found large spatial and temporal variations in the magnitudes of GPP(max) which were related to vegetation species composition and phenology rather than abiotic factors. Specifically, we identified vegetation phenology as the main driver of the seasonal GPP(max) trajectory. Abiotic anomalies (i.e. in air temperature and water table level), however, caused species-specific divergence between the trajectories of GPP(max) and plant development. Our study demonstrates that photosynthetically active biomass constrains the potential peatland photosynthesis while abiotic factors act as secondary modifiers. This further calls for a better representation of species-specific vegetation phenology in process-based peatland models to improve predictions of global change impacts on the peatland carbon cycle.
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| 7. |
- Serk, Henrik, 1980-, et al.
(författare)
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Global CO2 fertilization of Sphagnum peat mosses via suppression of photorespiration during the twentieth century
- 2021
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Natural peatlands contribute significantly to global carbon sequestration and storage of biomass, most of which derives from Sphagnum peat mosses. Atmospheric CO2 levels have increased dramatically during the twentieth century, from 280 to > 400 ppm, which has affected plant carbon dynamics. Net carbon assimilation is strongly reduced by photorespiration, a process that depends on the CO2 to O2 ratio. Here we investigate the response of the photorespiration to photosynthesis ratio in Sphagnum mosses to recent CO2 increases by comparing deuterium isotopomers of historical and contemporary Sphagnum tissues collected from 36 peat cores from five continents. Rising CO2 levels generally suppressed photorespiration relative to photosynthesis but the magnitude of suppression depended on the current water table depth. By estimating the changes in water table depth, temperature, and precipitation during the twentieth century, we excluded potential effects of these climate parameters on the observed isotopomer responses. Further, we showed that the photorespiration to photosynthesis ratio varied between Sphagnum subgenera, indicating differences in their photosynthetic capacity. The global suppression of photorespiration in Sphagnum suggests an increased net primary production potential in response to the ongoing rise in atmospheric CO2, in particular for mire structures with intermediate water table depths.
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| 8. |
- Yasui, Takao, et al.
(författare)
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Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications.
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| 9. |
- 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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