Biomimetic Cell Structures: Probing Induced pH-Feedback Loops and pH Self-Monitoring in Cytosol Using Binary Enzyme-Loaded Polymersomes in Proteinosome.

Structures and functions of eukaryotic cells with an outer permeable membrane, a cytoskeleton, functional organelles, and motility can be mimicked by giant multicompartment protocells containing various synthetic organelles. Herein, two kinds of artificial organelles with stimuli-triggered regulation ability, glucose oxidase-(GOx)-loaded pH-responsive polymersomes A (GOx-Psomes A) and urease-loaded pH-responsive polymersomes B (Urease-Psomes B), and a pH-sensor (Dextran-FITC) are encapsulated into proteinosomes via the Pickering emulsion method. Thus, a polymersomes-in-proteinosome system is constructed which is able to probe biomimetic pH homeostasis. Alternating fuels (glucose or urea) introduced from outside the protocell penetrate the membrane of proteinosomes and enter into GOx-Psomes A and Urease-Psomes B to produce chemical signals (gluconic acid or ammonia) resulting in pH-feedback loops (pH jump and pH drop). This will counteract the catalytic "switch on" or "switch off" of enzyme-loaded Psomes A and B owing to their different pH-responsive membranes. Dextran-FITC in the proteinosome allows self-monitoring of slight pH fluctuations in the lumen of protocells. Overall, this approach shows heterogeneous polymersome-in-proteinosome architectures with sophisticated features such as input-regulated pH changes mediated by negative and positive feedback in loops and cytosolic pH self-monitoring, features that are imperative for advanced protocell design.

Foliar water uptake in Pinus species depends on needle age and stomatal wax structures.

BACKGROUND AND AIMS: Foliar water uptake (FWU) has been documented in many species and is increasingly recognized as a non-trivial factor in plant-water relationships. However, it remains unknown whether FWU is a widespread phenomenon in Pinus species, and how it may relate to needle traits such as the form and structure of stomatal wax plugs. In this contribution, these questions were addressed by studying FWU in current-year and 1-year-old needles of seven Pinus species. METHODS: We monitored FWU gravimetrically and analysed the needle surface via cryo-scanning electron microscopy. Additionally, we considered the effect of artificial wax erosion by application of the surfactant Triton X-100, which is able to alter wax crystals. KEY RESULTS: The results show for all species that (1) FWU occurred, (2) FWU is higher in old needles compared to young needles and (3) there is substantial erosion of stomatal wax plugs in old needles. FWU was highest in Pinus canariensis, which has a thin stomatal wax plug. Surfactant treatment enhanced FWU. CONCLUSIONS: The results of this study provide evidence for (1) widespread FWU in Pinus, (2) the influence of stomatal wax plugs on FWU and (3) age-related needle surface erosion.

A Self-Assembled Matrix System for Cell-Bioengineering Applications in Different Dimensions, Scales, and Geometries.

Stem cell bioengineering and therapy require different model systems and materials in different stages of development. If a chemically defined biomatrix system can fulfill most tasks, it can minimize the discrepancy among various setups. By screening biomaterials synthesized through a coacervation-mediated self-assembling mechanism, a biomatrix system optimal for 2D human mesenchymal stromal cell (hMSC) culture and osteogenesis is identified. Its utility for hMSC bioengineering is further demonstrated in coating porous bioactive glass scaffolds and nanoparticle synthesis for esiRNA delivery to knock down the SOX-9 gene with high delivery efficiency. The self-assembled injectable system is further utilized for 3D cell culture, segregated co-culture of hMSC with human umbilical vein endothelial cells (HUVEC) as an angiogenesis model, and 3D bioprinting. Most interestingly, the coating of bioactive glass with the self-assembled biomatrix not only supports the proliferation and osteogenesis of hMSC in the 3D scaffold but also induces the amorphous bioactive glass (BG) scaffold surface to form new apatite crystals resembling bone-shaped plate structures. Thus, the self-assembled biomatrix system can be utilized in various dimensions, scales, and geometries for many different bioengineering applications.

Construction of Eukaryotic Cell Biomimetics: Hierarchical Polymersomes-in-Proteinosome Multicompartment with Enzymatic Reactions Modulated Protein Transportation.

