| 1. |
- Borro, Bruno C., et al.
(author)
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Microfluidics-based self-assembly of peptide-loaded microgels : Effect of three dimensional (3D) printed micromixer design
- 2019
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In: Journal of Colloid and Interface Science. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 0021-9797 .- 1095-7103. ; 538, s. 559-568
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Journal article (peer-reviewed)abstract
- In an effort to contribute to research in scalable production systems for polymeric delivery systems loaded with antimicrobial peptides (AMPS), we here investigate effects of hydrodynamic flow conditions on microfluidic particle generation. For this purpose, rapid prototyping using 3D printing was applied to prepare micromixers with three different geometric designs, which were used to prepare Ca2+-crosslinked alginate microgels loaded with the AMP polymyxin B in a continuous process. Based on fluid dynamic simulations, the hydrodynamic flow patterns in the micromixers were designed to be either (i) turbulent with chaotic disruption, (ii) laminar with convective mixing, or (iii) convective with microvortex formation. The physicochemical properties of the microgels prepared with these micromixers were characterized by photon correlation spectroscopy, laser-Doppler micro-electrophoresis, smallangle x-ray scattering, and ellipsometry. The particle size and compactness were found to depend on the micromixer geometry: From such studies, particle size and compactness were found to depend on micromixer geometry, the smallest and most compact particles were obtained by preparation involving microvortex flows, while larger and more diffuse microgels were formed upon laminar mixing. Polymyxin B was found to be localized in the particle interior and to cause particle growth with increasing peptide loading. Ca2+-induced cross-linking of alginate, in turn, results in particle contraction. The peptide encapsulation efficiency was found to be higher than 80% for all investigated micromixer designs; the highest encapsulation efficiency observed for the smallest particles generated by microvortexmediated self-assembly. Ellipsometry results for surface-immobilized microgels, as well as results on peptide encapsulation, demonstrated electrolyte-induced peptide release. Taken together, these findings demonstrate that rapid prototyping of microfluidics using 3D-printed micromixers offers promises for continuous manufacturing of AMP-loaded microgels. Although the micromixer combining turbulent flow and microvortexes was demonstrated to be the most efficient, all three micromixer designs were found to mediate self-assembly of small microgels displaying efficient peptide encapsulation. This demonstrates the robustness of employing 3D-printed micromixers for microfluidic assembly of AMP-loaded microgels during continuous production.
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| 2. |
- Bucciarelli, Saskia
(author)
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A scattering study of concentrated lens protein solutions and mixtures - Towards understanding the molecular origin of presbyopia
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Healthy eye lenses are transparent and flexible, able to adapt shape in order to focus on far away and close-by objects. With age, however, this flexibility is lost, leading to a condition known as presbyopia. This vision disorder where close-by objects appear blurred commonly starts around the age of 40. The aim of this thesis is to elucidate the molecular origin of the gradual hardening of the core of the eye lens, made of fibre cells filled with concentrated mixtures of proteins, the so-called crystallins, which leads to the formation of presbyopia. To this end, various scattering techniques, static and dynamic light scattering (SLS & DLS), small-angle X-ray scattering (SAXS) and neutron spin echo (NSE), are used to study solutions of crystallins at concentrations up to those present in the lens. The first part of this work deals with solutions of the individual proteins. We show that different techniques allow us to access solution dynamics on different length scales. While DLS probes the collective dynamics, NSE accesses length scales corresponding to the average distance between nearest neighbors in the solution. We find that the dynamic behavior of protein solutions depends on the nature of the protein interactions, as well as on their patchiness. For the largest of the lens proteins, α-crystallin, well described by a model of polydisperse (20%) colloidal hard spheres, we find a slowing down of the local dynamics over a large range of volume fractions
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| 3. |
- Bucciarelli, Saskia, et al.
(author)
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Dramatic influence of patchy attractions on short-time protein diffusion under crowded conditions
- 2016
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In: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 2:12, s. 1601432-1601432
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Journal article (peer-reviewed)abstract
- In the dense and crowded environment of the cell cytoplasm, an individual protein feels the presence of and interacts with all surrounding proteins. While we expect this to strongly influence the short-time diffusion coefficient Ds of proteins on length scales comparable to the nearest-neighbor distance, this quantity is difficult to assess experimentally. We demonstrate that quantitative information about Ds can be obtained from quasi-elastic neutron scattering experiments using the neutron spin echo technique. We choose two well-characterized and highly stable eye lens proteins, bovine α-crystallin and γB-crystallin, and measure their diffusion at concentrations comparable to those present in the eye lens. While diffusion slows down with increasing concentration for both proteins, we find marked variations that are directly linked to subtle differences in their interaction potentials. A comparison with computer simulations shows that anisotropic and patchy interactions play an essential role in determining the local short-time dynamics. Hence, our study clearly demonstrates the enormous effect that weak attractions can have on the short-time diffusion of proteins at concentrations comparable to those in the cellular cytosol.
