Anika S. Mostaert

Nanoscale Mechanical Characterisation of Amyloid Fibrils Discovered in a Natural Adhesive

Using the atomic force microscope, we have investigated the nanoscale mechanical response of the attachment adhesive of the terrestrial alga Prasiola linearis (Prasiolales, Chlorophyta). We were able to locate and extend highly ordered mechanical structures directly from the natural adhesive matrix of the living plant. The in vivo mechanical response of the structured biopolymer often displayed the repetitive sawtooth force-extension characteristics of a material exhibiting high mechanical strength at the molecular level. Mechanical and histological evidence leads us to propose a mechanism for mechanical strength in our sample based on amyloid fibrils. These proteinaceous, pleated β-sheet complexes are usually associated with neurodegenerative diseases. However, we now conclude that the amyloid protein quaternary structures detected in our material should be considered as a possible generic mechanism for mechanical strength in natural adhesives.

Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production.

This paper describes a microalgal cell lipid fluorescence enhancement method using BODIPY(505/515), which can be used to screen for lipids in wild-type microalgae and to monitor lipid content within microalgae production processes to determine optimal harvesting time. The study was based on four microalgae species (Dunaliella teteriolecta, Tetraselmis suecica, Nannochloropsis oculata, and Nannochloris atomus) selected because of their inherent high lipid content. An extended analysis was carried out with N. oculata due to the depressed fluorescence observed when compared with the other experimental strains. BODIPY(505/515) lipid fluorescence was determined for two solvent pre-treatment methods (DMSO and glycerol) and four staining condition parameters (analysis time, staining temperature, dye concentration, and algal cell concentration). It was found that lipid fluorescence of thick cell-walled microalgae, such as N. oculata, is significantly enhanced by both the pre-treatment methods and staining condition parameters, thereby significantly enhancing lipid fluorescence by ca. 800 times the base autofluorescence. The lipid fluorescence enhancement method provides a quick and simple index for in vivo Flow Cytometry quantification of total lipid contents for purposes of species screening or whole culture monitoring in biofuel-directed microalgae production.

Beneficial characteristics of mechanically functional amyloid fibrils evolutionarily preserved in natural adhesives

While biological systems are notorious for their complexity, nature sometimes displays mechanisms that are elegant in their simplicity. We have recently identified such a mechanism at work to enhance the mechanical properties of certain natural adhesives. The mechanism is simple because it utilizes a non-specific protein folding and subsequent aggregation process, now thought to be generic for any polypeptide under appropriate conditions. This non-specific folding forms proteinaceous crossed β-sheet amyloid fibrils, which are usually associated with neurodegenerative diseases. Here we show evidence for the beneficial mechanical characteristics of these fibrils discovered in natural adhesives. We suggest that amyloid protein quaternary structures should be considered as a possible generic mechanism for mechanical strength in a range of natural adhesives and other natural materials due to their many beneficial mechanical features and apparent ease of self-assembly.

Pigments and fatty acids of marine raphidophytes: A chemotaxonomic re-evaluation

The patterns of occurrence of photosynthetic pigments and fatty acids among seven available species (11 strains) of marine raphidophytes were determined and used as chemotaxonomic markers. All currently recognized genera of marine raphidophytes were included for analysis: that is, Chattonella, Fibrocapsa, Heterosigma, Olisthodiscus and Haramonas. The characteristic pigment composition was shown to be chlorophyll a, chlorophylls c1 and/or c2, fucoxanthin as the major carot‐enoid, β,β‐carotene and any or all of zeaxanthin, violaxanthin and an auroxanthin‐like pigment as the minor carotenoids. The carotenoid composition of all marine raphidophyte genera investigated was virtually the same, except in Fibrocapsa and Haramonas, which differed due to the occurrence of fucoxanthinol and 19′‐butanoyloxyfucoxanthin, respectively. These fucoxanthin derivatives, in addition to fucoxanthin, have potential chemotaxonomic use for differentiating the two species. In all 11 strains, 15 fatty acids (saturated, mono‐unsaturated and polyunsaturated) were determined. Significant taxonomic distinctions between genera were reflected by their fatty acid profiles. A rapid key for the differentiation of genera, in addition to morphological features, may be the absence of the 18:4 fatty acid in Olisthodiscus; presence of 18:5 in Heterosigma; the presence of fucoxanthinol in Fibrocapsa and presence of 19′‐butanoyloxyfucoxanthin in Haramonas.

Characterisation of Amyloid Nanostructures in the Natural Adhesive of Unicellular Subaerial Algae

The composition and nanoscale mechanical characteristics of the adhesive from two species of subaerial green unicellular microalgae (Chlorophyta), Coccomyxa sp. and Glaphyrella trebouxiodes, have been studied using Raman spectroscopy, chemical staining, and atomic force microscopy (AFM). Raman spectroscopy confirmed the adhesive proteins of both species to be predominantly in ß-sheet conformations and composed of a number of hydrophobic amino acid residues. Chemical staining with Congo red and thioflavin-T dyes further confirmed the presence of amyloid-like structures. Probing the adhesives with AFM revealed highly ordered and repetitive mechanical responses indicative of highly ordered structures within the adhesive. The repetitive nature of the sawtooth response is typical of a “sacrificial bond” and “hidden length” mechanism, and what we -propose is the result of mechanical manipulation of individual molecules within an intermolecular ß-sheet that makes up the generic amyloid structure. The mechanical data show how amyloid provides cohesive strength to the adhesives, and this intrinsic mechanical property of an amyloid-based adhesive explains the ecological success of attachment of these subaerial microalgae on various surfaces in urban environments. It is unknown to what extent amyloid fibrils occur in algal adhesives, but we postulate that the amyloid structure could provide a widespread mechanism for mechanical strength.

