| 1. |
- Castelain, Mickaël, et al.
(författare)
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Fast uncoiling kinetics of F1C pili expressed by uropathogenic Escherichia coli are revealed on a single pilus level using force-measuring optical tweezers
- 2011
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 40:3, s. 305-316
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Tidskriftsartikel (refereegranskat)abstract
- Uropathogenic Escherichia coli (UPEC) expressvarious kinds of organelles, so-called pili or fimbriae, thatmediate adhesion to host tissue in the urinary tract throughspecific receptor-adhesin interactions. The biomechanicalproperties of these pili have been considered important forthe ability of bacteria to withstand shear forces from rinsingurine flows. Force-measuring optical tweezers have beenused to characterize individual organelles of F1C typeexpressed by UPEC bacteria with respect to such properties.Qualitatively, the force-versus-elongation response wasfound to be similar to that of other types of helix-like piliexpressed by UPEC, i.e., type 1, P, and S, with force-inducedelongation in three regions, one of which represents theimportant uncoiling mechanism of the helix-like quaternarystructure. Quantitatively, the steady-state uncoiling forcewas assessed as 26.4 ±1.4 pN, which is similar to those ofother pili (which range from 21 pN for SI to 30 pN for type 1).The corner velocity for dynamic response (1,400 nm/s) wasfound to be larger than those of the other pili (400–700 nm/sfor S and P pili, and 6 nm/s for type 1). The kinetics werefound to be faster, with a thermal opening rate of 17 Hz, afew times higher than S and P pili, and three orders ofmagnitude higher than type 1. These data suggest that F1Cpili are, like P and S pili, evolutionarily selected to primarilywithstand the conditions expressed in the upper urinary tract.
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| 2. |
- Nilsson, Daniel P. G., et al.
(författare)
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Step-by-Step Guide to 3D Print Motorized Rotation Mounts for Optical Applications
- 2021
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Ingår i: Applied Optics. - : Optical Society of America. - 0003-6935 .- 1539-4522. ; 60:13, s. 3764-3771
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Tidskriftsartikel (refereegranskat)abstract
- Motorized rotation mounts and stages are versatile instruments that introduce computer control to optical systems, enabling automation and scanning actions. They can be used for intensity control and position adjustments, etc. However, these rotation mounts come with a hefty price tag, and this limits their use. This work shows how to build two different types of motorized rotation mounts for 1" optics, using a 3D printer and off-the-shelf components. The first is intended for reflective elements, like mirrors and gratings, and the second for transmissive elements, like polarizers and retarders. We evaluate and compare their performance to commercial systems based on velocity, resolution, precision, backlash, and axis wobble. Also, we investigate the angular stability using Allan variance analysis. The results show that our mounts perform similar to systems costing more than 2000 Euro, while also being quick to build and costing less than 200 Euro. As a proof of concept, we show how to control lasers used in an optical tweezers and Raman spectroscopy setup. When used for this, the 3D printed motorized rotational mounts provide intensity control with a resolution of 0.03 percentage points or better.
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| 3. |
- Alakpa, Enateri V., et al.
(författare)
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Bioprinted schwann and mesenchymal stem cell co-cultures for enhanced spatial control of neurite outgrowth
- 2023
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Ingår i: Gels. - : MDPI. - 2310-2861. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Bioprinting nerve conduits supplemented with glial or stem cells is a promising approach to promote axonal regeneration in the injured nervous system. In this study, we examined the effects of different compositions of bioprinted fibrin hydrogels supplemented with Schwann cells and mesenchymal stem cells (MSCs) on cell viability, production of neurotrophic factors, and neurite outgrowth from adult sensory neurons. To reduce cell damage during bioprinting, we analyzed and optimized the shear stress magnitude and exposure time. The results demonstrated that fibrin hydrogel made from 9 mg/mL of fibrinogen and 50IE/mL of thrombin maintained the gel’s highest stability and cell viability. Gene transcription levels for neurotrophic factors were significantly higher in cultures containing Schwann cells. However, the amount of the secreted neurotrophic factors was similar in all co-cultures with the different ratios of Schwann cells and MSCs. By testing various co-culture combinations, we found that the number of Schwann cells can feasibly be reduced by half and still stimulate guided neurite outgrowth in a 3D-printed fibrin matrix. This study demonstrates that bioprinting can be used to develop nerve conduits with optimized cell compositions to guide axonal regeneration.