The eukaryotic cell is a smart compartment containing an outer permeable membrane, a cytoskeleton, and functional organelles, presenting part structures for life. The integration of membrane-containing artificial organelles (=polymersomes) into a large microcompartment is a key step towards the establishment of exquisite cellular biomimetics with different membrane properties. Herein, an efficient way to construct a hierarchical multicompartment composed of a hydrogel-filled proteinosome hybrid structure with an outer homogeneous membrane, a smart cytoskeleton-like scaffold, and polymersomes is designed. Specially, this hybrid structure creates a micro-environment for pH-responsive polymersomes to execute a desired substance transport upon response to biological stimuli. Within the dynamic pH-stable skeleton of the protein hydrogels, polymersomes with loaded PEGylated insulin biomacromolecules demonstrate a pH-responsive reversible swelling-deswelling and a desirable, on-demand cargo release which is induced by the enzymatic oxidation of glucose to gluconic acid. This stimulus responsive behavior is realized by tunable on/off states through protonation of the polymersomes membrane under the enzymatic reaction of glucose oxidase, integrated in the skeleton of protein hydrogels. The integration of polymersomes-based hybrid structure into the proteinosome compartment and the stimuli-response on enzyme reactions fulfills the requirements of eukaryotic cell biomimetics in complex architectures and allows mimicking cellular transportation processes.

Cuticular Hydrocarbon Trails Released by Host Larvae Lose their Kairomonal Activity for Parasitoids by Solidification.

Successful host search by parasitic wasps is often mediated by host-associated chemical cues. The ectoparasitoid Holepyris sylvanidis is known to follow chemical trails released by host larvae of the confused flour beetle, Tribolium confusum, for short-range host location. Although the hexane-extractable trails consist of stable, long-chain cuticular hydrocarbons (CHCs) with low volatility, the kairomonal activity of a trail is lost two days after release. Here, we studied whether this loss of kairomonal activity is due to changes in the chemical trail composition induced by microbial activity. We chemically analyzed trails consisting of hexane extracts of T. confusum larvae after different time intervals past deposition under sterile and non-sterile conditions. GC-MS analyses revealed that the qualitative and quantitative pattern of the long-chain CHCs of larval trails did not significantly change over time, neither under non-sterile nor sterile conditions. Hence, our results show that the loss of kairomonal activity of host trails is not due to microbially induced changes of the CHC pattern of a trail. Interestingly, the kairomonal activity of trails consisting of host larval CHC extracts was recoverable after two days by applying hexane to them. After hexane evaporation, the parasitoids followed the reactivated host trails as they followed freshly laid ones. Cryo-scanning electron microscopy showed that the trails gradually formed filament-shaped microstructures within two days. This self-assemblage of CHCs was reversible by hexane application. Our study suggests that the long-chain CHCs of a host trail slowly undergo solidification by a self-assembling process, which reduces the accessibility of CHCs to the parasitoid's receptors as such that the trail is no longer eliciting trail-following behavior.

Evidence for a sexually selected function of the attachment system in bedbugs Cimex lectularius (Heteroptera, Cimicidae).

Attachment to surfaces is a major aspect of an animal's interaction with the environment. Consequently, shaping of the attachment system in relation to weight load and substrate is considered to have occurred mainly by natural selection. However, sexual selection may also be important because many animals attach to their partner during mating. The two hypotheses generate opposing predictions in species where males are smaller than females. Natural selection predicts that attachment ability will scale positively with load, and hence body size, and so will be larger in females than males. Sexual selection predicts attachment forces in males will be larger than those in females, despite the males' smaller size because males benefit from uninterrupted copulation by stronger attachment to the female. We tested these predictions in the common bedbug Cimex lectularius, a species in which both sexes, as well as nymphs, regularly carry large loads: blood meals of up to 3 times their body weight. By measuring attachment forces to smooth surfaces and analysing in situ fixed copulating pairs and the morphology of attachment devices, we show that: (i) males generate twice the attachment force of females, despite weighing 15% less; (ii) males adhere to females during copulation using hairy tibial adhesive pads; (iii) there are more setae, and more setae per unit area, in the pads of males than in those of females but there is no difference in the shape of the tarsal setae; and (iv) there is an absence of hairy tibial attachment pads and a low attachment force in nymphs. These results are consistent with a sexually selected function of attachment in bedbugs. Controlling sperm transfer and mate guarding by attaching to females during copulation may also shape the evolution of male attachment structures in other species. More generally, we hypothesise the existence of an arms race in terms of male attachment structures and female counterparts to impede attachment, which may result in a similar evolutionary diversification to male genitalia.

