Distinct functions of FASCILIN-LIKE ARABINOGALACTAN PROTEINS relate to domain structure.

The role of glycoproteins as key cell surface molecules during development and stress is well established; yet, the relationship between their structural features and functional mechanisms is poorly defined. FASCICLIN-LIKE ARABINOGALACTAN PROTEINs (FLAs), which impact plant growth and development, are an excellent example of a glycoprotein family with a complex multidomain structure. FLAs combine globular fasciclin-like (FAS1) domains with regions that are intrinsically disordered and contain glycomotifs for directing the addition of O-linked arabinogalactan (AG) glycans. Additional posttranslational modifications on FLAs include N-linked glycans in the FAS1 domains, a cleaved signal peptide at the N terminus, and often a glycosylphosphatidylinositol (GPI) anchor signal sequence at the C terminus. The roles of glycosylation, the GPI anchor, and FAS1 domain functions in the polysaccharide-rich extracellular matrix of plants remain unclear, as do the relationships between them. In this study, we examined sequence-structure-function relationships of Arabidopsis (Arabidopsis thaliana) FLA11, demonstrated to have roles in secondary cell wall (SCW) development, by introducing domain mutations and functional specialization through domain swaps with FLA3 and FLA12. We identified FAS1 domains as essential for FLA function, differentiating FLA11/FLA12, with roles in SCW development, from FLA3, specific to flowers and involved in pollen development. The GPI anchor and AG glycosylation co-regulate the cell surface location and release of FLAs into cell walls. The AG glycomotif sequence closest to the GPI anchor (AG2) is a major feature differentiating FLA11 from FLA12. The results of our study show that the multidomain structure of different FLAs influences their subcellular location and biological functions during plant development.

Spatiotemporal deposition of cell wall polysaccharides in oat endosperm during grain development.

Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3;1,4)-ß-D-glucan (mixed-linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in 2 Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularization phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days postemergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularization around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularization and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather than during cellularization. In addition, differences in the spatiotemporal patterning of MLG were also observed between the 2 varieties. The lower MLG-containing cultivar AC Morgan (3.5% w/w groats) was marked by the presence of a discontinuous pattern of MLG labeling, while labeling in the same walls in CDC Morrison (5.6% w/w groats) was mostly even and continuous. RNA-sequencing analysis revealed higher transcript levels of multiple MLG biosynthetic cellulose synthase-like F (CSLF) and CSLH genes during grain development in CDC Morrison compared with AC Morgan that likely contributes to the increased abundance of MLG at maturity in CDC Morrison. CDC Morrison was also observed to have smaller endosperm cells with thicker walls than AC Morgan from cellularization onwards, suggesting the processes controlling cell size and shape are established early in development. This study has highlighted that the molecular processes influencing MLG content and deposition are more complex than previously imagined.

Composite Bioprinted Hydrogels Containing Porous Polymer Microparticles Provide Tailorable Mechanical Properties for 3D Cell Culture.

The mechanical and architectural properties of the three-dimensional (3D) tissue microenvironment can have large impacts on cellular behavior and phenotype, providing cells with specialized functions dependent on their location. This is especially apparent in macrophage biology where the function of tissue resident macrophages is highly specialized to their location. 3D bioprinting provides a convenient method of fabricating biomaterials that mimic specific tissue architectures. If these printable materials also possess tunable mechanical properties, they would be highly attractive for the study of macrophage behavior in different tissues. Currently, it is difficult to achieve mechanical tunability without sacrificing printability, scaffold porosity, and a loss in cell viability. Here, we have designed composite printable biomaterials composed of traditional hydrogels [nanofibrillar cellulose (cellulose) or methacrylated gelatin (gelMA)] mixed with porous polymeric high internal phase emulsion (polyHIPE) microparticles. By varying the ratio of polyHIPEs to hydrogel, we fabricate composite hydrogels that mimic the mechanical properties of the neural tissue (0.1-0.5 kPa), liver (1 kPa), lungs (5 kPa), and skin (10 kPa) while maintaining good levels of biocompatibility to a macrophage cell line.

