| 1. |
- Albertsson, Ingrid, et al.
(author)
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Functional interactions between nitrite reductase and nitric oxide reductase from Paracoccus denitrificans
- 2019
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In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Journal article (peer-reviewed)abstract
- Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd(1) nitrite reductase (cd(1)NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd(1)NiR, presumably because cd(1)NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd(1)NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd(1)NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd(1)NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.
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| 2. |
- Ali, Amjad, et al.
(author)
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Photoisomerization of heptamethine cyanine (Cy7) dyes: A theoretical study
- 2024
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In: Dyes and pigments. - : Elsevier BV. - 0143-7208 .- 1873-3743. ; 230
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Journal article (peer-reviewed)abstract
- In this study, density functional theory (DFT) combined with time-dependent (TD) DFT calculations were employed to investigate the photoisomerization reaction kinetics of two near infrared (NIR) heptamethine cyanine (Cy7-NH3 and Cy7-SO3) dyes in the ground singlet state and the first excited singlet state. We found that the photoisomerization of the ground state all-trans Cy7 molecules results in at least one mono-cis and one all-cis species that demonstrate redshifted emission, in agreement with recently published transient state excitation modulation spectroscopy and fluorescence correlation spectroscopy measurements. The transition states were estimated for a whole photoisomerization pathway for both the ground singlet and first excited singlet state potential energy surfaces. We have found that all-cis isomers of the studied Cy7 dyes can be achieved through a sequential two-step photoisomerization within the excited singlet state potential energy surface, along the double CC bond adjacent to edge group (leading to mono-cis isomer 1) and along the double CC bond adjacent to the central-chain group (leading to mono-cis isomer 2). Computations show that all-trans→ mono-cis isomer 1→all-cis kinetics is limited by the first trans→ mono-cis isomer 1 stage, while the all-trans→ mono-cis isomer 2→all-cis pathway is limited by the second mono-cis isomer 2→all-cis stage. Accounting for the fact that mono-cis isomer 2 demonstrates red-shifted emission compared to the all-trans form and that this mono-cis isomer 2 is reachable through the energetically favorable all-trans→ mono-cis isomer 2 stage, we concluded that the experimentally observed red-shifted emission by Cy7-NH3 and Cy7-SO3 should be assigned to the formation of mono-cis isomer 2 species. If the all-cis isomer is populated through the double-step photoisomerization it can also be considered as a source of red-shifted emission. However, as follows from our simulations, the all-cis isomer is kinetically intricate to achieve compared to the mono-cis isomer 2.
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| 3. |
- Bagawath-Singh, Sunitha, et al.
(author)
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Cytokines Induce Faster Membrane Diffusion of MHC Class I and the Ly49A Receptor in a Subpopulation of Natural Killer Cells
- 2016
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In: Frontiers in Immunology. - : Frontiers. - 1664-3224. ; 7
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Journal article (peer-reviewed)abstract
- Cytokines have the potential to drastically augment immune cell activity. Apart from altering the expression of a multitude of proteins, cytokines also affect immune cell dynamics. However, how cytokines affect the molecular dynamics within the cell membrane of immune cells has not been addressed previously. Molecular movement is a vital component of all biological processes, and the rate of motion is, thus, an inherent determining factor for the pace of such processes. Natural killer (NK) cells are cytotoxic lymphocytes, which belong to the innate immune system. By fluorescence correlation spectroscopy, we investigated the influence of cytokine stimulation on the membrane density and molecular dynamics of the inhibitory receptor Ly49A and its ligand, the major histocompatibility complex class I allele H-2D(d), in freshly isolated murine NK cells. H-2D(d) was densely expressed and diffused slowly in resting NK cells. Ly49A was expressed at a lower density and diffused faster. The diffusion rate in resting cells was not altered by disrupting the actin cytoskeleton. A short-term stimulation with interleukin-2 or interferon- alpha + beta did not change the surface density of moving H-2D(d) or Ly49A, despite a slight upregulation at the cellular level of H-2D(d) by interferon-alpha + beta, and of Ly49A by IL-2. However, the molecular diffusion rates of both H-2D(d) and Ly49A increased significantly. A multivariate analysis revealed that the increased diffusion was especially marked in a subpopulation of NK cells, where the diffusion rate was increased around fourfold compared to resting NK cells. After IL-2 stimulation, this subpopulation of NK cells also displayed lower density of Ly49A and higher brightness per entity, indicating that Ly49A may homo-cluster to a larger extent in these cells. A faster diffusion of inhibitory receptors could enable a faster accumulation of these molecules at the immune synapse with a target cell, eventually leading to a more efficient NK cell response. It has previously been assumed that cytokines regulate immune cells primarily via alterations of protein expression levels or posttranslational modifications. These findings suggest that cytokines may also modulate immune cell efficiency by increasing the molecular dynamics early on in the response.
