| 1. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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Fluorescence Microscopy of Nanochannel-Confined DNA
- 2024
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Ingår i: Methods in Molecular Biology. - 1940-6029 .- 1064-3745. - 9781071633762 - 9781071633779 ; 2694, s. 175-202
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Bokkapitel (övrigt vetenskapligt/konstnärligt)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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| 2. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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High diversity of bla NDM-1 -encoding plasmids in Klebsiella pneumoniae isolated from neonates in a Vietnamese hospital
- 2022
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Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 1872-7913 .- 0924-8579. ; 59:2
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: The carbapenemase-encoding gene blaNDM-1 has been reported in Vietnam during the last 10 years, and blaNDM-producing Enterobacteriaceae are now silently and rapidly spreading. A key factor behind dissemination of blaNDM-1 is plasmids, mobile genetic elements that commonly carry antibiotic resistance genes and spread via conjugation. The diversity of blaNDM-1-encoding plasmids from neonates at a large Vietnamese hospital was characterized in this study. Methods: 18 fecal Klebsiella pneumoniae and Klebsiella quasipneumoniae isolates collected from 16 neonates at a large pediatric hospital in Vietnam were studied using optical DNA mapping (ODM) and next-generation sequencing (NGS). Plasmids carrying the blaNDM-1 gene were identified by combining ODM with Cas9 restriction. The plasmids in the isolates were compared to investigate whether the same plasmid was present in different patients. Results: Although the same plasmid was found in some isolates, ODM confirmed that there were at least 10 different plasmids encoding blaNDM-1 among the 18 isolates, thus indicating wide plasmid diversity. The ODM results concur with the NGS data. Interestingly, some isolates had two distinct plasmids encoding blaNDM-1 that could be readily identified with ODM. The coexistence of different plasmids carrying the same blaNDM-1 gene in a single isolate has rarely been reported, probably because of limitations in plasmid characterization techniques. Conclusions: The plasmids encoding the blaNDM-1 gene in this study cohort were diverse and may represent a similar picture in Vietnamese society. The study highlights important aspects of the usefulness of ODM for plasmid analysis.
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| 3. |
- Kumar Bikarolla, Santosh, 1985, et al.
(författare)
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Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
- 2019
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Ingår i: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the blaCTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the blaCTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying blaCTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings.IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.
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| 4. |
- Müller, Vilhelm, 1990, et al.
(författare)
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Cultivation-Free Typing of Bacteria Using Optical DNA Mapping
- 2020
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Ingår i: Acs Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 6:5, s. 1076-1084
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Tidskriftsartikel (refereegranskat)abstract
- A variety of pathogenic bacteria can infect humans, and rapid species identification is crucial for the correct treatment. However, the identification process can often be time-consuming and depend on the cultivation of the bacterial pathogen(s). Here, we present a stand-alone, enzyme-free, optical DNA mapping assay capable of species identification by matching the intensity profiles of large DNA molecules to a database of fully assembled bacterial genomes (>10 000). The assay includes a new data analysis strategy as well as a general DNA extraction protocol for both Gram-negative and Gram-positive bacteria. We demonstrate that the assay is capable of identifying bacteria directly from uncultured clinical urine samples, as well as in mixtures, with the potential to be discriminative even at the subspecies level. We foresee that the assay has applications both within research laboratories and in clinical settings, where the time-consuming step of cultivation can be minimized or even completely avoided.
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| 5. |
- Müller, Vilhelm, 1990, et al.
(författare)
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Enzyme-free optical DNA mapping of the human genome using competitive binding
- 2019
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 47:15
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Tidskriftsartikel (refereegranskat)abstract
- Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized.
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| 6. |
- 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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| 7. |
- Öz, Robin, 1992, et al.
(författare)
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Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:5, s. 2629-2641
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Tidskriftsartikel (refereegranskat)abstract
- We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.
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| 8. |
- Andersson, John, 1993, et al.