5-HT3 Receptors on Mitochondria Influence Mitochondrial Function.

The 5-hydroxytryptamine 3 (5-HT3) receptor belongs to the pentameric ligand-gated cation channel superfamily. Humans have five different 5-HT3 receptor subunits: A to E. The 5-HT3 receptors are located on the cell membrane, but a previous study suggested that mitochondria could also contain A subunits. In this article, we explored the distribution of 5-HT3 receptor subunits in intracellular and cell-free mitochondria. Organelle prediction software supported the localization of the A and E subunits on the inner membrane of the mitochondria. We transiently transfected HEK293T cells that do not natively express the 5-HT3 receptor with an epitope and fluorescent protein-tagged 5HT3A and 5HT3E subunits. Fluorescence microscopy and cell fractionation indicated that both subunits, A and E, localized to the mitochondria, while transmission electron microscopy revealed the location of the subunits on the mitochondrial inner membrane, where they could form heteromeric complexes. Cell-free mitochondria isolated from cell culture media colocalized with the fluorescent signal for A subunits. The presence of A and E subunits influenced changes in the membrane potential and mitochondrial oxygen consumption rates upon exposure to serotonin; this was inhibited by pre-treatment with ondansetron. Therefore, it is likely that the 5-HT3 receptors present on mitochondria directly impact mitochondrial function and that this may have therapeutic implications.

FLA11 and FLA12 glycoproteins fine-tune stem secondary wall properties in response to mechanical stresses.

Secondary cell walls (SCWs) in stem xylem vessel and fibre cells enable plants to withstand the enormous compressive forces associated with upright growth. It remains unclear if xylem vessel and fibre cells can directly sense mechanical stimuli and modify their SCW during development. We provide evidence that Arabidopsis SCW-specific Fasciclin-Like Arabinogalactan-proteins 11 (FLA11) and 12 (FLA12) are possible cell surface sensors regulating SCW development in response to mechanical stimuli. Plants overexpressing FLA11 (OE-FLA11) showed earlier SCW development compared to the wild-type (WT) and altered SCW properties that phenocopy WT plants under compression stress. By contrast, OE-FLA12 stems showed higher cellulose content compared to WT plants, similar to plants experiencing tensile stress. fla11, OE-FLA11, fla12, and OE-FLA12 plants showed altered SCW responses to mechanical stress compared to the WT. Quantitative polymerase chain reaction (qPCR) and RNA-seq analysis revealed the up-regulation of genes and pathways involved in stress responses and SCW synthesis and regulation. Analysis of OE-FLA11 nst1 nst3 plants suggests that FLA11 regulation of SCWs is reliant on classical transcriptional networks. Our data support the involvement of FLA11 and FLA12 in SCW sensing complexes to fine-tune both the initiation of SCW development and the balance of lignin and cellulose synthesis/deposition in SCWs during development and in response to mechanical stimuli.

Manipulating the Ordered Nanostructure of Self-Assembled Monoolein and Phytantriol Nanoparticles with Unsaturated Fatty Acids.

Mesophase structures of self-assembled lyotropic liquid crystalline nanoparticles are important factors that directly influence their ability to encapsulate and release drugs and their biological activities. However, it is difficult to predict and precisely control the mesophase behavior of these materials, especially in complex systems with several components. In this study, we report the controlled manipulation of mesophase structures of monoolein (MO) and phytantriol (PHYT) nanoparticles by adding unsaturated fatty acids (FAs). By using high throughput formulation and small-angle X-ray scattering characterization methods, the effects of FAs chain length, cis-trans isomerism, double bond location, and level of chain unsaturation on self-assembled systems are determined. Additionally, the influence of temperature on the phase behavior of these nanoparticles is analyzed. We found that in general, the addition of unsaturated FAs to MO and PHYT induces the formation of mesophases with higher Gaussian surface curvatures. As a result, a rich variety of lipid polymorphs are found to correspond with the increasing amounts of FAs. These phases include inverse bicontinuous cubic, inverse hexagonal, and discrete micellar cubic phases and microemulsion. However, there are substantial differences between the phase behavior of nanoparticles with trans FA, cis FAs with one double bond, and cis FAs with multiple double bonds. Therefore, the material library produced in this study will assist the selection and development of nanoparticle-based drug delivery systems with desired mesophase.

