The staying power of adhesion-associated antioxidant activity in Mytilus californianus

The California mussel, Mytilus californianus, adheres in the highly oxidizing intertidal zone with a fibrous holdfast called the byssus using 3, 4-dihydroxyphenyl-l-alanine (DOPA)-containing adhesive proteins. DOPA is susceptible to oxidation in seawater and, upon oxidation, loses adhesion. Successful mussel adhesion thus depends critically on controlling oxidation and reduction. To explore how mussels regulate redox during their functional adhesive lifetime, we tracked extractable protein concentration, DOPA content and antioxidant activity in byssal plaques over time. In seawater, DOPA content and antioxidant activity in the byssus persisted much longer than expected—50% of extractable DOPA and 30% of extractable antioxidant activity remained after 20 days. Antioxidant activity was located at the plaque–substrate interface, demonstrating that antioxidant activity keeps DOPA reduced for durable and dynamic adhesion. We also correlated antioxidant activity to cysteine and DOPA side chains of mussel foot proteins (mfps), suggesting that mussels use both cysteine and DOPA redox reservoirs for controlling interfacial chemistry. These data are discussed in the context of the biomaterial structure and properties of the marine mussel byssus.     

Ocean acidification alters the material properties of Mytilus edulis shells

Ocean acidification (OA) and the resultant changing carbonate saturation states is threatening the formation of calcium carbonate shells and exoskeletons of marine organisms. The production of biominerals in such organisms relies on the availability of carbonate and the ability of the organism to biomineralize in changing environments. To understand how biomineralizers will respond to OA the common blue mussel, Mytilus edulis, was cultured at projected levels of pCO₂ (380, 550, 750, 1000 µatm) and increased temperatures (ambient, ambient plus 2°C). Nanoindentation (a single mussel shell) and microhardness testing were used to assess the material properties of the shells. Young''s modulus (E), hardness (H) and toughness (K_IC) were measured in mussel shells grown in multiple stressor conditions. OA caused mussels to produce shell calcite that is stiffer (higher modulus of elasticity) and harder than shells grown in control conditions. The outer shell (calcite) is more brittle in OA conditions while the inner shell (aragonite) is softer and less stiff in shells grown under OA conditions. Combining increasing ocean pCO₂ and temperatures as projected for future global ocean appears to reduce the impact of increasing pCO₂ on the material properties of the mussel shell. OA may cause changes in shell material properties that could prove problematic under predation scenarios for the mussels; however, this may be partially mitigated by increasing temperature.     

In situ ATR-IR spectroscopic and electron microscopic analyses of settlement secretions of Undaria pinnatifida kelp spores

Knowledge about the settlement of marine organisms on substrates is important for the development of environmentally benign new methods for control of marine biofouling. The adhesion to substrates by spores of Undaria pinnatifida, a kelp species that is invasive to several countries, was studied by scanning electron and transmission electron microscopies (SEM/TEM) as well as by in situ attenuated total reflection infrared (ATR-IR) spectroscopy. The IR spectra showed that adhesive secretion began approximately 15 min after initial settlement and that the adhesive bulk material contained protein and anionic polysaccharides. Energy dispersive X-ray microanalysis of the adhesive identified sulphur and phosphorus as well as calcium and magnesium ions, which facilitate the gelation of the anionic polysaccharides in the sea water. The adhesive may be secreted from Golgi bodies in the spore, which were imaged by TEM of spore thin sections. Additionally, an in situ settlement study on TiO₂ particle film by ATR-IR spectroscopy revealed the presence of phosphorylated moieties directly binding the substrate. The presence of anionic groups dominating the adhesive suggests that inhibition of spore adhesion will be favoured by negatively charged surfaces.     