Functional morphology of tarsal adhesive pads and attachment ability in ticks Ixodes ricinus (Arachnida, Acari, Ixodidae).

The presence of well-developed, elastic claws on ticks and widely pilose hosts led us to hypothesise that ticks are mostly adapted to attachment and locomotion on rough, strongly corrugated and hairy, felt-like substrates. However, by using a combination of morphological and experimental approaches, we visualised the ultrastructure of attachment devices of Ixodes ricinus and showed that this species adheres more strongly to smooth surfaces than to rough ones. Between paired, elongated, curved, elastic claws, I. ricinus bears a large, flexible, foldable adhesive pad, which represents an adaptation to adhesion on smooth surfaces. Accordingly, ticks attached strongest to glass and to surface profiles similar to those of the human skin, generating safety factors (attachment force relative to body weight) up to 534 (females). Considerably lower attachment force was found on silicone substrates and as a result of thanatosis after jolting.

Plant pressure sensitive adhesives: similar chemical properties in distantly related plant lineages.

MAIN CONCLUSION: A mixture of resins based on aliphatic esters and carboxylic acids occurs in distantly related genera Peperomia and Roridula , serving different functions as adhesion in seed dispersal and prey capture. According to mechanical characteristics, adhesive secretions on both leaves of the carnivorous flypaper Roridula gorgonias and epizoochorous fruits of Peperomia polystachya were expected to be similar. The chemical analysis of these adhesives turned out to be challenging because of the limited available mass for analysis. Size exclusion chromatography and Fourier transform infrared spectroscopy were suitable methods for the identification of a mixture of compounds, most appropriately containing natural resins based on aliphatic esters and carboxylic acids. The IR spectra of the Peperomia and Roridula adhesive resemble each other; they correspond to that of a synthetic ethylene-vinyl acetate copolymer, but slightly differ from that of natural tree resins. Thus, the pressure sensitive adhesive properties of the plant adhesives are chemically proved. Such adhesives seem to appear independently in distantly related plant lineages, habitats, life forms, as well as plant organs, and serve different functions such as prey capture in Roridula and fruit dispersal in Peperomia. However, more detailed chemical analyses still remain challenging because of the small available volume of plant adhesive.

Adhesive properties of Aphrophoridae spittlebug foam.

Aphrophora alni spittlebug nymphs produce a wet foam from anal excrement fluid, covering and protecting themselves against numerous impacts. Foam fluid contact angles on normal (26°) and silanized glass (37°) suggest that the foam wets various substrates, including plant and arthropod surfaces. The pull-off force depends on the hydration state and is higher the more dry the fluid. Because the foam desiccates as fast as water, predators once captured struggle to free from drying foam, becoming stickier. The present study confirms that adhesion is one of the numerous foam characteristics resulting in multifunctional effects, which promote spittlebugs' survival and render the foam a smart, biocompatible material of biological, biomimetic and biomedical interest. The sustainable 'reuse' of large amounts of excrement for foam production and protection of the thin nymph integument suggests energetic and evolutionary advantages. Probably, that is why foam nests have evolved in different groups of organisms, such as spittlebugs, frogs and fish.

Integument and defence in larva and prepupa of a sawfly living on a semi-aquatic plant.

The larvae of the sawfly Rhadinoceraea micans live and feed on a semi-aquatic plant, Iris pseudacorus, and their integument is strongly hydrophobic. The hydrophobicity is part of a chemical defence strategy, easy bleeding, also known from congeners. The prepupae burrow into the soil where they form a cocoon in which they pupate, thus implying different micro-environmental conditions. The cuticle structure and wetting defensive effectiveness of R. micans were compared between larvae and prepupae. The two stages were similarly well defended against attacking ants by the bleeding of a deterrent hemolymph, whereas they were dissimilar in the cuticle surface that presented sculptures and wax crystals at the larval stage only. The integument of prepupae was less structured, and hydrophilic. Larvae of R. micans exhibit, among sawflies, an exceptional cuticle structuring and we assume that they occupy this particular niche of a semi-aquatic environment to avoid encounters with ground-dwelling predators whereas prepupae may benefit from the chemical defence acquired at larval stage.