Amphiphilic brush polymers produced using the RAFT polymerisation method stabilise and reduce the cell cytotoxicity of lipid lyotropic liquid crystalline nanoparticles.

Self-assembled lipid lyotropic liquid crystalline nanoparticles such as hexosomes and cubosomes contain internal anisotropic and isotropic nanostructures, respectively. Despite the remarkable potential of such nanoparticles in various biomedical applications, the stabilisers used in formulating the nanoparticles are often limited to commercially available polymers such as the Pluronic block copolymers. This study explored the potential of using Reversible Addition-Fragmentation chain Transfer (RAFT) technology to design amphiphilic brush-type polymers for the purpose of stabilising phytantriol and monoolein-based lipid dispersions. The synthesised brush-type polymers consisted of a hydrophobic C12 short chain and a hydrophilic poly(ethylene glycol)methyl ether acrylate (PEGA) long chain with multiple 9-unit poly(ethylene oxide) (PEO) brushes with various molecular weights. It was observed that increasing the PEO brush density and thus the length of the hydrophilic component improved the stabilisation effectiveness for phytantriol and monoolein-based cubosomes. Synchrotron small-angle X-ray scattering (SAXS) experiments confirmed that the RAFT polymer-stabilised cubosomes had an internal double-diamond cubic phase with tunable water channel sizes. These properties were dependent on the molecular weight of the polymers, which were considered in some cases to be anisotropically distributed within the cubosomes. The in vitro toxicity of the cubosomes was assessed by cell viability of two human adenocarcinoma cell lines and haemolytic activities to mouse erythrocytes. The results showed that phytantriol cubosomes stabilised by the RAFT polymers were less toxic compared to their Pluronic F127-stabilised analogues. This study provides valuable insight into designing non-linear amphiphilic polymers for the effective stabilisation and cellular toxicity improvement of self-assembled lipid lyotropic liquid crystalline nanoparticles.

Morphology of the cornea and iris in the Australian lungfish Neoceratodus forsteri (Krefft 1870) (Dipnoi): Functional and evolutionary perspectives of transitioning from an aquatic to a terrestrial environment.

The Australian lungfish, Neoceratodus forsteri (Krefft 1870), is the sole extant member of the Ceratodontidae within the Dipnoi, a small order of sarcopterygian (lobe-finned) fishes, that is thought to be the earliest branching species of extant lungfishes, having changed little over the last 100 million years. To extend studies on anatomical adaptations associated with the fish-tetrapod transition, the ultrastructure of the cornea and iris is investigated using light and electron (transmission and scanning) microscopy to investigate structure-function relationships and compare these to other vertebrate corneas (other fishes and tetrapods). In contrast to previous studies, the cornea is found to have only three main components, comprising an epithelium with its basement membrane, a stroma with a Bowman's layer and an endothelium, and is not split into a dermal (secondary) spectacle and a scleral cornea. The epithelial cells are large, relatively low in density and similar to many species of non-aquatic tetrapods and uniquely possess numerous surface canals that contain and release mucous granules onto the corneal surface to avoid desiccation. A Bowman's layer is present and, in association with extensive branching and anastomosing of the collagen fibrils, may be an adaptation for the inhibition of swelling and/or splitting of the stroma during its amphibious lifestyle. The dorsal region of the stroma possesses aggregations of pigment granules that act as a yellow, short wavelength-absorbing filter during bright light conditions. Desçemet's membrane is absent and replaced by an incomplete basement membrane overlying a monocellular endothelium. The iris is pigmented, well-developed, vascularised and contractile containing reflective crystals anteriorly. Based upon its ultrastructure and functional adaptations, the cornea of N. forsteri is more similar to amphibians than to other bony fishes and is well-adapted for an amphibious lifestyle.

