Analysis of age-related left ventricular collagen remodeling in living donors: Implications in arrhythmogenesis

Age-related fibrosis in the left ventricle (LV) has been mainly studied in animals by assessing collagen content. Using second-harmonic generation microscopy and image processing, we evaluated amount, aggregation and spatial distribution of LV collagen in young to old pigs, and middle-age and elder living donors. All collagen features increased when comparing adult and old pigs with young ones, but not when comparing adult with old pigs or middle-age with elder individuals. Remarkably, all collagen parameters strongly correlated with lipofuscin, a biological age marker, in humans. By building patient-specific models of human ventricular tissue electrophysiology, we confirmed that amount and organization of fibrosis modulated arrhythmia vulnerability, and that distribution should be accounted for arrhythmia risk assessment. In conclusion, we characterize the age-associated changes in LV collagen and its potential implications for ventricular arrhythmia development. Consistency between pig and human results substantiate the pig as a relevant model of age-related LV collagen dynamics. © 2022 The Author(s

Inter-laboratory comparison of nanoparticle size measurements using dynamic light scattering and differential centrifugal sedimentation

Nanoparticle in vitro toxicity studies often report contradictory results with one main reason being insufficient material characterization. In particular the characterization of nanoparticles in biological media remains challenging. Our aim was to provide robust protocols for two of the most commonly applied techniques for particle sizing, i.e. dynamic light scattering (DLS) and differential centrifugal sedimentation (DCS) that should be readily applicable also for users not specialized in nanoparticle physico-chemical characterization. A large number of participants (40, although not all participated in all rounds) were recruited for a series of inter-laboratory comparison (ILC) studies covering many different instrument types, commercial and custom-built, as another possible source of variation. ILCs were organized in a consecutive manner starting with dispersions in water employing well-characterized near-spherical silica nanoparticles (nominal 19 nm and 100 nm diameter) and two types of functionalized spherical polystyrene nanoparticles (nominal 50 nm diameter). At first each laboratory used their in-house established procedures. In particular for the 19 nm silica particles, the reproducibility of the methods was unacceptably high (reported results were between 10 nm and 50 nm). When comparing the results of the first ILC round it was observed that the DCS methods performed significantly worse than the DLS methods, thus emphasizing the need for standard operating procedures (SOPs). SOPs have been developed by four expert laboratories but were tested for robustness by a larger number of independent users in a second ILC (11 for DLS and 4 for DCS). In a similar approach another SOP for complex biological fluids, i.e. cell culture medium containing serum was developed, again confirmed via an ILC with 8 participating laboratories. Our study confirms that well-established and fit-for-purpose SOPs are indispensable for obtaining reliable and comparable particle size data. Our results also show that these SOPs must be optimized with respect to the intended measurement system (e.g. particle size technique, type of dispersant) and that they must be sufficiently detailed (e.g. avoiding ambiguity regarding measurand definition, etc.). SOPs may be developed by a small number of expert laboratories but for their widespread applicability they need to be verified by a larger number of laboratories

Transient loading of CD34+ hematopoietic progenitor cells with polystyrene nanoparticles

Sarah Deville,1,2 Wahyu Wijaya Hadiwikarta,1 Nick Smisdom,1,2 Bart Wathiong,1,3 Marcel Ameloot,2 Inge Nelissen,1 Jef Hooyberghs1,3 1VITO, Flemish Institute for Technological Research, Mol, Belgium; 2Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium; 3Theoretical Physics, Hasselt University, Diepenbeek, Belgium Abstract: CD34+ hematopoietic progenitor cells (HPCs) offer great opportunities to develop new treatments for numerous malignant and non-malignant diseases. Nanoparticle (NP)-based strategies can further enhance this potential, and therefore a thorough understanding of the loading behavior of HPCs towards NPs is essential for a successful application. The present study focusses on the interaction kinetics of 40 nm sized carboxylated polystyrene (PS) NPs with HPCs. Interestingly, a transient association of the NPs with HPCs is observed, reaching a maximum within 1 hour and declining afterwards. This behavior is not seen in dendritic cells (CD34-DCs) differentiated from HPCs, which display a monotonic increase in NP load. We demonstrate that this transient interaction requires an energy-dependent cellular process, suggesting active loading and release of NPs by HPCs. This novel observation offers a unique approach to transiently equip HPCs. A simple theoretical approach modeling the kinetics of NP loading and release is presented, contributing to a framework of describing this phenomenon. Keywords: nanoparticles, hematopoietic progenitor cells, dendritic cells, uptake, releas

Interplay between flow and diffusion in capillary alginate hydrogels

Alginate gels with naturally occurring macroscopic capillaries have been used as a model system to study the interplay between laminar flow and diffusion of nanometer-sized solutes in real time. Calcium alginate gels that contain homogeneously distributed parallel-aligned capillary structures were formed by external addition of crosslinking ions to an alginate sol. The effects of different flow rates (0, 1, 10, 50 and 100 mu l min(-1)) and three different probes (fluorescein, 10 kDa and 500 kDa fluorescein isothiocyanate-dextran) on the diffusion rates of the solutes across the capillary wall and in the bulk gel in between the capillaries were investigated using confocal laser scanning microscopy. The flow in the capillaries was produced using a syringe pump that was connected to the capillaries via a tube. Transmission electron microscopy revealed an open aggregated structure close to the capillary wall, followed by an aligned network layer and the isotropic network of the bulk gel. The most pronounced effect was observed for the 1 nm-diameter fluorescein probe, for which an increase in flow rate increased the mobility of the probe in the gel. Fluorescence recovery after photobleaching confirmed increased mobility close to the channel, with increasing flow rate. Mobility maps derived using raster image correlation spectroscopy showed that the layer with the lowest mobility corresponded to the anisotropic layer of ordered network chains. The combination of microscopy techniques used in the present study elucidates the flow and diffusion behaviors visually, qualitatively and quantitatively, and represents a promising tool for future studies of mass transport in non-equilibrium systems

Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane

The spatio-temporal membrane behavior of glycine receptors (GlyR) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR {alpha}3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms implies that membrane behavior is crucial in determining GlyR {alpha}3 physiology. Therefore diffusion and aggregation of homomeric {alpha}3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR {alpha}3L and the non-clustering GlyR {alpha}3K cannot be explained by normal diffusion. SPT measurements indicate that the {alpha}3L receptors undergo transient trapping and directed motion, while the GlyR {alpha}3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane

Linearly polarized second harmonic generation microscopy reveals chirality (vol 18, pg 8286, 2010)

An erratum is presented to clarify both the direction of light polarization in our experiments and the unit cell geometry of the samples. Additionally, the spectral dependence of the second harmonic generation response in the G-shaped nanostructures made of gold is corrected. These changes do not affect our previous conclusions. (C) 2011 Optical Society of Americ

Fluorescence recovery after photobleaching in material and life sciences: Putting theory into practice

Fluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure-interaction-diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials