Practical hardware for evolvable robots.

The evolutionary robotics field offers the possibility of autonomously generating robots that are adapted to desired tasks by iteratively optimising across successive generations of robots with varying configurations until a high-performing candidate is found. The prohibitive time and cost of actually building this many robots means that most evolutionary robotics work is conducted in simulation, but to apply evolved robots to real-world problems, they must be implemented in hardware, which brings new challenges. This paper explores in detail the design of an example system for realising diverse evolved robot bodies, and specifically how this interacts with the evolutionary process. We discover that every aspect of the hardware implementation introduces constraints that change the evolutionary space, and exploring this interplay between hardware constraints and evolution is the key contribution of this paper. In simulation, any robot that can be defined by a suitable genetic representation can be implemented and evaluated, but in hardware, real-world limitations like manufacturing/assembly constraints and electrical power delivery mean that many of these robots cannot be built, or will malfunction in operation. This presents the novel challenge of how to constrain an evolutionary process within the space of evolvable phenotypes to only those regions that are practically feasible: the viable phenotype space. Methods of phenotype filtering and repair were introduced to address this, and found to degrade the diversity of the robot population and impede traversal of the exploration space. Furthermore, the degrees of freedom permitted by the hardware constraints were found to be poorly matched to the types of morphological variation that would be the most useful in the target environment. Consequently, the ability of the evolutionary process to generate robots with effective adaptations was greatly reduced. The conclusions from this are twofold. 1) Designing a hardware platform for evolving robots requires different thinking, in which all design decisions should be made with reference to their impact on the viable phenotype space. 2) It is insufficient to just evolve robots in simulation without detailed consideration of how they will be implemented in hardware, because the hardware constraints have a profound impact on the evolutionary space.

Synthesis and characterization of a Noble metal Enhanced Optical Nanohybrid (NEON): a high brightness detection platform based on a dye-doped silica nanoparticle.

A highly bright and photostable, fluorescent nanohybrid particle is presented which consists of gold nanoparticles (GNPs) embedded in dye-doped silica in a core-shell configuration. The dye used is the near-infrared emitting 4,5-benzo-5'-(iodoacetaminomethyl)-1',3,3,3',3'-pentamethyl-1-(4-sulfobutyl) indodicarbo cyanine. The nanohybrid architecture comprises a GNP core which is separated from a layer of dye molecules by a 15 nm buffer layer and has an outer protective, undoped silica shell. Using this architecture, a brightness factor of 550 has been achieved compared to the free dye. This hybrid system, referred to as Noble metal Enhanced Optical Nanohybrid (NEON) in this paper, is the first nanohybrid construct to our knowledge which demonstrates such tunable fluorescence property. NEON has enhanced photostability compared to the free dye and compared to a control particle without GNPs. Furthermore, the NEON particle, when used as a fluorescent label in a model bioassay, shows improved performance over assays using a conventional single dye molecule label.

Synthesis and characterization of model silica-gold core-shell nanohybrid systems to demonstrate plasmonic enhancement of fluorescence.

In this work, gold-silica plasmonic nanohybrids have been synthesized as model systems which enable tuning of dye fluorescence enhancement/quenching interactions. For each system, a dye-doped silica core is surrounded by a 15 nm spacer region, which in turn is surrounded by gold nanoparticles (GNPs). The GNPs are either covalently conjugated via mercapto silanization to the spacer or encapsulated in a separate external silica shell. The intermediate spacer region can be either dye doped or left undoped to enable quenching and plasmonic enhancement effects respectively. The study indicates that there is a larger enhancement effect when GNPs are encapsulated in the outer shell compared to the system of external conjugation. This is due to the environmental shielding provided by shell encapsulation compared to the exposure of the GNPs to the solvent environment for the externally conjugated system. The fluorescence signal enhancement of the nanohybrid systems was evaluated using a standard HRP-anti-HRP fluorescence based assay platform.

Label-free optical characterization methods for detecting amine silanization-driven gold nanoparticle self-assembly.