WALL-ASSOCIATED KINASE Like 14 regulates vascular tissue development in Arabidopsis and tomato.

Initiation of plant vascular tissue is regulated by transcriptional networks during development and in response to environmental stimuli. The WALL-ASSOCIATED KINASES (WAKs) and WAK-likes (WAKLs) are cell surface receptors involved in cell expansion and defence in cells with primary walls, yet their roles in regulation of vascular tissue development that contain secondary walls remains unclear. In this study, we showed tomato (Solanum lycopersicum) SlWAKL2 and the orthologous gene in Arabidopsis thaliana, AtWAKL14, were specifically expressed in vascular tissues. SlWAKL2-RNAi tomato plants displayed smaller fruit size with fewer seeds and vascular bundles compared to wild-type (WT) and over-expression (OE) lines. RNA-seq data showed that SlWAKL2-RNAi fruits down-regulated transcript levels of genes related to vascular tissue development compared to WT. Histological analysis showed T-DNA insertion mutant wakl14-1 had reduced plant stem length with fewer number of xylem vessels and interfascicular fibres compared to WT, with no significant differences in cellulose and lignin content. Mutant wakl14-1 also showed reduced number of vascular bundles in fruit. A proWAKL14::mCherry-WAKL14 fusion protein was able to complement wakl14-1 phenotypes and showed mCherry-WAKL14 associated with the plasma membrane. In vitro binding assays showed both SlWAKL2 and AtWAKL14 can interact with pectin and oligogalacturonides. Our results reveal novel roles of WAKLs in regulating vascular tissue development.

Cell invasive amyloid assemblies from SARS-CoV-2 peptides can form multiple polymorphs with varying neurotoxicity.

The neurological symptoms of COVID-19, often referred to as neuro-COVID include neurological pain, memory loss, cognitive and sensory disruption. These neurological symptoms can persist for months and are known as Post-Acute Sequalae of COVID-19 (PASC). The molecular origins of neuro-COVID, and how it contributes to PASC are unknown, however a growing body of research highlights that the self-assembly of protein fragments from SARS-CoV-2 into amyloid nanofibrils may play a causative role. Previously, we identified two fragments from the SARS-CoV-2 proteins, Open Reading Frame (ORF) 6 and ORF10, that self-assemble into neurotoxic amyloid assemblies. Here we further our understanding of the self-assembly mechanisms and nano-architectures formed by these fragments and their biological responses. By solubilising the peptides in a fluorinated solvent, we eliminate insoluble aggregates in the starting materials (seeds) that change the polymorphic landscape of the assemblies. The resultant assemblies are dominated by structures with higher free energies (e.g. ribbons and amorphous aggregates) that are less toxic to cultured neurons but do affect their mitochondrial respiration. We also show the first direct evidence of cellular uptake of viral amyloids. This work highlights the importance of understanding the polymorphic behaviour of amyloids and the correlation to neurotoxicity, particularly in the context of neuro-COVID and PASC.

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.

Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.

The functional anatomy of the cornea of the Shorthead lamprey, Mordacia mordax (Mordaciidae, Agnatha): A comparison between downstream and upstream migrants.