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| 4. |
- BUCCIARELLI, SASKIA, et al.
(author)
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Extended Law of Corresponding States Applied to Solvent Isotope Effect on a Globular Protein
- 2016
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In: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 7:9, s. 1610-1615
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Journal article (peer-reviewed)abstract
- Investigating proteins with techniques such as NMR or neutron scattering frequently requires the partial or complete substitution of D2O for H2O as a solvent, often tacitly assuming that such a solvent substitution does not significantly alter the properties of the protein. Here, we report a systematic investigation of the solvent isotope effect on the phase diagram of the lens protein gamma B-crystallin in aqueous solution as a model system exhibiting liquid-liquid phase separation. We demonstrate that the observed strong variation of the critical temperature T-c can be described by the extended law of corresponding states for all H2O/D2O ratios, where scaling of the temperature by T-c or the reduced second virial coefficient accurately reproduces the binodal, spinodal, and osmotic compressibility. These findings highlight the impact of H2O/D2O substitution on gamma B-crystallin properties and warrant further investigations into the universality of this phenomenon and its underlying mechanisms.
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| 5. |
- Bucciarelli, Saskia, et al.
(author)
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Unusual dynamics of concentration fluctuations in solutions of weakly attractive globular proteins
- 2015
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In: Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185 .- 1089-5639. ; 6:22, s. 4470-4474
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Journal article (peer-reviewed)abstract
- The globular protein γB-crystallin exhibits a complex phase behavior, where liquid–liquid phase separation characterized by a critical volume fraction ϕc = 0.154 and a critical temperature Tc = 291.8 K coexists with dynamical arrest on all length scales at volume fractions around ϕ ≈ 0.3–0.35, and an arrest line that extends well into the unstable region below the spinodal. However, although the static properties such as the osmotic compressibility and the static correlation length are in quantitative agreement with predictions for binary liquid mixtures, this is not the case for the dynamics of concentration fluctuations described by the dynamic structure factor S(q,t). Using a combination of dynamic light scattering and neutron spin echo measurements, we demonstrate that the competition between critical slowing down and dynamical arrest results in a much more complex wave vector dependence of S(q,t) than previously anticipated.
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| 6. |
- Cardinaux, Frederic, et al.
(author)
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Cluster-Driven Dynamical Arrest in Concentrated Lysozyme Solutions
- 2011
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In: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 115:22, s. 7227-7237
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Journal article (peer-reviewed)abstract
- We present a detailed experimental and numerical study of the structural and dynamical properties of salt-free lysozyme solutions. In particular, by combining small-angle X-ray scattering (SAXS) data with neutron spin echo (NSE) and rheology experiments, we are able to identify that an arrest transition takes place at intermediate densities, driven by the slowing down of the cluster motion. Using an effective pair potential among proteins, based on the combination of short-range attraction and long-range repulsion, we account remarkably well for the peculiar volume fraction dependence of the effective structure factor measured by SAXS. We show that a transition from a monomer to a cluster-dominated fluid happens at volume fractions larger than phi greater than or similar to 0.05 where the close agreement between NSE measurements and Brownian dynamics simulations confirms the transient nature of the clusters. Clusters even stay transient above the geometric percolation found in simulation at phi > 0.15, though NSE reveals a cluster lifetime that becomes increasingly large and indicates a divergence of the diffusivity at phi greater than or similar to 0.26. Macroscopic measurements of the viscosity confirm this transition where the long-lived-nature of the clusters is at the origin of the simultaneous dynamical arrest at all length scales.
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| 7. |
- Foffi, Giuseppe, et al.
(author)
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Hard sphere-like glass transition in eye lens α-crystallin solutions.
- 2014
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In: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 111:47, s. 16748-16753
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Journal article (peer-reviewed)abstract
- We study the equilibrium liquid structure and dynamics of dilute and concentrated bovine eye lens α-crystallin solutions, using small-angle X-ray scattering, static and dynamic light scattering, viscometry, molecular dynamics simulations, and mode-coupling theory. We find that a polydisperse Percus-Yevick hard-sphere liquid-structure model accurately reproduces both static light scattering data and small-angle X-ray scattering liquid structure data from α-crystallin solutions over an extended range of protein concentrations up to 290 mg/mL or 49% vol fraction and up to ca. 330 mg/mL for static light scattering. The measured dynamic light scattering and viscosity properties are also consistent with those of hard-sphere colloids and show power laws characteristic of an approach toward a glass transition at α-crystallin volume fractions near 58%. Dynamic light scattering at a volume fraction beyond the glass transition indicates formation of an arrested state. We further perform event-driven molecular dynamics simulations of polydisperse hard-sphere systems and use mode-coupling theory to compare the measured dynamic power laws with those of hard-sphere models. The static and dynamic data, simulations, and analysis show that aqueous eye lens α-crystallin solutions exhibit a glass transition at high concentrations that is similar to those found in hard-sphere colloidal systems. The α-crystallin glass transition could have implications for the molecular basis of presbyopia and the kinetics of molecular change during cataractogenesis.