(författare)
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Polymer Brushes on Silica Nanostructures Prepared by Aminopropylsilatrane Click Chemistry: Superior Antifouling and Biofunctionality
- 2023
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Ingår i: ACS Applied Materials & Interfaces. - : American Chemical Society (ACS). - 1944-8252 .- 1944-8244. ; 15:7, s. 10228-10239
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Tidskriftsartikel (refereegranskat)abstract
- In nanobiotechnology, the importance of controlling interactions between biological molecules and surfaces is paramount. In recent years, many devices based on nanostructured silicon materials have been presented, such as nanopores and nanochannels. However, there is still a clear lack of simple, reliable, and efficient protocols for preventing and controlling biomolecule adsorption in such structures. In this work, we show a simple method for passivation or selective biofunctionalization of silica, without the need for polymerization reactions or vapor-phase deposition. The surface is simply exposed stepwise to three different chemicals over the course of ∼1 h. First, the use of aminopropylsilatrane is used to create a monolayer of amines, yielding more uniform layers than conventional silanization protocols. Second, a cross-linker layer and click chemistry are used to make the surface reactive toward thiols. In the third step, thick and dense poly(ethylene glycol) brushes are prepared by a grafting-to approach. The modified surfaces are shown to be superior to existing options for silica modification, exhibiting ultralow fouling (a few ng/cm2) after exposure to crude serum. In addition, by including a fraction of biotinylated polymer end groups, the surface can be functionalized further. We show that avidin can be detected label-free from a serum solution with a selectivity (compared to nonspecific binding) of more than 98% without the need for a reference channel. Furthermore, we show that our method can passivate the interior of 150 nm × 100 nm nanochannels in silica, showing complete elimination of adsorption of a sticky fluorescent protein. Additionally, our method is shown to be compatible with modifications of solid-state nanopores in 20 nm thin silicon nitride membranes and reduces the noise in the ion current. We consider these findings highly important for the broad field of nanobiotechnology, and we believe that our method will be very useful for a great variety of surface-based sensors and analytical devices.
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| 9. |
- Frykholm, Karolin, 1977, et al.
(författare)
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DNA in Nanochannels - Theory and Applications
- 2022
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Ingår i: Quarterly Reviews of Biophysics. - 0033-5835 .- 1469-8994. ; 55
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Forskningsöversikt (refereegranskat)abstract
- Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is relatively easy to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA-protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides kilobase pair resolution of the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, rather than recent applications in human genetics, including the use of enzyme-based labeling and affinity-based labeling to produce the genomic maps. The second is DNA-protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with the chain ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology and biophysics.
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| 10. |
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| 11. |
- Jiang, Kai, 1988, et al.
(författare)
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Alpha-Synuclein Modulates the Physical Properties of DNA
- 2018
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Ingår i: Chemistry - A European Journal. - : Wiley. - 1521-3765 .- 0947-6539. ; 24:58, s. 15685-15690
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Tidskriftsartikel (refereegranskat)abstract
- Published by Wiley-VCH Verlag GmbH & Co. KGaA. Fundamental research on Parkinson's disease (PD) most often focuses on the ability of α-synuclein (aS) to form oligomers and amyloids, and how such species promote brain cell death. However, there are indications that aS also plays a gene-regulatory role in the cell nucleus. Here, the interaction between monomeric aS and DNA in vitro has been investigated with single-molecule techniques. Using a nanofluidic channel system, it was discovered that aS binds to DNA and by studying the DNA–protein complexes at different confinements we determined that aS binding increases the persistence length of DNA from 70 to 90 nm at high coverage. By atomic force microscopy it was revealed that at low protein-to-DNA ratio, the aS binding occurs as small protein clusters scattered along the DNA; at high protein-to-DNA ratio, the DNA is fully covered by protein. As DNA-aS interactions may play roles in PD, it is of importance to characterize biophysical properties of such complexes in detail.
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| 12. |
- Jiang, Kai, 1988, et al.
(författare)
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Annealing of ssDNA and compaction of dsDNA by the HIV-1 nucleocapsid and Gag proteins visualized using nanofluidic channels
- 2019
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Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 52, s. e2-e2
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Tidskriftsartikel (refereegranskat)abstract
- The nucleocapsid protein NC is a crucial component in the human immunodeficiency virus type 1 life cycle. It functions both in its processed mature form and as part of the polyprotein Gag that plays a key role in the formation of new viruses. NC can protect nucleic acids (NAs) from degradation by compacting them to a dense coil. Moreover, through its NA chaperone activity, NC can also promote the most stable conformation of NAs. Here, we explore the balance between these activities for NC and Gag by confining DNA-protein complexes in nanochannels. The chaperone activity is visualized as concatemerization and circularization of long DNA via annealing of short single-stranded DNA overhangs. The first ten amino acids of NC are important for the chaperone activity that is almost completely absent for Gag. Gag condenses DNA more efficiently than mature NC, suggesting that additional residues of Gag are involved. Importantly, this is the first single DNA molecule study of full-length Gag and we reveal important differences to the truncated Δ-p6 Gag that has been used before. In addition, the study also highlights how nanochannels can be used to study reactions on ends of long single DNA molecules, which is not trivial with competing single DNA molecule techniques.
