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  • 1. Ahrentorp, Fredrik
    et al.
    Blomgren, Jakob
    Jonasson, Christian
    Sarwe, Anna
    Sepehri, Sobhan
    Eriksson, Emil
    Kalaboukhov, Alexei
    Jesorka, Aldo
    Winkler, Dag
    Schneiderman, Justin F.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Albert, Jan
    Gómez de la Torre, Teresa Zardán
    Strømme, Maria
    Johansson, Christer
    Sensitive magnetic biodetection using magnetic multi-core nanoparticles and RCA coils2017In: Journal of Magnetism and Magnetic Materials, ISSN 0304-8853, E-ISSN 1873-4766, Vol. 427, p. 14-18Article in journal (Refereed)
    Abstract [en]

    We use functionalized iron oxide magnetic multi-core particles of 100 nm in size (hydrodynamic particle diameter) and AC susceptometry (ACS) methods to measure the binding reactions between the magnetic nanoparticles (MNPs) and bio-analyte products produced from DNA segments using the rolling circle amplification (RCA) method. We use sensitive induction detection techniques in order to measure the ACS response. The DNA is amplified via RCA to generate RCA coils with a specific size that is dependent on the amplification time. After about 75 min of amplification we obtain an average RCA coil diameter of about 1 mu m. We determine a theoretical limit of detection (LOD) in the range of 11 attomole (corresponding to an analyte concentration of 55 fM for a sample volume of 200 mu L) from the ACS dynamic response after the MNPs have bound to the RCA coils and the measured ACS readout noise. We also discuss further possible improvements of the LOD.

  • 2. Asp, Michaela
    et al.
    Giacomello, Stefania
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Larsson, Ludvig
    Wu, Chenglin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Fürth, Daniel
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wärdell, Eva
    Custodio, Joaquin
    Reimegård, Johan
    Salmén, Fredrik
    Österholm, Cecilia
    Ståhl, Patrik L.
    Sundström, Erik
    Åkesson, Elisabet
    Bergmann, Olaf
    Bienko, Magda
    Månsson-Broberg, Agneta
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Sylvén, Christer
    Lundeberg, Joakim
    A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart2019In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 179, no 7, p. 1647-1660Article in journal (Refereed)
    Abstract [en]

    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. Ayoglu, Burcu
    et al.
    Birgersson, Elin
    Mezger, Anja
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Uhlén, Mathias
    Nilsson, Peter
    Schwenk, Jochen M.
    Multiplexed protein profiling by sequential affinity capture2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 8, p. 1251-1256Article in journal (Refereed)
    Abstract [en]

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capturebead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond.

  • 4. Barisic, Ivan
    et al.
    Schoenthaler, Silvia
    Ke, Rongqin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Noehammer, Christa
    Wiesinger-Mayr, Herbert
    Multiplex detection of antibiotic resistance genes using padlock probes2013In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 77, no 2, p. 118-125Article in journal (Refereed)
    Abstract [en]

    The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse beta-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the beta-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10(4) DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of beta-lactamase genes in dilutions of 10(7) cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.

  • 5. Beghini, Alessandro
    et al.
    Corlazzoli, Francesca
    Del Giacco, Luca
    Re, Matteo
    Lazzaroni, Francesca
    Brioschi, Matteo
    Valentini, Giorgio
    Ferrazzi, Fulvia
    Ghilardi, Anna
    Righi, Marco
    Turrini, Mauro
    Mignardi, Marco
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Cesana, Clara
    Bronte, Vincenzo
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Morra, Enrica
    Cairoli, Roberto
    Regeneration-associated WNT Signaling Is Activated in Long-term Reconstituting AC133(bright) Acute Myeloid Leukemia Cells2012In: Neoplasia, ISSN 1522-8002, E-ISSN 1476-5586, Vol. 14, no 12, p. 1236-+Article in journal (Refereed)
    Abstract [en]

    Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/beta-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT 10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated beta-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)gamma c(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration. Neoplasia (2012) 14, 1236-1248

  • 6. Ben Aissa, Alejandra
    et al.
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Indian Institute of Technology, India.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Pividori, Maria Isabel
    Electrochemical Genosensing of E. coli Based on Padlock Probes and Rolling Circle Amplification2021In: Sensors, E-ISSN 1424-8220, Vol. 21, no 5, article id 1749Article in journal (Refereed)
    Abstract [en]

    Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of E. coli based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment.

  • 7. Boije, Henrik
    et al.
    Ring, Henrik
    Fard, Shahrzad Shirazi
    Grundberg, Ida
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University .
    Hallbook, Finn
    Alternative Splicing of the Chromodomain Protein Morf4l1 Pre-mRNA Has Implications on Cell Differentiation in the Developing Chicken Retina2013In: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 51, no 2, p. 615-628Article in journal (Refereed)
    Abstract [en]

    The proliferation, cell cycle exit and differentiation of progenitor cells are controlled by several different factors. The chromodomain protein mortality factor 4-like 1 (Morf4l1) has been ascribed a role in both proliferation and differentiation. Little attention has been given to the existence of alternative splice variants of the Morf4l1 mRNA, which encode two Morf41l isoforms: a short isoform (S-Morf4l1) with an intact chromodomain and a long isoform (L-Morf4l1) with an insertion in or in the vicinity of the chromodomain. The aim of this study was to investigate if this alternative splicing has a function during development. We analysed the temporal and spatial distribution of the two mRNAs and over-expressed both isoforms in the developing retina. The results showed that the S-Morf4l1 mRNA is developmentally regulated. Over-expression of S-Morf4l1 using a retrovirus vector produced a clear phenotype with an increase of early-born neurons: retinal ganglion cells, horizontal cells and cone photoreceptor cells. Over-expression of L-Morf4l1 did not produce any distinguishable phenotype. The over-expression of S-Morf4l1 but not L-Morf4l1 also increased apoptosis in the infected regions. Our results suggest that the two Morf4l1 isoforms have different functions during retinogenesis and that Morf4l1 functions are fine-tuned by developmentally regulated alternative splicing. The data also suggest that Morf4l1 contributes to the regulation of cell genesis in the retina.