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| 4. |
- Bagheri, Niusha, et al.
(author)
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Change in the emission saturation and kinetics of upconversion nanoparticles under different light irradiations
- 2019
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In: Optical materials (Amsterdam). - : Elsevier. - 0925-3467 .- 1873-1252. ; 97
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Journal article (peer-reviewed)abstract
- Nd3+-sensitized upconversion nanoparticles (UCNPs) can be excited by both 980 and 808 nm light, which is regarded as a particularly advantageous property of these particles. In this work, we demonstrate that the nanoparticles can exhibit significantly different response when excited at these two excitation wavelengths, showing dependence on the intensity of the excitation light and the way it is distributed in time. Specifically, with 808 nm excitation saturation in the emitted luminescence is more readily reached with increasing excitation intensities than upon 980 nm excitation. This is accompanied by delayed upconversion luminescence (UCL) kinetics and weaker UCL intensities. The different luminescence response at 808 and 980 nm excitation reported in this work is relevant in a manifold of applications using UCNPs as labels and sensors. This could also open new possibilities for multi-wavelength excitable UCNPs for upconversion color display and in laser-scanning microscopy providing selective readouts and sub-sectioning of samples.
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| 5. |
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| 7. |
- Bergstrand, Jan, et al.
(author)
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Fast, streamlined fluorescence nanoscopy resolves rearrangements of SNARE and cargo proteins in platelets co-incubated with cancer cells
- 2022
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In: Journal of Nanobiotechnology. - : Springer Nature. - 1477-3155. ; 20:1
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Journal article (peer-reviewed)abstract
- Background Increasing evidence suggests that platelets play a central role in cancer progression, with altered storage and selective release from platelets of specific tumor-promoting proteins as a major mechanism. Fluorescence-based super-resolution microscopy (SRM) can resolve nanoscale spatial distribution patterns of such proteins, and how they are altered in platelets upon different activations. Analysing such alterations by SRM thus represents a promising, minimally invasive strategy for platelet-based diagnosis and monitoring of cancer progression. However, broader applicability beyond specialized research labs will require objective, more automated imaging procedures. Moreover, for statistically significant analyses many SRM platelet images are needed, of several different platelet proteins. Such proteins, showing alterations in their distributions upon cancer progression additionally need to be identified. Results A fast, streamlined and objective procedure for SRM platelet image acquisition, analysis and classification was developed to overcome these limitations. By stimulated emission depletion SRM we imaged nanoscale patterns of six different platelet proteins; four different SNAREs (soluble N-ethylmaleimide factor attachment protein receptors) mediating protein secretion by membrane fusion of storage granules, and two angiogenesis regulating proteins, representing cargo proteins within these granules coupled to tumor progression. By a streamlined procedure, we recorded about 100 SRM images of platelets, for each of these six proteins, and for five different categories of platelets; incubated with cancer cells (MCF-7, MDA-MB-231, EFO-21), non-cancer cells (MCF-10A), or no cells at all. From these images, structural similarity and protein cluster parameters were determined, and probability functions of these parameters were generated for the different platelet categories. By comparing these probability functions between the categories, we could identify nanoscale alterations in the protein distributions, allowing us to classify the platelets into their correct categories, if they were co-incubated with cancer cells, non-cancer cells, or no cells at all. Conclusions The fast, streamlined and objective acquisition and analysis procedure established in this work confirms the role of SNAREs and angiogenesis-regulating proteins in platelet-mediated cancer progression, provides additional fundamental knowledge on the interplay between tumor cells and platelets, and represent an important step towards using tumor-platelet interactions and redistribution of nanoscale protein patterns in platelets as a basis for cancer diagnostics.