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| 13. |
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| 14. |
- Jiang, Kai, 1988, et al.
(författare)
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The HIV-1 nucleocapsid chaperone protein forms locally compacted globules on long double-stranded DNA
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:8, s. 4550-4563
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Tidskriftsartikel (refereegranskat)abstract
- The nucleocapsid (NC) protein plays key roles in Human Immunodeficiency Virus 1 (HIV-1) replication, notably by condensing and protecting the viral RNA genome and by chaperoning its reverse transcription into double-stranded DNA (dsDNA). Recent findings suggest that integration of viral dsDNA into the host genome, and hence productive infection, is linked to a small subpopulation of viral complexes where reverse transcription was completed within the intact capsid. Therefore, the synthesized dsDNA has to be tightly compacted, most likely by NC, to prevent breaking of the capsid in these complexes. To investigate NC's ability to compact viral dsDNA, we here characterize the compaction of single dsDNA molecules under unsaturated NC binding conditions using nanofluidic channels. Compaction is shown to result from accumulation of NC at one or few compaction sites, which leads to small dsDNA condensates. NC preferentially initiates compaction at flexible regions along the dsDNA, such as AT-rich regions and DNA ends. Upon further NC binding, these condensates develop into a globular state containing the whole dsDNA molecule. These findings support NC's role in viral dsDNA compaction within the mature HIV-1 capsid and suggest a possible scenario for the gradual dsDNA decondensation upon capsid uncoating and NC loss.
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| 15. |
- Kang, Evan S. H., et al.
(författare)
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Organic Anisotropic Excitonic Optical Nanoantennas
- 2022
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Ingår i: Advanced Science. - : Wiley. - 2198-3844 .- 2198-3844. ; 9:23
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Tidskriftsartikel (refereegranskat)abstract
- Optical nanoantennas provide control of light at the nanoscale, which makes them important for diverse areas ranging from photocatalysis and flat metaoptics to sensors and biomolecular tweezing. They have traditionally been limited to metallic and dielectric nanostructures that sustain plasmonic and Mie resonances, respectively. More recently, nanostructures of organic J-aggregate excitonic materials have been proposed capable of also supporting nanooptical resonances, although their advance has been hampered from difficulty in nanostructuring. Here, the authors present the realization of organic J-aggregate excitonic nanostructures, using nanocylinder arrays as model system. Extinction spectra show that they can sustain both plasmon-like resonances and dielectric resonances, owing to the material providing negative and large positive permittivity regions at the different sides of its exciton resonance. Furthermore, it is found that the material is highly anisotropic, leading to hyperbolic and elliptic permittivity regions. Nearfield analysis using optical simulation reveals that the nanostructures therefore support hyperbolic localized surface exciton resonances and elliptic Mie resonances, neither of which has been previously demonstrated for this type of material. The anisotropic nanostructures form a new type of optical nanoantennas, which combined with the presented fabrication process opens up for applications such as fully organic excitonic metasurfaces.
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| 16. |
- Karami, Nahid, 1959, et al.
(författare)
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Identity of bla ctx-m carrying plasmids in sequential esbl-e. Coli isolates from patients with recurrent urinary tract infections
- 2021
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Ingår i: Microorganisms. - : MDPI AG. - 2076-2607. ; 9:6
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Tidskriftsartikel (refereegranskat)abstract
- Plasmid-mediated multidrug resistance in E. coli is becoming increasingly prevalent. Considering this global threat to human health, it is important to understand how plasmid-mediated resistance spreads. From a cohort of 123 patients with recurrent urinary tract infections (RUTI) due to extended spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL E. coli), only five events with a change of ESBL E. coli strain between RUTI episodes were identified. Their blaCTX-M encoding plasmids were compared within each pair of isolates using optical DNA mapping (ODM) and PCR-based replicon typing. Despite similar blaCTX-M genes and replicon types, ODM detected only one case with identical plasmids in the sequential ESBL E. coli strains, indicating that plasmid transfer could have occurred. For comparison, plasmids from seven patients with the same ESBL E. coli strain reoccurring in both episodes were analyzed. These plasmids (encoding blaCTX-M-3, blaCTX-M-14, and blaCTX-M-15 ) were unaltered for up to six months between recurrent infections. Thus, transmission of blaCTX-M plasmids appears to be a rare event during the course of RUTI. Despite the limited number (n = 23) of plasmids investigated, similar blaCTX-M-15 plasmids in unrelated isolates from different patients were detected, suggesting that some successful plasmids could be associated with specific strains, or are more easily transmitted.
