Electrochemical Properties of Silver Nanoparticles in Mesoporous Silica and Titania Films: Specific Behavior of Titania Composite.

Electrochemical behavior of silver nanoparticles in mesoporous oxides electrodes is investigated. Mesoporous SiO2 and TiO2 films deposited on FTO (fluorine-doped tin oxide) and containing Ag nanoparticles (NPs) are used as electrodes. The study of voltammetric curves (CVs) and the diffusion of Ag+ ions out of the films highlight the importance of the retention of Ag+ ions by the TiO2 films. By varying several factors such as the speed rate or the initial potential, we observe the existence of the two potentials' anodic peaks. These are explained by the nature of two silver NP populations created in two distinct areas in the film and with different size distributions, as shown by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations. The size distributions of the two NP populations allow the position and shape of each of the oxidation peaks in the CVs to be adequately simulated.

A caveolin-1 dependent glucose-6-phosphatase trafficking contributes to hepatic glucose production.

OBJECTIVE: Deregulation of hepatic glucose production is a key driver in the pathogenesis of diabetes, but its short-term regulation is incompletely deciphered. According to textbooks, glucose is produced in the endoplasmic reticulum by glucose-6-phosphatase (G6Pase) and then exported in the blood by the glucose transporter GLUT2. However, in the absence of GLUT2, glucose can be produced by a cholesterol-dependent vesicular pathway, which remains to be deciphered. Interestingly, a similar mechanism relying on vesicle trafficking controls short-term G6Pase activity. We thus investigated whether Caveolin-1 (Cav1), a master regulator of cholesterol trafficking, might be the mechanistic link between glucose production by G6Pase in the ER and glucose export through a vesicular pathway. METHODS: Glucose production from fasted mice lacking Cav1, GLUT2 or both proteins was measured in vitro in primary culture of hepatocytes and in vivo by pyruvate tolerance tests. The cellular localization of Cav1 and the catalytic unit of glucose-6-phosphatase (G6PC1) were studied by western blotting from purified membranes, immunofluorescence on primary hepatocytes and fixed liver sections and by in vivo imaging of chimeric constructs overexpressed in cell lines. G6PC1 trafficking to the plasma membrane was inhibited by a broad inhibitor of vesicular pathways or by an anchoring system retaining G6PC1 specifically to the ER membrane. RESULTS: Hepatocyte glucose production is reduced at the step catalyzed by G6Pase in the absence of Cav1. In the absence of both GLUT2 and Cav1, gluconeogenesis is nearly abolished, indicating that these pathways can be considered as the two major pathways of de novo glucose production. Mechanistically, Cav1 colocalizes but does not interact with G6PC1 and controls its localization in the Golgi complex and at the plasma membrane. The localization of G6PC1 at the plasma membrane is correlated to glucose production. Accordingly, retaining G6PC1 in the ER reduces glucose production by hepatic cells. CONCLUSIONS: Our data evidence a pathway of glucose production that relies on Cav1-dependent trafficking of G6PC1 to the plasma membrane. This reveals a new cellular regulation of G6Pase activity that contributes to hepatic glucose production and glucose homeostasis.

Passive limitation of surface contamination by perFluoroDecylTrichloroSilane coatings in the ISS during the MATISS experiments.

Future long-duration human spaceflight will require developments to limit biocontamination of surface habitats. The MATISS (Microbial Aerosol Tethering on Innovative Surfaces in the international Space Station) experiments allowed for exposing surface treatments in the ISS (International Space Station) using a sample-holder developed to this end. Three campaigns of FDTS (perFluoroDecylTrichloroSilane) surface exposures were performed over monthly durations during distinct periods. Tile scanning optical microscopy (×3 and ×30 magnifications) showed a relatively clean environment with a few particles on the surface (0.8 to 7 particles per mm2). The varied densities and shapes in the coarse area fraction (50-1500 µm2) indicated different sources of contamination in the long term, while the bacteriomorph shapes of the fine area fraction (0.5-15 µm2) were consistent with microbial contamination. The surface contamination rates correlate to astronauts' occupancy rates on board. Asymmetric particles density profiles formed throughout time along the air-flow. The higher density values were located near the flow entry for the coarse particles, while the opposite was the case for the fine particles, probably indicating the hydrophobic interaction of particles with the FDTS surface.

A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch.

Optogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the AsLOV2 photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizing mutations in its N-terminal and C-terminal domains. LiCre can be activated within minutes of illumination with blue light without the need of additional chemicals. When compared to existing photoactivatable Cre recombinases based on two split units, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark. LiCre was efficient both in yeast, where it allowed us to control the production of ß-carotene with light, and human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses.

In a biologist's toolkit, the Cre protein holds a special place. Naturally found in certain viruses, this enzyme recognises and modifies specific genetic sequences, creating changes that switch on or off whatever gene is close by. Genetically engineering cells or organisms so that they carry Cre and its target sequences allows scientists to control the activation of a given gene, often in a single tissue or organ. However, this relies on the ability to activate the Cre protein 'on demand' once it is in the cells of interest. One way to do so is to split the enzyme into two pieces, which can then reassemble when exposed to blue light. Yet, this involves the challenging step of introducing both parts separately into a tissue. Instead, Duplus-Bottin et al. engineered LiCre, a new system where a large section of the Cre protein is fused to a light sensor used by oats to detect their environment. LiCre is off in the dark, but it starts to recognize and modify Cre target sequences when exposed to blue light. Duplus-Bottin et al. then assessed how LiCre compares to the two-part Cre system in baker's yeast and human kidney cells. This showed that the new protein is less 'incorrectly' active in the dark, and can switch on faster under blue light. The improved approach could give scientists a better tool to study the role of certain genes at precise locations and time points, but also help them to harness genetic sequences for industry or during gene therapy.

Microglia modulate gliotransmission through the regulation of VAMP2 proteins in astrocytes.

Vesicular release is one of the release mechanisms of various signaling molecules. In neurons, the molecular machinery involved in vesicular release has been designed through evolution to trigger fast and synchronous release of neurotransmitters. Similar machinery with a slower kinetic and a slightly different molecular assembly allows astrocytes to release various transmitters such as adenosine triphosphate (ATP), glutamate, and D-serine. Astrocytes are important modulators of neurotransmission through gliotransmitter release. We recently demonstrated that microglia, another type of glia, release ATP to modulate synaptic transmission using astrocytes as intermediate. We now report that microglia regulate astrocytic gliotransmission through the regulation of SNARE proteins in astrocytes. Indeed, we found that gliotransmission triggered by P2Y1 agonist is impaired in slices from transgenic mice devoid of microglia. Using total internal reflection fluorescence imaging, we found that the vesicular release of gliotransmitter by astrocytes was different in cultures lacking microglia compared to vesicular release in astrocytes cocultured with microglia. Quantification of the kinetic of vesicular release indicates that the overall release appears to be faster in pure astrocyte cultures with more vesicles close to the membrane when compared to astrocytes cocultured with microglia. Finally, biochemical investigation of SNARE protein expression indicates an upregulation of VAMP2 in absence of microglia. Altogether, these results indicate that microglia seems to be involved in the regulation of an astrocytic phenotype compatible with proper gliotransmission. The mechanisms described in this study could be of importance for central nervous system diseases where microglia are activated.

Towards a passive limitation of particle surface contamination in the Columbus module (ISS) during the MATISS experiment of the Proxima Mission.

Future long-duration human spaceflight calls for developments to limit biocontamination of the surface habitats. The MATISS experiment tests surface treatments in the ISS's atmosphere. Four sample holders were mounted with glass lamella with hydrophobic coatings, and exposed in the Columbus module for ~6 months. About 7800 particles were detected by tile scanning optical microscopy (×3 and ×30 magnification) indicating a relatively clean environment (a few particles per mm2), but leading to a significant coverage-rate (>2% in 20 years). Varied shapes were displayed in the coarse (50-1500 µm2) and fine (0.5-50 µm2) area fractions, consistent with scale dices (tissue or skin) and microbial cells, respectively. The 200-900 µm2 fraction of the coarse particles was systematically higher on FDTS and SiOCH than on Parylene, while the opposite was observed for the <10 µm2 fraction of the fine particles. This trend suggests two biocontamination sources and a surface deposition impacted by hydrophobic coatings.

Tumble Kinematics of Escherichia coli near a Solid Surface.

