The survey and reference assisted assembly of the Octopus vulgaris genome.

The common octopus, Octopus vulgaris, is an active marine predator known for the richness and plasticity of its behavioral repertoire, and remarkable learning and memory capabilities. Octopus and other coleoid cephalopods, cuttlefish and squid, possess the largest nervous system among invertebrates, both for cell counts and body to brain size. O. vulgaris has been at the center of a long-tradition of research into diverse aspects of its biology. To leverage research in this iconic species, we generated 270 Gb of genomic sequencing data, complementing those available for the only other sequenced congeneric octopus, Octopus bimaculoides. We show that both genomes are similar in size, but display different levels of heterozygosity and repeats. Our data give a first quantitative glimpse into the rate of coding and non-coding regions and support the view that hundreds of novel genes may have arisen independently despite the close phylogenetic distance. We furthermore describe a reference-guided assembly and an open genomic resource (CephRes-gdatabase), opening new avenues in the study of genomic novelties in cephalopods and their biology.

Formation of pit-spanning phospholipid bilayers on nanostructured silicon dioxide surfaces for studying biological membrane events.

Zwitterionic phospholipid vesicles are known to adsorb and ultimately rupture on flat silicon dioxide (SiO2) surfaces to form supported lipid bilayers. Surface topography, however, alters the kinetics and mechanistic details of vesicles adsorption, which under certain conditions may be exploited to form a suspended bilayer. Here we describe the use of nanostructured SiO2 surfaces prepared by the colloidal lithography technique to scrutinize the formation of suspended 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers from a solution of small unilamellar lipid vesicles (SUVs). Atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) were employed to characterize nanostructure fabrication and lipid bilayer assembly on the surface.

Unfolding dynamics of the mucin SEA domain probed by force spectroscopy suggest that it acts as a cell-protective device.

MUC1 and other membrane-associated mucins harbor long, up to 1 µm, extended highly glycosylated mucin domains and sea urchin sperm protein, enterokinase and agrin (SEA) domains situated on their extracellular parts. These mucins line luminal tracts and organs, and are anchored to the apical cell membrane by a transmembrane domain. The SEA domain is highly conserved and undergoes a molecular strain-dependent autocatalytic cleavage during folding in the endoplasmic reticulum, a process required for apical plasma membrane expression. To date, no specific function has been designated for the SEA domain. Here, we constructed a recombinant protein consisting of three SEA domains in tandem and used force spectroscopy to assess the dissociation force required to unfold individual, folded SEA domains. Force-distance curves revealed three peaks, each representing unfolding of a single SEA domain. Fitting the observed unfolding events to a worm-like chain model yielded an average contour length of 32 nm per SEA domain. Analysis of forces applied on the recombinant protein revealed an average unfolding force of 168 pN for each SEA domain at a loading rate of 25 nN·s(-1). Thus, the SEA domain may act as a breaking point that can dissociate before the plasma membrane is breached when mechanical forces are applied to cell surfaces.

Diffraction from arrays of plasmonic nanoparticles with short-range lateral order.

We have measured the angular distribution of light scattered off 2D plasmonic Al nanoparticle ensembles. We created these samples with disk-like nanoparticles, 175 and 500 nm in diameter, respectively, using hole-mask colloidal lithography and electron beam lithography. The nanoparticle arrangements in the samples display the short-range order (but no long-range order) characteristic for an ensemble formed by random sequential adsorption. As a consequence of this, the ensemble scattering patterns can be quantitatively well described by combining the single-particle scattering pattern with a static structure factor that carries information about the diffraction effects caused by the short-range order of the ensemble. We also performed sensing experiments in which we monitored changes in the angle-resolved scattering intensity for a fixed wavelength as a function of the thickness of an ultrathin SiO(2) coating covering the Al nanoparticles. The data show that the angle and strength of the main diffraction peak vary linearly with SiO(2) coating thickness in the range 1.5-4.5 nm and suggest that measurements of the scattering profile could be a competitive alternative to traditional transmission measurements in terms of sensitivity.

Mechanistic insight into the structure and dynamics of entangled and hydrated λ-phage DNA.

Intrinsic dynamics of DNA plays a crucial role in DNA-protein interactions and has been emphasized as a possible key component for in vivo chromatin organization. We have prepared an entangled DNA microtube above the overlap concentration by exploiting the complementary cohesive ends of λ-phage DNA, which is confirmed by atomic force microscopy and agarose gel electrophoresis. Photon correlation spectroscopy further confirmed that the entangled solutions are found to exhibit the classical hydrodynamics of a single chain segment on length scales smaller than the hydrodynamic length scale of single λ-phage DNA molecule. We also observed that in 41.6% (gm water/gm DNA) hydrated state, λ-phage DNA exhibits a dynamic transition temperature (T(dt)) at 187 K and a crossover temperature (T(c)) at 246 K. Computational insight reveals that the observed structure and dynamics of entangled λ-phage DNA are distinctively different from the behavior of the corresponding unentangled DNA with open cohesive ends, which is reminiscent with our experimental observation.

