Hierarchically structured bioinspired nanocomposites.

Next-generation structural materials are expected to be lightweight, high-strength and tough composites with embedded functionalities to sense, adapt, self-repair, morph and restore. This Review highlights recent developments and concepts in bioinspired nanocomposites, emphasizing tailoring of the architecture, interphases and confinement to achieve dynamic and synergetic responses. We highlight cornerstone examples from natural materials with unique mechanical property combinations based on relatively simple building blocks produced in aqueous environments under ambient conditions. A particular focus is on structural hierarchies across multiple length scales to achieve multifunctionality and robustness. We further discuss recent advances, trends and emerging opportunities for combining biological and synthetic components, state-of-the-art characterization and modelling approaches to assess the physical principles underlying nature-inspired design and mechanical responses at multiple length scales. These multidisciplinary approaches promote the synergetic enhancement of individual materials properties and an improved predictive and prescriptive design of the next era of structural materials at multilength scales for a wide range of applications.

The Elephant Evolved p53 Isoforms that Escape MDM2-Mediated Repression and Cancer.

The p53 tumor suppressor is a transcription factor with roles in cell development, apoptosis, oncogenesis, aging, and homeostasis in response to stresses and infections. p53 is tightly regulated by the MDM2 E3 ubiquitin ligase. The p53-MDM2 pathway has coevolved, with MDM2 remaining largely conserved, whereas the TP53 gene morphed into various isoforms. Studies on prevertebrate ancestral homologs revealed the transition from an environmentally induced mechanism activating p53 to a tightly regulated system involving cell signaling. The evolution of this mechanism depends on structural changes in the interacting protein motifs. Elephants such as Loxodonta africana constitute ideal models to investigate this coevolution as they are large and long-living as well as having 20 copies of TP53 isoformic sequences expressing a variety of BOX-I MDM2-binding motifs. Collectively, these isoforms would enhance sensitivity to cellular stresses, such as DNA damage, presumably accounting for strong cancer defenses and other adaptations favoring healthy aging. Here we investigate the molecular evolution of the p53-MDM2 system by combining in silico modeling and in vitro assays to explore structural and functional aspects of p53 isoforms retaining the MDM2 interaction, whereas forming distinct pools of cell signaling. The methodology used demonstrates, for the first time that in silico docking simulations can be used to explore functional aspects of elephant p53 isoforms. Our observations elucidate structural and mechanistic aspects of p53 regulation, facilitate understanding of complex cell signaling, and suggest testable hypotheses of p53 evolution referencing Peto's Paradox.

Dynamic environments do not appear to constrain spider web building behaviour.

Many laboratory experiments demonstrate how orb-web spiders change the architecture of their webs in response to prey, surroundings and wind loading. The overall shape of the web and a range of other web parameters are determined by frame and anchor threads. In the wild, unlike the lab, the anchor threads are attached to branches and leaves that are not stationary but move, which affects the thread tension field. Here we experimentally test the effect of a moving support structure on the construction behaviour and web-parameters of the garden cross spider Araneus diadematus. We found no significant differences in building behaviour between rigid and moving anchors in total time spent and total distance covered nor in the percentage of the total time spent and distance covered to build the three major web components: radials, auxiliary and capture spirals. Moreover, measured key parameters of web-geometry were equally unaffected. These results call for re-evaluation of common understanding of spider webs as thread tensions are often considered to be a major factor guiding the spider during construction and web-operation.

Hard X-ray nano-holotomography with a Fresnel zone plate.

X-ray phase contrast nanotomography enables imaging of a wide range of samples with high spatial resolution in 3D. Near-field holography, as one of the major phase contrast techniques, is often implemented using X-ray optics such as Kirkpatrick-Baez mirrors, waveguides and compound refractive lenses. However, these optics are often tailor-made for a specific beamline and challenging to implement and align. Here, we present a near-field holography setup based on Fresnel zone plates which is fast and easy to align and provides a smooth illumination and flat field. The imaging quality of different types of Fresnel zone plates is compared in terms of the flat-field quality, the achievable resolution and exposure efficiency i.e. the photons arriving at the detector. Overall, this setup is capable of imaging different types of samples at high spatial resolution of below 100 nm in 3D with access to the quantitative phase information.