Laser processing of natural mussel adhesive protein thin films

A novel laser processing technique is presented for depositing mussel adhesive protein thin films. Synthetic adhesives (e.g., acrylics, cyanoacrylates, epoxies, phenolics, polyurethanes, and silicones) have largely displaced natural adhesives in the automotive, aerospace, biomedical, electronic, and marine equipment industries over the past century. However, rising concerns over the environmental and health effects of solvents, monomers, and additives used in synthetic adhesives have led the adhesives community to seek natural alternatives. Marine mussel adhesive protein is a formaldehyde-free natural adhesive that demonstrates excellent adhesion to several classes of materials, including pure metals, metal oxides, polymers, and glasses. We have demonstrated the deposition of Mytilus edulis foot protein-1 thin films using matrix assisted pulsed laser evaporation (MAPLE). The Fourier transform infrared spectrum data suggest that the matrix assisted pulsed laser evaporation process does not cause significant damage to the chemical structure of M. edulis foot protein-1. In addition, matrix assisted pulsed laser evaporation appears to provide a better control over film thickness and film roughness than conventional solvent-based thin film processing techniques. MAPLE-deposited mussel adhesive protein thin films have numerous potential electronic, medical, and marine applications.     

Comprehensive Study of the Influence of Altered Gravity on the Oxidative Burst of Mussel (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) Hemocytes

Microgravity induces alterations in the functioning of immune cell; however, the underlying mechanisms have not yet been identified. In this study, hemocytes (blood cells) of the blue mussel Mytilus edulis were investigated under altered gravity conditions. The study was conducted on the ground in preparation for the BIOLAB TripleLux-B experiment, which will be performed on the International Space Station (ISS). On-line kinetic measurements of reactive oxygen species (ROS) production during the oxidative burst and thus cellular activity of isolated hemocytes were performed in a photomultiplier (PMT)-clinostat (simulated microgravity) and in the 1g operation mode of the clinostat in hypergravity on the Short-Arm Human Centrifuge (SAHC) as well as during parabolic flights. In addition to studies with isolated hemocytes, the effect of altered gravity conditions on whole animals was investigated. For this purpose, whole mussels were exposed to hypergravity (1.8 g) on a multi-sample incubator centrifuge (MuSIC) or to simulated microgravity in a submersed clinostat. After exposure for 48 h, hemocytes were taken from the mussels and ROS production was measured under 1 g conditions. The results from the parabolic flights and clinostat studies indicate that mussel hemocytes respond to altered gravity in a fast and reversible manner. Hemocytes (after cryo-conservation) exposed to simulated microgravity (μ g), as well as fresh hemocytes from clinorotated animals, showed a decrease in ROS production. Measurements during a permanent exposure of hemocytes to hypergravity (SAHC) show a decrease in ROS production. Hemocytes of mussels measured after the centrifugation of whole mussels did not show an influence to the ROS response at all. Hypergravity during parabolic flights led to a decrease but also to an increase in ROS production in isolated hemocytes, whereas the centrifugation of whole mussels did not influence the ROS response at all. This study is a good example how ground-based facility experiments can be used to prepare for an upcoming ISS experiment, in this case the TRIPLE LUX B experiment.     

Adhesive secretions of live mussels observed in situ by attenuated total reflection-infrared spectroscopy

The chemical species involved in the adhesion of blue mussels (Mytilus galloprovincialis) and greenshell mussels (Perna canaliculus) to surfaces has been investigated using in situ attenuated total reflection infrared (ATR-IR) spectroscopy. Mussel spat ranging in size from 0.5 to 25 mm were placed in a flow cell containing a ZnSe multiple internal reflection prism and supplied with temperature-controlled seawater. Distinctively different absorption spectra were obtained when the mussels were predominantly moving across the surface or forming permanent bonds. With limited movement, the absorption spectrum was characteristic of protein with peaks near 1647 cm-1 (amide I), 1543 cm-1 (amide II), and 1235 cm-1 (amide III). When the mussels were observed to be moving across the surface of the ATR-IR crystal there was a strong broad absorption maximum around 1200-900 cm-1 (carbohydrate polymers), presumably due to the secretion of a weakly acidic mucopolysaccharide. Distinct differences in the spectra obtained from the adhesive secretions of blue or greenshell mussels were not observed. The data presented is the first reported use of IR spectroscopy to obtain in situ, real-time, chemical data on the interactions between invertebrates and substrates immersed in sea water.     

Mussel (Mytilus edulis) byssus deposition in response to variations in surface wettability.