Oscillatory optical response of an amorphous two-dimensional array of gold nanoparticles.

The optical response of metallic nanoparticle arrays is dominated by localized surface plasmon excitations and is the sum of individual particle contributions modified by interparticle coupling that depends on specific array geometry. We demonstrate a so far unexplored distinct oscillatory behavior of the plasmon peak position, full width at half maximum, and extinction efficiency in large area amorphous arrays of Au nanodisks, which depend on the minimum particle center-to-center distance in the array. Amorphous arrays exhibit short-range order and are completely random at long distances. In our theoretical analysis we introduce a film of dipoles approach, within the framework of the coupled dipole approximation, which describes the array as an average particle surrounded by a continuum of dipoles with surface densities determined by the pair correlation function of the array.

Solar hydrogen production with semiconductor metal oxides: new directions in experiment and theory.

An overview of a collaborative experimental and theoretical effort toward efficient hydrogen production via photoelectrochemical splitting of water into di-hydrogen and di-oxygen is presented here. We present state-of-the-art experimental studies using hematite and TiO(2) functionalized with gold nanoparticles as photoanode materials, and theoretical studies on electro and photo-catalysis of water on a range of metal oxide semiconductor materials, including recently developed implementation of self-interaction corrected energy functionals.

A multifaceted approach to hydrogen storage.

The widespread adoption of hydrogen as an energy carrier could bring significant benefits, but only if a number of currently intractable problems can be overcome. Not the least of these is the problem of storage, particularly when aimed at use onboard light-vehicles. The aim of this overview is to look in depth at a number of areas linked by the recently concluded HYDROGEN research network, representing an intentionally multi-faceted selection with the goal of advancing the field on a number of fronts simultaneously. For the general reader we provide a concise outline of the main approaches to storing hydrogen before moving on to detailed reviews of recent research in the solid chemical storage of hydrogen, and so provide an entry point for the interested reader on these diverse topics. The subjects covered include: the mechanisms of Ti catalysis in alanates; the kinetics of the borohydrides and the resulting limitations; novel transition metal catalysts for use with complex hydrides; less common borohydrides; protic-hydridic stores; metal ammines and novel approaches to nano-confined metal hydrides.

Bioreducible insulin-loaded nanoparticles and their interaction with model lipid membranes.

To improve design processes in the field of nanomedicine, in vitro characterization of nanoparticles with systematically varied properties is of great importance. In this study, surface sensitive analytical techniques were used to evaluate the responsiveness of nano-sized drug-loaded polyelectrolyte complexes when adsorbed to model lipid membranes. Two bioreducible poly(amidoamine)s (PAAs) containing multiple disulfide linkages in the polymer backbone (SS-PAAs) were synthesized and used to form three types of nanocomplexes by self-assembly with human insulin, used as a negatively charged model protein at neutral pH. The resulting nanoparticles collapsed on top of negatively charged model membranes upon adsorption, without disrupting the membrane integrity. These structural rearrangements may occur at a cell surface which would prevent uptake of intact nanoparticles. By the addition of glutathione, the disulfide linkages in the polymer backbone of the SS-PAAs were reduced, resulting in fragmentation of the polymer and dissociation of the adsorbed nanoparticles from the membrane. A decrease in ambient pH also resulted in destabilization of the nanoparticles and desorption from the membrane. These mimics of intracellular environments suggest dissociation of the drug formulation, a process that releases the protein drug load, when the nanocomplexes reaches the interior of a cell.

Electroplate and lift lithography for patterned micro/nanowires using ultrananocrystalline diamond (UNCD) as a reusable template.

A fast, simple, scalable technique is described for the controlled, solution-based, electrochemical synthesis of patterned metallic and semiconducting nanowires from reusable, nonsacrificial, ultrananocrystalline diamond (UNCD) templates. This enables the repeated fabrication of arrays of complex patterns of nanowires, potentially made of any electrochemically depositable material. Unlike all other methods of patterning nanowires, this benchtop technique quickly mass-produces patterned nanowires whose diameters are not predefined by the template, without requiring intervening vacuum or clean room processing. This technique opens a pathway for studying nanoscale phenomena with minimal equipment, allowing the process-scale development of a new generation of nanowire-based devices.

Gold, platinum, and aluminum nanodisk plasmons: material independence, subradiance, and damping mechanisms.