  • 8. Carinelli, S.
    et al.
    Kühnemund, M.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Pividori, M. I.
    Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification2017In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 93, p. 65-71Article in journal (Refereed)
    Abstract [en]

    This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries.

  • 9. Carow, Berit
    et al.
    Hauling, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kramnik, Igor
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Rottenberg, Martin E.
    Spatial and temporal localization of immune transcripts defines hallmarks and diversity in the tuberculosis granuloma2019In: Nature Communications, E-ISSN 2041-1723, Vol. 10, article id 1823Article in journal (Refereed)
    Abstract [en]

    Granulomas are the pathological hallmark of tuberculosis (TB) and the niche where bacilli can grow and disseminate or the immunological microenvironment in which host cells interact to prevent bacterial dissemination. Here we show 34 immune transcripts align to the morphology of lung sections from Mycobacterium tuberculosis-infected mice at cellular resolution. Colocalizing transcript networks at <10 mu m in C57BL/6 mouse granulomas increase complexity with time after infection. B-cell clusters develop late after infection. Transcripts from activated macrophages are enriched at subcellular distances from M. tuberculosis. Encapsulated C3HeB/FeJ granulomas show necrotic centers with transcripts associated with immunosuppression (Foxp3, Il10), whereas those in the granuloma rims associate with activated T cells and macrophages. We see highly diverse networks with common interactors in similar lesions. Different immune landscapes of M. tuberculosis granulomas depending on the time after infection, the histopathological features of the lesion, and the proximity to bacteria are here defined.

  • 10.
    Ciftci, Sibel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Canovas, Rocio
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Paulraj, Thomas
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Crespo, Gaston A.
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    The sweet detection of rolling circle amplification: Glucose-based electrochemical genosensor for the detection of viral nucleic acid2020In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 151, article id 112002Article in journal (Refereed)
    Abstract [en]

    Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125 mu g mL(-1) of GOx with 5 mM glucose using phosphate buffer saline (PBS), monitored for 1 mM were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings.

  • 11.
    Ciftci, Sibel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Abdurahman, Samir
    Appelberg, K. Sofia
    Mirazimi, Au
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Digital Rolling Circle Amplification-Based Detection of Ebola and Other Tropical Viruses2020In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 22, no 2, p. 272-283Article in journal (Refereed)
    Abstract [en]

    Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent in 2018 to 2019 in Congo. Thus, immediate diagnostic attention is demanded at the point of care in resource-limited settings, because the performance and the operational parameters of conventional EBOV testing are Limited. Especially, their sensitivity, specificity, and coverage of other tropical disease viruses make them unsuitable for diagnostic at the point of care. Here, a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV is presented. For this, a set of PLPs, separately targeting the viral RNA and complementary RNA of all seven EBOV genes, was used in the RCA assay and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous detection of both EBOV RNA types was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. Because PLPs and RCA are inherently multiplexable, we demonstrate the extension of the probe panel for the simultaneous detection of the tropical viruses Ebola, Zika, and Dengue. The demonstrated high specificity, sensitivity, and multiplexing capability in combination with the digital quantification rendered the assay a promising diagnostic tool toward tropical virus detection at the point of care.

  • 12.
    Ciftci, Sibel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Abdurahman, Samir
    Appelberg, Sofia
    Mirazimi, Ali
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Multiplexed rolling circle amplification detection of Ebola, Zika and Dengue towards point-of-care diagnosticsManuscript (preprint) (Other academic)
    Abstract [en]

    Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent 2018-19 outbreak in Congo. Immediate diagnostic attention is demanded, and most importantly at the point-of-care in resource-limited settings. The performance and the operational parameters of conventional EBOV testing are limited by either their sensitivity, specificity, or both, and often do not cover other tropical disease viruses. We present a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV from cell culture isolates as well as clinical samples obtained from patients of West Africa outbreak. For this, a set of PLPs, separately targeting the vRNA and cRNA of all the seven genes of EBOV, were used in the RCA and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous duplex detection of both EBOV vRNA and cRNA was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. As PLPs and RCA are inherently mulitplexable, we demonstrate the extension of the probe panel to the simultaneous detection of the tropical viruses Ebola, Zika and Dengue. The simple, rapid, specific and multiplexable isothermal assay developed for tropical virus detection suits the point-of-care needs, bringing RCA a step closer to bedside diagnostics.

  • 13.
    Ciftci, Sibel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hakhverdyan, Mikhayil
    Bálint, Ádám
    Herthnek, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses2019In: Scientific Reports, E-ISSN 2045-2322, Vol. 9, article id 2872Article in journal (Refereed)
    Abstract [en]

    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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  • 14.
    Ciftci, Sibel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Paulraj, Thomas
    Crespo, Gaston
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    The sweet detection of rolling circle amplification: Glucose-based electrochemical detection of virus nucleic acidManuscript (preprint) (Other academic)
    Abstract [en]

    Infectious diseases remain a constant threat on a global scale by recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralizing healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. Nucleic acid amplification technology (NAAT) has proven to be a powerful tool for the control of outbreak with high sensitivity and specificity. However, NAAT is mostly based on amplification methods that require specialized instrumentation and trained personnel, such as PCR with sophisticated detectors. Here, we present an isothermal padlock probe-based assay for the detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical approach as the read-out. The assay design is based on rolling circle amplification (RCA) upon magnetic beads, connecting the RCA products (RCPs) via streptavidin-biotin bridges to GOx needed for the electrochemical measurement with externally provided glucose. The RCPs forming on the surface of beads are imaged using scanning electron microscopy, and the presence of the GOx to the RCP complex is confirmed using atomic force microscopy. Parameters such as the choice of buffers, concentrations of glucose and GOx and measurement time were optimized, as well as the mode of addition of glucose was tested. 125 μg/mL of GOx with 5 mM glucose using PBS as washing buffer, monitored for 15 min were chosen as the optimized conditions. The effect of temperature was tested and found to be critical at 37 °C for enhanced performance of the sensor. Finally, we evaluate the analytical performance of our sensor system by using cell culture isolate and clinical samples of Ebola virus. The study provides a proof-of-concept of simple and portable molecular diagnostics for emerging pathogens, beneficial especially for resource-limited settings. 