Functional flexibility in a spider's orb web.

Web spiders rely on vibrations propagated via their web to identify, locate and capture entangled prey. Here, we experimentally tested the robustness of the orb weaver's predation strategy when webs are severely distorted and silk tensions are drastically altered throughout the web, a common occurrence in the wild. We assessed prey identification efficiency by comparing the spider's initial reaction times towards a fruit fly trapped in the web, we measured location efficiency by comparing times and number of tugging bouts performed, and we determined capture efficiency by comparing capture times. It emerged that spiders are capable of identifying, locating and capturing prey in distorted webs, albeit taking somewhat longer to do so.

Structural Diversity of Native Major Ampullate, Minor Ampullate, Cylindriform, and Flagelliform Silk Proteins in Solution.

The foundations of silk spinning, the structure, storage, and activation of silk proteins, remain highly debated. By combining solution small-angle neutron and X-ray scattering (SANS and SAXS) alongside circular dichroism (CD), we reveal a shape anisotropy of the four principal native spider silk feedstocks from Nephila edulis. We show that these proteins behave in solution like elongated semiflexible polymers with locally rigid sections. We demonstrated that minor ampullate and cylindriform proteins adopt a monomeric conformation, while major ampullate and flagelliform proteins have a preference for dimerization. From an evolutionary perspective, we propose that such dimerization arose to help the processing of disordered silk proteins. Collectively, our results provide insights into the molecular-scale processing of silk, uncovering a degree of evolutionary convergence in protein structures and chemistry that supports the macroscale micellar/pseudo liquid crystalline spinning mechanisms proposed by the community.

Movement reveals reproductive tactics in male elephants.

Long-term bio-logging has the potential to reveal how movements, and hence life-history trade-offs, vary over a lifetime. Reproductive tactics in particular may vary as individuals' trade-off current investment versus lifetime fitness. Male African savanna elephants (Loxodona africana) provide a telling example of balancing body growth with reproductive fitness due to the combination of indeterminate growth and strongly delineated periods of sexual activity (musth), which results in reproductive tactics that alter with age. Our study aims to quantify the extent to which male elephants alter their movement patterns, and hence energetic allocation, in relation to (a) reproductive state and (b) age, and (c) to determine whether musth periods can be detected directly from GPS tracking data. We used a combination of GPS tracking data and visual observations of 25 male elephants ranging in age from 20 to 52 years to examine the influence of reproductive state and age on movement. We then used a three-state hidden Markov model (HMM) to detect musth behaviour in a subset of sequential tracking data. Our results demonstrate that male elephants increased their daily mean speed and range size with age and in musth. Furthermore, non-musth speed decreased with age, presumably reflecting a shift towards energy acquisition during non-musth. Thus, despite similar speeds and marginally larger ranges between reproductive states at age 20, by age 50, males were travelling 2.0 times faster in a 3.5 times larger area in musth relative to non-musth. The distinctiveness of musth periods over age 35 meant the three-state HMM could automatically detect musth movement with high sensitivity and specificity, but could not for the younger age class. We show that male elephants increased their energetic allocation into reproduction with age as the probability of reproductive success increases. Given that older male elephants tend to be both the target of legal trophy hunting and illegal poaching, man-made interference could drive fundamental changes in elephant reproductive tactics. Bio-logging, as our study reveals, has the potential both to quantify mature elephant reproductive tactics remotely and to be used to institute proactive management strategies around the reproductive behaviour of this charismatic keystone species.

In-drop capillary spooling of spider capture thread inspires hybrid fibers with mixed solid-liquid mechanical properties.

