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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, therefore a thorough understanding of the loading behavior of HPCs towards NPs is essential to a successful application. 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 one 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 modelling NP loading and release is presented, contributing to a framework to describe this phenomenon. The authors would like to thank Birgit Bare and Pascale Berckmans for their contribution in the flow cytometry experiments. This work was funded by INTERREG-IV A (Bio-MiMedics), BOF (Hasselt University), a tUL impulse grant Phase II by the Province of Limburg (Belgium), FP7 project QualityNano (Grant Agreement No INFRA-2010-262163). SD was supported by a fellowship of VITO and Hasselt University (BOF). NS was supported by a post-doctoral scholarship of Research Foundation Flanders (FWO Vlaanderen) and VITO. BW was supported by a fellowship of VITO.

Additional file 1. The additional data includes information regarding the TEM protocol, TEM images of the PS NPs and HPCs, data on NP characterization and cell viability assessment. Furthermore, flow cytometry gating strategies are included.

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 America

De glycinereceptor (GlyR) vervult een belangrijke rol in de snelle, inhibitorische neuronale signaaloverdracht in het centrale zenuwstelsel van vertebraten. De GlyR is een pentameer dat kan bestaan uit verschillende subeenheden (α1, α2, α3, α4, β). Clustering van de heteropentamere GlyR α1β ter hoogte van het postsynaptisch membraan is essentieel om de signaaloverdracht efficiënt uit te voeren. De regulatie van deze clustering werd lange tijd beschouwd als een statisch gegeven waarbij voornamelijk de endo- en exocytose van aggregaten van GlyRs bepalend werd geacht. Fluorescentiemicroscopie van de receptor in levende cellen heeft deze visie grondig veranderd.

Transfected Chinese hamster ovary cells were cultured on bare uncoated chemical vapor deposited thin nano- and microcrystalline diamond surfaces, hydrophobic hydrogenand hydrophilic oxygen-terminated. Optical and biochemical analyses show that compared to glass controls, growth and viability were not significantly affected (one-way-analysis of variance, ANOVA). Based on two-way ANOVA analyses, neither grain size nor surface termination had a significant influence until 5 days post-seeding. The authors thank Mrs. R. Beenaerts, Mr. J. Janssen, Mr. P. Pirotte, Mr. J. Soogen, Mr. R. van Werde for expert laboratory assistance, Prof. Dr. I. Lambrichts and Mr.M. Jans for logistics and fixation procedures, Mr. K. Vanstreels and Mr. B. Ruttens for assistance with the scanning electron microscope, Prof. Dr. R. Valcke for the use of his spectrophotometers, Mr. J. Duchateau, Mr. F. Horemans, Mrs. V. Vrindts for cleaning the diamond substrates, Ir. L. Naelaerts (KHLIM) for lending stereomicroscopes. S. W. is a post-doctoral research fellow of the Research Foundation – Flanders (FWO-Vlaanderen). This work was funded by the Research Council of the UHasselt, tUL, the K.U. Leuven (GOA/2006/02) and by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). Support by IAP P6/27 Functional Supramolecular Systems (BELSPO) and by the FWOonderzoeksgemeenschap “Scanning andWide Field Microscopy of (Bio)-organic Systems” is gratefully acknowledged.

In this report both qualitative and quantitative results on cell viability of Chinese hamster ovary (CHO) cells cultured on bare, well-characterized nano- and microcrystalline diamond surfaces, either hydrogen or oxygen terminated, are presented and discussed. CHO cells stably transfected with homomeric embryonic a2 glycine receptors are chosen since they offer a simplified cellular model. A systematic investigation of the viability is justified since earlier published reports are limited to more elaborate coated, single-crystal diamond surfaces. Qualitative information was derived from visual inspection of the CHO cells on the microwave plasma-enhanced chemical vapor deposited (MPE-CVD) thin diamond films - deposited on silicon - using both scanning electron microscopy and optical reflection microscopy. Compared with control glass substrates, no apparent difference existed either in cell morphology or in cell density on all substrates tested. In order to quantify these observations, other methods were applied. These consisted of different biochemical assays: the MTT cell proliferation assay (testing for proliferation and metabolic activity), the Bradford assay (total protein content), the [3H]-thymidine cell proliferation assay (proliferation) and flow cytometry (living/necrotic/apoptotic cells). Consistent with the results obtained from visual inspection, no significant differences were found using one-way ANOVA. Yet, after 7 days post seeding, the MTT result did indicate significant differences between the tested substrates. However, this is not yet reproduced. Based on these results, it can reasonably be conclude that, at least for the CHO cells used, bare nano- and microcrystalline diamond surfaces poorly affect, if at all, the growth and the viability of cultured cells. As a result, the diamond surfaces which are easiest to grow and to modify, with the best characteristics and the best shelf life, can be applied as substrate to develop non-invasive interfaces between biological cells on the one hand, and microelectronics on the other.