  • 15. Clausson, Carl-Magnus
    et al.
    Arngården, Linda
    Ishaq, Omer
    Klaesson, Axel
    Kühnemund, Malte
    Grannas, Karin
    Koos, Björn
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ranefall, Petter
    Krzywkowski, Tomasz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Brismar, Hjalmar
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wählby, Carolina
    Söderberg, Ola
    Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio2015In: Scientific Reports, E-ISSN 2045-2322, Vol. 5, article id 12317Article in journal (Refereed)
    Abstract [en]

    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.

  • 16. de Miranda, Noel F. C. C.
    et al.
    Peng, Roujun
    Georgiou, Konstantinos
    Wu, Chenglin
    Sörqvist, Elin Falk
    Berglund, Mattias
    Chen, Longyun
    Gao, Zhibo
    Lagerstedt, Kristina
    Lisboa, Susana
    Roos, Fredrik
    van Wezel, Tom
    Teixeira, Manuel R.
    Rosenquist, Richard
    Sundström, Christer
    Enblad, Gunilla
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Zeng, Yixin
    Kipling, David
    Pan-Hammarström, Qiang
    DNA repair genes are selectively mutated in diffuse large B cell lymphomas2013In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 210, no 9, p. 1729-1742Article in journal (Refereed)
    Abstract [en]

    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.

  • 17. Donolato, Marco
    et al.
    Antunes, Paula
    Bejhed, Rebecca S.
    Gómez de la Torre, Teresa Zardán
    Österberg, Frederik W.
    Strömberg, Mattias
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Strømme, Maria
    Svedlindh, Peter
    Hansen, Mikkel F.
    Vavassori, Paolo
    Novel Readout Method for Molecular Diagnostic Assays Based on Optical Measurements of Magnetic Nanobead Dynamics2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 3, p. 1622-1629Article in journal (Refereed)
    Abstract [en]

    We demonstrate detection of DNA coils formed from a Vibrio cholerae DNA target at picomolar concentrations using a novel optomagnetic approach exploiting the dynamic behavior and optical anisotropy of magnetic nanobead (MNB) assemblies. We establish that the complex second harmonic optical transmission spectra of MNB suspensions measured upon application of a weak uniaxial AC magnetic field correlate well with the rotation dynamics of the individual MNBs. Adding a target analyte to the solution leads to the formation of permanent MNB clusters, namely, to the suppression of the dynamic MNB behavior. We prove that the optical transmission spectra are highly sensitive to the formation of permanent MNB clusters and, thereby to the target analyte concentration. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition and isothermal rolling circle amplification and benchmark against a commercial equipment. The results demonstrate the fast optomagnetic readout of rolling circle products from bacterial DNA utilizing the dynamic properties of MNBs in a miniaturized and low-cost platform requiring only a transparent window in the chip.

  • 18.
    Dou, Dan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wang, Hao
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Östbye, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Thiele, Swantje
    Resa-Infante, Patricia
    Mounogou Kouassi, Nancy
    Sender, Vicky
    Hentrich, Karina
    Mellroth, Peter
    Henriques-Normark, Birgitta
    Gabriel, Gülsah
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Daniels, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method2017In: Cell Reports, E-ISSN 2211-1247, Vol. 20, no 1, p. 251-263Article in journal (Refereed)
    Abstract [en]

    Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

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  • 19. El-Heliebi, Amin
    et al.
    Hille, Claudia
    Laxman, Navya
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Svedlund, Jessica
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Haudum, Christoph
    Ercan, Erkan
    Kroneis, Thomas
    Chen, Shukun
    Smolle, Maria
    Rossmann, Christopher
    Krzywkowski, Tomasz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ahlford, Annika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Devyser AB, Sweden.
    Darai, Evangelia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    von Amsberg, Gunhild
    Alsdorf, Winfried
    König, Frank
    Löhr, Matthias
    de Kruijff, Inge
    Riethdorf, Sabine
    Gorges, Tobias M.
    Pantel, Klaus
    Bauernhofer, Thomas
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Sedlmayr, Peter
    In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells2018In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 64, no 3, p. 536-546Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms.

    METHODS: We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wildtype (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients.

    RESULTS: In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts.

    CONCLUSIONS: Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

  • 20. Engström, Anna
    et al.
    Gómez de la Torre, Teresa Zardán
    Strømme, Maria
    Nilsson, Mats E.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Herthnek, David
    Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout2013In: PLOS ONE, E-ISSN 1932-6203, Vol. 8, no 4, article id e62015Article in journal (Refereed)
    Abstract [en]

    Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.