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| 8. |
- Bergstrand, Jan, et al.
(author)
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On the decay time of upconversion luminescence
- 2019
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In: Nanoscale. - : Royal Society of Chemistry. - 2040-3364 .- 2040-3372. ; 11:11, s. 4959-4969
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Journal article (peer-reviewed)abstract
- In this study, we systematically investigate the decay characteristics of upconversion luminescence (UCL) under anti-Stokes excitation through numerical simulations based on rate-equation models. We find that a UCL decay profile generally involves contributions from the sensitizer's excited-state lifetime, energy transfer and cross-relaxation processes. It should thus be regarded as the overall temporal response of the whole upconversion system to the excitation function rather than the intrinsic lifetime of the luminescence emitting state. Only under certain conditions, such as when the effective lifetime of the sensitizer's excited state is significantly shorter than that of the UCL emitting state and of the absence of cross-relaxation processes involving the emitting energy level, the UCL decay time approaches the intrinsic lifetime of the emitting state. Subsequently, Stokes excitation is generally preferred in order to accurately quantify the intrinsic lifetime of the emitting state. However, possible cross-relaxation between doped ions at high doping levels can complicate the decay characteristics of the luminescence and even make the Stokes-excitation approach fail. A strong cross-relaxation process can also account for the power dependence of the decay characteristics of UCL.
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| 9. |
- Bergstrand, Jan, et al.
(author)
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Scanning inverse fluorescence correlation spectroscopy
- 2014
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In: Optics Express. - : Optical Society of America. - 1094-4087. ; 22:11, s. 13073-13090
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Journal article (peer-reviewed)abstract
- Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross-and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 +/- 12 nm diameter, and 40 nm domains were estimated to 65 +/- 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.
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| 10. |
- Bergstrand, Jan
(author)
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Super resolution fluorescence imaging : analyses, simulations and applications
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Fluorescence methods offer extraordinary sensitivity and specificity, and are extensively used in the life sciences. In recent years, super resolution fluorescence imaging techniques have developed strongly, uniquely combining ~10 nm sub diffraction resolution and specific labeling with high efficiency. This thesis explores this potential, with a major focus on Stimulated Emission Depletion, STED, microscopy, applications thereof, image analyses and simulation studies. An additional theme in this thesis is development and use of single molecule fluorescence correlation spectroscopy, FCS, and related techniques, as tools to study dynamic processes at the molecular level. In paper I the proteins cytochrome-bo3 and ATP-synthase are studied with fluorescence cross-correlation spectroscopy, FCCS. These two proteins are a part of the energy conversion process in E. coli, converting ADP into ATP. We found that an increased interaction between these proteins, detected by FCCS, correlates with an increase in the ATP production. In paper II an FCS-based imaging method is developed, capable to determine absolute sizes of objects, smaller than the resolution limit of the microscope used. Combined with STED, this may open for studies of membrane nano-domains, such as those investigated by simulations in paper VII. In paper III and paper IV super resolution STED imaging was applied on Streptococcus Pneumoniae, revealing information about function and distribution of proteins involved in the defense mechanism of the bacteria, as well as their role in bacterial meningitis. In paper V, we used STED imaging to investigate protein distributions in platelets. We then found that the adhesion protein P-selectin changes its distribution pattern in platelets incubated with tumor cells, and with machine learning algorithms and classical image analysis of the STED images it is possible to automatically distinguish such platelets from platelets activated by other means. This could provide a strategy for minimally invasive diagnostics of early cancer development, and deeper understanding of the role of platelets in cancer development. Finally, this thesis presents Monte-Carlo simulations of biological processes and their monitoring by FCS. In paper VI, a combination of FCCS and simulations was applied to resolve the interactions between a transcription factor (p53) and an oncoprotein (MDM2) inside live cells. In paper VII, the feasibility of FCS techniques for studying nano-domains in membranes is investigated purely by simulations, identifying the conditions under which such nano-domains would be possible to detect by FCS. In paper VIII, proton exchange dynamics at biological membranes were simulated in a model, verifying experimental FCS data and identifying fundamental mechanisms by which membranes mediate proton exchange on a local (~10nm) scale.
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