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| 17. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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A parallelized nanofluidic device for high-throughput optical dna mapping of bacterial plasmids
- 2021
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Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 12:10
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Tidskriftsartikel (refereegranskat)abstract
- Optical DNA mapping (ODM) has developed into an important technique for DNA anal-ysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids—circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae, we demon-strate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plas-mids in clinical diagnostics.
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| 18. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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Identification and characterization of plasmids carrying the mobile colistin resistance gene mcr-1 using optical DNA mapping
- 2023
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Ingår i: JAC-Antimicrobial Resistance. - : Oxford University Press (OUP). - 2632-1823. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- Objectives Colistin is a last-resort antibiotic, but there has been a rapid increase in colistin resistance, threatening its use in the treatment of infections with carbapenem-resistant Enterobacterales (CRE). Plasmid-mediated colistin resistance, in particular the mcr-1 gene, has been identified and WGS is the go-to method in identifying plasmids carrying mcr-1 genes. The goal of this study is to demonstrate the use of optical DNA mapping (ODM), a fast, efficient and amplification-free technique, to characterize plasmids carrying mcr-1. Methods ODM is a single-molecule technique, which we have demonstrated can be used for identifying plasmids harbouring antibiotic resistance genes. We here applied the technique to plasmids isolated from 12 clinical Enterobacterales isolates from patients at a major hospital in Thailand and verified our results using Nanopore long-read sequencing. Results We successfully identified plasmids encoding the mcr-1 gene and, for the first time, demonstrated the ability of ODM to identify resistance gene sites in small (similar to 30 kb) plasmids. We further identified bla(CTX-M) genes in different plasmids than the ones encoding mcr-1 in three of the isolates studied. Finally, we propose a cut-and-stretch assay, based on similar principles, but performed using surface-functionalized cover slips for DNA immobilization and an inexpensive microscope with basic functionalities, to identify the mcr-1 gene in a plasmid sample. Conclusions Both ODM and the cut-and-stretch assay developed could be very useful in identifying plasmids encoding antibiotic resistance in hospitals and healthcare facilities. The cut-and-stretch assay is particularly useful in low- and middle-income countries, where existing techniques are limited.
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| 19. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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Multiplexed optical DNA mapping to identify plasmids and their resistance genes in fecal samples
- 2019
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Ingår i: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. ; , s. 856-857
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Konferensbidrag (refereegranskat)abstract
- Multi-drug resistant bacteria are a major health threat worldwide. Resistance is frequently horizontally transferred among bacteria via mobile genetic elements, such as plasmids. We have developed a one-step staining protocol to reveal sequence-correlated fluorescence profiles along stretched plasmids. In this report, we demonstrate an automation approach to increase the throughput of nanofluidic optical DNA mapping(ODM). Using plasmid samples derived from patients' feces, the results demonstrate an improved throughput, both regarding the number of plasmids analyzed per sample as well as the number of samples analyzed. By combining the ODM with Cas9/CRISPR, plasmids containing the antibiotic resistance gene are readily identified.
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| 20. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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Optical dna mapping of plasmids reveals clonal spread of carbapenem-resistant klebsiella pneumoniae in a large thai hospital
- 2021
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Ingår i: Antibiotics. - : MDPI AG. - 2079-6382. ; 10:9
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Tidskriftsartikel (refereegranskat)abstract
- Carbapenem-resistant Klebsiella pneumoniae (CR-KP) in patients admitted to hospitals pose a great challenge to treatment. The genes causing resistance to carbapenems are mostly found in plasmids, mobile genetic elements that can spread easily to other bacterial strains, thus exacerbating the problem. Here, we studied 27 CR-KP isolates collected from different types of samples from 16 patients admitted to the medical ward at Siriraj Hospital in Bangkok, Thailand, using next generation sequencing (NGS) and optical DNA mapping (ODM). The majority of the isolates belonged to sequence type (ST) 16 and are described in detail herein. Using ODM, we identified the plasmid carrying the blaNDM-1 gene in the ST16 isolates and the plasmids were very similar, highlighting the possibility of using ODM of plasmids as a surrogate marker of nosocomial spread of bacteria. We also demonstrated that ODM could identify that the blaCTX-M-15 and blaOXA-232 genes in the ST16 isolates were encoded on separate plasmids from the blaNDM-1 gene and from each other. The other three isolates belonged to ST147 and each of them had distinct plasmids encoding blaNDM-1 .
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| 21. |
- Kesarimangalam, Sriram, 1983, et al.