Bacteria tumble periodically, following environmental cues. Whether they can tumble near a solid surface is a basic issue for the inception of infection or mineral biofouling. Observing freely swimming Escherichia coli near and parallel to a glass surface imaged at high magnification (×100) and high temporal resolution (500 Hz), we identified tumbles as events starting (or finishing, respectively) in abrupt deceleration (or reacceleration, respectively) of the body motion. Selected events show an equiprobable clockwise (CW) or counterclockwise change in direction that is superimposed on a surface CW path because of persistent propulsion. These tumbles follow a common long (about 300 ± 100 ms, N = 52) deceleration-reorientation-acceleration pattern. A wavelet transform multiscale analysis shows these tumbles cause in-plane diffusive reorientations with 1.5 rad2/s rotational diffusivity, a value that compares with that measured in bulk tumbles. In half of the cases, additional few-millisecond bursts of an almost equiprobable CW or counterclockwise change of direction (12 ± 90°, N = 89) occur within the reorientation stage. The highly dispersed absolute values of change of direction (70 ± 66°, N = 89) of only a few bursts destabilize the cell-swimming direction. These first observations of surface tumbles set a foundation for statistical models of run-and-tumble surface motion different from that in bulk and lend support for chemotaxis near solid surface.

Innovative particle standards and long-lived imaging for 2D and 3D dSTORM.

Direct stochastic optical reconstruction microscopy (dSTORM), developed in the last decade, has revolutionised optical microscopy by enabling scientists to visualise objects beyond the resolution provided by conventional microscopy (200 nm). We developed an innovative method based on blinking particle standards and conditions for long-lived imaging over several weeks. Stable localisation precisions within the 10 nm-range were achieved for single virions and in cellulo 2D imaging of centrosomes, as well as their reliable reconstruction in 3D dSTORM.

Host cell surfaces induce a Type IV pili-dependent alteration of bacterial swimming.

For most pathogenic bacteria, flagellar motility is recognized as a virulence factor. Here, we analysed the swimming behaviour of bacteria close to eukaryotic cellular surfaces, using the major opportunistic pathogen Pseudomonas aeruginosa as a model. We delineated three classes of swimming trajectories on both cellular surfaces and glass that could be differentiated by their speeds and local curvatures, resulting from different levels of hydrodynamic interactions with the surface. Segmentation of the trajectories into linear and curved sections or pause allowed us to precisely describe the corresponding swimming patterns near the two surfaces. We concluded that (i) the trajectory classes were of same nature on cells and glass, however the trajectory distribution was strikingly different between surface types, (ii) on cell monolayers, a larger fraction of bacteria adopted a swimming mode with stronger bacteria-surface interaction mostly dependent upon Type IV pili. Thus, bacteria swim near boundaries with diverse patterns and importantly, Type IV pili differentially influence swimming near cellular and abiotic surfaces.

Expression of Nucleolin Affects Microtubule Dynamics.

Nucleolin is present in diverse cellular compartments and is involved in a variety of cellular processes from nucleolar structure and function to intracellular trafficking, cell adhesion and migration. Recently, nucleolin has been localized at the mature centriole where it is involved in microtubule nucleation and anchoring. Although this new function of nucleolin linked to microtubule regulation has been identified, the global effects of nucleolin on microtubule dynamics have not been addressed yet. In the present study, we analyzed the roles of nucleolin protein levels on global microtubule dynamics by tracking the EB3 microtubule plus end binding protein in live cells. We have found that during microtubule growth phases, nucleolin affects both the speed and life time of polymerization and by analyzing catastrophe events, we showed that nucleolin reduces catastrophe frequency. This new property of nucleolin was then confirmed in a cold induced microtubule depolymerization experiment in which we have found that cold resistant microtubules were totally destabilized in nucleolin depleted cells. Altogether, our data demonstrate a new function of nucleolin on microtubule stabilization, thus bringing novel insights into understanding the multifunctional properties of nucleolin in healthy and cancer cells.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses.

Podosome rings generate forces that drive saltatory osteoclast migration.

Podosomes are dynamic, actin-containing adhesion structures that collectively self-organize as rings. In this study, we first show by observing osteoclasts plated on bead-seeded soft substrates that podosome assemblies, such as rings, are involved in tension forces. During the expansion of a podosome ring, substrate displacement is oriented outward, suggesting that podosomal structures push the substrate away. To further elucidate the function of forces generated by podosomes, we analyze osteoclast migration. Determining the centers of mass of the whole cell (G) and of actin (P), we demonstrate that osteoclasts migrate by "jumps" and that the trajectories of G and P are strongly correlated. The velocity of the center of mass as a function of time reveals that osteoclasts rapidly catch up with podosomal structures in a periodic pattern. We conclude that actin dynamics inside the cell are not only correlated with cell migration, but drive it.