A self-assembled dynamic extracellular matrix-like hydrogel system with multi-scale structures for cell bioengineering applications.

Extracellular matrix (ECM) provides various types of direct interactions with cells and a dynamic environment, which can be remodeled through different assembly/degradation mechanisms to adapt to different biological processes. Herein, through introducing polyphosphate-modified hyaluronic acid and bioactive glass (BG) nano-fibril into a self-assembled hydrogel system with peptide-polymer conjugate, we can realize many new ECM-like functions in a synthetic polymer network. The hydrogel network formation is mediated by coacervation, followed by a gradual transition of peptide structure from  α-helix to ß-sheet. The ECM-like hydrogels can be degraded through a number of orthogonal mechanisms, including treatments with protease, hyaluronidase, alkaline phosphatase, and calcium ion. As 2D coating, the ECM-like hydrogels can be used to modify the planar surface to promote the adhesion of mesenchymal stromal cells, or to coat the cell surface in a layer-by-layer fashion to shield the interaction with the substrate. As ECM-like hydrogels for 3D cell culture, the system is compatible with injection and cell encapsulation. Upon incorporating fragmented electrospun bioactive glass nano-fibril into the hydrogels, the synergetic effects of soft hydrogel and stiff reinforcement nanofibers on recapitulating ECM functions result in reduced cell circularity in 3D. Finally, by injecting the ECM-like hydrogels into mice, gradual degradations over a time period of one month and high biocompatibility have been shown in vivo. The contribution of complex network dynamics and hierarchical structures to cell-biomatrix interaction can be investigated multi-dimensionally, as many mechanisms are orthogonal to each other and can be regulated individually. STATEMENT OF SIGNIFICANCE: A list of native ECM features has attracted the most interest and attention in the research of synthetic biomaterials. In this research, we have described a simple ECM-like hydrogel system in which the complex and elegant functions of native ECM can be recapitulated in a chemically defined synthetic system. The ECM-like hydrogel systems were developed to avoid undesired features of biological substances (e.g., ethical concerns, batch-to-batch variation, immunogenicity, and potential risk of contamination), as well as gaining new functions to facilitate bioengineering applications (e.g., 3D cell culture, injection, and high stability). To this end, we have developed an ECM-like hydrogel system and provide evidence that this purely synthetic biomaterial is a promising candidate for cell bioengineering applications.

Leaf beetle attachment on wrinkles: isotropic friction on anisotropic surfaces.

The influence of surface roughness on the attachment ability of insects has been repeatedly reported. In previous experiments, complex surface topographies were used as test substrates, whereas periodical structures have so far been neglected. In the present study, traction experiments with adult beetles Gastrophysa viridula and Leptinotarsa decemlineata were carried out to study the influence of surfaces, structured with periodical wrinkles, on insect attachment. Force measurements were carried out on male and female insects, both intact and after removal of claws, performing tethered walking on five polydimethylsiloxane substrates: (i) smooth, non-structured (control), (ii-v) structured with wrinkles of different wavelengths (366, 502, 911 and 25,076 nm). In two test series, beetles walked either perpendicular or parallel to the wrinkle alignment. Adults of G. viridula produced generally higher forces than those of L. decemlineata. The results show that the alignment of wrinkles had no significant influence on the force generation by beetles, probably because of the skewed position of their tarsomeres relative to the substrates. In both sexes, the highest force values were obtained on surfaces with wrinkles of 25 µm wavelength. On other wrinkled substrates, forces were significantly reduced in both males and females compared with the smooth, flat control, with the minimum force achieved on wrinkles with a wavelength of 911 nm.

Always on the bright side: the climbing mechanism of Galium aparine.