The Shorthead lamprey Mordacia mordax (Mordaciidae, Agnatha) represents one of the earliest stages of vertebrate evolution. This study investigates the ultrastructural anatomy of the cornea, iris and anterior chamber in the eyes of this species in both the downstream and upstream migrant phases of its protracted lifecycle to assess the morphological and quantitative changes associated with growth, corneal function and vision. Using light and both scanning and transmission electron microscopy, the cornea is found to be divided into dermal and scleral components separated by a mucoid layer. A range of distinguishing corneal features are compared in the two adult phases of the lifecycle, including epithelial microprojections, mucus-secreting epithelial cells, the number, thickness, formation and level of branching and anastomosing of collagen lamellae, the type and distribution of vertical sutures, the structure of the mucoid layer and annular ligament and the number and distribution of a large number of basement membranes throughout the cornea. Significant differences are found between the two phases, which are thought to reflect adaptations to the variable environmental conditions encountered throughout this species' lifecycle. The study provides insights into the evolutionary pressures on extant representatives of the earliest stages in the evolution of the vertebrate eye.

Promoter and domain structures regulate FLA12 function during Arabidopsis secondary wall development.

Introduction: Fasciclin-like arabinogalactan-proteins (FLAs) are a family of multi-domain glycoproteins present at the cell surface and walls of plants. Arabidopsis thaliana FLA12 and homologs in cotton, Populus, and flax have been shown to play important functions regulating secondary cell wall (SCW) development. FLA12 has been shown to have distinct roles from the closely related FLA11 that also functions during SCW development. The promoter and domain features of FLA12 that regulate functional specificity have not been well characterized. Methods: In this study, promoter swap experiments of FLA11 and FLA12 were investigated. Mutation of proposed functional regions within FLA12 were used to investigate the role of post-translational modifications on sub-cellular location and trafficking. Domain swap experiments between FLA11 and FLA12 were performed to identify regions of functional specificity. Results: Promote swap experiments showed that FLA12 is differentially expressed in both stem and rosette leaves compared to FLA11. Post-translational modifications, in particular addition of the glycosylphosphatidylinositol-anchor (GPI-anchor), were shown to be important for FLA12 location at the plasma membrane (PM)/cell wall interface. Domain swap experiments between FLA11 and FLA12 showed that the C-terminal arabinogalactan (AG) glycan motif acts as a key regulatory region differentiating FLA12 functions from FLA11. Discussion: Understanding of FLA12 promoter and functional domains has provided new insights into the regulation of SCW development and functional specificity of FLAs for plant growth and development.

Metabolomic Analysis of Extracellular Vesicles from the Cereal Fungal Pathogen Fusarium graminearum.

Fusarium graminearum (F. graminearum) is a filamentous fungus that infects cereals such as corn, wheat, and barley, with serious impact on yield as well as quality when the grain is contaminated with mycotoxins. Despite the huge impact of F. graminearum on food security and mammalian health, the mechanisms used by F. graminearum to export virulence factors during infection are not fully understood and may involve non-classical secretory pathways. Extracellular vesicles (EVs) are lipid-bound compartments produced by cells of all kingdoms that transport several classes of macromolecules and are implicated in cell-cell communication. EVs produced by human fungal pathogens carry cargo that facilitate infection, leading us to ask whether plant fungal pathogens also deliver molecules that increase virulence via EVs. We examined the metabolome of the EVs produced by F. graminearum to determine whether they carry small molecules that could modulate plant-pathogen interactions. We discovered that EVs from F. graminearum were produced in liquid medium-containing inducers of trichothecene production, but in lower quantities compared to other media. Nanoparticle tracking analysis and cryo-electron microscopy revealed that the EVs were morphologically similar to EVs from other organisms; hence, the EVs were metabolically profiled using LC-ESI-MS/MS. This analysis revealed that EVs carry 2,4-dihydroxybenzophenone (BP-1) and metabolites that have been suggested by others to have a role in host-pathogen interactions. BP-1 reduced the growth of F. graminearum in an in vitro assay, suggesting that F. graminearum might use EVs to limit metabolite self-toxicity.

Highly aqueously stable C60-polymer nanoparticles with excellent photodynamic property for potential cancer treatment.

