Crown-of-thorns starfish spines secrete defence proteins.

Background: The crown-of-thorns starfish (COTS; Acanthaster species) is a slow-moving corallivore protected by an extensive array of long, sharp toxic spines. Envenomation can result in nausea, numbness, vomiting, joint aches and sometimes paralysis. Small molecule saponins and the plancitoxin proteins have been implicated in COTS toxicity. Methods: Brine shrimp lethality assays were used to confirm the secretion of spine toxin biomolecules. Histological analysis, followed by spine-derived proteomics helped to explain the source and identity of proteins, while quantitative RNA-sequencing and phylogeny confirmed target gene expression and relative conservation, respectively. Results: We demonstrate the lethality of COTS spine secreted biomolecules on brine shrimp, including significant toxicity using aboral spine semi-purifications of >10 kDa (p > 0.05, 9.82 µg/ml), supporting the presence of secreted proteins as toxins. Ultrastructure observations of the COTS aboral spine showed the presence of pores that could facilitate the distribution of secreted proteins. Subsequent purification and mass spectrometry analysis of spine-derived proteins identified numerous secretory proteins, including plancitoxins, as well as those with relatively high gene expression in spines, including phospholipase A2, protease inhibitor 16-like protein, ependymin-related proteins and those uncharacterized. Some secretory proteins (e.g., vitellogenin and deleted in malignant brain tumor protein 1) were not highly expressed in spine tissue, yet the spine may serve as a storage or release site. This study contributes to our understanding of the COTS through functional, ultrastructural and proteomic analysis of aboral spines.

Identification of Gene Biomarkers for Tigilanol Tiglate Content in Fontainea picrosperma.

Tigilanol tiglate (EBC-46) is a small-molecule natural product under development for the treatment of cancers in humans and companion animals. The drug is currently produced by purification from the Australian rainforest tree Fontainea picrosperma (Euphorbiaceae). As part of a selective-breeding program to increase EBC-46 yield from F. picrosperma plantations, we investigated potential gene biomarkers associated with biosynthesis of EBC-46. Initially, we identified individual plants that were either high (>0.039%) or low EBC-46 (<0.008%) producers, then assessed their differentially expressed genes within the leaves and roots of these two groups by quantitative RNA sequencing. Compared to low EBC-46 producers, high-EBC-46-producing plants were found to have 145 upregulated genes and 101 downregulated genes in leaves and 53 upregulated genes and 82 downregulated genes in roots. Most of these genes were functionally associated with defence, transport, and biosynthesis. Genes identified as expressed exclusively in either the high or low EBC-46-producing plants were further validated by quantitative PCR, showing that cytochrome P450 94C1 in leaves and early response dehydration 7.1 and 2-alkenal reductase in roots were consistently and significantly upregulated in high-EBC-46 producers. In summary, this study has identified biomarker genes that may be used in the selective breeding of F. picrosperma.

The byssal-producing glands and proteins of the silverlip pearl oyster Pinctada maxima (Jameson, 1901).

Pinctada maxima are most well known for their production of high-quality natural pearls. They also generate another natural material, the byssus, an adhesive thread critical for steadfast attachment underwater. Herein, P. maxima byssal threads were analysed via proteotranscriptomics to reveal 49 proteins. Further characterisation was undertaken on five highly expressed genes: glycine-rich thread protein (GRT; also known as PUF3), apfp1/perlucin-like protein (Pmfp1); peroxidase; thrombospondin 1, and Balbiani ring 3 (BR3), which showed localised tissue expression. The spatial distribution of GRT and Pmfp1 via immunodetection combined with histology helped to identify glandular regions of the foot that contribute to byssal thread production: the byssal gland, the duct gland, and two thread-forming glands of basophilic and acidophilic serous-like cells. This work advanced primary knowledge on the glands involved in the creation of byssal threads and the protein composition of the byssus for P. maxima, providing a platform for the design of marine biopolymers.

First report of Kudoa thunni and Kudoa musculoliquefaciens affecting the quality of commercially harvested yellowfin tuna and broadbill swordfish in Eastern Australia.