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| 4. |
- Andersson, Magnus, et al.
(författare)
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A structural basis for sustained bacterial adhesion : Biomechanical properties of CFA/I Pili
- 2012
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 415:5, s. 918-928
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Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized at single organelle level the intrinsic biomechanical properties and kinetics of individual CFA/I pili, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P-pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix, and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.
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| 5. |
- Andersson, Magnus, et al.
(författare)
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Force measuring optical tweezers system for long time measurements of P pili stability
- 2006
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Ingår i: Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IV. - : SPIE. - 081946130X - 9780819461308 ; , s. 608810-
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Konferensbidrag (refereegranskat)abstract
- A force-measuring optical tweezers instrumentation and long time measurements of the elongation and retraction of bacterial fimbriae from Uropathogenic E. coli (UPEC) under strain are presented. The instrumentation is presented in some detail. Special emphasis is given to measures taken to reduce the influence of noise and drifts in the system and from the surrounding, which makes long term force measurements possible. Individual P pili from UPEC bacteria were used as a biological model system for repetitive unfolding and refolding cycles of bacterial fimbriae under equilibrium conditions. P pili have evolved into a three-dimensional helix-like structure, the PapA rod, that can be successively and significantly elongated and/or unfolded when exposed to external forces. The instrumentation is used for characterization of the force-vs.-elongation response of the PapA rod of individual P pili, with emphasis on the long time stability of the forced unfolding and refolding of the helical structure of the PapA rod. The results show that the PapA rod is capable of withstanding extensive strain, leading to a complete unfolding of the helical structure, repetitive times during the life cycle of a bacterium without any noticeable alteration of the mechanical properties of the P pili. This function is believed to be importance for UPEC bacteria in vivo since it provides a close contact to a host cell (which is an initial step of invasion) despite urine cleaning attempts.
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| 6. |
- Baker, Joseph, et al.
(författare)
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Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress
- 2021
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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. - 0027-8424 .- 1091-6490. ; 118:21
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Tidskriftsartikel (refereegranskat)abstract
- Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that Class 1a pili of uropathogenic E. coli (UPEC), as well as Class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC Class 5 pili. Examining structural and steered molecular dynamics simulation data, we find this difference in Class 1 pili subunit behavior originates from an alpha-helical motif that can unfold when exposed to force. A disulfide bond cross-linking beta-strands in Class 1 pili stabilizes subunits, allowing them to tolerate higher forces than Class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, Class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of Class 5 ETEC pili.
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| 7. |
- Baker, Joseph, et al.
(författare)
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Unveiling the Contributions of Secondary Structure and Disulfide Bonds for Bacterial Adhesion Pili Extension using a Multiscale Approach
- 2021
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bacterial adhesion pili are essential virulence factors for many pathogenic Escherichia coli, including bacteria that cause urinary tract infections (UPEC) and diarrheal diseases (ETEC). To sustain adhesion under forces similar to those in the fluid environments of the urinary tract and gastrointestinal tract, these pili (also called fimbriae) can extend to over seven times their original length. Both UPEC and ETEC can uncoil their quaternary structure under pulling force and re-coil to their helical form when the force is reduced, as observed using optical tweezers. However, after extension to a linear polymer UPEC undergo an additional reversible conformational change, that is not seen in ETEC. The mechanism for this conformational change in UPEC is not known. Therefore, to obtain a comprehensive picture of pilus extension we have taken a synergistic approach that combines optical tweezer experiments, structural data from cryo-EM, and steered molecular dynamics simulations to investigate the response of pilin subunits to force.Our multi-faceted approach provides novel molecular-scale insights into the structural changes that occur in UPEC and ETEC pili under pulling forces. We find that the conformational change observed in UPEC pili in optical tweezer experiments is correlated with the presence of an alpha helix. In addition, structural analysis and steered molecular dynamics simulations show that there is a disulfide bond that provides additional stability of UPEC pilin subunits that is not observed in ETEC pilins, which lack cysteine residues. Together, these results suggest that the mechanism of extension of bacterial adhesion pili is related to their environmental niche, and the magnitude of fluid forces in the urinary tract versus the GI tract.
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| 8. |
- Barbercheck Epler, Chelsea R., et al.
