| 1. |
- Daemen, Sabine, et al.
(författare)
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Label-free CARS microscopy reveals similar triacylglycerol acyl chain length and saturation in myocellular lipid droplets of athletes and individuals with type 2 diabetes
- 2020
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 1432-0428 .- 0012-186X. ; 63:12, s. 2654-2664
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Tidskriftsartikel (refereegranskat)abstract
- Aims/hypothesis: Intramyocellular lipid (IMCL) content associates with development of insulin resistance, albeit not in insulinsensitive endurance-trained athletes (trained). Qualitative and spatial differences in muscle lipid composition may underlie this so-called athlete’s paradox. Here we studied triacylglycerol (TAG) composition of individual myocellular lipid droplets (LDs) in trained individuals and individuals with type 2 diabetes mellitus. Methods: Trained (˙V O2max 71.0 ± 1.6 ml O2 [kg lean body mass (LBM)]−1 min−1), normoglycaemic (fasting glucose 5.1 ± 0.1 mmol/l) individuals and untrained (V O2max 36.8 ± 1.5 ml O2 [kg LBM]−1 min−1) individuals with type 2 diabetes (fasting glucose 7.4 ± 0.5 mmol/l), with similar IMCL content (3.5 ± 0.7% vs 2.5 ± 0.3%, p = 0.241), but at opposite ends of the insulin sensitivity spectrum(glucose infusion rate 93.8 ± 6.6 vs 25.7 ± 5.3 μmol [kg LBM]−1 min−1 for trained individuals and those with type 2 diabetes, respectively) were included from our database in the present study. We applied in situ label-free broadband coherent anti-Stokes Raman scattering (CARS) microscopy to sections from skeletal muscle biopsies to measure TAG acyl chain length and saturation of myocellular LDs. This approach uniquely permits examination of individual LDs in their native environment, in a fibre-type-specific manner, taking into account LD size and subcellular location. Results: Despite a significant difference in insulin sensitivity, we observed remarkably similar acyl chain length and saturation in trained and type 2 diabetic individuals (chain length: 18.12 ± 0.61 vs 18.36 ± 0.43 number of carbons; saturation: 0.37 ± 0.05 vs 0.38 ± 0.06 number of C=C bonds). Longer acyl chains or higher saturation (lower C=C number) could be detected in subpopulations of LDs, i.e. large LDs (chain length: 18.11 ± 0.48 vs 18.63 ± 0.57 carbon number) and subsarcolemmal LDs (saturation: 0.34 ± 0.02 vs 0.36 ± 0.04 C=C number), which are more abundant in individuals with type 2 diabetes. Conclusions/interpretation: In contrast to reports of profound differences in the lipid composition of lipids extracted from skeletal muscle from trained and type 2 diabetic individuals, our in situ, LD-specific approach detected only modest differences in TAG composition in LD subpopulations, which were dependent on LD size and subcellular location. If, and to what extent, these modest differences can impact insulin sensitivity remains to be elucidated.
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| 2. |
- Lau, Hang Kuen, et al.
(författare)
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Microstructured Elastomer-PEG Hydrogels via Kinetic Capture of Aqueous Liquid–Liquid Phase Separation
- 2018
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Ingår i: Advanced Science. - : Wiley. - 2198-3844 .- 2198-3844. ; 5:6
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Tidskriftsartikel (refereegranskat)abstract
- Heterogeneous hydrogels with desired matrix complexity are studied for a variety of biomimetic materials. Despite the range of such microstructured materials described, few methods permit independent control over microstructure and microscale mechanics by precisely controlled, single-step processing methods. Here, a phototriggered crosslinking methodology that traps microstructures in liquid–liquid phase-separated solutions of a highly elastomeric resilin-like polypeptide (RLP) and poly(ethylene glycol) (PEG) is reported. RLP-rich domains of various diameters can be trapped in a PEG continuous phase, with the kinetics of domain maturation dependent on the degree of acrylation. The chemical composition of both hydrogel phases over time is assessed via in situ hyperspectral coherent Raman microscopy, with equilibrium concentrations consistent with the compositions derived from NMR-measured coexistence curves. Atomic force microscopy reveals that the local mechanical properties of the two phases evolve over time, even as the bulk modulus of the material remains constant, showing that the strategy permits control of mechanical properties on micrometer length scales, of relevance in generating mechanically robust materials for a range of applications. As one example, the successful encapsulation, localization, and survival of primary cells are demonstrated and suggest the potential application of phase-separated RLP-PEG hydrogels in regenerative medicine applications.
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| 3. |
- Paul, Alexandra, 1988, et al.
(författare)
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Biomechanical Dependence of SARS-CoV-2 Infections
- 2022
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Ingår i: ACS Applied Bio Materials. - : American Chemical Society (ACS). - 2576-6422. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- Older people have been disproportionately vulnerable to the current SARS-CoV-2 pandemic, with an increased risk of severe complications and death compared to other age groups. A mix of underlying factors has been speculated to give rise to this differential infection outcome including changes in lung physiology, weakened immunity, and severe immune response. Our study focuses on the impact of biomechanical changes in lungs that occur as individuals age, that is, the stiffening of the lung parenchyma and increased matrix fiber density. We used hydrogels with an elastic modulus of 0.2 and 50 kPa and conventional tissue culture surfaces to investigate how infection rate changes with parenchymal tissue stiffness in lung epithelial cells challenged with SARS-CoV-2 Spike (S) protein pseudotyped lentiviruses. Further, we employed electrospun fiber matrices to isolate the effect of matrix density. Given the recent data highlighting the importance of alternative virulent strains, we included both the native strain identified in early 2020 and an early S protein variant (D614G) that was shown to increase the viral infectivity markedly. Our results show that cells on softer and sparser scaffolds, closer resembling younger lungs, exhibit higher infection rates by the WT and D614G variant. This suggests that natural changes in lung biomechanics do not increase the propensity for SARS-CoV-2 infection and that other factors, such as a weaker immune system, may contribute to increased disease burden in the elderly.