Counterclockwise circular motion of bacteria swimming at the air-liquid interface.

Flagellar propulsion of swimming Escherichia coli produces circling clockwise motions near planar solid surfaces. Counterclockwise motion was first reported near air-TN medium interfaces, showing that slip at the interface is a key parameter of bacterial swimming.

A tightly regulated molecular motor based upon T7 RNA polymerase.

Controlled movement of materials or molecules within the nanometer range is essential in many applications of nanotechnology. Here we report the capture, movement, and release of cargo molecules along DNA by a modified form of T7 RNA polymerase (RNAP) in a manner that is controlled by the sequence of the DNA. Using single-molecule methods, we visualize the assembly and manipulation of nanodevices and the ability to harness rotary and linear forces of the RNAP motor.

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study.

A new method based on combined atomic force microscopy (AFM) and fluorescence microscopy observations, is proposed to visualize the insertion of glycosylphosphatidyl inositol (GPI) anchored alkaline phosphatase from buffer solutions into supported phospholipid bilayers. The technique involves the use of 27 nm diameter fluorescent latex beads covalently coupled to the amine groups of proteins. Fluorescence microscopy allows the estimation of the relative protein coverage into the membrane and also introduces a height amplification for the detection of protein/bead complexes with the AFM. The coupling of the beads with the amine groups is not specific; this new and simple approach opens up new ways to investigate proteins into supported membrane systems.

A model of sequence-dependent protein diffusion along DNA.

We introduce a probabilistic model for protein sliding motion along DNA during the search of a target sequence. The model accounts for possible effects due to sequence-dependent interaction between the nonspecific DNA and the protein. Hydrogen bonds formed at the target site are used as the main sequence-dependent interaction between protein and DNA. The resulting dynamical properties and the possibility of an experimental verification are discussed in details. We show that, while at large times the process reaches a linear diffusion regime, it initially displays a sub-diffusive behavior. The sub-diffusive regime can last sufficiently long to be of biological interest.

Interaction of the C1 complex of complement with sulfated polysaccharide and DNA probed by single molecule fluorescence microscopy.

The complex C1 triggers the activation of the Complement classical pathway through the recognition and binding of antigen-antibody complex by its subunit C1q. The globular region of C1q is responsible for C1 binding to the immune complex. C1q can also bind nonimmune molecules such as DNA and sulfated polysaccharides, leading either to the activation or inhibition of Complement. The binding site of these nonimmune ligands is debated in the literature, and it has been proposed to be located either in the globular region or in the collagen-like region of C1q, or in both. Using single molecule fluorescence microscopy and DNA molecular combing as reporters of interactions, we have probed the C1q binding properties of T4 DNA and of fucoidan, an algal sulfated fucose-based polysaccharide endowed with potent anticomplementary activity. We have been able to visualize the binding of C1q as well as of C1 and of the isolated collagen-like region to individual DNA strands, indicating that the collagen-like region is the main binding site of DNA. From binding assays with C1r, one of the protease components of C1, we concluded that the DNA binding site on the collagen-like region is located within the stalk part. Competition experiments between fucoidan and DNA for the binding of C1q showed that fucoidan binds also to the collagen-like region part of C1q. Unlike DNA, the binding of fucoidan to collagen-like region involves interactions with the hinge region that accommodate the catalytic tetramer C1r2-C1s2 of C1. This binding property of fucoidan to C1q provides a mechanistic basis for the anticomplementary activity of the sulfated polysaccharide.

Crystallographic, Electrochemical, and Pulsed EPR Study of Copper(II) Polyimidazole Complexes Relevant to the Metal Sites of Copper Proteins(,).