Localized surface plasmon resonances (LSPR) are collective electronic excitations in metallic nanoparticles. The LSPR spectral peak position, as a function of nanoparticle size and material, is known to depend primarily on dynamic depolarization and electron structure related effects. The former gives rise to the well-known spectral red shift with increasing nanoparticle size. A corresponding understanding of the LSPR spectral line width for a wide range of nanoparticle sizes and different metals does, however, not exist. In this work, the radiative and nonradiative damping contributions to the LSPR line width over a broad nanoparticle size range (40-500 nm) for a selection of three metals with fundamentally different bulk dielectric properties (Au, Pt, and Al) are explored experimentally and theoretically. Excellent agreement was obtained between the observed experimental trends and the predictions based on electrostatic spheroid theory (MLWA), and the obtained results were successfully related to the specific band structure of the respective metal. Moreover, for the first time, a clear transition from a radiation damping dominated to a quenched radiation damping regime (subradiance) in large nanoparticles was observed and probed by varying the electron density through appropriate material choice. To minimize inhomogeneous broadening (commonly present in ensemble-based spectroscopic measurements), a novel, electron-beam lithography (EBL)-based nanofabrication method was developed. The method generates large-area 2D patterns of randomly distributed nanodisks with well-defined size and shape, narrow size distribution, and tunable (minimum) interparticle distance. In order to minimize particle-particle coupling effects, sparse patterns with a large interparticle distance (center-to-center ≥6 particle diameters) were considered.

Electrodeless QCM-D for lipid bilayer applications.

An electrodeless quartz crystal microbalance with dissipation monitoring (QCM-D) setup is used to monitor the formation of supported lipid bilayers (SLBs) on bare quartz crystal sensor surfaces. The kinetic behavior of the formation of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) SLB on SiO(2) surfaces is discussed and compared for three cases: (i) a standard SiO(2) film deposited onto the gold electrode of a quartz crystal, (ii) an electrodeless quartz crystal with a sputter-coated SiO(2) film, and (iii) an uncoated electrodeless quartz crystal sensor surface. We demonstrate, supported by imaging the SLB on an uncoated electrodeless surface using atomic force microscopy (AFM), that a defect-free, completely covering bilayer is formed in all three cases. Differences in the kinetics of the SLB formation on the different sensor surfaces are attributed to differences in surface roughness. The latter assumption is supported by imaging the different surfaces using AFM. We show furthermore that electrodeless quartz crystal sensors can be used not only for the formation of neutral SLBs but also for positively and negatively charged SLBs. Based on our results we propose electrodeless QCM-D to be a valuable technique for lipid bilayer and related applications providing several advantages compared to electrode-coated surfaces like optical transparency, longer lifetime, and reduced costs.

Vesicle adsorption and phospholipid bilayer formation on topographically and chemically nanostructured surfaces.

We have investigated the influence of combined nanoscale topography and surface chemistry on lipid vesicle adsorption and supported bilayer formation on well-controlled model surfaces. To this end, we utilized colloidal lithography to nanofabricate pitted Au-SiO(2) surfaces, where the top surface and the walls of the pits consisted of silicon dioxide whereas the bottom of the pits was made of gold. The diameter and height of the pits were fixed at 107 and 25 nm, respectively. Using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique and atomic force microscopy (AFM), we monitored the processes occurring upon exposure of these nanostructured surfaces to a solution of extruded unilamellar 1-palmitolyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles with a nominal diameter of 100 nm. To scrutinize the influence of surface chemistry, we studied two cases: (1) the bare gold surface at the bottom of the pits and (2) the gold passivated by biotinamidocaproyl-labeled bovine serum albumin (BBSA) prior to vesicle exposure. As in our previous work on pitted silicon dioxide surfaces, we found that the pit edges promote bilayer formation on the SiO(2) surface for the vesicle size used here in both cases. Whereas in the first case we observed a slow, continuous adsorption of intact vesicles onto the gold surface at the bottom of the pits, the presence of BBSA in the second case prevented the adsorption of intact vesicles into the pits. Instead, our experimental results, together with free energy calculations for various potential membrane configurations, indicate the formation of a continuous, supported lipid bilayer that spans across the pits. These results are significantly important for various biotechnology applications utilizing patterned lipid bilayers and highlight the power of the combined QCM-D/AFM approach to study the mechanism of lipid bilayer formation on nanostructured surfaces.

Combined QCM-D and EIS study of supported lipid bilayer formation and interaction with pore-forming peptides.