  • 21. Floriddia, Elisa M.
    et al.
    Lourenco, Tania
    Zhang, Shupei
    van Bruggen, David
    Hilscher, Markus M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Cartana AB, Sweden.
    Kukanja, Petra
    dos Santos, Joao P. Goncalves
    Altinkok, Muge
    Yokota, Chika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Llorens-Bobadilla, Enric
    Mulinyawe, Sara B.
    Graos, Mario
    Sun, Lu O.
    Frisen, Jonas
    Nilsson, Mats
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Castelo-Branco, Goncalo
    Distinct oligodendrocyte populations have spatial preference and different responses to spinal cord injury2020In: Nature Communications, E-ISSN 2041-1723, Vol. 11, no 1, article id 5860Article in journal (Refereed)
    Abstract [en]

    Mature oligodendrocytes (MOLs) show transcriptional heterogeneity, the functional consequences of which are unclear. MOL heterogeneity might correlate with the local environment or their interactions with different neuron types. Here, we show that distinct MOL populations have spatial preference in the mammalian central nervous system (CNS). We found that MOL type 2 (MOL2) is enriched in the spinal cord when compared to the brain, while MOL types 5 and 6 (MOL5/6) increase their contribution to the OL lineage with age in all analyzed regions. MOL2 and MOL5/6 also have distinct spatial preference in the spinal cord regions where motor and sensory tracts run. OL progenitor cells (OPCs) are not specified into distinct MOL populations during development, excluding a major contribution of OPC intrinsic mechanisms determining MOL heterogeneity. In disease, MOL2 and MOL5/6 present different susceptibility during the chronic phase following traumatic spinal cord injury. Our results demonstrate that the distinct MOL populations have different spatial preference and different responses to disease.

  • 22. Geny, Sylvain
    et al.
    Moreno, Pedro M. D.
    Krzywkowski, Tomasz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Gissberg, Olof
    Andersen, Nicolai K.
    Isse, Abdirisaq J.
    El-Madani, Amro M.
    Lou, Chenguang
    Pabon, Y. Vladimir
    Anderson, Brooke A.
    Zaghloul, Eman M.
    Zain, Rula
    Hrdlicka, Patrick J.
    Jørgensen, Per T.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lundin, Karin E.
    Pedersen, Erik B.
    Wengel, Jesper
    Smith, C. I. Edvard
    Next-generation bis-locked nucleic acids with stacking linker and 2 '-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 5, p. 2007-2019Article in journal (Refereed)
    Abstract [en]

    Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.

  • 23. Grundberg, Ida
    et al.
    Kiflemariam, Sara
    Mignardi, Marco
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Imgenberg-Kreuz, Juliana
    Edlund, Karolina
    Micke, Patrick
    Sundström, Magnus
    Sjöblom, Tobias
    Botling, Johan
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics2013In: Oncotarget, E-ISSN 1949-2553, Vol. 4, no 12, p. 2407-2418Article in journal (Refereed)
    Abstract [en]

    Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.

  • 24. Guo, Maoxiang
    et al.
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    van der Wijngaart, Wouter
    Efficient DNA-assisted synthesis of trans-membrane gold nanowires2018In: microsystems and nanoengineering, ISSN 2055-7434, Vol. 4, article id UNSP 17084Article in journal (Refereed)
    Abstract [en]

    Whereas electric circuits and surface-based (bio) chemical sensors are mostly constructed in-plane due to ease of manufacturing, 3D microscale and nanoscale structures allow denser integration of electronic components and improved mass transport of the analyte to (bio) chemical sensor surfaces. This work reports the first out-of-plane metallic nanowire formation based on stretching of DNA through a porous membrane. We use rolling circle amplification (RCA) to generate long single-stranded DNA concatemers with one end anchored to the surface. The DNA strands are stretched through the pores in the membrane during liquid removal by forced convection. Because the liquid-air interface movement across the membrane occurs in every pore, DNA stretching across the membrane is highly efficient. The stretched DNA molecules are transformed into trans-membrane gold nanowires through gold nanoparticle hybridization and gold enhancement chemistry. A 50 fM oligonucleotide concentration, a value two orders of magnitude lower than previously reported for flat surface-based nanowire formation, was sufficient for nanowire formation. We observed nanowires in up to 2.7% of the membrane pores, leading to an across-membrane electrical conductivity reduction from open circuit to <20 Omega. The simple electrical read-out offers a high signal-to-noise ratio and can also be extended for use as a biosensor due to the high specificity and scope for multiplexing offered by RCA.

  • 25.
    Gyllborg, Daniel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mattsson Langseth, Christoffer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Choi, Eunkyoung
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Marco Salas, Sergio
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hilscher, Markus M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lein, Ed S.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue2020In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 48, no 19, article id e112Article in journal (Refereed)
    Abstract [en]

    Visualization of the transcriptome in situ has proven to be a valuable tool in exploring single-cell RNA-sequencing data, providing an additional spatial dimension to investigate multiplexed gene expression, cell types, disease architecture or even data driven discoveries. In situ sequencing (ISS) method based on padlock probes and rolling circle amplification has been used to spatially resolve gene transcripts in tissue sections of various origins. Here, we describe the next iteration of ISS, HybISS, hybridization-based in situ sequencing. Modifications in probe design allows for a new barcoding system via sequence-by-hybridization chemistry for improved spatial detection of RNA transcripts. Due to the amplification of probes, amplicons can be visualized with standard epifluorescence microscopes for high-throughput efficiency and the new sequencing chemistry removes limitations bound by sequence-by-ligation chemistry of ISS. HybISS design allows for increased flexibility and multiplexing, increased signal-to-noise, all without compromising throughput efficiency of imaging large fields of view. Moreover, the current protocol is demonstrated to work on human brain tissue samples, a source that has proven to be difficult to work with image-based spatial analysis techniques. Overall, HybISS technology works as a targeted amplification detection method for improved spatial transcriptomic visualization, and importantly, with an ease of implementation.

  • 26. Haniffa, Muzlifah
    et al.
    Taylor, Deanne
    Linnarsson, Sten
    Aronow, Bruce J.
    Bader, Gary D.
    Barker, Roger A.
    Camara, Pablo G.
    Camp, J. Gray
    Chedotal, Alain
    Copp, Andrew
    Etchevers, Heather C.
    Giacobini, Paolo
    Gottgens, Berthold
    Guo, Guoji
    Hupalowska, Ania
    James, Kylie R.
    Kirby, Emily
    Kriegstein, Arnold
    Lundeberg, Joakim
    Marioni, John C.
    Meyer, Kerstin B.
    Niakan, Kathy K.
    Nilsson, Mats
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Olabi, Bayanne
    Pe'er, Dana
    Regev, Aviv
    Rood, Jennifer
    Rozenblatt-Rosen, Orit
    Satija, Rahul
    Teichmann, Sarah A.
    Treutlein, Barbara
    Vento-Tormo, Roser
    Webb, Simone
    A roadmap for the Human Developmental Cell Atlas2021In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 597, no 7875, p. 196-205Article in journal (Refereed)
    Abstract [en]

    This Perspective outlines the Human Developmental Cell Atlas initiative, which uses state-of-the-art technologies to map and model human development across gestation, and discusses the early milestones that have been achieved. The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.