Fluorescence lifetime correlation spectroscopy (FLCS) is presented as a single-step label-free detection method for probing the amine silanization-driven spontaneous 3D self-assembly of freestanding gold nanoparticles (GNPs) in solution. Unlike the conventional methods of studying self-assembly, for example, UV-vis spectroscopy and electron microscopy, FLCS utilizes the intrinsic gold fluorescence. The significance of this approach is to amalgamate the measurement of optical and hydrodynamic size properties simultaneously to achieve a more coherent description of the self-assembly pathway. GNP self-assembly has two-stage kinetics. Electrostatic interaction drives the initial amine silanization, and this is followed by siloxane bond formation between hydrolyzed ethoxy groups of GNP-attached APTES, resulting in the formation of micrometer-sized superstructures. The self-assembly has resulted in a 5-fold increase in the fluorescence lifetime (FL), and the FLCS study has shown an 8- to 10-fold increase in the diffusion coefficient using the pure diffusion model. This result is consistent with the transmission electron microscopy (TEM) observation, which shows a few hundred fold increase in the diameter due to assembly formation by the GNPs.

Dextran-coated silica nanoparticles for calcium-sensing.

This article describes the synthesis and characterisation of fluorescent composite nanoparticles consisting of a silica core and a dextran shell. The silica core contains a rhodamine-based reference dye, which allows ratiometric measurements and the dextran shell is labelled with the Ca(2+)-sensitive dye Fluo-4. The nanoparticles have an average hydrodynamic diameter of 95 nm, good colloidal stability and show a 2.9-fold increase in fluorescence intensity upon binding to Ca(2+) ions. The apparent dissociation constant of K'(d) ≈ 520 nM is well suited for measurements in the physiological range.

Fluorescence lifetime analysis and fluorescence correlation spectroscopy elucidate the internal architecture of fluorescent silica nanoparticles.

Fluorescence lifetime (FL) analysis and fluorescence correlation spectroscopy (FCS) have been successfully employed to reveal detailed information about the internal architecture of fluorescent silica nanoparticles (NPs). The dual-component lifetime behavior shows a two-domain dye distribution in the NP as a function of solvent accessibility. The introduction of an undoped silica shell serves to stabilize the outer dye fraction that is manifest as an increase in lifetime. The FCS not only shows a size-dependent increase in the cross-correlation decay constant but also demonstrates a significant relaxation in the FCS signal with the introduction of an undoped silica shell.

Novel multiparametric approach to elucidate the surface amine-silanization reaction profile on fluorescent silica nanoparticles.

This Article addresses the important issue of the characterization of surface functional groups for optical bioassay applications. We use a model system consisting of spherical dye-doped silica nanoparticles (NPs) that have been functionalized with amine groups whereby the encapsulated cyanine-based near-infrared dye fluorescence acts as a probe of the NP surface environment. This facilitates the identification of the optimum deposition parameters for the formation of a stable ordered amine monolayer and also elucidates the functionalization profile of the amine-silanization process. Specifically, we use a novel approach where the techniques of fluorescence correlation spectroscopy (FCS) and fluorescence lifetime measurement (FL) are used in conjunction with the more conventional analytical techniques of zeta potential measurement and Fourier transfer infrared spectroscopy (FTIR). The dynamics of the ordering of the amine layer in different stages of the reaction have been characterized by FTIR, FL, and FCS. The results indicate an optimum reaction time for the formation of a stable amine layer, which is optimized for further biomolecular conjugation, whereas extended reaction times lead to a disordered cross-linked layer. The results have been validated using an immunoglobulin (IgG) plate-based direct binding assay where the maximum number of IgG-conjugated aminated NPs were captured by immobilized anti-IgG antibodies for the NP sample corresponding to the optimized amine-silanization condition. Importantly, these results point to the potential of FCS and FL as useful analytical tools in diverse fields such as characterization of surface functionalization.

Potentiation of exocytosis by phospholipase C-coupled G-protein-coupled receptors requires the priming protein Munc13-1.