(författare)
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Optical DNA mapping using nanochannels for identification of plasmids carrying carbapenemase blandm-1 gene from patients admitted to a Vietnamese hospital
- 2019
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Ingår i: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. ; , s. 805-806
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Konferensbidrag (refereegranskat)abstract
- Antibiotic misuse has resulted in a rapidly increasing prevalence of multidrug resistant (MDR) bacteria, posing serious threat to human health. Bacterial plasmids play a significant role in spreading antibiotic resistance genes between strains and species. Rapid identification and characterization of plasmids, and knowledge about the presence of resistance genes, thus becomes essential. Here, we use optical DNA mapping (ODM), to identify the plasmid carrying blaNDM-1 gene in samples collected from patients in Vietnam National Children's Hospital (VNCH) in Hanoi, Veitnam, and investigate if a single plasmid is responsible for spreading of the blaNDM-1 gene across different strains of K. pneumonia.
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| 22. |
- Lee, Seunghyun, et al.
(författare)
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Plasmonic polymer nanoantenna arrays for electrically tunable and electrode-free metasurfaces
- 2023
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Ingår i: Journal of Materials Chemistry A. - : ROYAL SOC CHEMISTRY. - 2050-7488 .- 2050-7496. ; 11:40, s. 21569-21576
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Tidskriftsartikel (refereegranskat)abstract
- Electrically tunable metasurfaces and interrelated nanofabrication techniques are essential for metasurface-based optoelectronic applications. We present a nanofabrication method suitable for various types of plasmonic polymer metasurfaces including inverted arrays of nanoantennas. Inverted metasurfaces are of particular interest since the metasurface itself can work as an electrode due to its interconnected nature, which enables electrical control without adopting an additional electrode. In comparison with inverted nanodisk arrays that support relatively weak resonance features, we show that inverted nanorod arrays can possess stronger resonances, even comparable with those of nanorod arrays. The origin of plasmon resonances in inverted arrays is systematically investigated using finite-difference time-domain (FDTD) simulations. Further, we demonstrate electrically tunable electrode-free metasurface devices using polymer inverted nanorod arrays, which can operate in the full spectral range of the material including the mid-infrared region. Electrically tunable and electrode-free metasurfaces using plasmonic polymer inverted nanoantenna arrays can operate across the entire spectral range of the material, including the mid-infrared region.
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| 23. |
- Levin, Sune, 1991, et al.
(författare)
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Nanofluidic Trapping of Faceted Colloidal Nanocrystals for Parallel Single-Particle Catalysis
- 2022
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Ingår i: Acs Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 16:9, s. 15206-15214
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Tidskriftsartikel (refereegranskat)abstract
- Catalyst activity can depend distinctly on nano -particle size and shape. Therefore, understanding the structure sensitivity of catalytic reactions is of fundamental and technical importance. Experiments with single-particle resolution, where ensemble-averaging is eliminated, are required to study it. Here, we implement the selective trapping of individual spherical, cubic, and octahedral colloidal Au nanocrystals in 100 parallel nanofluidic channels to determine their activity for fluorescein reduction by sodium borohydride using fluorescence microscopy. As the main result, we identify distinct structure sensitivity of the rate-limiting borohydride oxidation step originating from different edge site abundance on the three particle types, as confirmed by first -principles calculations. This advertises nanofluidic reactors for the study of structure-function correlations in catalysis and identifies nanoparticle shape as a key factor in borohydride-mediated catalytic reactions.
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| 24. |
- Lin, Yii Lih, 1978, et al.
(författare)
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Nanofluidic optical DNA mapping for rapid identification of antibiotics resistant plasmids
- 2018
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Ingår i: 22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018. ; 3, s. 1822-1825
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Konferensbidrag (refereegranskat)abstract
- Bacterial resistance to antibiotics has become a major threat to health worldwide. In 2014, the World Health Organization (WHO) classified antibiotic resistance as one of the major threats to human health.1 As the resistance genes are frequently transmitted via mobile genetic elements, such as plasmids, a method to rapidly detect and identify plasmids is needed. This paper reports an improved nanofluidic optical mapping strategy to identify plasmids and locate the antibiotic resistance genes via the Cas9/CRISPR technique. We used the assay to analyze clinical samples from a potential nosocomial outbreak in an Ethiopian hospital, tracing the transmission routes among wards.
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| 25. |
- Lin, Yii Lih, 1978, et al.