An essential element in the web-trap architecture, the capture silk spun by ecribellate orb spiders consists of glue droplets sitting astride a silk filament. Mechanically this thread presents a mixed solid-liquid behavior unknown to date. Under extension, capture silk behaves as a particularly stretchy solid, owing to its molecular nanosprings, but it totally switches behavior in compression to now become liquid-like: It shrinks with no apparent limit while exerting a constant tension. Here, we unravel the physics underpinning the unique behavior of this "liquid wire" and demonstrate that its mechanical response originates in the shape-switching of the silk filament induced by buckling within the droplets. Learning from this natural example of geometry and mechanics, we manufactured programmable liquid wires that present previously unidentified pathways for the design of new hybrid solid-liquid materials.

Drop-on-coilable-fibre systems exhibit negative stiffness events and transitions in coiling morphology.

We investigate the mechanics of elastic fibres carrying liquid droplets. In such systems, buckling may localize inside the drop cavity if the fibre is thin enough. This so-called drop-on-coilable-fibre system exhibits a surprising liquid-like response under compression and a solid-like response under tension. Here we analyze this unconventional behavior in further detail and find theoretical, numerical and experimental evidence of negative stiffness events. We find that the first and main negative stiffness regime owes its existence to the transfer of capillary-stored energy into mechanical curvature energy. The following negative stiffness events are associated with changes in the coiling morphology of the fibre. Eventually coiling becomes tightly locked into an ordered phase where liquid and solid deformations coexist.

Spider Silk: Mother Nature's Bio-Superlens.

It was recently discovered that transparent microspheres and cylinders can function as a super-resolution lens (i.e., superlens) to focus light beyond the diffraction limit. A number of high-resolution applications based on these lenses have been successfully demonstrated and span nanoscopy, imaging, and spectroscopy. Fabrication of these superlenses, however, is often complex and requires sophisticated engineering processes. Clearly an easier model candidate, such as a naturally occurring superlens, is highly desirable. Here, we report for the first time a biological superlens provided by nature: the minor ampullate spider silk spun from the Nephila spider. This natural biosuperlens can distinctly resolve 100 nm features under a conventional white-light microscope with peak wavelength at 600 nm, attaining a resolution of λ/6 that is well beyond the classical limit. Thus, our work opens a new door to develop biology-based optical systems that may provide a new solution to integrating optics in biological systems.

The complexity of silk under the spotlight of synthetic biology.

For centuries silkworm filaments have been the focus of R&D innovation centred on textile manufacture with high added value. Most recently, silk research has focused on more fundamental issues concerning bio-polymer structure-property-function relationships. This essay outlines the complexity and fundamentals of silk spinning, and presents arguments for establishing this substance as an interesting and important subject at the interface of systems biology (discovery) and synthetic biology (translation). It is argued that silk is a generic class of materials where each type of silk presents a different embodiment of emergent properties that combine genetically determined (anticipatory) and environmentally responsive components. In spiders' webs the various silks have evolved to form the interactive components of an intricate fabric that provides an extended phenotype to the spider's body morphology.

Dry-Spun Silk Produces Native-Like Fibroin Solutions.

Silk's outstanding mechanical properties and energy efficient solidification mechanisms provide inspiration for biomaterial self-assembly as well as offering a diverse platform of materials suitable for many biotechnology applications. Experiments now reveal that the mulberry silkworm Bombyx mori secretes its silk in a practically "unspun" state that retains much of the solvent water and exhibits a surprisingly low degree of molecular order (ß-sheet crystallinity) compared to the state found in a fully formed and matured fiber. These new observations challenge the general understanding of silk spinning and in particular the role of the spinning duct for structure development. Building on this discovery we report that silk spun in low humidity appears to arrest a molecular annealing process crucial for ß-sheet formation. This, in turn, has significant positive implications, enabling the production of a high fidelity reconstituted silk fibroin with properties akin to the gold standard of unspun native silk.

Glass transitions in native silk fibres studied by dynamic mechanical thermal analysis.