Mussels (Mytilus edulis) are economically important in their role as an aquaculture species and also with regard to marine biofouling. They attach tenaciously to a wide variety of submerged surfaces by virtue of collagenous attachment threads termed 'byssi'. The aim of this study was to characterize the spreading of the byssal attachment plaque, which mediates attachment to the surface, on a range of surfaces in response to changes in wettability. To achieve this, well characterized self-assembled monolayers of omega-terminated alkanethiolates on gold were used, allowing correlation of byssal plaque spreading with a single surface characteristic--wettability. The present results were inconsistent with those from previous studies, in that there was a positive correlation between plaque size and surface wettability; a trend which is not explained by conventional wetting theory for a three-phase system. A recent extension to wetting theory with regard to hydrophilic proteins is discussed and the results of settlement assays are used to attempt reconciliation of these results with those of similar previous studies and, also, with recent data presented for the spreading of Ulva linza spore adhesive.     

The importance of accurate muscle modelling for biomechanical analyses: a case study with a lizard skull

Computer-based simulation techniques such as multi-body dynamics analysis are becoming increasingly popular in the field of skull mechanics. Multi-body models can be used for studying the relationships between skull architecture, muscle morphology and feeding performance. However, to be confident in the modelling results, models need to be validated against experimental data, and the effects of uncertainties or inaccuracies in the chosen model attributes need to be assessed with sensitivity analyses. Here, we compare the bite forces predicted by a multi-body model of a lizard (Tupinambis merianae) with in vivo measurements, using anatomical data collected from the same specimen. This subject-specific model predicts bite forces that are very close to the in vivo measurements and also shows a consistent increase in bite force as the bite position is moved posteriorly on the jaw. However, the model is very sensitive to changes in muscle attributes such as fibre length, intrinsic muscle strength and force orientation, with bite force predictions varying considerably when these three variables are altered. We conclude that accurate muscle measurements are crucial to building realistic multi-body models and that subject-specific data should be used whenever possible.     

Antibiotic-decorated titanium with enhanced antibacterial activity through adhesive polydopamine for dental/bone implant

Implant-associated infections, which are normally induced by microbial adhesion and subsequent biofilm formation, are a major cause of morbidity and mortality. Therefore, practical approaches to prevent implant-associated infections are in great demand. Inspired by adhesive proteins in mussels, here we have developed a novel antibiotic-decorated titanium (Ti) material with enhanced antibacterial activity. In this study, Ti substrate was coated by one-step pH-induced polymerization of dopamine followed by immobilization of the antibiotic cefotaxime sodium (CS) onto the polydopamine-coated Ti through catechol chemistry. Contact angle measurement and X-ray photoelectron spectroscopy confirmed the presence of CS grafted on the Ti surface. Our results demonstrated that the antibiotic-grafted Ti substrate showed good biocompatibility and well-behaved haemocompatibility. In addition, the antibiotic-grafted Ti could effectively prevent adhesion and proliferation of Escherichia coli (Gram-negative) and Streptococcus mutans (Gram-positive). Moreover, the inhibition of biofilm formation on the antibiotic-decorated Ti indicated that the grafted CS could maintain its long-term antibacterial activity. This modified Ti substrate with enhanced antibacterial activity holds great potential as implant material for applications in dental and bone graft substitutes.     

Fabrication of a novel micro-nano fibrous nonwoven scaffold with Antheraea assama silk fibroin for use in tissue engineering

The success of developing artificial organs by tissue engineering depends on scaffold properties and architecture. Here, we describe the fabrication of an Antheraea assama fibroin based novel micro-nano fibrous nonwoven scaffold. The morphological and chemical characterization was done by scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) respectively, which demonstrated the formation of scaffold with micro-nano architecture. The biocompatibility was assessed in vitro by haemolysis and cytotoxicity assays, whereby the scaffold was found to be nontoxic and efficient in supporting cell adhesion and growth.     