  • 27.
    Hernández-Neuta, Iván
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Magoulopoulou, Anastasia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Pineiro, Flor
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lisby, Jan Gorm
    Gulberg, Mats
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Highly multiplexed targeted sequencing strategy for infectious disease surveillance2023In: BMC Biotechnology, E-ISSN 1472-6750, Vol. 23, article id 31Article in journal (Refereed)
    Abstract [en]

    Background Global efforts to characterize diseases of poverty are hampered by lack of affordable and comprehensive detection platforms, resulting in suboptimal allocation of health care resources and inefficient disease control. Next generation sequencing (NGS) can provide accurate data and high throughput. However, shotgun and metagenome-based NGS approaches are limited by low concentrations of microbial DNA in clinical samples, requirements for tailored sample and library preparations plus extensive bioinformatics analysis. Here, we adapted molecular inversion probes (MIPs) as a cost-effective target enrichment approach to characterize microbial infections from blood samples using short-read sequencing. We designed a probe panel targeting 2 bacterial genera, 21 bacterial and 6 fungi species and 7 antimicrobial resistance markers (AMRs).

    Results Our approach proved to be highly specific to detect down to 1 in a 1000 pathogen DNA targets contained in host DNA. Additionally, we were able to accurately survey pathogens and AMRs in 20 out of 24 samples previously profiled with routine blood culture for sepsis.

    Conclusions Overall, our targeted assay identifies microbial pathogens and AMRs with high specificity at high throughput, without the need for extensive sample preparation or bioinformatics analysis, simplifying its application for characterization and surveillance of infectious diseases in medium- to low- resource settings.

  • 28.
    Hernández-Neuta, Iván
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Brightmeyer, J.
    Tis, T. Ba
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wei, Q.
    Ozcan, A.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Smartphone-based clinical diagnostics: towards democratization of evidence-based health care2019In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 285, no 1, p. 19-39Article, review/survey (Refereed)
    Abstract [en]

    Recent advancements in bioanalytical techniques have led to the development of novel and robust diagnostic approaches that hold promise for providing optimal patient treatment, guiding prevention programs and widening the scope of personalized medicine. However, these advanced diagnostic techniques are still complex, expensive and limited to centralized healthcare facilities or research laboratories. This significantly hinders the use of evidence-based diagnostics for resource-limited settings and the primary care, thus creating a gap between healthcare providers and patients, leaving these populations without access to precision and quality medicine. Smartphone-based imaging and sensing platforms are emerging as promising alternatives for bridging this gap and decentralizing diagnostic tests offering practical features such as portability, cost-effectiveness and connectivity. Moreover, towards simplifying and automating bioanalytical techniques, biosensors and lab-on-a-chip technologies have become essential to interface and integrate these assays, bringing together the high precision and sensitivity of diagnostic techniques with the connectivity and computational power of smartphones. Here, we provide an overview of the emerging field of clinical smartphone diagnostics and its contributing technologies, as well as their wide range of areas of application, which span from haematology to digital pathology and rapid infectious disease diagnostics.

  • 29.
    Hernández-Neuta, Iván
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Pereiro, Iago
    Ahlford, Annika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ferraro, Davide
    Zhang, Qiongdi
    Viovy, Jean-Louis
    Descroix, Stéphanie
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode2018In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 102, p. 531-539Article in journal (Refereed)
    Abstract [en]

    Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120 mu L of DNA dilution at flow rates ranging from 1 to 5 mu L/min without compromising performance. The fluidized bed was 20-50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics.

  • 30.
    Hernández-Neuta, Iván
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Pereiro, Iago
    Ahlford, Annika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ferraro, Davide
    Zhang, Qiongdi
    Viovy, Jean-Louis
    Descroix, Stéphanie
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Microfluidic magnetic fluidized bed for DNA analysis in continuous flow modeManuscript (preprint) (Other academic)
    Abstract [en]

    Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA processing in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification (RCA). This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions and high throughput capabilities, with flow rates up to 5 L/min without compromising performance. The obtained efficiency values using the fluidized bed were superior to a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based micro arrayand tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostic systems.

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  • 31.
    Hilscher, Markus M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mattsson Langseth, Christoffer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kukanja, Petra
    Yokota, Chika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Castelo‑Branco, Gonçalo
    Spatial and temporal heterogeneity in the lineage progression of fine oligodendrocyte subtypes2022In: BMC Biology, E-ISSN 1741-7007, Vol. 20, no 1, article id 122Article in journal (Refereed)
    Abstract [en]

    Background: Oligodendrocytes are glial cells that support and insulate axons in the central nervous system through the production of myelin. Oligodendrocytes arise throughout embryonic and early postnatal development from oligodendrocyte precursor cells (OPCs), and recent work demonstrated that they are a transcriptional heterogeneous cell population, but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of the corpus callosum, cingulate, motor, and somatosensory cortex in the brain, as well as gray matter (GM) and white matter (WM) regions in the spinal cord, at postnatal (P10), juvenile (P20), and young adult (P60) stages. We systematically compare the abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations.

    Results: We observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain, corroborating with previous studies. We found myelination still ongoing in the adult corpus callosum while it was more advanced in the cortex. Interestingly, we also observed a lateral-to-medial gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. The ISS experiments also exposed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM, and we found that neighboring preferences of some oligodendroglia populations are altered from the juvenile to the adult CNS.