New methods reveal oldest known fossil epiphyllous moss: Bryiidites utahensis gen. et sp. nov. (Bryidae)

PREMISE OF THE STUDY Epiphyllous bryophytes are a highly characteristic feature of many humid tropical forest ecosystems. In contrast to the extensive fossil record for the leaves of their host plants, the record is virtually nonexistent for the epiphylls themselves, despite a fossil record for mosses that begins in the Middle Carboniferous Period, 330 million years ago. METHODS Epifluorescence optical microscopy, scanning electron microscopy, and atomic force microscopy were employed to investigate an intimate association between a newly discovered epiphyllous moss and a Lauraceae plant host from the middle Cretaceous. KEY RESULTS We describe the oldest fossil specimen of an epiphyllous moss, Bryiidites utahensis gen. et sp. nov., identified from an individual specimen only 450 µm long, situated on an approximately one millimeter square fossil leaf fragment. The moss epiphyll is exquisitely preserved as germinating spores and short-celled protonemata with transverse and oblique cross-walls closely matching those of extant epiphyllous mosses on the surface of the plant-leaf hosts. CONCLUSIONS The extension of the epiphyll record back to the middle Cretaceous provides fossil evidence for the appearance of epiphyllous mosses during the diversification of flowering plants, at least 95 million years ago. It also provides substantive evidence for a tropical maritime climate in central North America during the middle Cretaceous.

Osmoprotectors in some species of Japanese mangrove macroalgae

The macroalgae asSociated with the mangrove vegetation of the Japanese Islands Okinawa, Ishigaki and Iriomote were investigated. The flora includes members of the red algal genera Bostrychia, Caloglossa and Catenella, as well as the brown alga Dictyotopsis propagulifera Troll, which may be considered typical of mangrove forests. The distribution of the low molecular weight carbohydrates sorbitol, dulcitol, mannitol and floridoside was studied in the mangrove algae. Their physiological role as osmoprotectors was assessed by investigating the effect of salinity on the intracellular sorbitol and dulcitol concentration in Bostrychia pinnata J. Tanaka et Chihara and on the mannitol content in D. propagulifera. In both species the polyol values increased with increasing salinity.

The dispersion of SWCNT bundles on interaction with p-terphenyl

The interaction and dispersion of single-walled carbon nanotube (SWCNT) bundles were investigated. SWCNTs were produced by arc discharge and by the high pressure decomposition of carbon monoxide (HiPco method), in the presence of the molecule p-terphenyl. The dispersion of SWCNT bundles and their interaction with p-terphenyl in their as-produced state and after purification were compared. A number of spectroscopic and microscopic techniques were used to probe the SWCNTs and their interaction with p-terphenyl. X-ray energy dispersive analysis was used to give an elemental analysis of the SWCNT samples before and after purification. Fluorescence and atomic force microscopy are used as techniques to assess the degree of interaction and dispersion of the SWCNT bundles. Results show that the extent of bundle dispersion and the degree of interaction with p-terphenyl is related to the purity of the SWCNTs.

Asymmetric spatula heads combined with lateral forces provide a mechanism for controlling the adhesive attachment of a range of spider species

Dry adhesion is a common strategy utilized throughout nature, allowing attachment of animals to a large range of surfaces. Crucially, it enables the animal to switch rapidly and safely between attachment and detachment in order to facilitate motion. Here we investigate the magnitude and directionality of the dry adhesion systems of a number of spider species. Atomic force microscopy (AFM) was used to measure the normal adhesion strength of a single setule. Secondly, scanning electron microscopy (SEM) was used to directly observe the interaction of a spider seta with an AFM cantilever tip and then, based on the known stiffness of the cantilever and the deflection distance, to estimate the adhesion force between them. The AFM-measured values for normal adhesion of single spider setules to a surface (9–16 nN) were similar to values predicted by the Johnson–Kendall–Roberts (JKR) model. In contrast, the force of adhesion, estimated from the SEM images, between the seta and the cantilever reached values as high as 10 μN, much higher than predicted by the JKR theory alone. With spiders, as with geckos, the principle of contact splitting leads to an increase in adhesion force. In addition, it was observed that significantly higher adhesion occurred when the setules were dragged laterally across a spherical surface in the proximal direction (i.e. towards the spiders tarsus). When pushed in the opposite direction, adhesion was greatly reduced. This confirms that the spider dry adhesion system is another example of directional adhesion.

Qualitative and Quantitative Study of Spiny Starfish (Marthasterias Glacialis) Footprints Using Atomic Force Microscopy

Starfish produce a strong, temporary adhesive from their tube feet. To detach from a substrate surface, they secrete a deadhesive, leaving behind the adhesive in the form of a footprint. Atomic force microscopy was used to investigate the physical and mechanical properties of the footprints produced by the spiny starfish, Marthasterias glacialis, using height imaging and energy dissipation. The adhesive material was found to be multi‐layered, composed of a homogeneous film and spongy meshwork of similar mechanical properties, covered with a gel‐like matrix supported by a web of interweaving fibres. Contrast in mechanical properties between the meshwork and the gel‐like matrix was detected, as well as within the matrix between the fibres and the surrounding material. The results presented here correlate well with previous research investigating the heterogeneous structure of starfish adhesive footprints. The structure is complex at the micro‐ and nanoscale, and the use of quantitative and qualitative atomic force microscopy techniques, under native conditions, has provided insight into the function of the secretion as a whole.