STUDYING OF INTRACELLULAR PROCESSES BY RECORDING THE DYNAMIC SPECKLE

The objective of this work was to use a method of dynamic speckle interferometry for studying the cellular metabolism by averaging the data obtained from a small number of cells, as well as inside individual cells. The design of the experimental technique is presented. The technique allows assessing the activity of biological processes in various parts of the cell

Myosin V regulates synaptopodin clustering and localization in the dendrites of hippocampal neurons

The spine apparatus (SA) is an endoplasmic reticulum-related organelle that is present in a subset of dendritic spines in cortical and pyramidal neurons, and plays an important role in Ca2+ homeostasis and dendritic spine plasticity. The protein synaptopodin is essential for the formation of the SA and is widely used as a maker for this organelle. However, it is still unclear which factors contribute to its localization at selected synapses, and how it triggers local SA formation. In this study, we characterized development, localization and mobility of synaptopodin clusters in hippocampal primary neurons, as well as the molecular dynamics within these clusters. Interestingly, synaptopodin at the shaftassociated clusters is less dynamic than at spinous clusters. We identify the actin-based motor proteins myosin V (herein referring to both the myosin Va and Vb forms) and VI as novel interaction partners of synaptopodin, and demonstrate that myosin V is important for the formation and/or maintenance of the SA. We found no evidence of active microtubule-based transport of synaptopodin. Instead, new clusters emerge inside spines, which we interpret as the SA being assembled on-site

Results from the intercalibration of optical low light calibration sources 2011

Following the 38th Annual European Meeting on Atmospheric Studies by Optical Methods in Siuntio in Finland, an intercalibration workshop for optical low light calibration sources was held in Sodankylä, Finland. The main purpose of this workshop was to provide a comparable scale for absolute measurements of aurora and airglow. All sources brought to the intercalibration workshop were compared to the Fritz Peak reference source using the Lindau Calibration Photometer built by Wilhelm Barke and Hans Lauche in 1984. The results were compared to several earlier intercalibration workshops. It was found that most sources were fairly stable over time, with errors in the range of 5–25%. To further validate the results, two sources were also intercalibrated at UNIS, Longyearbyen, Svalbard. Preliminary analysis indicates agreement with the intercalibration in Sodankylä within about 15–25%

Use of facile mechanochemical method to functionalize carbon nanofibers with nanostructured polyaniline and their electrochemical capacitance

A facile approach to functionalize carbon nanofibers [CNFs] with nanostructured polyaniline was developed via in situ mechanochemical polymerization of polyaniline in the presence of chemically treated CNFs. The nanostructured polyaniline grafting on the CNF was mainly in a form of branched nanofibers as well as rough nanolayers. The good dispersibility and processability of the hybrid nanocomposite could be attributed to its overall nanostructure which enhanced its accessibility to the electrolyte. The mechanochemical oxidation polymerization was believed to be related to the strong Lewis acid characteristic of FeCl3 and the Lewis base characteristic of aniline. The growth mechanism of the hierarchical structured nanofibers was also discussed. After functionalization with the nanostructured polyaniline, the hybrid polyaniline/CNF composite showed an enhanced specific capacitance, which might be related to its hierarchical nanostructure and the interaction between the aromatic polyaniline molecules and the CNFs

Effects of Proteins from Culture Medium on Surface Property of Silanes- Functionalized Magnetic Nanoparticles

Monodisperse magnetic nanoparticles (MNPs) were synthesized by thermal decomposition of iron-oleate and functionalized with silanes bearing various functional groups such as amino group (NH2), short-chain poly(ethylene glycol) (PEG), and carboxylic group (COOH). Then, silanes-functionalized magnetic nanoparticles (silanes-MNPs) were incubated in cell culture medium plus fetal calf serum to investigate the effects of proteins from culture medium on surface property of MNPs. Zeta potential measurements showed that although surface charges of silanes-MNPs were different, they exhibited negative charges at neutral pH and approximate isoelectric points after they were incubated in cell culture medium. The reason was that silanes-MNPs could easily adsorb proteins from culture medium via non-covalent binding, resulting in the formation of protein-silanes-MNPs conjugates. Moreover, silanes-MNPs with various functional groups had different adsorption capacity to proteins, as confirmed by Coomassie blue fast staining method. The in vitro cell experiments showed that protein-silanes-MNPs had higher cellular uptake by cancer cells than silanes-MNPs

Exploiting nanobodies and Affimers for superresolution imaging in light microscopy

Antibodies have long been the main approach used for localizing proteins of interest by light microscopy. In the past 5 yr or so, and with the advent of superresolution microscopy, the diversity of tools for imaging has rapidly expanded. One main area of expansion has been in the area of nanobodies, small single-chain antibodies from camelids or sharks. The other has been the use of artificial scaffold proteins, including Affimers. The small size of nanobodies and Affimers compared with the traditional antibody provides several advantages for superresolution imaging

Periodic actin structures in neuronal axons are required to maintain microtubules

Axons are the cable-like neuronal processes wiring the nervous system. They contain parallel bundles of microtubules as structural backbones, surrounded by regularly-spaced actin rings termed the periodic membrane skeleton (PMS). Despite being an evolutionarily-conserved, ubiquitous, highly-ordered feature of axons, the function of PMS is unknown. Here we studied PMS abundance, organisation and function, combining versatile Drosophila genetics with super-resolution microscopy and various functional readouts. Analyses with 11 different actin regulators and 3 actin-targeting drugs suggest PMS to contain short actin filaments which are depolymerisation resistant and sensitive to spectrin, adducin and nucleator deficiency - consistent with microscopy-derived models proposing PMS as specialised cortical actin. Upon actin removal we observed gaps in microtubule bundles, reduced microtubule polymerisation and reduced axon numbers suggesting a role of PMS in microtubule organisation. These effects become strongly enhanced when carried out in neurons lacking the microtubule-stabilising protein Short stop (Shot). Combining the aforementioned actin manipulations with Shot deficiency revealed a close correlation between PMS abundance and microtubule regulation, consistent with a model in which PMS-dependent microtubule polymerisation contributes to their maintenance in axons. We discuss potential implications of this novel PMS function along axon shafts for axon maintenance and regeneration

Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells

Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues

Genetically enhanced asynapsis of autosomal chromatin promotes transcriptional dysregulation and meiotic failure

During meiosis, pairing of homologous chromosomes and their synapsis are essential prerequisites for normal male gametogenesis. Even limited autosomal asynapsis often leads to spermatogenic impairment, the mechanism of which is not fully understood. The present study was aimed at deliberately increasing the size of partial autosomal asynapsis and analysis of its impact on male meiosis. For this purpose, we studied the effect of t12 haplotype encompassing four inversions on chromosome 17 on mouse autosomal translocation T(16;17)43H (abbreviated T43H). The T43H/T43H homozygotes were fully fertile in both sexes, while +/T43H heterozygous males, but not females, were sterile with meiotic arrest at late pachynema. Inclusion of the t12 haplotype in trans to the T43H translocation resulted in enhanced asynapsis of the translocated autosome, ectopic phosphorylation of histone H2AX, persistence of RAD51 foci, and increased gene silencing around the translocation break. Increase was also on colocalization of unsynapsed chromatin with sex body. Remarkably, we found that transcriptional silencing of the unsynapsed autosomal chromatin precedes silencing of sex chromosomes. Based on the present knowledge, we conclude that interference of meiotic silencing of unsynapsed autosomes with meiotic sex chromosome inactivation is the most likely cause of asynapsis-related male sterility