Galium aparine is a herbaceous climbing plant that attaches to host plants mainly via its leaves, which are covered by hooked trichomes. Although such hooks are found on both leaf surfaces, the leaves of G. aparine are mainly positioned upon the leaves of supporting plants and rarely beneath. In order to understand the mechanism underlying this observation, we have studied structural and mechanical properties of single leaf hooks, frictional properties of leaf surfaces, turgor pressure in different leaf tissues and bending properties of the leaves in different directions. Abaxial and adaxial leaf hooks differ significantly in orientation, distribution, structure and mechanical properties. In accordance with these differences, friction properties of leaves depend on the direction of the applied force and differ significantly between both leaf surfaces. This results in a ratchet mechanism. Abaxial leaf hooks provide strong attachment upon the leaves of adjacent plants, whereas adaxial hooks cause a gliding-off from the underside of the leaves of host plants. Thus, the leaves of G. aparine can function as attachment organs, and simultaneously orient themselves advantageously for their photosynthetic function. Further adaptations in turgor pressure or concerning an anisotropy of the flexural stiffness of the leaves have not been found.

Conductive Hydrogels with Dynamic Reversible Networks for Biomedical Applications.

Conductive hydrogels (CHs) are emerging as a promising and well-utilized platform for 3D cell culture and tissue engineering to incorporate electron signals as biorelevant physical cues. In conventional covalently crosslinked conductive hydrogels, the network dynamics (e.g., stress relaxation, shear shining, and self-healing) required for complex cellular functions and many biomedical utilities (e.g., injection) cannot be easily realized. In contrast, dynamic conductive hydrogels (DCHs) are fabricated by dynamic and reversible crosslinks. By allowing for the breaking and reforming of the reversible linkages, DCHs can provide dynamic environments for cellular functions while maintaining matrix integrity. These dynamic materials can mimic some properties of native tissues, making them well-suited for several biotechnological and medical applications. An overview of the design, synthesis, and engineering of DCHs is presented in this review, focusing on the different dynamic crosslinking mechanisms of DCHs and their biomedical applications.

Convergent synthesis of diversified reversible network leads to liquid metal-containing conductive hydrogel adhesives.

Many features of extracellular matrices, e.g., self-healing, adhesiveness, viscoelasticity, and conductivity, are associated with the intricate networks composed of many different covalent and non-covalent chemical bonds. Whereas a reductionism approach would have the limitation to fully recapitulate various biological properties with simple chemical structures, mimicking such sophisticated networks by incorporating many different functional groups in a macromolecular system is synthetically challenging. Herein, we propose a strategy of convergent synthesis of complex polymer networks to produce biomimetic electroconductive liquid metal hydrogels. Four precursors could be individually synthesized in one to two reaction steps and characterized, then assembled to form hydrogel adhesives. The convergent synthesis allows us to combine materials of different natures to generate matrices with high adhesive strength, enhanced electroconductivity, good cytocompatibility in vitro and high biocompatibility in vivo. The reversible networks exhibit self-healing and shear-thinning properties, thus allowing for 3D printing and minimally invasive injection for in vivo experiments.

Egg attachment of the asparagus beetle Crioceris asparagi to the crystalline waxy surface of Asparagus officinalis.

Plant surfaces covered with crystalline epicuticular waxes are known to be anti-adhesive, hardly wettable and preventing insect attachment. But there are insects that are capable of gluing their eggs to these surfaces by means of proteinaceous secretions. In this study, we analysed the bonding region between the eggs of Crioceris asparagi and the plant surface of Asparagus officinalis using light and cryo-scanning electron microscopy. The wettability of the plant surface by egg secretion was compared with that by Aqua Millipore water, aqueous sugar solution and chicken egg white. Furthermore, the force required to remove C. asparagi eggs from the plant surface was measured, in order to evaluate the egg's bonding strength. Mean pull-off force was 14.7 mN, which is about 8650 times higher than the egg weight. Egg glue was observed spreading over the wax crystal arrays on the plant cladophyll and wetting them. Similar wetting behaviour on the A. officinalis surface was observed for chicken egg white. Our results support the hypothesis that the mechanism of insect egg adhesion on micro- and nanostructured hydrophobic plant surfaces is related to the proteinaceous nature of adhesive secretions of insect eggs. The secretion wets superhydrophobic surfaces and after solidifying builds up a composite, consisting of the solidified glue and wax crystals, at the interface between the egg and plant cuticle.

Desiccation resistance of adhesive secretion in the protocarnivorous plant Roridula gorgonias as an adaptation to periodically dry environment.