    Conclusions: Overall, our ISS experiments reveal spatial heterogeneity of oligodendrocyte lineage progression in the brain and spinal cord and uncover differences in the timing of oligodendrocyte differentiation and myelination, which could be relevant to further investigate functional heterogeneity of oligodendroglia, especially in the context of injury or disease.

  • 32. Horta, Sara
    et al.
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Yeh, S.
    Mattson Langseth, Christoffer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kagro, Kadri
    Breukers, Jolien
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Geukens, Nick
    Lammertyn, Jeroen
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Vanhoorelbeke, Karen
    Evaluation of immuno-rolling circle amplification for multiplexed detection and profiling of antigen-specific antibody isotypesManuscript (preprint) (Other academic)
    Abstract [en]

    Antibody characterization has become essential for diagnosis and development of therapeutic solutions in autoimmune, cardiovascular and infectious diseases. The specificity, affinity and isotype are crucial information for antibody studies and can be obtained from screening plasma samples or populations of B cells. Current technologies are mainly focusing on the discovery of abundant immunoglobulins, namely IgG, and are based on bulk measurements. In this study, we present a digital screening platform utilizing rolling circle amplification (RCA) for the detection of antigen-specific antibody isotypes in solution or secreted from single cells. To validate this approach, the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) was used as model disease and anti-ADAMTS13 antibodies were the target molecules. Antibody-oligonucleotide conjugates (AOCs) were designed for the multiplexed detection of human antibody isotypes IgA, IgG and IgM. Then, ADAMTS13 fragments were coated on glass slides and subsequently, target antibodies identified by specific AOC binding and visualized via an RCA assay. First, we validated the method by characterizing the assay specificity and limit of detection (LoD). When seeding different isotypes, we confirmed the high specificity of the assay (> 90%) and detection of monoclonal anti-ADAMTS13 IgG down to 0.3 ng/mL. A dilution series of a plasma sample of iTTP patient confirmed the multiplexed detection of the three isotypes with higher sensitivity compared to ELISA. Finally, we performed single cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving, achieving a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.

  • 33. Horta, Sara
    et al.
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Yeh, Shu-hao
    Mattsson Langseth, Christoffer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kangro, Kadri
    Breukers, Jolien
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Geukens, Nick
    Vanhoorelbeke, Karen
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lammertyn, Jeroen
    Evaluation of Immuno-Rolling Circle Amplification for Multiplex Detection and Profiling of Antigen-Specific Antibody Isotypes2021In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 93, no 15, p. 6169-6177Article in journal (Refereed)
    Abstract [en]

    Antibody characterization is essential for understanding the immune system and development of diagnostics and therapeutics. Current technologies are mainly focusing on the detection of antigen-specific immunoglobulin G (IgG) using bulk singleplex measurements, which lack information on other isotypes and specificity of individual antibodies. Digital immunoassays based on nucleic acid amplification have demonstrated superior performance by allowing the detection of single molecules in a multiplex and sensitive manner. In this study, we demonstrate for the first time an immuno-rolling circle amplification (immunoRCA) assay for the multiplex detection of three antigen-specific antibody isotypes (IgG, IgA, and IgM) and its integration with microengraving. To validate this approach, we used the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) as the model disease with anti-ADAMTS13 autoantibodies as the diagnostic target molecules. To identify the anti-ADAMTS13 autoantibody isotypes, we designed a pool of three unique antibody-oligonucleotide conjugates for identification and subsequent amplification and visualization via RCA. To validate this approach, we first confirmed an assay specificity of >88% and a low limit of detection of 0.3 ng/mL in the spiked buffer. Subsequently, we performed a dilution series of an iTTP plasma sample for the multiplex detection of the three isotypes with higher sensitivity compared to an enzyme-linked immunosorbent assay. Finally, we demonstrated single-cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving and achieved a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.

  • 34. Johansson, Martin M.
    et al.
    Lundin, Elin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mirzazadeh, Mohammadreza
    Halvardson, Jonatan
    Darj, Elisabeth
    Feuk, Lars
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Jazin, Elena
    Spatial sexual dimorphism of X and Y homolog gene expression in the human central nervous system during early male development2016In: Biology of Sex Differences, ISSN 2042-6410, Vol. 7Article in journal (Refereed)
    Abstract [en]

    Background: Renewed attention has been directed to the functions of the Y chromosome in the central nervous system during early human male development, due to the recent proposed involvement in neurodevelopmental diseases. PCDH11Y and NLGN4Y are of special interest because they belong to gene families involved in cell fate determination and formation of dendrites and axon. Methods: We used RNA sequencing, immunocytochemistry and a padlock probing and rolling circle amplification strategy, to distinguish the expression of X and Y homologs in situ in the human brain for the first time. To minimize influence of androgens on the sex differences in the brain, we focused our investigation to human embryos at 8-11 weeks post-gestation. Results: We found that the X- and Y-encoded genes are expressed in specific and heterogeneous cellular sub-populations of both glial and neuronal origins. More importantly, we found differential distribution patterns of X and Y homologs in the male developing central nervous system. Conclusions: This study has visualized the spatial distribution of PCDH11X/Y and NLGN4X/Y in human developing nervous tissue. The observed spatial distribution patterns suggest the existence of an additional layer of complexity in the development of the male CNS.

  • 35. Ke, Rongqin
    et al.
    Mignardi, Marco
    Hauling, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Fourth Generation of Next-Generation Sequencing Technologies: Promise and Consequences2016In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 37, no 12, p. 1363-1367Article, review/survey (Refereed)
    Abstract [en]

    In this review, we discuss the emergence of the fourth-generation sequencing technologies that preserve the spatial coordinates of RNA and DNA sequences with up to subcellular resolution, thus enabling back mapping of sequencing reads to the original histological context. This information is used, for example, in two current large-scale projects that aim to unravel the function of the brain. Also in cancer research, fourth-generation sequencing has the potential to revolutionize the field. Cancer Research UK has named Mapping the molecular and cellular tumor microenvironment in order to define new targets for therapy and prognosis one of the grand challenges in tumor biology. We discuss the advantages of sequencing nucleic acids directly in fixed cells over traditional next-generation sequencing (NGS) methods, the limitations and challenges that these new methods have to face to become broadly applicable, and the impact that the information generated by the combination of in situ sequencing and NGS methods will have in research and diagnostics.