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| 8. |
- Parra-Ortiz, Elisa, et al.
(author)
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Effects of oxidation on the physicochemical properties of polyunsaturated lipid membranes
- 2019
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In: Journal of Colloid and Interface Science. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 0021-9797 .- 1095-7103. ; 538, s. 404-419
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Journal article (peer-reviewed)abstract
- The exposure of biological membranes to reactive oxygen species (ROS) plays an important role in many pathological conditions such as inflammation, infection, or sepsis. ROS also modulate signaling processes and produce markers for damaged tissue. Lipid peroxidation, mainly affecting polyunsaturated phospholipids, results in a complex mixture of oxidized products, which may dramatically alter membrane properties. Here, we have employed a set of biophysical and surface-chemical techniques, including neutron and X-ray scattering, to study the structural, compositional, and stability changes due to oxidative stress on phospholipid bilayers composed of lipids with different degrees of polyunsaturation. In doing so, we obtained real-time information about bilayer degradation under in situ UV exposure using neutron reflectometry. We present a set of interrelated physicochemical effects, including gradual increases in area per molecule, head group and acyl chain hydration, as well as bilayer thinning, lateral phase separation, and defect formation leading to content loss upon membrane oxidation. Such effects were observed to depend on the presence of polyunsaturated phospholipids in the lipid membrane, suggesting that these may also play a role in the complex oxidation processes occurring in cells.
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| 9. |
- Roosen-Runge, Felix, et al.
(author)
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Crowding in the Eye Lens : Modeling the Multisubunit Protein β-Crystallin with a Colloidal Approach
- 2020
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In: Biophysical Journal. - : Elsevier BV. - 0006-3495. ; 119:12, s. 2483-2496
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Journal article (peer-reviewed)abstract
- We present a multiscale characterization of aqueous solutions of the bovine eye lens protein βH crystallin from dilute conditions up to dynamical arrest, combining dynamic light scattering, small-angle x-ray scattering, tracer-based microrheology, and neutron spin echo spectroscopy. We obtain a comprehensive explanation of the observed experimental signatures from a model of polydisperse hard spheres with additional weak attraction. In particular, the model predictions quantitatively describe the multiscale dynamical results from microscopic nanometer cage diffusion over mesoscopic micrometer gradient diffusion up to macroscopic viscosity. Based on a comparative discussion with results from other crystallin proteins, we suggest an interesting common pathway for dynamical arrest in all crystallin proteins, with potential implications for the understanding of crowding effects in the eye lens.
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| 10. |
- Zhou, Yuedan, et al.
(author)
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HMGB1 binds to the rs7903146 locus in TCF7L2 in human pancreatic islets.
- 2016
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In: Molecular and Cellular Endocrinology. - : Elsevier BV. - 1872-8057 .- 0303-7207. ; 430, s. 138-145
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Journal article (peer-reviewed)abstract
- The intronic SNP rs7903146 in the T-cell factor 7-like 2 gene (TCF7L2) is the common genetic variant most highly associated with Type 2 diabetes known to date. The risk T-allele is located in an open chromatin region specific to human pancreatic islets of Langerhans, thereby accessible for binding of regulatory proteins. The risk T-allele locus exhibits stronger enhancer activity compared to the non-risk C-allele. The aim of this study was to identify transcriptional regulators that bind the open chromatin region in the rs7903146 locus and thereby potentially regulate TCF7L2 expression and activity. Using affinity chromatography followed by Edman sequencing, we identified one candidate regulatory protein, i.e. high-mobility group protein B1 (HMGB1). The binding of HMGB1 to the rs7903146 locus was confirmed in pancreatic islets from human deceased donors, in HCT116 and in HEK293 cell lines using: (i) protein purification on affinity columns followed by Western blot, (ii) chromatin immunoprecipitation followed by qPCR and (iii) electrophoretic mobility shift assay. The results also suggested that HMGB1 might have higher binding affinity to the C-allele of rs7903146 compared to the T-allele, which was supported in vitro using Dynamic Light Scattering, possibly in a tissue-specific manner. The functional consequence of HMGB1 depletion in HCT116 and INS1 cells was reduced insulin and TCF7L2 mRNA expression, TCF7L2 transcriptional activity and glucose stimulated insulin secretion. These findings suggest that the rs7903146 locus might exert its enhancer function by interacting with HMGB1 in an allele dependent manner.
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