(författare)
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Optical maps of plasmids as a proxy for clonal spread of MDR bacteria: A case study of an outbreak in a rural Ethiopian hospital
- 2020
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 75:10, s. 2804-2811
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: MDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements.We believe that this method has the potential to be a tool of pivotal importance for MDR control. Methods: Twenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria. Results: ODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles. Conclusions: ODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low-and middle-income countries.
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| 26. |
- Lindblom, Anna, et al.
(författare)
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Interspecies plasmid transfer appears rare in sequential infections with extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae
- 2019
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Ingår i: Diagnostic Microbiology and Infectious Disease. - : Elsevier BV. - 0732-8893 .- 1879-0070. ; 93:4, s. 380-385
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Tidskriftsartikel (refereegranskat)abstract
- From a cohort of 1836 Swedish patients infected with ESBL-producing Enterobacteriaceae (EPE) during 2004-2014, 513 patients with recurrent EPE infection were identified. Only in 14 of the 513 patients was a change of species (ESBL-E. coli to ESBL-K. pneumoniae or vice versa) found between the index and subsequent infection. Eleven sequential urine isolates from 5 of the 14 patients were available for further analysis of possible transfer of ESBL-carrying plasmids. The plasmid content was studied using optical DNA mapping (ODM), PCR-based replicon typing, and ESBL gene sequencing. ODM allowed us to directly compare whole plasmids between isolates and found similar ESBL-carrying plasmids in 3 out of the 5 patients. The ODM results and the rarity in shift of species between ESBL-E. coli and ESBL-K. pneumoniae imply that in recurrent EPE infections interspecies plasmid transfer is uncommon. (C) 2018 Elsevier Inc. All rights reserved.
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| 27. |
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| 28. |
- Lubart, Quentin, 1989, et al.
(författare)
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High throughput size-determination and multiplexed fluorescence analysis of single biological particles in a nanofluidic device
- 2019
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Ingår i: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. ; , s. 1420-1421
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Konferensbidrag (refereegranskat)abstract
- Biological nanoparticles, such as exosomes and viruses, are responsible for a multitude of important functions, but methods to characterize them on the single particle level are rare. We here present a nanofluidic platform for multi-parametric characterization of biological nanoparticles with high throughput. The device consists of feeding microchannels and an array of ~100 nanochannels where the nanoparticles can be characterized. We determine the size by analyzing the Brownian motion of the particles and quantify their content based on fluorescence imaging of up to three different colors. We successfully benchmark our method against existing techniques, such as Nanoparticle Tracking Analysis (NTA).
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| 29. |
- Möller, Carl Ivar, 1990, et al.
(författare)
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Nanofluidics highlights importance of CtIP's tetrameric structure for DNA repair
- 2024
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 123:3, s. 148A-148A
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The initial steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The activity of these proteins in nucleolytic DSB resection that commits the repair to homologous recombination (HR) are well studied, but their role in tethering and bridging the DNA ends is less understood. We have previously shown, on the single molecule level, how phosphorylated CtIP can bridge the ends of single long (∼50 kb) DNA molecules using nanofluidic channels. The study revealed that the way which CtIP bridges DNA is dependent on its oligomeric state. The tetrameric wild-type CtIP almost exclusively generated circular products as an effect of intramolecular bridging while the tetramerization-deficient CtIPL27E mutant showed results in line with intermolecular bridging. Additionally, truncated mutants showed that the bridging by CtIP is promoted by domains distinct from those that stimulate the nuclease activity of MRN. In this study we use nanofluidics to build on these results and characterize how CtIP bridges long DNA molecules with ends of different design. The method is based on the confinement of long DNA molecules in nanochannels. In contrast to tethering one or both ends to a substrate, as in most common single DNA molecule methods, the DNA is completely free in the nanochannels, meaning that reaction on the DNA ends can be conveniently studied. By utilizing customizable long DNA substrates in conjunction with a set of truncated mutants we seek to investigate CtIP’s ability to bridge DNA with differing modifications and the importance of CtIP’s oligomeric and phosphorylated state to further understand the mechanism behind DNA bridging by CtIP.
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| 30. |
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| 31. |
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| 32. |
- Möller, Carl Ivar, 1990, et al.