  • 36.
    Ke, Rongqin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University .
    Mignardi, Marco
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University .
    Pacureanu, Alexandra
    Svedlund, Jessica
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Botling, Johan
    Wählby, Carolina
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University.
    In situ sequencing for RNA analysis in preserved tissue and cells2013In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 10, no 9, p. 857-+Article in journal (Refereed)
    Abstract [en]

    Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue. We demonstrate in situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections.

  • 37.
    Ke, Rongqin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Nong, Rachel Yuan
    Fredriksson, Simon
    Landegren, Ulf
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection2013In: PLOS ONE, E-ISSN 1932-6203, Vol. 8, no 7, article id e69813Article in journal (Refereed)
    Abstract [en]

    Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.

  • 38. Kiflemariam, Sara
    et al.
    Mignardi, Marco
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ali, Muhammad Akhtar
    Bergh, Anders
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöblom, Tobias
    In situ sequencing identifies TMPRSS2-ERG fusion transcripts, somatic point mutations and gene expression levels in prostate cancers2014In: Journal of Pathology, ISSN 0022-3417, E-ISSN 1096-9896, Vol. 234, no 2, p. 253-261Article in journal (Refereed)
    Abstract [en]

    Translocations contribute to the genesis and progression of epithelial tumours and in particular to prostate cancer development. To better understand the contribution of fusion transcripts and visualize the clonal composition of multifocal tumours, we have developed a technology for multiplex in situ detection and identification of expressed fusion transcripts. When compared to immunohistochemistry, TMPRSS2-ERG fusion-negative and fusion-positive prostate tumours were correctly classified. The most prevalent TMPRSS2-ERG fusion variants were visualized, identified, and quantitated in human prostate cancer tissues, and the ratio of the variant fusion transcripts could for the first time be directly determined by in situ sequencing. Further, we demonstrate concurrent in situ detection of gene expression, point mutations, and gene fusions of the prostate cancer relevant targets AMACR, AR, TP53, and TMPRSS2-ERG. This unified approach to in situ analyses of somatic mutations can empower studies of intra-tumoural heterogeneity and future tissue-based diagnostics of mutations and translocations.

  • 39. Koos, Björn
    et al.
    Kamali-Moghaddam, Masood
    David, Leonor
    Sobrinho-Simões, Manuel
    Dimberg, Anna
    Nilsson, Mats
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wählby, Carolina
    Söderberg, Ola
    Next-Generation Pathology-Surveillance of Tumor Microecology2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 11, p. 2013-2022Article, review/survey (Refereed)
    Abstract [en]

    A tumor is a heterogeneous population of cells that provides an environment in which every cell resides in a microenvironmental niche. Microscopic evaluation of tissue sections, based on histology and immunohistochemistry, has been a cornerstone in pathology for decades. However, the dawn of novel technologies to investigate genetic aberrations is currently adopted in routine molecular pathology. We herein describe our view on how recent developments in molecular technologies, focusing on proximity ligation assay and padlock probes, can be applied to merge the two branches of pathology, allowing molecular profiling under histologic observation. We also discuss how the use of image analysis will be pivotal to obtain information at a cellular level and to interpret holistic images of tissue sections. By understanding the cellular communications in the microecology of tumors, we will be at a better position to predict disease progression and response to therapy.

  • 40.
    Krzywkowski, Tomasz
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ciftci, Sibel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Assadian, Farzaneh
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Punga, Tanel
    Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection2017In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 91, no 11, article id e00166-17Article in journal (Refereed)
    Abstract [en]

    An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.

  • 41.
    Krzywkowski, Tomasz
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kühnemund, Malte
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection2019In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 25, no 1, p. 82-89Article in journal (Refereed)
    Abstract [en]

    Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3'-OH RNA/5'-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5' flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide poly-morphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.

  • 42.
    Krzywkowski, Tomasz
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kühnemund, Malte
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Detection of miRNAs using chimeric DNA/RNA iLock probes utilizing novel activity of PBCV-1 DNA ligase: RNA-templated ligation of ssRNAManuscript (preprint) (Other academic)
    Abstract [en]

    Accurate detection of miRNAs with complementary probes is challenging due to the short target size, and often high sequence similarity between isoforms belonging to the same miRNA family. Ligation based methods can provide powerful discrimination of subtle sequence variation among target sequences, but they have been difficult to implement for direct RNA analysis due to the sloppiness and inefficiency of most DNA ligases on RNA substrates. In this work, we have studied if RNA substitutions in padlock probes can provide higher catalytic efficiencies for PBCV-1 DNA ligase on RNA substrates. We also characterise end-joining fidelity for Chlorella virus DNA ligase (PBCV DNA ligase 1) and T4RNA ligase 2 (T4Rnl2) in RNA-templated 3'-OH RNA/5’-pDNA chimeric probe ligation. Although we observed considerable ligation efficiency improvement towards short miRNA targets for PBCV-1 ligated chimeric probes, it showed no sequence specificity towards mismatches at the ligation junction. T4Rnl2 showed some base discrimination, but not satisfactory for robust RNA sequence analysis. To increase end-joining fidelity in PBCV-1 DNA ligase catalysed direct RNA detection assays (iLock probes), we have recently introduced an alternative ligation assay design in which ligation probes first undergo sequence- specific 5’ FLAP removal in order to create ligatable substrates. We have tested various chimeric iLock probe designs where RNA substitutions were introduced at different positions in the FLAP and at the ligation junction. We defined two particular nucleotide positions in the iLock probe sequence that when substituted with RNA, significantly increased iLock probe activation and ligation. We further characterized the end-joining fidelity of PBCV-1 and T4Rnl2 catalysed iLock reactions. Both enzymes showed high ligation fidelities for single nucleotide polymorphisms on RNA and miRNA. Finally, we demonstrate a multiplexed chimeric iLock probe miRNA profiling assay using sequencing-by-ligation as readout. 