Silks are a family of semi-crystalline structural materials, spun naturally by insects, spiders and even crustaceans. Compared to the characteristic ß-sheet crystalline structure in silks, the non-crystalline structure and its composition deserves more attention as it is equally critical to the filaments' high toughness and strength. Here we further unravel the structure-property relationship in silks using Dynamic Mechanical Thermal Analysis (DMTA). This technique allows us to examine the most important structural relaxation event of the disordered structure the disordered structure, the glass transition (GT), in native silk fibres of the lepidopteran Bombyx mori and Antheraea pernyi and the spider Nephila edulis. The measured glass transition temperature Tg, loss tangent tan δ and dynamic storage modulus are quantitatively modelled based on Group Interaction Modelling (GIM). The "variability" issue in native silks can be conveniently explained by the different degrees of structural disorder as revealed by DMTA. The new insights will facilitate a more comprehensive understanding of the structure-property relations for a wide range of biopolymers.

Identification and classification of silks using infrared spectroscopy.

Lepidopteran silks number in the thousands and display a vast diversity of structures, properties and industrial potential. To map this remarkable biochemical diversity, we present an identification and screening method based on the infrared spectra of native silk feedstock and cocoons. Multivariate analysis of over 1214 infrared spectra obtained from 35 species allowed us to group silks into distinct hierarchies and a classification that agrees well with current phylogenetic data and taxonomies. This approach also provides information on the relative content of sericin, calcium oxalate, phenolic compounds, poly-alanine and poly(alanine-glycine) ß-sheets. It emerged that the domesticated mulberry silkmoth Bombyx mori represents an outlier compared with other silkmoth taxa in terms of spectral properties. Interestingly, Epiphora bauhiniae was found to contain the highest amount of ß-sheets reported to date for any wild silkmoth. We conclude that our approach provides a new route to determine cocoon chemical composition and in turn a novel, biological as well as material, classification of silks.

Silk Reconstitution Disrupts Fibroin Self-Assembly.

Using atomic force microscopy, we present the first molecular-scale comparison of two of the most important silk dopes, native (NSF) and reconstituted (RSF) silkworm fibroin. We found that both systems depended on shear to show self-assembly. Significant differences in the nature of self-assembly between NSF and RSF were shown. In the highest studied concentration of 1000 mg/L, NSF exhibited assembly into 20-30 nm-wide nanofibrils closely resembling the surface structures found in natural silk fibers. RSF, in contrast, showed no self-assembly whatsoever at the same concentration, which suggests that the reconstitution process significantly disrupts silk's inherent self-assembly capability. At lower concentrations, both RSF and NSF formed fibrils under shear, apparently denatured by the substrate. Using image analysis, we quantified the properties of these self-assembled fibrils as a function of concentration and found low-concentration fibrils of NSF to form larger continuous structures than those of RSF, further supporting NSF's superior self-assembly capabilities.

Spiders spinning electrically charged nano-fibres.

Most spider threads are on the micrometre and sub-micrometre scale. Yet, there are some spiders that spin true nano-scale fibres such as the cribellate orb spider, Uloborus plumipes. Here, we analyse the highly specialized capture silk-spinning system of this spider and compare it with the silk extrusion systems of the more standard spider dragline threads. The cribellar silk extrusion system consists of tiny, morphologically basic glands each terminating through exceptionally long and narrow ducts in uniquely shaped silk outlets. Depending on spider size, hundreds to thousands of these outlet spigots cover the cribellum, a phylogenetically ancient spinning plate. We present details on the unique functional design of the cribellate gland-duct-spigot system and discuss design requirements for its specialist fibrils. The spinning of fibres on the nano-scale seems to have been facilitated by the evolution of a highly specialist way of direct spinning, which differs from the aqua-melt silk extrusion set-up more typical for other spiders.

Adhesion of dry and wet electrostatic capture silk of uloborid spider.