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| 4. |
- Paul, Alexandra, 1988, et al.
(författare)
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Comparing lipid remodeling of brown adipose tissue, white adipose tissue, and liver after one-week high fat diet intervention with quantitative Raman microscopy
- 2023
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Ingår i: Journal of Cellular Biochemistry. - : Wiley. - 0730-2312 .- 1097-4644. ; 124:3, s. 382-395
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Tidskriftsartikel (refereegranskat)abstract
- Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, nonchronic dietary intervention (a 1-week interval) on WAT and interscapular BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that 1 week of high fat diet (HFD) intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after 1 week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance.
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| 5. |
- Paul, Alexandra, 1988, et al.
(författare)
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Effect of ambient temperature on respiratory tract cells exposed to SARS-CoV-2 viral mimicking nanospheres - An experimental study
- 2021
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Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- The novel coronavirus caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached more than 160 countries and has been declared a pandemic. SARS-CoV-2 infects host cells by binding to the angiotensin-converting enzyme 2 (ACE-2) surface receptor via the spike (S) receptor-binding protein (RBD) on the virus envelope. Global data on a similar infectious disease spread by SARS-CoV-1 in 2002 indicated improved stability of the virus at lower temperatures facilitating its high transmission in the community during colder months (December–February). Seasonal viral transmissions are strongly modulated by temperatures, which can impact viral trafficking into host cells; however, an experimental study of temperature-dependent activity of SARS-CoV-2 is still lacking. We mimicked SARS-CoV-2 with polymer beads coated with the SARS-CoV-2 S protein to study the effect of seasonal temperatures on the binding of virus-mimicking nanospheres to lung epithelia. The presence of the S protein RBD on nanosphere surfaces led to binding by Calu-3 airway epithelial cells via the ACE-2 receptor. Calu-3 and control fibroblast cells with S-RBD-coated nanospheres were incubated at 33 and 37 °C to mimic temperature fluctuations in the host respiratory tract, and we found no temperature dependence in contrast to nonspecific binding of bovine serum ablumin-coated nanospheres. Moreover, the ambient temperature changes from 4 to 40 °C had no effect on S-RBD-ACE-2 ligand-receptor binding and minimal effect on the S-RBD protein structure (up to 40 °C), though protein denaturing occurred at 51 °C. Our results suggest that ambient temperatures from 4 to 40 °C have little effect on the SARS-CoV-2-ACE-2 interaction in agreement with the infection data currently reported.
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| 6. |
- Paul, Alexandra, 1988, et al.
(författare)
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Molecular Microscopy of Oil Body and Lipid Droplet Chemistry In Situ with Physiologically-Relevant Readouts
- 2020
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 118:3, s. 468A-468A
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Konferensbidrag (refereegranskat)abstract
- Spatial heterogeneity at the molecular scale is a ubiquitous feature of all biological tissues, which is fundamentally linked to their native functions and to pathology. Probing the local chemistry of complex biological tissues requires the development and application of imaging tools that can identify the intrinsic molecular features in a sample without sacrificing high spatial resolution. In this talk, I will describe our efforts to tackle this challenge for measuring lipid inclusion chemistry and morphology in situ. We have developed nonlinear label-free microscopy and associated analytical tools to determine the biochemistry of lipid droplets and oil bodies with high spatial resolution in a variety of samples. Importantly, our method yields physiologically-relevant quantities (chain length and saturation) as opposed to physical chemical ratios. I will show how we have used this ability to map how lipid droplet chemical composition in brown and white adipose tissue adapts to high fat dietary intervention. Going forward, we want to expand the disease pathologies studied and I will present early results on our work on lipid droplets in neurological diseases.
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| 7. |
- Paul, Alexandra, 1988, et al.
(författare)
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Quantitative Mapping of Triacylglycerol Chain Length and Saturation Using Broadband CARS Microscopy
- 2019
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 116:12, s. 2346-2355
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Tidskriftsartikel (refereegranskat)abstract
- Lipid droplets (LDs), present in many cell types, are highly dynamic organelles that store neutral lipids, primarily triacylglycerols (TAGs). With the discovery of new LD functions (e.g., in immune response, protein clearage, and occurrence with disease), new methods to study LD chemical composition in situ are necessary. We present an approach for in situ, quantitative TAG analysis using label-free, coherent Raman microscopy that allows deciphering LD TAG composition in different biochemically complex samples with submicrometer spatial resolution. Employing a set of standard TAGs, we generate a spectral training matrix capturing the variation caused in Raman-like spectra by TAG backbone, chain length, and number of double bonds per chain, as well as the presence of proteins or other diluting molecules. Comparing our fitting approach to gas chromatography measurements for mixtures of standard TAGs and food oils, we find the root mean-square error for the prediction of TAG chemistry to be 0.69 CH2 and 0.15 #C=C. When progressing to more complex samples such as oil emulsions and LDs in various eukaryotic cells, we find good agreement with bulk gas chromatography measurements. For differentiated adipocytes, we find a significant increase in the number of double bonds in small LDs (below 2 μm in diameter) compared to large LDs (above 2 μm in diameter). Coupled with a relatively limited sample preparation requirement, this approach should enable rapid and accurate TAG LD analysis for a variety of cell biology and technological applications.
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