  • 43.
    Krzywkowski, Tomasz
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kühnemund, Malte
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Reverse-transcriptase activity of Phi29 DNA polymeraseManuscript (preprint) (Other academic)
    Abstract [en]

    F29 (Phi29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real-time (SMRT) sequencing. Here we report the ability of F29 DNA polymerase to amplify partially RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions support a similar amplification rate as pure DNA substrates. We observed that increasing the number of consecutive RNA substitutions in the circular templates suppress replication, and cannot be recovered by addition of M-MuLV reverse-transcriptase. In summary, this novel ability of F29 to accept RNA-containing substrates broadens the spectrum of applications for F29 mediated RCA. Applications include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes. 

  • 44.
    Krzywkowski, Tomasz
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kühnemund, Malte
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wu, Di
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Limited reverse transcriptase activity of phi29 DNA polymerase2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 7, p. 3625-3632Article in journal (Refereed)
    Abstract [en]

    Phi29 (Phi 29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.

  • 45.
    Krzywkowski, Tomasz
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 18, article id e161Article in journal (Refereed)
    Abstract [en]

    Ligation-based nucleic acid detection methods are primarily limited to DNA, since they exhibit poor performance on RNA. This is attributed to reduced end-joining efficiency and/or fidelity of ligases. Interestingly, chlorella virus DNA ligase (PBCV-1 DNA ligase) has recently been shown to possess high RNA-templated DNA end-joining activity; however, its fidelity has not yet been systematically evaluated. Herein, we characterized PBCV-1 ligase for its RNA-templated end-joining fidelity at single base mismatches in 3′ and 5′ DNA probe termini and found an overall limited end-joining fidelity. To improve the specificity in PBCV-1 ligase-driven RNA detection assays, we utilized structure-specific 5′ exonucleolytic activity of Thermus aquaticus DNA polymerase, used in the invader assay. In the iLock (invader padLock) probe assay, padlock probe molecules are activated prior ligation thus the base at the probe ligation junction is read twice in order to aid successful DNA ligation: first, during structure-specific invader cleavage and then during sequence-specific DNA ligation. We report two distinct iLock probe activation mechanisms and systematically evaluate the assay specificity, including single nucleotide polymorphisms on RNA, mRNA and miRNA. We show significant increase in PBCV-1 ligation fidelity in the iLock probe assay configuration for RNA detection.

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  • 46. Kuhnemund, M.
    et al.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore2015In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 67, p. 11-17Article in journal (Refereed)
    Abstract [en]

    Novel portable, sensitive and selective DNA sensor methods for bio-sensing applications are required that can rival conventionally used non-portable and expensive fluorescence-based sensors. In this paper, rolling circle amplification (RCA) products are detected in solution and on magnetic particles using a resistive pulse sensing (RPS) nanopore. Low amounts of DNA molecules are detected by padlock probes which are circularized in a strictly target dependent ligation reaction. The DNA-padlock probe-complex is captured on magnetic particles by sequence specific capture oligonucleotides and amplified by a short RCA. Subsequent RPS analysis is used to identify individual particles with single attached RCA products from blank particles. This proof of concept opens up for a novel non-fluorescent digital DNA quantification method that can have many applications in bio-sensing and diagnostic approaches.

  • 47. Kuhnemund, Malte
    et al.
    Witters, Daan
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lammertyn, Jeroen
    Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection2014In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, no 16, p. 2983-2992Article in journal (Refereed)
    Abstract [en]

    We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rotting circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products mu L-1 as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics.

  • 48. Kukanja, Petra
    et al.
    Mattsson Langseth, Christoffer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Rodríguez-Kirby, Leslie A. Rubio
    Agirre, Eneritz
    Zheng, Chao
    Raman, Amitha
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Yokota, Chika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Avenel, Christophe
    Tiklová, Katarína
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Guerreiro-Cacais, Andre O.
    Olsson, Tomas
    Hilscher, Markus M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Castelo-Branco, Gonçalo
    Cellular architecture of evolving neuroinflammatory lesions and multiple sclerosis pathology2024In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 187, no 8, p. 1990-2009Article in journal (Refereed)
    Abstract [en]

    Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single-cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single-cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease-associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single-cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS.

  • 49. Kuroda, Arisa
    et al.
    Ishigaki, Yuri
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sato, Kiichi
    Sato, Kae
    Microfluidics-based in situ Padlock/Rolling Circle Amplification System for Counting Single DNA Molecules in a Cell2014In: Analytical Sciences, ISSN 0910-6340, E-ISSN 1348-2246, Vol. 30, no 12, p. 1107-1112Article in journal (Refereed)
    Abstract [en]

    In situ padlock/rolling circle amplification (RCA) is a method used to amplify, visualize, and quantify target DNA molecules in cells. However, the multiple reaction steps involved make this technique costly and cumbersome. We developed a novel, simplified, automated microfluidic system for RCA, and demonstrated its effectiveness by counting amplified mitochondria' DNA fragments in HeLa cells. After optimizing the volume of the reaction solutions and washing buffer composition, the product yield was equal to that obtained by the conventional manual method. The required volume of reagents was reduced to 10 mu L, which is less than half the volume used in the conventional method. To the best of our knowledge, this is the first report of an automated microfluidic method for in situ padlock/RCA, which can be useful for making highly efficient pathological diagnoses.

  • 50.
    Kühnemund, Malte
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Sharif, Mohd Istiaq
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Cornaglia, Matteo
    Gijs, Martin A. M.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 8, article id e59Article in journal (Refereed)
    Abstract [en]

    Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.

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