(författare)
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Xrs2/NBS1 promote end-bridging activity of the MRE11-RAD50 complex
- 2024
-
Ingår i: Biochemical and Biophysical Research Communications. - 1090-2104 .- 0006-291X. ; 695
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Tidskriftsartikel (refereegranskat)abstract
- DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close proximity to enable ligation by non-homologous end-joining (NHEJ), while the more precise, but less available, repair by homologous recombination (HR) requires close proximity of a sister chromatid. The human MRE11-RAD50-NBS1 (MRN) complex, Mre11-Rad50-Xrs2 (MRX) in yeast, is involved in both repair pathways. Here we use nanofluidic channels to study, on the single DNA molecule level, how MRN, MRX and their constituents interact with long DNA and promote DNA bridging. Nanofluidics is a suitable method to study reactions on DNA ends since no anchoring of the DNA end(s) is required. We demonstrate that NBS1 and Xrs2 play important, but differing, roles in the DNA tethering by MRN and MRX. NBS1 promotes DNA bridging by MRN consistent with tethering of a repair template. MRX shows a “synapsis-like” DNA end-bridging, stimulated by the Xrs2 subunit. Our results highlight the different ways MRN and MRX bridge DNA, and the results are in agreement with their key roles in HR and NHEJ, respectively, and contribute to the understanding of the roles of NBS1 and Xrs2 in DSB repair.
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| 33. |
- Nyblom, My, 1995, et al.
(författare)
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Bacterial identification by optical mapping of genomic DNA in nanofluidic channels
- 2019
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Ingår i: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - 9781733419000 ; , s. 821-822
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Konferensbidrag (refereegranskat)abstract
- A variety of pathogenic bacteria can infect humans and the increase in bacteria resistant to common antibiotics is a large threat to human health worldwide. This work presents a method, based on optical DNA mapping (ODM) in nanofluidic channels, that can detect the type of bacterial present in a sample by matching the obtained maps of large DNA molecules to a database of fully assembled bacterial genomes. The extraction and labelling protocol has been designed to work for both Gram-positive and Gram-negative bacteria, not requiring any prior knowledge about the sample content.
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| 34. |
- 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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| 35. |
- 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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| 36. |
- Sewunet, Tsegaye, et al.
(författare)
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Fecal carriage and clonal dissemination of blaNDM-1 carrying Klebsiella pneumoniae sequence type 147 at an intensive care unit in Lao PDR
- 2022
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 17:10
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Carbapenemase-producing Enterobacterales (CPE) are high priority targets of global antimicrobial surveillance. Herein, we determined the colonization rate of CPE on admission to intensive care units in Vientiane, Lao PDR in August-September 2019. METHODS: Data regarding clinical conditions, infection control, and antibiotic usage were collected during admission. Rectal swab samples (n = 137) collected during admission were inoculated to selective chromogenic agars, followed by confirmatory tests for extended-spectrum beta-lactamases and carbapenemases. All CPE isolates were sequenced on Illumina (HiSeq2500), reads assembled using SPAdes 3.13, and the draft genomes used to query a database (https://www.genomicepidemiology.org) for resistome, plasmid replicons, and sequence types (ST). Optical DNA mapping (ODM) was used to characterize plasmids and to determine location of resistance genes. Minimum spanning tree was generated using the Bacterial Isolate Genome Sequence database (BIGSdb) and annotated using iTOL. RESULT: From 47 Enterobacterales isolated on selective agars, K. pneumoniae (25/47) and E. coli (12/47) were the most prevalent species, followed by K aerogenes (2/47), K. variicola (1/47), and K. oxytoca (1/47). The overall prevalence of ESBLs was 51.0%; E. coli 83.3% (10/12) and Klebsiella spp. 41.3% (12/29). Twenty percent of the K. pneumoniae (5/25) isolates were carbapenem-resistant, and 4/5 contained the blaNDM-1 gene. All blaNDM-1 isolates belonged to ST147 and were indistinguishable with cgMLST. ODM showed that the blaNDM-1 gene was located on identical plasmids in all isolates. CONCLUSION: The prevalence of ESBL-producing Enterobacterales was high, while carbapenemases were less common. However, the detection of clonal dissemination of blaNDM-1-producing K. pneumoniae isolates in one of the intensive care units calls for vigilance. Stringent infection prevention and antimicrobial stewardship strategies are highly important measures.
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| 37. |
- Sharma, Rajhans, 1990, et al.
(författare)
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Real-time compaction of nanoconfined DNA by an intrinsically disordered macromolecular counterion
- 2020
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 533:1, s. 175-180
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate how a recently developed nanofluidic device can be used to study protein-induced compaction of genome-length DNA freely suspended in solution. The protein we use in this study is the hepatitis C virus core protein (HCVcp), which is a positively charged, intrinsically disordered protein. Using nanofluidic devices in combination with fluorescence microscopy, we observe that protein-induced compaction preferentially begins at the ends of linear DNA. This observation would be difficult to make with many other single-molecule techniques, which generally require the DNA ends to be anchored to a substrate. We also demonstrate that this protein-induced compaction is reversible and can be dynamically modulated by exposing the confined DNA molecules to solutions containing either HCVcp (to promote compaction) or Proteinase K (to disassemble the compact nucleo-protein complex). Although the natural binding partner for HCVcp is genomic viral RNA, the general biophysical principles governing protein-induced compaction of DNA are likely relevant for a broad range of nucleic acid-binding proteins and their targets.