Fullerenes are a class of carbon nanomaterials that find a wide range of applications in biomedical fields, especially for photodynamic cancer therapy because of its photosensitive effect. However, hydrophobic fullerenes can only be dispersed in organic solvents which hinders their biomedical applications. Here, we report a facile method to prepare highly water-dispersible fullerene (C60)-polymer nanoparticles with hydrodynamic sizes of 50-70 nm. Hydrophilic random copolymers containing different ratios of polyethylene glycol methyl ether methacrylate and 2-aminoethylmethacrylamide were synthesized for conjugating with C60 molecules through efficient nucleophilic Michael addition reaction between amine groups from hydrophilic polymer and carbon-carbon double bonds from C60. As a result, the amphiphilic C60-polymer conjugates could be well dispersed and nano-assembled in water with a C60 concentration as high as 7.8 mg/mL, demonstrating a significant improvement for the solubility of C60 in an aqueous system. Owing to the high C60 content, the C60-polymer nanoparticles showed a strong photodynamic therapy effect on human lung cancer cells (A549) under light irradiation (450 nm) in both 2D cell culture and 3D spheroid culture, while demonstrating ignorable cytotoxicity under dark. This highly efficient and convenient method to prepare water-dispersible C60-polymer conjugates may have a great impact on the future biomedical applications of fullerenes.

WAKL8 Regulates Arabidopsis Stem Secondary Wall Development.

Wall-associated kinases/kinase-likes (WAKs/WAKLs) are plant cell surface sensors. A variety of studies have revealed the important functions of WAKs/WAKLs in regulating cell expansion and defense in cells with primary cell walls. Less is known about their roles during the development of the secondary cell walls (SCWs) that are present in xylem vessel (XV) and interfascicular fiber (IF) cells. In this study, we used RNA-seq data to screen Arabidopsis thaliana WAKs/WAKLs members that may be involved in SCW development and identified WAKL8 as a candidate. We obtained T-DNA insertion mutants wakl8-1 (inserted at the promoter region) and wakl8-2 (inserted at the first exon) and compared the phenotypes to wild-type (WT) plants. Decreased WAKL8 transcript levels in stems were found in the wakl8-2 mutant plants, and the phenotypes observed included reduced stem length and thinner walls in XV and IFs compared with those in the WT plants. Cell wall analysis showed no significant changes in the crystalline cellulose or lignin content in mutant stems compared with those in the WT. We found that WAKL8 had alternative spliced versions predicted to have only extracellular regions, which may interfere with the function of the full-length version of WAKL8. Our results suggest WAKL8 can regulate SCW thickening in Arabidopsis stems.

Neurotoxic amyloidogenic peptides in the proteome of SARS-COV2: potential implications for neurological symptoms in COVID-19.

COVID-19 is primarily known as a respiratory disease caused by SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, severe headaches, and even stroke are reported in up to 30% of cases and can persist even after the infection is over (long COVID). These neurological symptoms are thought to be produced by the virus infecting the central nervous system, however we don't understand the molecular mechanisms triggering them. The neurological effects of COVID-19 share similarities to neurodegenerative diseases in which the presence of cytotoxic aggregated amyloid protein or peptides is a common feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we identified two peptides from the SARS-CoV-2 proteome that self-assemble into amyloid assemblies. Furthermore, these amyloids were shown to be highly toxic to neuronal cells. We suggest that cytotoxic aggregates of SARS-CoV-2 proteins may trigger neurological symptoms in COVID-19.

Cathelicidin-3 Associated With Serum Extracellular Vesicles Enables Early Diagnosis of a Transmissible Cancer.

