| 1. |
- Karempudi, Praneeth
(författare)
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Microfluidics and AI for single-cell microbiology
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Most of the biological sciences deal with understanding the relationships between phenotypes and the underlying molecular mechanisms of organisms. This thesis is an engineering, computational, and experimental exercise in expanding the scope and scale of phenotype-genotype mapping techniques in single-cell microbiology using microscopy, microfluidics, and image processing. To this end, we use mother-machine-based microfluidic devices together with recently developed techniques in deep learning and optics. We use optical microscopes to observe cells of different genotypes, physically move cells, and image molecules inside them.We have designed a novel microfluidic device to expand the throughput of single-cell lineage tracing an order of magnitude compared to existing methods. We demonstrate the ability to isolate single cells from such a device using optical tweezers after phenotypic characterization in real time. We have developed analysis algorithms of various kinds with the prime intention of performing high-throughput real-time image processing in conjunction with experimental runs to identify interesting cells for further investigation.We have also developed an experimental protocol for bacterial species identification using fluorescence-in-situ hybridization (FISH) in microfluidic chips to complement an existing phenotype-based antibiotic-susceptibility test (AST). We apply this method together with deep-learning-based cell segmentation and tracking algorithms, and image classification methods to perform species-ID of up to 10 species in 2-3 hrs.Lastly, we have developed a 3D dot localization method to investigate how the chromosome structure changes during the E. coli cell cycle. Different loci on the E. coli chromosome were labeled using DNA-binding fluorescent proteins and imaged using an optical setup with an astigmatic point-spread-function. Mother-machine devices were used to constrain the movement of cells to the lateral plane during growth. A deep-learning-based single-molecule localization method was adapted for this application and used to map the chromosomal loci’s physical position in 3D as a function of cell size during the E. coli cell cycle.
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| 2. |
- Brandis, Gerrit, 1985-, et al.
(författare)
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Antibiotic perseverance increases the risk of resistance development
- 2023
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 120:2
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Tidskriftsartikel (refereegranskat)abstract
- The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular responses. Using single-cell imaging, we studied the growth response of Escherichia coli to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for three out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the population size and led to an up to 40-fold increase in the frequency of antibiotic resistance mutations in gram-negative and gram-positive species. We conclude that antibiotic perseverance is a common phenomenon that has the potential to impact antibiotic resistance development across pathogenic bacteria.
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| 3. |
- Mahmutovic, Anel
(författare)
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Reaction-Diffusion kinetics of Protein DNA Interactions
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transcription factors need to rapidly find one specific binding site among millions of nonspecific sites on the chromosomal DNA. In this thesis I use various aspects of reaction-diffusion theory to investigate the interaction between proteins and DNA and to explain the searching, finding and binding to specific operator sites. Using molecular dynamics methods we calculate the free energy profile for the model protein LacI as it leaves a nonspecific stretch of DNA and as it slides along DNA. Based on the free energy profiles we estimate the microscopic dissociation rate constant, kdmicro ~1.45×104s-1, and the 1D diffusion coefficient, D1 ~ 0.05-0.29 μm2s-1 (2-40μs to slide 1 basepair (bp)). At a non-atomistic level of detail we estimate the number of microscopic rebindings before a macroscopic dissociation occurs which leads to the macroscopic residence time, τDmacro ~ 48±12ms resulting in a in vitro sliding length estimate of 135-345bp.When we fit the DNA interaction parameters for in vivo conditions to recent single molecule in vivo experiments we conclude that neither hopping nor intersegment transfer contribute to the target search for the LacI dimer, that it appears to bind the specific Osym operator site as soon as it slides into it, and that the sliding length is around 40bp in the cell. The estimated in vivo D1 ~ 0.025 μm2s-1 is higher than expected from estimates of D1 based on viscosity and the atomistic simulations. Surprisingly, we were also forced to conclude that the nonspecific association for the LacI dimer appeared reaction limited which is in conflict with the free energy profile. This inconsistency is resolved by allowing for steric effects. Using reaction-diffusion theory and simulations we show that an apparent reaction limited association can be diffusion limited if geometry and steric effects are taken into account. Furthermore, the simulations show that a protein binds ~2 times faster to a DNA molecule with a helical reactive patch than to a stripe patch running along the length of the DNA. This facilitated binding has a direct impact on the search time especially in the presence of other DNA binding proteins.
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| 4. |
- Marklund, Emil, et al.