(författare)
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Bacterial adhesion pili
- 2018
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Ingår i: Membrane protein complexes. - Singapore : Springer Publishing Company. - 9789811077579 - 9789811077562 ; , s. 1-18
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Bokkapitel (refereegranskat)abstract
- Escherichia coli bacterial cells produce multiple types of adhesion pili that mediate cell-cell and cell-host attachments. These pili (also called 'fimbriae') are large biopolymers that are comprised of subunits assembled via a sophisticated micro-machinery into helix-like structures that are anchored in the bacterial outer membrane. They are commonly essential for initiation of disease and thus provide a potential target for antibacterial prevention and treatment. To develop new therapeutics for disease prevention and treatment we need to understand the molecular mechanisms and the direct role of adhesion pili during pathogenesis. These helix-like pilus structures possess fascinating and unique biomechanical properties that have been thoroughly investigated using high-resolution imaging techniques, force spectroscopy and fluid flow chambers. In this chapter, we first discuss the structure of pili and the micro-machinery responsible for the assembly process. Thereafter, we present methods for measurement of the biomechanics of adhesion pili, including optical tweezers. Data demonstrate unique biomechanical properties of pili that allow bacteria to sustain binding during in vivo fluid shear forces. We thereafter summarize the current biomechanical findings related to adhesion pili and show that pili biomechanical properties are niche-specific. That is, the data suggest that there is an organ-specific adaptation of pili that facilitates infection of the bacteria's target tissue. Thus, pilus biophysical properties are an important part of Escherichia coli pathogenesis, allowing bacteria to overcome hydrodynamic challenges in diverse environments.
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| 9. |
- Björnham, Oscar, et al.
(författare)
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Theory for nonlinear dynamic force spectroscopy
- 2017
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 46:3, s. 225-233
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Tidskriftsartikel (refereegranskat)abstract
- Dynamic force spectroscopy (DFS) is an experimental technique that is commonly used to assess information on the strength, energy landscape, and lifetime of noncovalent bio-molecular interactions. DFS traditionally requires an applied force that increases linearly with time so that the bio-complex under investigation is exposed to a constant loading rate. However, tethers or polymers can modulate the applied force in a nonlinear manner. For example, bacterial adhesion pili and polymers with worm-like chain properties are structures that show nonlinear force responses. In these situations, the theory for traditional DFS cannot be readily applied. In this work, we expand the theory for DFS to also include nonlinear external forces while still maintaining compatibility with the linear DFS theory. To validate the theory, we modeled a bio- complex expressed on a stiff, an elastic, and a worm-like chain polymer, using Monte Carlo methods, and assessed the corresponding rupture force spectra. It was found that the nonlinear DFS (NLDFS) theory correctly predicted the numerical results. We also present a protocol suggesting an experimental approach and analysis method of the data to estimate the bond length and the thermal off-rate.
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| 10. |
- Castelain, Mickaël, et al.
(författare)
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Characterization of the Biomechanical Properties of T4 Pili Expressed by Streptococcus pneumoniae – A Comparison between Helix-like and Open Coil-like Pili
- 2009
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Ingår i: ChemPhysChem. - : Wiley. - 1439-4235 .- 1439-7641. ; 10:9-10, s. 1533-1540
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial adhesion organelles, known as fimbria or pili, are expressed by Gram–positive as well as Gram–negative bacteria families. These appendages play a key role in the first steps of the invasion and infection processes, and they therefore provide bacteria with pathogenic abilities. To improve the knowledge of pili-mediated bacterial adhesion to host cells and how these pili behave under the presence of an external force, we first characterize, using force measuring optical tweezers, open coil-like T4 pili expressed by Gram–positive Streptococcus pneumoniae with respect to their biomechanicalproperties. It is shown that their elongation behavior can be well described by the worm-like chain model and that they possess a large degree of flexibility. Their properties are then compared with those of helix-like pili expressed by Gram–negative uropathogenic Escherichia coli (UPEC), which have different pili architecture. The differences suggest that these two types of pili have distinctly dissimilar mechanisms to adhere and sustain external forces. Helix-like pili expressed by UPEC bacteria adhere to host cells by single adhesins located at the distal end of the pili while their helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow and to increase the cooperativity of the pili ensemble. Open coil-like pili expressed by S. pneumoniae adhere to cells by a multitude of adhesins distributed along the pili. It is hypothesized that these two types of pili represent different strategies of adhering to host cells in the presence of external forces. When exposed to significant forces, bacteria expressing helix-like pili remain attached bydistributing the external force among a multitude of pili, whereas bacteria expressing open coil-like pili sustain large forces primarily by their multitude of binding adhesins.
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