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| 38. |
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| 39. |
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| 40. |
- Wranne, Moa, 1986, et al.
(författare)
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Comparison of CTX-M encoding plasmids present during the early phase of the ESBL pandemic in western Sweden
- 2024
-
Ingår i: Scientific Reports. - 2045-2322 .- 2045-2322. ; 14:1, s. 11880-
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Tidskriftsartikel (refereegranskat)abstract
- Plasmids encoding blaCTX-M genes have greatly shaped the evolution of E. coli producing extended-spectrum beta-lactamases (ESBL-E. coli) and adds to the global threat of multiresistant bacteria by promoting horizontal gene transfer (HGT). Here we screened the similarity of 47 blaCTX-M -encoding plasmids, from 45 epidemiologically unrelated and disperse ESBL-E. coli strains, isolated during the early phase (2009-2014) of the ESBL pandemic in western Sweden. Using optical DNA mapping (ODM), both similar and rare plasmids were identified. As many as 57% of the plasmids formed five ODM-plasmid groups of at least three similar plasmids per group. The most prevalent type (28%, IncIl, pMLST37) encoded blaCTX-M-15 (n = 10), blaCTX-M-3 (n = 2) or blaCTX-M-55 (n = 1). It was found in isolates of various sequence types (STs), including ST131. This could indicate ongoing local HGT as whole-genome sequencing only revealed similarities with a rarely reported, IncIl plasmid. The second most prevalent type (IncFII/FIA/FIB, F1:A2:B20) harboring blaCTX-M-27, was detected in ST131-C1-M27 isolates, and was similar to plasmids previously reported for this subclade. The results also highlight the need for local surveillance of plasmids and the importance of temporospatial epidemiological links so that detection of a prevalent plasmid is not overestimated as a potential plasmid transmission event in outbreak investigations.
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| 41. |
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| 42. |
- Öz, Robin, 1992, et al.
(författare)
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A nanofluidic device for real-time visualization of DNA–protein interactions on the single DNA molecule level
- 2019
-
Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3372 .- 2040-3364. ; 11:4, s. 2071-2078
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Tidskriftsartikel (refereegranskat)abstract
- Single DNA molecule techniques have revolutionized our understanding of DNA-protein interactions. Traditional techniques for such studies have the major drawback that the DNA molecule studied is attached to a bead or a surface. Stretching of DNA molecules in nanofluidic channels has enabled single-molecule studies of DNA-protein interactions without the need of tethering the molecule to a foreign entity. This in turn allows for studying reactions along the whole extension of the molecule, including the free DNA ends. However, existing studies either rely on measurements where all components are mixed before introduction into the nanochannels or where passive diffusion brings the reagents to the confined DNA molecule. We here present a new generation of nanofluidic devices, where active exchange of the local environment within the nanofluidic channel is possible, while keeping the DNA molecule stretched and in confinement. To demonstrate the functionality of this novel device we added different analytes, such as SDS, spermidine and DNase I, to YOYO-1 stained DNA and studied the response in real time. We also performed a FRET-based reaction, where two different analytes were added sequentially to the same DNA molecule. We believe that this design will enable in situ mapping of complex biochemical processes, involving multiple proteins and cofactors, on single DNA molecules as well as other biomacromolecules.
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| 43. |
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| 44. |
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| 45. |
- Öz, Robin, 1992, et al.
(författare)
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Phosphorylated CtIP bridges DNA to promote annealing of broken ends
- 2020
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:35, s. 21403-21412
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Tidskriftsartikel (refereegranskat)abstract
- The DNA of our cells is constantly exposed to various types of damaging agents. One of the most critical types of damage is when both strands of the DNA break, and thus such breaks need to be efficiently repaired. It is known that CtIP promotes nucleases in DNA break repair. Here we show that CtIP can also hold the two DNA strands together in solution when DNA is free to move, using novel methodology that allows the monitoring of thousands of single DNA molecules in nanofabricated devices. DNA bridging likely facilitates the enzymatic repair steps and identifies novel CtIP functions that are crucial for repairing broken DNA.
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