| 51. |
- Nyblom, My, 1995, et al.
(författare)
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Strain-level bacterial typing directly from patient samples using optical DNA mapping
- 2023
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Ingår i: COMMUNICATIONS MEDICINE. - : Springer Science and Business Media LLC. - 2730-664X. ; 3:31
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Tidskriftsartikel (refereegranskat)abstract
- For bacterial infections, it is important to rapidly and accurately identify and characterize the type of bacteria involved so that optimal antibiotic treatment can be given quickly to the patient. However, current diagnostic methods are sometimes slow and cannot be used for mixtures of bacteria. We have, therefore, developed a method to identify bacteria directly from patient samples. The method was tested on two common species of disease-causing bacteria - Escherichia coli and Klebsiella pneumoniae - and it could correctly identify the bacterial strain or subtype in both urine samples and mixtures. Hence, the method has the potential to provide fast diagnostic information for choosing the most suited antibiotic, thereby reducing the risk of death and suffering. Nyblom, Johnning et al. develop an optical DNA mapping approach for bacterial strain typing of patient samples. They demonstrate rapid identification of clinically relevant E. coli and K. pneumoniae strains, without the need for cultivation. BackgroundIdentification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.MethodsIn this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections. Results We demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids. Conclusion The optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management.
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| 52. |
- Persson, Fredrik, 1979, et al.
(författare)
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Lipid-Based Passivation in Nanofluidics
- 2012
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 12:5, s. 2260-2265
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Tidskriftsartikel (refereegranskat)abstract
- Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. 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 demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA-DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein-DNA interactions with high spatial and temporal resolution.
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| 53. |
- Persson, Fredrik, 1979, et al.
(författare)
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Local conformation of confined DNA studied using emission polarization anisotropy
- 2010
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Ingår i: Biophysical Society 54th Annual Meeting.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- When confined in nanochannels with dimensions smaller than the DNA radius of gyration, DNA will extend along the channel. We investigate long DNA confined in nanochannels, using fluorescence microscopy and intercalated dyes. Studies of the dynamics and statics of DNA in such nanoscale confinements as a function of e.g. degree of confinement and ionic strength have yielded new insights into the physical properties of DNA with relevance for applications in genomics as well as fundamental understanding of DNA packaging in vivo. Our work extends the field by not only studying the location of the emitting dyes along a confined DNA molecule but also monitoring the polarization of the emitted light. By measuring the emission polarized parallel and perpendicular to the extension axis of the stretched DNA, information on the local spatial distribution of the DNA backbone can be obtained. Comparing polarizations in two directions for DNA confined in channels of effective diameters of 85 nm and 170 nm reveals a striking difference. Whereas the DNA in the larger channels shows an isotropic polarization of the emitted light, the light is to a large extent polarized perpendicular to the elongation of the DNA in the smaller channels. We expect this technique to have a large impact on the studies of changes in DNA conformation induced by protein binding or during DNA compactation as well as in fundamental polymer physics studies of DNA in confined environments, for example in bacterial spores and viruses.
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| 54. |
- Persson, Fredrik, 1979, et al.
(författare)
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Local conformation of confined DNA studied using emission polarization anisotropy
- 2010
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Ingår i: NanoBioTech-Montreux 2009.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- In nanochannels with dimensions smaller than the DNA radius of gyration, DNA will extend along the channel. We investigate long DNA confined in nanochannels using fluorescence microscopy and intercalated dyes. Studies of the dynamics and statics of the DNA extension or position in such nanoscale confinements as a function of e.g. DNA contour length, degree and shape of confinement as well as ionic strength have yielded new insights in the physical properties of DNA with relevance for applications in genomics as well as fundamental understanding of DNA packaging in vivo. Our work extends the field by not only studying the location of the emitting dyes along a confined DNA molecule but also monitoring the polarization of the emitted light. We use intercalating dyes (YOYO-1) whose emission is polarized perpendicular to the DNA extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched DNA, information on the local spatial distribution of the DNA backbone can be obtained. The results obtained are analogous to linear dichroism (LD) but on a single-molecule level, and obtained in a highly parallel fashion. We will discuss results in shallow (60 nm) and deep (180 nm) channels and describe an example of how the technique can be used to investigate non-uniform stretching of DNA on the single molecule level. Comparing polarizations in two directions for DNA confined in channels of effective diameters of 85 nm and 170 nm reveals a striking difference. Whereas the DNA in the larger channels shows an isotropic polarization of the emitted light, the light is to a large extent polarized perpendicular to the elongation of the DNA in the smaller channels. The ratio of the polarization parallel and perpendicular to the elongation direction, I|| / I⊥, is a measure of the relative local orientation of the DNA backbone. We believe that this technique will have a large impact on the studies of changes in DNA conformation induced by protein binding or during DNA compactation as well as in fundamental polymer physics studies of DNA in confined environments, for example in bacterial spores and viruses.
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| 55. |
- Sasanian, Nima, 1993, et al.