We demonstrate the impressive adhesive qualities of uloborid spider orb-web capture when dry, which are lost when the nano-filament threads are wetted. A force sensor with a 50 nN-1 mN detection sensitively allowed us to measure quantitatively the stress-strain characteristics of native silk threads in both the original dry state and after wetting by controlled application of water mist with droplet sizes ranging between 3 and 5 µm and densities ranging between 10(4) and 10(5) per mm(3). Stress forces of between 1 and 5 µN/µm(2) in the native, dry multifilament thread puffs were reduced to between 0.1 and 0.5 µN/µm(2) in the wetted collapsed state, with strain displacements reducing from between 2 and 5 mm in the dry to 0.10-0.12 mm in the wetted states. We conclude that wetting cribellate threads reduce their van der Waals adhesion with implications on the thread's adhesive strength under tension. This should be considered when discussing the evolutionary transitions of capture silks from the ancestral dry-state nano-filaments of the cribellate spider taxa to the wet-state glue-droplets of the ecribellate taxa.

Structure and properties of silk from the African wild silkmoth Gonometa postica reared indoors.

African wild silkmoth, Gonometa postica Walker (Lepidoptera: Lasiocampidae), were reared indoors in order to examine the influence of rearing conditions on the structure and properties of silk cocoon shells and degummed fibers by using a scanning electron microscope, an Instron tensile tester, and a thermogravimetric analyzer. The cocoons reared indoors showed inferior quality in weight, length, width, and cocoon shell ratio compared to cocoons reared outdoors. There were no differences in cocoon shell and fiber surfaces and cross sectional structures. Cocoon shells were covered with calcium oxalate crystals with few visible fibers on their surface. Degummed fibers were smooth with minimum unfractured surfaces and globular to triangular cross sections. Indoor-reared cocoon shells had a significantly higher breaking strain, while the breaking stress was higher for cocoons reared outdoors. Fibers from indoor cocoons had a significantly higher breaking stress while outdoor fibers had higher breaking strain. Thermogravimetric analysis curves showed two main thermal reactions revealing the dehydration of water molecules and ir-reversible decomposition of the crystallites in both cocoons and fibers reared indoors and outdoors. Cocoon shells underwent additional peaks of decomposition with increased temperature. The total weight loss was higher for cocoon shells and degummed fibers from indoors. Rearing conditions (temperature and relative humidity), feeding method used, changes in total life span, days to molting, and spinning might have influenced the variation in the properties observed.The ecological and commercial significances of indoor rearing of G. posticaare discussed.

Water mobility, denaturation and the glass transition in proteins.

A quantitative mechanism is presented that links protein denaturation and the protein-water glass transition through an energy criterion for the onset of mobility of strong protein-water bonds. Differences in the zero point vibrational energy in the ordered and disordered bonded states allow direct prediction of the two transition temperatures. While the onset of water mobility induces the same change in heat capacity for both transitions, the order-disorder transition of denaturation also predicts the observed excess enthalpy gain. The kinetics of the water and protein components through the glass transition are predicted and compared with dielectric spectroscopy observations. The energetic approach provides a consistent mechanism for processes such as refolding and aggregation of proteins involved in protein maintenance and adaptability, as the conformational constraints of strong water-amide bonds are lost with increased molecular mobility. Moreover, we suggest that the ordered state of peptide-water bonds is induced at the point of protein synthesis and could play a key role in the function of proteins through the enhancement of electronic activity by ferroelectric domains in the protein hydration shell, which is lost upon denaturation.

The impact behaviour of silk cocoons.

Silk cocoons, constructed by silkmoths (Lepidoptera), are protective structural composites. Some cocoons appear to have evolved towards structural and material optimisation in order to sustain impact strikes from predators and hinder parasite ingress. This study investigates the protective properties of silk cocoons with different morphologies by evaluating their impact resistance and damage tolerance. Finite element analysis was used to analyse empirical observations of the quasi-static impact response of the silk cocoons, and to evaluate the separate benefits of the structures and materials through the deformation and damage mechanism. We use design principles from composite engineering in order to understand the structure-property-function relationship of silkworm cocoons. Understanding the highly evolved survival strategies of the organisms building natural cocoons will hopefully lead to inspiration that in turn could lead to improved composite design.