Predicting connectivity of green turtles at Palmyra Atoll,central Pacific: a focus on mtDNA and dispersal modelling

Population connectivity and spatial distribution are fundamentally related to ecology, evolution and behaviour. Here, we combined powerful genetic analysis with simulations of particle dispersal in a high-resolution ocean circulation model to investigate the distribution of green turtles foraging at the remote Palmyra Atoll National Wildlife Refuge, central Pacific. We analysed mitochondrial sequences from turtles (n = 349) collected there over 5 years (2008–2012). Genetic analysis assigned natal origins almost exclusively (approx. 97%) to the West Central and South Central Pacific combined Regional Management Units. Further, our modelling results indicated that turtles could potentially drift from rookeries to Palmyra Atoll via surface currents along a near-Equatorial swathe traversing the Pacific. Comparing findings from genetics and modelling highlighted the complex impacts of ocean currents and behaviour on natal origins. Although the Palmyra feeding ground was highly differentiated genetically from others in the Indo-Pacific, there was no significant differentiation among years, sexes or stage-classes at the Refuge. Understanding the distribution of this foraging population advances knowledge of green turtles and contributes to effective conservation planning for this threatened species.     

Effects of extrusion and heat treatment on the microstructure and tensile properties of in situ TiBw/Ti6Al4V composite with a network architecture

In situ TiB whisker reinforced Ti6Al4V (TiBw/Ti64) composites with a network architecture were extruded and heat treated in order to further improve their mechanical properties. The microstructure results show that the equiaxed network architecture was extruded to column network architecture and TiB whisker to alignment distribution. The transformed β phase is formed and the residual stress generated during extrusion obviously decreases after water quenching and aging processes. The tensile test results show that the strength, elastic modulus and ductility of the composites can be significantly improved by the subsequent extrusion, and then, the strength can be further improved by water quenching and aging processes after hot extrusion deformation. The elastic modulus of the as-sintered composites with a novel network microstructure follows the upper bound of Hashin-Shtrikman (H-S) theory before extrusion, while that of the as-extruded composites with a column network microstructure agrees well with the prediction from Halpin-Tsai equation.     

Contrasts between organic participation in apatite biomineralization in brachiopod shell and vertebrate bone identified by nuclear magnetic resonance spectroscopy

Unusually for invertebrates, linguliform brachiopods employ calcium phosphate mineral in hard tissue formation, in common with the evolutionarily distant vertebrates. Using solid-state nuclear magnetic resonance spectroscopy (SSNMR) and X-ray powder diffraction, we compare the organic constitution, crystallinity and organic matrix–mineral interface of phosphatic brachiopod shells with those of vertebrate bone. In particular, the organic–mineral interfaces crucial for the stability and properties of biomineral were probed with SSNMR rotational echo double resonance (REDOR). Lingula anatina and Discinisca tenuis shell materials yield strikingly dissimilar SSNMR spectra, arguing for quite different organic constitutions. However, their fluoroapatite-like mineral is highly crystalline, unlike the poorly ordered hydroxyapatite of bone. Neither shell material shows ¹³C{³¹P} REDOR effects, excluding strong physico-chemical interactions between mineral and organic matrix, unlike bone in which glycosaminoglycans and proteins are composited with mineral at sub-nanometre length scales. Differences between organic matrix of shell material from L. anatina and D. tenuis, and bone reflect evolutionary pressures from contrasting habitats and structural purposes. The absence of organic–mineral intermolecular associations in brachiopod shell argues that biomineralization follows different mechanistic pathways to bone; their details hold clues to the molecular structural evolution of phosphatic biominerals, and may provide insights into novel composite design.     

Bone tissue reactions to biomimetic ion-substituted apatite surfaces on titanium implants

The aim of this study was to evaluate the bone tissue response to strontium- and silicon-substituted apatite (Sr-HA and Si-HA) modified titanium (Ti) implants. Sr-HA, Si-HA and HA were grown on thermally oxidized Ti implants by a biomimetic process. Oxidized implants were used as controls. Surface properties, i.e. chemical composition, surface thickness, morphology/pore characteristics, crystal structure and roughness, were characterized with various analytical techniques. The implants were inserted in rat tibiae and block biopsies were prepared for histology, histomorphometry and scanning electron microscopy analysis. Histologically, new bone formed on all implant surfaces. The bone was deposited directly onto the Sr-HA and Si-HA implants without any intervening soft tissue. The statistical analysis showed significant higher amount of bone–implant contact (BIC) for the Si-doped HA modification (P = 0.030), whereas significant higher bone area (BA) for the Sr-doped HA modification (P = 0.034), when compared with the non-doped HA modification. The differences were most pronounced at the early time point. The healing time had a significant impact for both BA and BIC (P < 0.001). The present results show that biomimetically prepared Si-HA and Sr-HA on Ti implants provided bioactivity and promoted early bone formation.     

