| 1. |
- Xavier, Miguel, et al.
(author)
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Label-free enrichment of primary human skeletal progenitor cells using deterministic lateral displacement
- 2019
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In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 19:3, s. 513-523
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Journal article (peer-reviewed)abstract
- Skeletal stem cells (SSCs) are present in bone marrow (BM) and offer great potential for bone regenerative therapies. However, in the absence of a unique marker, current sorting approaches remain challenging in the quest for simple strategies to deliver SSCs with consistent regeneration and differentiation capacities. Microfluidics offers the possibility to sort cells marker-free, based on intrinsic biophysical properties. Recent studies indicate that SSCs are stiffer than leukocytes and are contained within the larger cell fraction in BM. This paper describes the use of deterministic lateral displacement (DLD) to sort SSCs based on cell size and stiffness. DLD is a technology that uses arrays of micropillars to sort cells based on their diameter. Cell deformation within the device can change the cell size and affect sorting - here evidenced using human cell lines and by fractionation of expanded SSCs. Following sorting, SSCs remained viable and retained their capacity to form clonogenic cultures (CFU-F), indicative of stem cell potential. Additionally, larger BM cells showed enhanced capacity to form CFU-F. These findings support the theory that SSCs are more abundant within the larger BM cell fraction and that DLD, or other size-based approaches, could be used to provide enriched SSC populations with significant implications for stem cell research and translation to the clinic.
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| 2. |
- Krog, Jens, et al.
(author)
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Stochastic unfolding of nanoconfined DNA: Experiments, model and Bayesian analysis
- 2018
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In: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 149:21
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Journal article (peer-reviewed)abstract
- Nanochannels provide a means for detailed experiments on the effect of confinement on biomacro-molecules, such as DNA. Here we introduce a model for the complete unfolding of DNA from the circular to linear configuration. Two main ingredients are the entropic unfolding force and the friction coefficient for the unfolding process, and we describe the associated dynamics by a non-linear Langevin equation. By analyzing experimental data where DNA molecules are photo-cut and unfolded inside a nanochannel, our model allows us to extract values for the unfolding force as well as the friction coefficient for the first time. In order to extract numerical values for these physical quantities, we employ a recently introduced Bayesian inference framework. We find that the determined unfolding force is in agreement with estimates from a simple Flory-type argument. The estimated friction coefficient is in agreement with theoretical estimates for motion of a cylinder in a channel. We further validate the estimated friction constant by extracting this parameter from DNA's center-of -mass motion before and after unfolding, yielding decent agreement. We provide publically available software for performing the required image and Bayesian analysis. Published by AIP Publishing.
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| 3. |
- Nyberg, Lena, 1979, et al.
(author)
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A single-step competitive binding assay for mapping of single DNA molecules
- 2012
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In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 417:1, s. 404-408
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Journal article (peer-reviewed)abstract
- Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.
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| 4. |
- Werner, Erik, et al.
(author)
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Monomer distributions and intrachain collisions of a polymer confined to a channel
- 2013
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In: Macromolecules. - : American Chemical Society (ACS). - 0024-9297 .- 1520-5835. ; 46:16, s. 6644-6650
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Journal article (peer-reviewed)abstract
- We study the conformations of a self-avoiding polymer confined to a channel by computing the cross-sectional distributions of the positions of its monomers. By means of Monte Carlo simulations for a self-avoiding, freely jointed chain, we determine how the cross-sectional distribution for a given monomer depends on its location in the polymer and how strongly this distribution is affected by self-avoidance. To this end we analyze how the frequency of intrachain collisions between monomers depends on their spatial position in the channel and on their location within the polymer. We show that most collisions occur between closely neighboring monomers. As a consequence, the collision probability depends only weakly on the spatial position of the monomers. Our results explain why the effect of self-avoidance on the monomer distributions is weaker than predicted by mean-field theory. We discuss the relevance of our results for studies of DNA conformations in nanofluidic channels.
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| 5. |
- Werner, Erik, et al.
(author)
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Orientational correlations in confined DNA
- 2012
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In: Physical Review E. - 1539-3755 .- 2470-0045 .- 2470-0053. ; 86:4
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Journal article (peer-reviewed)abstract
- We study how the orientational correlations of DNA confined to nanochannels depend on the channel diameter D by means of Monte Carlo simulations and a mean-field theory. This theory describes DNA conformations in the experimentally relevant regime where Flory-de Gennes theory does not apply. We show how local correlations determine the dependence of the end-to-end distance of the DNA molecule upon D. Tapered nanochannels provide the necessary resolution in D to study experimentally how the extension of confined DNA molecules depends upon D. Our experimental and theoretical results are in qualitative agreement.
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| 6. |
- Kesarimangalam, Sriram, 1983, et al.
(author)
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Fluorescence Microscopy of Nanochannel-Confined DNA
- 2024
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In: Methods in Molecular Biology. - 1940-6029 .- 1064-3745. - 9781071633762 - 9781071633779 ; 2694, s. 175-202
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Book chapter (other academic/artistic)abstract
- Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level, and the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments, and analyze the data.
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| 7. |
- Bogas, Diana, et al.
(author)
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Applications of optical DNA mapping in microbiology
- 2017
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In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 62:6, s. 255-267
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Research review (peer-reviewed)abstract
- Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence-based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches. In addition, designing lab-on-a-chip systems based on OM is now feasible and will allow the integrated and automated microbiological analysis of biological fluids. Here, we review the basic technology of OM, detail the current state of the art of the field, and look ahead to possible future developments in OM technology for microbiological applications.
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| 8. |
- Fritzsche, Joachim, 1977, et al.
(author)
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A lipid-based passivation scheme for nanofluidics
- 2012
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In: 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012; Okinawa; Japan; 28 October 2012 through 1 November 2012. - 9780979806452 ; , s. 1876-1878
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Conference paper (peer-reviewed)abstract
- Stretching DNA in nanochannels allows for direct, visual studies of genomic DNA at the single molecule level. In order to facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We show long-term passivation of nanochannel surfaces to several relevant reagents and demonstrate that the performance of the lipid bilayer is significantly better compared to standard bovine serum albumin-based passivation. Moreover, we demonstrate how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation.
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| 9. |
- Alizadehheidari, Mohammadreza, 1987, et al.
(author)
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Unfolding of nanoconfined circular DNA
- 2015
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In: BIOPHYSICAL JOURNAL. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 108:2 Supplement 1
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Journal article (other academic/artistic)
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| 10. |
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