To analyse the change of mass in the resinous secretion of Roridula gorgonias, we carried out long-term measurements using a computerised ultra microbalance. In contrast to the aqueous mucilage of other carnivorous flypaper traps, this water-insoluble secretion does not desiccate even in dead and formalin-preserved plants. We found no significant desiccation of secretory drops within 10 h of continuous weighing. After 100 days of the secretion exposure to dry conditions, only about 8% of the mass was lost. Interestingly, returning these samples to humid conditions resulted in a mass recovery to the initial amount. This property of the secretion is discussed as an ecological adaptation to the periodically dry Fynbos habitat of the plant.

Robust, universal, and persistent bud secretion adhesion in horse-chestnut trees.

Buds of horse-chestnut trees are covered with a viscous fluid, which remains sticky after long-term exposure to heat, frost, radiation, precipitation, deposition of aerosols and particles, attacks by microbes and arthropods. The present study demonstrates that the secretion does not dry out under arid conditions, not melt at 50 °C, and not change significantly under UV radiation or frost at a microscopic level. It is slightly swellable under wet conditions; and, it universally wets and adheres to substrates having different polarities. Measured pull-off forces do not differ between hydrophilic and lipophilic surfaces, ranging between 58 and 186 mN, and resulting in an adhesive strength up to 204 kPa. The mechanical and chemical properties of secretion resemble those of pressure-sensitive adhesives. The Raman, infrared, and nuclear magnetic resonance spectra show the clear presence of saturated aliphatic hydrocarbons, esters, free carboxylic acids, as well as minor amounts of amides and aromatic compounds. We suggest a multi-component material (aliphatic hydrocarbon resin), including alkanes, fatty acids, amides, and tackifying terpenoids embedded in a fluid matrix (fatty acids) comprising nonpolar and polar portions serving the universal and robust adhesive properties. These characteristics matter for ecological-evolutionary aspects and can inspire innovative designs of multifunctional, biomimetic pressure-sensitive adhesives and varnishes.

A vegetable oil-based biopesticide with ovicidal activity against the two-spotted spider mite, Tetranychus urticae Koch.

A recently developed biopesticide made of safflower and cottonseed oils has excellent ovicidal activity against the hard-to-control spider mite Tetranychus urticae Koch (Acari: Tetranychidae). It has attracted attention as a sustainable treatment for controlling T. urticae because it has low potential for promoting resistance and little effect on the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae), which is an important natural enemy of spider mites. Here, we investigated the mechanism of its ovicidal activity against T. urticae. The oil droplets in the oil-in-water emulsion of the biopesticide strongly adhered to T. urticae eggs, seeped through the chorion being cut during hatching, and inhibited the embryonic rotational movement necessary for cutting and hatching. No adverse effect was observed on N. californicus eggs even in undiluted biopesticide. We conclude that this biopesticide and N. californicus can be used simultaneously in the integrated management of T. urticae in oily biopesticide-tolerant plant species.

Hierarchical organisation of the trap in the protocarnivorous plant Roridula gorgonias (Roridulaceae).

The flypaper trap of the protocarnivorous plant Roridula gorgonias is known to capture various insects, even those having a considerable body size, by using an adhesive, visco-elastic resinous secretion released by glandular trichomes of different dimensions. However, recent experimental studies have shown that the adhesion of long tentacle-shaped trichome secretion is not as strong as previously assumed. One may then ask why this flypaper trap is so highly effective. In the present study, we compared geometry, flexibility and the adhesive properties of secretion in different sized trichomes. We have analysed the gross morphology of the plant and its surfaces using light and cryo-scanning electron microscopy. Trichomes' stiffness and the adhesive properties of their secretion on different surfaces were measured. A combination of structural and experimental results, presented in this study, let us suggest that R. gorgonias represents a three-dimensional trap consisting of three functional hierarchical levels (plant, leaves and trichomes). According to their size, we classified three types of trichomes having a particular arrangement on the leaf. The longest trichomes are more flexible and less adhesive compared with the shortest ones. The latter are 48 times stiffer and their secretion has a 9 times higher adhesive strength. Our data support the hypothesis that the shortest trichomes are adapted to strong, long-term adherence to prey insects, and that the longest trichomes are responsible for initial trapping and entanglement function.