(författare)
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Probing physical properties of single amyloid fibrils using nanofluidic channels
- 2023
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Ingår i: European Biophysics Journal. - 1432-1017 .- 0175-7571. ; 52:SUPPL 1, s. S205-S205
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Amyloid fibrils formation via protein misfolding and aggregation is associated with many diseases, including neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. The presence of polymorphism phenomenon in a single protein sample makes it important to analyze amyloid fibrils on the single fibril level. We here introduce the concept of nanofluidic channel analysis to the study of single, fluorescently labeled amyloid fibrils. The confinement of amyloid fibrils in nanochannels makes it possible to measure their extension at each width as well as their emission intensity. We used Odijk’s theory for strongly confined polymers to determine the persistence length of each fibril. A majority of the persistence lengths were in the 1-10 m regime and for both the Alzheimer’s protein amyloid- (1-42) and the Parkinson’s protein-synuclein we find at least two populations of fibrils with different persistence lengths, indicating the coexistence of polymorphs with different physical properties. We foresee that the nanofluidics methodology that we have established here will be a useful future tool to study amyloid fibrils on the single fibril level to inform on heterogeneity in their physical properties and interactions.
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| 56. |
- Sasanian, Nima, 1993, et al.
(författare)
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Probing physical properties of single amyloid fibrils using nanofluidic channels
- 2023
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Ingår i: Nanoscale. - 2040-3372 .- 2040-3364. ; 15:46, s. 18737-18744
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid fibril formation is central to the pathology of many diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Amyloid fibrils can also have functional and scaffolding roles, for example in bacterial biofilms, and have also been exploited as useful biomaterials. Despite being linear protein homopolymers, amyloid fibrils can exhibit significant structural and morphological polymorphism, making it relevant to study them on the single fibril level. We here introduce the concept of nanofluidic channel analysis to the study of single, fluorescently-labeled amyloid fibrils in solution, monitoring the extension and emission intensity of individual fibrils confined in nanochannels with a depth of 300 nm and a width that gradually increases from 300 to 3000 nm. The change in fibril extension with channel width permitted accurate determination of the persistence length of individual fibrils using Odijk's theory for strongly confined polymers. The technique was applied to amyloid fibrils prepared from the Alzheimer's related peptide amyloid-β(1-42) and the Parkinson's related protein α-synuclein, obtaining mean persistence lengths of 5.9 ± 4.5 μm and 3.0 ± 1.6 μm, respectively. The broad distributions of fibril persistence lengths indicate that amyloid fibril polymorphism can manifest in their physical properties. Interestingly, the α-synuclein fibrils had lower persistence lengths than the amyloid-β(1-42) fibrils, despite being thicker. Furthermore, there was no obvious within-sample correlation between the fluorescence emission intensity per unit length of the labelled fibrils and their persistence lengths, suggesting that stiffness may not be proportional to thickness. We foresee that the nanofluidics methodology established here will be a useful tool to study amyloid fibrils on the single fibril level to gain information on heterogeneity in their physical properties and interactions.
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| 57. |
- Solgi, Hamid, et al.
(författare)
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Molecular Epidemiology of OXA-48 and NDM-1 Producing Enterobacterales Species at a University Hospital in Tehran, Iran, Between 2015 and 2016
- 2020
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Carbapenem-resistant Enterobacterales (CRE) is an increasing problem worldwide. Here, we examined the clonal relatedness of 71 non-repetitive CRE isolates collected in a university hospital in Tehran, Iran, between February 2015 and March 2016. Pulsed-field gel electrophoresis (PFGE) and MLST were used for epidemiological analysis. Screening for antibiotic resistance genes, PCR-based replicon typing, conjugation experiments, and optical DNA mapping were also performed. Among all 71 isolates, 47 isolates of Klebsiella pneumoniae (66.2%), eight Escherichia coli (11.2%), five Serratia marcescens (7%), and two Enterobacter cloacae (2.8%) harbored blaNDM–1 and blaOXA–48 genes together or alone. PFGE analysis revealed that most of the OXA-48- and NDM-1-producing K. pneumoniae and all of OXA-48-producing S. marcescens were clonally related, while all eight E. coli and two E. cloacae isolates were clonally unrelated. The predominant clones of carbapenemase-producing K. pneumoniae associated with outbreaks within the hospital were ST147 (n = 13) and ST893 (n = 10). Plasmids carrying blaNDM–1 and blaOXA–48 were successfully transferred to an E. coli K12-recipient strain. The blaOXA–48 gene was located on an IncL/M conjugative plasmid, while the blaNDM–1 gene was located on both IncFII ∼86-kb to ∼140-kb and IncA/C conjugative plasmids. Our findings provide novel epidemiologic data on carbapenemase-producing Enterobacterales (CPE) in Iran and highlight the importance of horizontal gene transfer in the dissemination of blaNDM–1 and blaOXA–48 genes. The occurrence and transmission of distinct K. pneumoniae clones call for improved infection control to prevent further spread of these pathogens in Iran.
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| 58. |
- Sundar Rajan, Vinoth, 1987, et al.
(författare)
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Mechanical characterization of base analogue modified nucleic acids by force spectroscopy
- 2021
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Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 23:26, s. 14151-14155
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Tidskriftsartikel (refereegranskat)abstract
- We use mechanical unfolding of single DNA hairpins with modified bases to accurately assess intra- and intermolecular forces in nucleic acids. As expected, the modification stabilizes the hybridized hairpin, but we also observe intriguing stacking interactions in the unfolded hairpin. Our study highlights the benefit of using base-modified nucleic acids in force-spectroscopy.
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| 59. |
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| 60. |
- Werner, Erik, et al.
(författare)
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Orientational correlations in confined DNA
- 2012
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Ingår i: Physical Review E. - 1539-3755 .- 2470-0045 .- 2470-0053. ; 86:4
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Tidskriftsartikel (refereegranskat)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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