The identification of practical early diagnostic biomarkers is a cornerstone of improved prevention and treatment of cancers. Such a case is devil facial tumor disease (DFTD), a highly lethal transmissible cancer afflicting virtually an entire species, the Tasmanian devil (Sarcophilus harrisii). Despite a latent period that can exceed one year, to date DFTD diagnosis requires visual identification of tumor lesions. To enable earlier diagnosis, which is essential for the implementation of effective conservation strategies, we analyzed the extracellular vesicle (EV) proteome of 87 Tasmanian devil serum samples using data-independent acquisition mass spectrometry approaches. The antimicrobial peptide cathelicidin-3 (CATH3), released by innate immune cells, was enriched in serum EV samples of both devils with clinical DFTD (87.9% sensitivity and 94.1% specificity) and devils with latent infection (i.e., collected while overtly healthy, but 3-6 months before subsequent DFTD diagnosis; 93.8% sensitivity and 94.1% specificity). Although high expression of antimicrobial peptides has been mostly related to inflammatory diseases, our results suggest that they can be also used as accurate cancer biomarkers, suggesting a mechanistic role in tumorous processes. This EV-based approach to biomarker discovery is directly applicable to improving understanding and diagnosis of a broad range of diseases in other species, and these findings directly enhance the capacity of conservation strategies to ensure the viability of the imperiled Tasmanian devil population.

The Functional Anatomy of the Cornea and Anterior Chamber in Lampreys: Insights From the Pouched Lamprey, Geotria australis (Geotriidae, Agnatha).

Extant lampreys (Petromyzontiformes) are one of two lineages of surviving jawless fishes or agnathans, and are therefore of critical importance to our understanding of vertebrate evolution. Anadromous lampreys undergo a protracted lifecycle, which includes metamorphosis from a larval ammocoete stage to an adult that moves between freshwater and saltwater with exposure to a range of lighting conditions. Previous studies have revealed that photoreception differs radically across the three extant families with the Pouched lamprey Geotria australis possessing a complex retina with the potential for pentachromacy. This study investigates the functional morphology of the cornea and anterior chamber of G. australis, which is specialised compared to its northern hemisphere counterparts. Using light microscopy, scanning and transmission electron microscopy and microcomputed tomography, the cornea is found to be split into a primary spectacle (dermal cornea) and a scleral cornea (continuous with the scleral eyecup), separated by a mucoid layer bounded on each side by a basement membrane. A number of other specialisations are described including mucin-secreting epithelial cells and microholes, four types of stromal sutures for the inhibition of stromal swelling, abundant anastomosing and branching of collagen lamellae, and a scleral endothelium bounded by basement membranes. The structure and function of the cornea including an annular and possibly a pectinate ligament and iris are discussed in the context of the evolution of the eye in vertebrates.

Cocultivation of an ultrasmall environmental parasitic bacterium with lytic ability against bacteria associated with wastewater foams.

Many wastewater treatment plants around the world suffer from the operational problem of foaming. This is characterized by a persistent stable foam that forms on the aeration basin, which reduces effluent quality. The foam is often stabilized by a highly hydrophobic group of Actinobacteria known as the Mycolata1. Gordonia amarae is one of the most frequently reported foaming members1. With no currently reliable method for treating foams, phage biocontrol has been suggested as an attractive treatment strategy2. Phages isolated from related foaming bacteria can destabilize foams at the laboratory scale3,4; however, no phage has been isolated that lyses G. amarae. Here, we assemble the complete genomes of G. amarae and a previously undescribed species, Gordonia pseudoamarae, to examine mechanisms that encourage stable foam production. We show that both of these species are recalcitrant to phage infection via a number of antiviral mechanisms including restriction, CRISPR-Cas and bacteriophage exclusion. Instead, we isolate and cocultivate an environmental ultrasmall epiparasitic bacterium from the phylum Saccharibacteria that lyses G. amarae and G. pseudoamarae and several other Mycolata commonly associated with wastewater foams. The application of this parasitic bacterium, 'Candidatus Mycosynbacter amalyticus', may represent a promising strategy for the biocontrol of bacteria responsible for stabilizing wastewater foams.