Copper(II) complexes of the following polyimidazole ligands have been synthesized: bis(imidazol-2-yl)methane (BIM), bis(imidazol-2-yl) ketone (BIK), 4-(imidazol-4-ylmethyl)-2-(imidazol-2-ylmethyl)imidazole (TRIM), bis[4-(imidazol-4-ylmethyl)imidazol-2-yl]methane (TIM), and bis[4-(imidazol-4-ylmethyl)imidazol-2-yl] ketone (TIK). Their crystal structures have been determined using X-ray diffraction. [Cu(ClO(4))(2)(BIM)(2)], 1, belongs to the triclinic space group P&onemacr; system, a = 7.161(4) Å, b = 7.986(6) Å, c = 9.865(3) Å, alpha = 76.73(5) degrees, beta = 71.18(3) degrees, gamma = 76.44(5) degrees, Z = 1, T = 291 K; R = 0.035, R(w) = 0.036 for 1668 reflections; Cu-N = 1.998(3) and 2.001(2) Å, Cu-O = 2.574(4) Å, in a tetragonal geometry. [Cu(BIK)(2)](ClO(4))(2), 2, belongs to the monoclinic space group C2/c system, a = 9.029(3) Å, b = 12.497(2)Å, c = 19.197(2) Å, beta = 94.59(2) degrees, Z = 4, T = 291 K; R = 0.056, R(w) = 0.061 for 1052 reflections; Cu-N = 1.961(7) and 1.954(7) Å, in a distorted tetrahedral geometry. [CuCl(TRIM)(CH(3)OH)]Cl, 6, belongs to the monoclinic space group P2(1)/n system, a = 14.192(5) Å, b = 13.832(5) Å, c = 7.913(3) Å, beta = 90.55(4) degrees, Z = 4, T = 291 K; R = 0.062, R(w) = 0.057 for 1377 reflections; Cu-N = 1.987(7), 2.007(7) and 2.007(6) Å, Cu-O = 2.521(7) Å, Cu-Cl = 2.298(2) Å, in a square pyramidal geometry. [Cu(ClO(4))(TIM)](ClO(4)), 4, belongs to the triclinic space group P&onemacr; system, a = 9.604(4) Å, b = 11.508(6) Å, c = 12.003(8) Å, alpha = 58.79(4) degrees, beta = 94.59(2) degrees, gamma = 67.43(3) degrees, Z = 2, T = 291 K; R = 0.057, R(w) = 0.062 for 2084 reflections; Cu-N = 1.985(7), 1.964(7), 1.967(7), and 1.966(7) Å, Cu-O = 2.553(8) Å, in a distorted square pyramidal geometry. [CuCl(TIK)](ClO(4)), 7, belongs to the triclinic space group P&onemacr; system, a = 7.432(3) Å, b = 12.573(3) Å, c = 12.945(2) Å, alpha = 114.94(4) degrees, beta = 92.46(2) degrees, gamma = 103.49(3) degrees, Z = 2, T = 291 K; R = 0.043, R(w) = 0.049 for 2305 reflections; Cu-N = 1.984(5), 1.989(5), 2.012(5), and 1.979(5) Å, Cu-Cl = 2.796(2) Å, in a distorted bipyramidal geometry. In methanol solution, the perchlorato complexes 1, 2, Cu(TRIM)(ClO(4))(2) (3), 4, and Cu(TIK)(ClO(4))(2) (5) exhibited redox potentials from -215 to +284 mV vs NHE together with a visible absorption from 604 to 728 nm. Electron spin-echo envelope modulation (ESEEM) spectroscopy data, particularly the nuclear quadrupole interaction (NQI) parameters e(2)qQ and eta of the remote nitrogen (N1H), were analyzed and interpreted according to the model devised by Jiang et al. (J. Am. Chem. Soc. 1990, 112, 9035) with reference to Cu(HIm)(4)(ClO(4))(2). The results are the following: (i) C2 substitution of the imidazole ring, next to the remote nitrogen (1, 2) decreases the asymmetry parameter eta to ca. 0.75 compared to 1.00 for Cu(HIm)(4)(ClO(4))(2); this effect of C2 substitution on the symmetry of the electric field gradient at N1H appears similar for both the electron-donating methylene substituents (1) and the electron-withdrawing carbonyl group (2). (ii) The electron-donating or -withdrawing properties of the substituent are reflected by the variation of the e(2)qQ parameter, increasing from 1.43 to 1.75 MHz (1) or decreasing to 1.38 MHz (2), and by the nu(+) transition shifting toward higher frequencies from 1.49 to 1.65 MHz (1, 3, 4) or to lower frequencies to 1.29 MHz (2, 5). The use of the eta and nu(+) parameters to assign the Ndelta vs Nepsilon coordination of histidine to the metal and to detect modified histidine in copper-binding proteins is discussed.