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. This work was supported by Ministerio de Ciencia e Innovacio´ n (Spain) (PID2019-105674RB-I00), by Gobierno de Arago´ n (LMP94_21) and (LMP128_21), by the European Research Council through grant ERC-StG 638284 and by European Social Fund and by Gobierno de Arago´ n (Reference Group BSICoS T39-20R) cofunded by FEDER 2014–2020 ‘‘Building Europe from Aragon‘‘. L.G-M was supported by a predoctoral fellowship from the Departamento de Ciencia, Universidad y Sociedad del Conocimiento from the Gobierno de Arago´ n 2016–2020 cofounded by Programa Operativo del Fondo Social Europeo Arago´ n (C150/2016), EMBO Short-Term fellowship (7710), and Ibercaja-CAI Estancias de Investigacio´ n IT18/18. Authors would like to acknowledge the Experimental Surgery Service of Aragon Health Sciences Institute (CIBA, Zaragoza, Spain), Mercazaragoza slaughterhouse (Zaragoza, Spain), and The Pink Pig slaughterhouse (Zuera, Spain) for their collaboration in the collection of young, adult, and boar tissue specimens, respectively.

Objective: Febrile seizures (FS) are fever-associated convulsions, being the most common seizure disorder in early childhood. A subgroup of these children later develops epilepsy characterized by a hyperexcitable neuronal network in the hippocampus. Hippocampal excitability is regulated by the hippocampal dentate gyrus (DG) where postnatal neurogenesis occurs. Experimental FS increase the survival of newborn hippocampal dentate granule cells (DGCs), yet the significance of this neuronal subpopulation to the hippocampal network remains unclear. In the current study, we characterized the temporal maturation and structural integration of these post-FS born DGCs in the DG. Methods: Experimental FS were induced in 10-day-old rat pups. The next day, retroviral particles coding for enhanced green fluorescent protein (eGFP) were stereotactically injected in the DG to label newborn cells. Histochemical analyses of eGFP expressing DGCs were performed one, 4, and 8 weeks later and consisted of the following: (1) colocalization with neurodevelopmental markers doublecortin, calretinin, and the mature neuronal marker NeuN; (2) quantification of dendritic complexity; and (3) quantification of spine density and morphology. Results: At neither time point were neurodevelopmental markers differently expressed between FS animals and normothermia (NT) controls. One week after treatment, DGCs from FS animals showed dendrites that were 66% longer than those from NT controls. At 4 and 8 weeks, Sholl analysis of the outer 83% of the molecular layer showed 20-25% more intersections in FS animals than in NT controls (p < 0.01). Although overall spine density was not affected, an increase in mushroom-type spines was observed after 8 weeks. Significance: Experimental FS increase dendritic complexity and the number of mushroom- type spines in post-FS born DGCs, demonstrating a more mature phenotype and suggesting increased incoming excitatory information. The consequences of this hyperconnectivity to signal processing in the DG and the output of the hippocampus remain to be studied. We greatly appreciate the kindness of H. van Praag for providing plasmids used for virus production. Furthermore, we would like to thank Rik Paesen for his help with the confocal microscope and Petra Bex and Rosette Beenaerts for their assistance with the virus production and immunohisto-chemistry. This research was financially supported by a 'Bijzonder Onderzoeksfonds' grant from Hasselt University. Nick Smisdom was supported by a post-doctoral scholarship of the Research Foundation - Flanders (FWO-Vlaanderen).

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.

The spatio-temporal membrane behavior of glycine receptors (GlyRs) 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 imply 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. (C) 2012 Elsevier B.V. All rights reserved. We would like to thank Ir. Ben De Clercq for providing us with the proper settings of the Becker and Hickl detection system. GlyR expression constructs were obtained thanks to the funding by the Helmholtz Association (VH-NG-246 to J.C.M.). S.R. and J.H. acknowledge financial support of the "Fonds voor Wetenschappelijk Onderzoek FWO" (grants G.0402.09, G.0413.10, G.0697.11), the K. U. Leuven Research Fund (GOA 2011/03), the Flemish government (long-term structural funding: Methusalem funding CASASMETH/08/04). S.R., J.H. and MA. thank the Federal Science Policy of Belgium (IAP-VI/27). M.A. recognizes the Province of Limburg (Belgium) for the financial support within the tUL IMPULS FASE II program allowing for upgrading the laser source used in this work. The support by the FWO-onderzoeksgemeenschap "Scanning and Wide Field Microscopy of (Bio)-organic Systems" is gratefully acknowledged by J.H. and M.A.