(författare)
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Sequence specificity in DNA binding is mainly governed by association
- 2022
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 375:6579, s. 442-445
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Tidskriftsartikel (refereegranskat)abstract
- Sequence-specific binding of proteins to DNA is essential for accessing genetic information. We derive a model that predicts an anticorrelation between the macroscopic association and dissociation rates of DNA binding proteins. We tested the model for thousands of different lac operator sequences with a protein binding microarray and by observing kinetics for individual lac repressor molecules in single-molecule experiments. We found that sequence specificity is mainly governed by the efficiency with which the protein recognizes different targets. The variation in probability of recognizing different targets is at least 1.7 times as large as the variation in microscopic dissociation rates. Modulating the rate of binding instead of the rate of dissociation effectively reduces the risk of the protein being retained on nontarget sequences while searching.
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| 5. |
- Liao, Qinghua, et al.
(författare)
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Long Time-Scale Atomistic Simulations of the Structure and Dynamics of Transcription Factor-DNA Recognition
- 2019
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 123:17, s. 3576-3590
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Tidskriftsartikel (refereegranskat)abstract
- Recent years have witnessed an explosion of interest in computational studies of DNA binding proteins, including both coarse grained and atomistic simulations of transcription factor-DNA recognition, to understand how these transcription factors recognize their binding sites on the DNA with such exquisite specificity. The present study performs microsecond time scale all-atom simulations of the dimeric form of the lactose repressor (Lad), both in the absence of any DNA and in the presence of both specific and nonspecific complexes, considering three different DNA sequences. We examine, specifically, the conformational differences between specific and nonspecific protein DNA interactions, as well as the behavior of the helix-turn-helix motif of Lad when interacting with the DNA. Our simulations suggest that stable Lad binding occurs primarily to bent A-form DNA, with a loss of Lad conformational entropy and optimization of correlated conformational equilibria across the protein. In addition, binding to the specific operator sequence involves a slightly larger number of stabilizing DNA protein hydrogen bonds (in comparison to nonspecific complexes), which may account for the experimentally observed specificity for this operator. In doing so, our simulations provide a detailed atomistic description of potential structural drivers for LacI selectivity.
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| 6. |
- Lindén, Martin, 1976-, et al.
(författare)
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Simulated single molecule microscopy with SMeagol
- 2016
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Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1367-4811. ; 32:15, s. 2394-2395
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Tidskriftsartikel (refereegranskat)abstract
- Summary: SMeagol is a software tool to simulate highly realistic microscopy data based on spatial systems biology models, in order to facilitate development, validation and optimization of advanced analysis methods for live cell single molecule microscopy data.Availability and implementation: SMeagol runs on Matlab R2014 and later, and uses compiled binaries in C for reaction–diffusion simulations. Documentation, source code and binaries for Mac OS, Windows and Ubuntu Linux can be downloaded from http://smeagol.sourceforge.net.
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| 7. |
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| 8. |
- Balzarotti, Francisco, et al.
(författare)
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Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes
- 2017
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 355:6325, s. 606-612
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Tidskriftsartikel (refereegranskat)abstract
- We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolutionmicroscopy, MINFLUXattained similar to 1-nanometer precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.
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| 9. |
- Bashardanesh, Zahedeh, et al.
(författare)
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Rotational and Translational Diffusion of Proteins as a Function of Concentration
- 2019
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Ingår i: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 4:24, s. 20654-20664
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Tidskriftsartikel (refereegranskat)abstract
- Atomistic simulations of three different proteins at different concentrations are performed to obtain insight into protein mobility as a function of protein concentration. We report on simulations of proteins from diluted to the physiological water concentration (about 70% of the mass). First, the viscosity was computed and found to increase by a factor of 7-9 going from pure water to the highest protein concentration, in excellent agreement with in vivo nuclear magnetic resonance results. At a physiological concentration of proteins, the translational diffusion is found to be slowed down to about 30% of the in vitro values. The slow-down of diffusion found here using atomistic models is slightly more than that of a hard sphere model that neglects the electrostatic interactions. Interestingly, rotational diffusion of proteins is slowed down somewhat more (by about 80-95% compared to in vitro values) than translational diffusion, in line with experimental findings and consistent with the increased viscosity. The finding that rotation is retarded more than translation is attributed to solvent-separated clustering. No direct interactions between the proteins are found, and the clustering can likely be attributed to dispersion interactions that are stronger between proteins than between protein and water. Based on these simulations, we can also conclude that the internal dynamics of the proteins in our study are affected only marginally under crowding conditions, and the proteins become somewhat more stable at higher concentrations. Simulations were performed using a force field that was tuned for dealing with crowding conditions by strengthening the protein-water interactions. This force field seems to lead to a reproducible partial unfolding of an alpha-helix in one of the proteins, an effect that was not observed in the unmodified force field.
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| 10. |
- Berg, Otto G., et al.
(författare)
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The helical structure of DNA facilitates binding
- 2016
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Ingår i: Journal of Physics A. - : IOP Publishing. - 1751-8113 .- 1751-8121. ; 9:36
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.
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