Fracture R-curve characterization of toughened epoxy adhesives

The R-curve behavior of two different rubber-toughened epoxy adhesives was measured as a function of the mode ratio. A bilinear model was used to characterize the fracture resistance (R-curve) behavior from crack initiation to steady-state crack propagation. Experiments showed that the model parameters depended strongly on the loading mode ratio and the adhesive bondline thickness, but were largely independent of the crack initiation geometry. The results are relevant to the prediction of the crack initiation load and ultimate strength of adhesive joints having relatively short overlap lengths such that a steady-state damage zone cannot develop prior to rupture.     

Fatigue behaviour of tensile steel/CFRP joints

In this paper the fatigue performance of tensile steel/CFRP (Carbon Fibre Reinforced Polymer) double shear lap joints is discussed. Joints were realized with two steel plates and two CFRP strips bonded using epoxy adhesive. Fatigue tests were performed on 16 specimens under constant stress range loading cycles. Two stress ratios (R = 0.1 and R = 0.4) were considered to investigate their influence on the fatigue lifetime. Debonding was observed to occur at stress concentration zones and propagate along the CFRP/adhesive interfaces. The stiffness degradation of the steel joint due to progressive debonding of the adhesive represents an index for the subsequent and progressive global failure. S–N curves are defined and compared to the fatigue resistance of welded detail categories of the Eurocode 3. The tests showed that the stress ratio, R, has a marginal influence on the fatigue lifetime of the steel/CFRP double shear lap joints. Finally, a fatigue limit corresponding to a stress range in the steel plate equal to 75 MPa was conservatively estimated during the tests. The fatigue limit seems to be insensitive to the stress ratio R.     

Directional reversals enable Myxococcus xanthus cells to produce collective one-dimensional streams during fruiting-body formation

The formation of a collectively moving group benefits individuals within a population in a variety of ways. The surface-dwelling bacterium Myxococcus xanthus forms dynamic collective groups both to feed on prey and to aggregate during times of starvation. The latter behaviour, termed fruiting-body formation, involves a complex, coordinated series of density changes that ultimately lead to three-dimensional aggregates comprising hundreds of thousands of cells and spores. How a loose, two-dimensional sheet of motile cells produces a fixed aggregate has remained a mystery as current models of aggregation are either inconsistent with experimental data or ultimately predict unstable structures that do not remain fixed in space. Here, we use high-resolution microscopy and computer vision software to spatio-temporally track the motion of thousands of individuals during the initial stages of fruiting-body formation. We find that cells undergo a phase transition from exploratory flocking, in which unstable cell groups move rapidly and coherently over long distances, to a reversal-mediated localization into one-dimensional growing streams that are inherently stable in space. These observations identify a new phase of active collective behaviour and answer a long-standing open question in Myxococcus development by describing how motile cell groups can remain statistically fixed in a spatial location.     

Morphogenesis of an extended phenotype: four-dimensional ant nest architecture

Animals produce a variety of structures to modify their environments adaptively. Such structures represent extended phenotypes whose development is rarely studied. To begin to rectify this, we used micro-computed tomography (CT) scanning and time-series experiments to obtain the first high-resolution dataset on the four-dimensional growth of ant nests. We show that extrinsic features within the environment, such as the presence of planes between layers of sediment, influence the architecture of Lasius flavus nests, with ants excavating horizontal tunnels along such planes. Intrinsically, the dimensions of the tunnels are associated with individual colonies, the dynamics of excavation can be explained by negative feedback and the angular distribution of tunnels is probably a result of local competition among tunnels for miners. The architecture and dynamics of ant nest excavation therefore result from local interactions of ants with one another and templates inherent in the environment. The influence of the environment on the form of structures has been documented across both biotic and abiotic domains. Our study opens up the utility of CT scanning as a technique for observing the morphogenesis of such structures.