A novel set-up combining the quartz crystal microbalance with dissipation monitoring technique (QCM-D) and electrochemical impedance spectroscopy (EIS) under flow conditions was successfully used to follow supported lipid bilayer (SLB) formation on SiO(2). This study demonstrates the simultaneous detection, in real time, of both the electrical and the structural properties of the SLB. The combination of the two techniques provided novel insights regarding the mechanism of SLB formation: we found indications for an annealing process of the lipid alkyl chains after the mass corresponding to complete bilayer coverage had been deposited. Moreover, the interaction of the SLB with the pore-forming toxin, gramicidin D (grD) was studied for grD concentrations ranging from 0.05 to 40 mg L(-1). Membrane properties were altered depending on the toxin concentration. For low grD concentrations, the electrical properties of the SLB changed upon insertion of active ion channels. For higher concentrations, the QCM-D data showed dramatic changes in the viscoelastic properties of the membrane while the EIS spectra did not change. AFM confirmed significant structural changes of the membrane at higher grD concentrations. Thus, the application of combined QCM-D and EIS detection provides complementary information about the system under study. This information will be particularly important for the continued detailed investigation of interactions at model membrane surfaces.

Thermodynamic and kinetic supercooling of liquid in a wedge pore.

Cyclohexane allowed to capillary condense from vapor in an annular wedge pore of mica in a surface force apparatus (SFA) remains liquid down to at least 14 K below the bulk melting-point T(m). This is an example of supercooling of a liquid due to confinement, like melting-point depression in porous media. In the wedge pore, however, the supercooled liquid is in equilibrium with vapor, and the amount of liquid (and thereby the radius of curvature r of the liquid-vapor interface) depends on the surface tension gamma(LV) of the liquid, not the interfacial tension between the solid and liquid. At coexistence r is inversely proportional to the temperature depression DeltaT below T(m), in accordance with a recently proposed model [P. Barber, T. Asakawa, and H. K. Christenson, J. Phys. Chem. C 111, 2141 (2007)]. We have now extended this model to include effects due to the temperature dependence of both the surface tension and the enthalpy of melting. The predictions of the improved model have been quantitatively verified in experiments using both a Mark IV SFA and an extended surface force apparatus (eSFA). The three-layer interferometer formed by the two opposing, backsilvered mica surfaces in a SFA was analyzed by conventional means (Mark IV) and by fast spectral correlation of up to 40 fringes (eSFA). We discuss the absence of freezing in the outermost region of the wedge pore down to 14 K below T(m) and attribute it to nonequilibrium (kinetic) supercooling, whereas the inner region of the condensate is thermodynamically supercooled.

Influence of nanotopography on phospholipid bilayer formation on silicon dioxide.

We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicle-to-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipid-membrane-based applications using rough or topographically structured surfaces.

Localized surface plasmon resonance sensing of lipid-membrane-mediated biorecognition events.

Supported phospholipid bilayers (SPBs) have emerged as important model systems for studies of the natural cell membrane and its components, which are essential for the integrity and function of cells in all living organisms, and also constitute common targets for therapeutic drugs and in disease diagnosis. However, the preferential occurrence of spontaneous SPB formation on silicon-based substrates, but not on bare noble-metal surfaces, has so far excluded the use of the localized surface plasmon resonance (LSPR) sensing principle for studies of lipid-membrane-mediated biorecognition reactions. This is because the LSPR phenomenon is associated with, and strongly confined to, the interfacial region of nanometric noble-metal particles. This problem has been overcome in this study by a self-assembly process utilizing localized rupture of phospholipid vesicles on silicon dioxide in the bottom of nanometric holes in a thin gold film. The hole-induced localization of the LSPR field to the voids of the holes is demonstrated to provide an extension of the LSPR sensing concept to studies of reactions confined exclusively to SPB-patches supported on SiO2. In particular, we emphasize the possibility of performing label-free studies of lipid-membrane-mediated reaction kinetics, including the compatibility of the assay with array-based reading (approximately 7 x 7 microm2) and detection of signals originating from bound protein in the zeptomole regime.

Metal nanowire arrays by electrodeposition.

We describe two related methods for preparing arrays of nanowires composed of molybdenum, copper, nickel, gold, and palladium. Nanowires were obtained by selectively electrodepositing either a metal oxide or a metal at the step edges present on the basal plane of highly oriented pyrolytic graphite (HOPG) electrodes. If a metal oxide was electrodeposited, then nanowires of the parent metal were obtained by reduction at elevated temperature in hydrogen. The resulting nanowires were organized in parallel arrays of 100-1000 wires. These nanowires were long (some > 500 microns), polycrystalline, and approximately hemicylindrical in cross-section. The nanowire arrays prepared by electrodeposition were also "portable": After embedding the nanowires in a polymer or cyanoacrylate film, arrays of nanowires could be lifted off the graphite surface thereby facilitating the incorporation of metal nanowire arrays into devices such as sensors.