The vesicle priming protein Munc13-1 is regulated by diacylglycerol (DAG) and is therefore hypothesized to play a role in the control of neurotransmitter release by phospholipase C (PLC)-coupled receptors. We combined voltage-clamp recordings of voltage-gated Ca2+ channels (VGCCs) and high-resolution capacitance measurements to investigate the mechanism of receptor-mediated modulation of exocytosis in bovine chromaffin cells. Activation of endogenous H1 G(q)-protein-coupled receptors (G(q)PCRs) by histamine potentiated stimulus-coupled secretion despite concurrently inhibiting Ca2+ influx through VGCCs. Histamine increased the size of the readily releasable pool of vesicles and in particular a subpool of fusion-competent vesicles localized in close proximity to VGCCs. Pharmacological characterization showed that potentiation of exocytosis depended on the activation of PLC but not protein kinase C. Overexpression of wild-type Munc13-1 by adenoviral infection had no effect on histamine-induced potentiation per se, whereas DAG-insensitive Munc13-1(H567K) completely abolished it. This is the first endogenous mammalian G(q)PCR signaling pathway identified that engages Munc13-1 to increase stimulus-coupled secretion by recruiting vesicles to the immediately releasable pool. G(q)PCRs are therefore able to control exocytosis at the level of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex formation to produce rapid, short-term potentiation of the secretory output of neurons and endocrine cells.

Casting protocols for the production of open cell aluminum foams by the replication technique and the effect on porosity.

Metal foams are interesting materials from both a fundamental understanding and practical applications point of view. Uses have been proposed, and in many cases validated experimentally, for light weight or impact energy absorbing structures, as high surface area heat exchangers or electrodes, as implants to the body, and many more. Although great progress has been made in understanding their structure-properties relationships, the large number of different processing techniques, each producing material with different characteristics and structure, means that understanding of the individual effects of all aspects of structure is not complete. The replication process, where molten metal is infiltrated between grains of a removable preform material, allows a markedly high degree of control and has been used to good effect to elucidate some of these relationships. Nevertheless, the process has many steps that are dependent on individual "know-how", and this paper aims to provide a detailed description of all stages of one embodiment of this processing method, using materials and equipment that would be relatively easy to set up in a research environment. The goal of this protocol and its variants is to produce metal foams in an effective and simple way, giving the possibility to tailor the outcome of the samples by modifying certain steps within the process. By following this, open cell aluminum foams with pore sizes of 1-2.36 mm diameter and 61% to 77% porosity can be obtained.

Electron transfer mechanism in Shewanella loihica PV-4 biofilms formed at graphite electrode.

Electron transfer mechanisms in Shewanella loihica PV-4 viable biofilms formed at graphite electrodes were investigated in potentiostat-controlled electrochemical cells poised at oxidative potentials (0.2V vs. Ag/AgCl). Chronoamperometry (CA) showed a repeatable biofilm growth of S. loihica PV-4 on graphite electrode. CA, cyclic voltammetry (CV) and its first derivative shows that both direct electron transfer (DET) mediated electron transfer (MET) mechanism contributes to the overall anodic (oxidation) current. The maximum anodic current density recorded on graphite was 90 µA cm(-2). Fluorescence emission spectra shows increased concentration of quinone derivatives and riboflavin in the cell-free supernatant as the biofilm grows. Differential pulse voltammetry (DPV) show accumulation of riboflavin at the graphite interface, with the increase in incubation period. This is the first study to observe a gradual shift from DET to MET mechanism in viable S. loihica PV-4 biofilms.

Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation.

BACKGROUND: Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive. RESULTS: In this work, we identify a novel role for CLCs in the regulated endocytosis of specific cargoes. Small interfering RNA-mediated knockdown of either CLCa or CLCb inhibits the uptake of GPCRs. Moreover, we demonstrate that phosphorylation of Ser204 in CLCb is required for efficient endocytosis of a subset of GPCRs and identify G protein-coupled receptor kinase 2 (GRK2) as a kinase that can phosphorylate CLCb on Ser204. Overexpression of CLCb(S204A) specifically inhibits the endocytosis of those GPCRs whose endocytosis is GRK2-dependent. CONCLUSIONS: Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs.

Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins.

Green fluorescent protein and its variants are frequently used as Förster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (

Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site.

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp(501) in the relay loop.

Engineering Dictyostelium discoideum myosin II for the introduction of site-specific fluorescence probes.

Dictyostelium discoideum is a useful host for the production of constructs for the analysis of structure-function relationships of myosin. Here we describe the use of myosin II constructs containing a single tryptophan residue, at different locations, for probing events at the nucleotide binding site, the relay loop and the communication path between them. GFP fusions have also been expressed at the N- and C-termini of the myosin motor to provide sensitive probes of the actomyosin dissociation reaction in microscope-based kinetic assays. We report on the fluorescence anisotropy of these constructs in the context of their use as resonance energy transfer probes.