Recent anecdotal reports from seafood processors in eastern Australia have described an increased occurrence of post-mortem myoliquefaction ('jellymeat') in broadbill swordfish Xiphias gladius, and macroscopic cysts throughout the musculature of yellowfin tuna Thunnus albacares. A genus of parasitic cnidarians, Kudoa (Myxosporea, Multivalvulida), species of which are known to occur in economically important wild-caught fish species globally, can cause similar quality-deterioration issues. However, Kudoa sp. epizootiology within commercially harvested, high-value fish caught within Australia is poorly understood, despite the parasite's economic importance. To determine the causative agent responsible for the observed quality deterioration in swordfish and yellowfin tuna, muscle-tissue samples from seafood processors in Mooloolaba, Australia, collected from October 2019-February 2020, were examined for parasitic infection. Kudoid myxospores were identified from both hosts and were subquadrate in shape, with four equal-sized polar capsules. The SSU rDNA sequences from both fish shared > 99% identity to Kudoa species. Kudoa musculoliquefaciens was isolated from 87.1% of swordfish sampled, suggesting that it is a widespread parasite in swordfish from the southwest Pacific Ocean. This study provides the first molecular and morphological characterisation of Kudoa thunni in yellowfin tuna and K. musculoliquefaciens in swordfish harvested from the waters of eastern Australia, expanding the geographical distribution of K. thunni and K. musculoliquefaciens to include the Coral and Tasman Seas. We demonstrate that not all infected swordfish progress to jellymeat, show the usefulness of molecular tools for reliably identifying infection by Kudoa spp., and add to the overall knowledge of kudoid epizootiology in wild-caught fish.

The P450 multigene family of Fontainea and insights into diterpenoid synthesis.

BACKGROUND: Cytochrome P450s (P450s) are enzymes that play critical roles in the biosynthesis of physiologically important compounds across all organisms. Although they have been characterised in a large number of plant species, no information relating to these enzymes are available from the genus Fontainea (family Euphorbiaceae). Fontainea is significant as the genus includes species that produce medicinally significant epoxy-tigliane natural products, one of which has been approved as an anti-cancer therapeutic. RESULTS: A comparative species leaf metabolome analysis showed that Fontainea species possess a chemical profile different from various other plant species. The diversity and expression profiles of Fontainea P450s were investigated from leaf and root tissue. A total of 103 and 123 full-length P450 genes in Fontainea picrosperma and Fontainea venosa, respectively (and a further 127/125 partial-length) that were phylogenetically classified into clans, families and subfamilies. The majority of P450 identified are most active within root tissue (66.2% F. picrosperma, 65.0% F. venosa). Representatives within the CYP71D and CYP726A were identified in Fontainea that are excellent candidates for diterpenoid synthesis, of which CYP726A1, CYP726A2 and CYP71D1 appear to be exclusive to Fontainea species and were significantly more highly expressed in root tissue compared to leaf tissue. CONCLUSION: This study presents a comprehensive overview of the P450 gene family in Fontainea that may provide important insights into the biosynthesis of the medicinally significant epoxy-tigliane diterpenes found within the genus.

Transcriptome analysis of the medicinally significant plant Fontainea picrosperma (Euphorbiaceae) reveals conserved biosynthetic pathways.

Euphorbiaceae is a large and diverse family of herbs, shrubs and trees that includes a number of species of considerable economic importance as sources of food, medicines and raw materials. One member of this family, Fontainea picrosperma, is the source plant for the diterpene ester tigilanol tiglate, a natural product recently approved as a treatment for canine mast cell tumours. Here we report the development of reference transcriptomes from root and leaf tissues of F. picrosperma, which include core diterpene biosynthesis genes. A total of ~12 Gb of combined clean reads were generated for assembly into 167,566 contigs with a GC (guanine-cytosine) content of ~41%. Gene ontology showed that 2286 and 2504 transcripts were enriched in the cellular process and 2369 and 2529 transcripts were enriched in the metabolic process categories in leaf and root tissue, respectively. The reference transcriptome contains genes coding for core enzymes involved in common secondary metabolite biosynthetic pathways, including the diterpene biosynthesis pathway within the mevalonate (MVA) and 2-C-methyl-D-erythritol 4- phosphate (MEP) pathways. A phylogenetic analysis using these genes found that F. picrosperma clustered most closely to Jatropha curcas. We found a significantly higher concentration of tigilanol tiglate in F. picrosperma root tissue, which correlated with higher levels of gene expression for enzymes associated with the MVA (6 genes) and MEP (7 genes) pathways, and we hypothesise that the initial stages of tigilanol tiglate biosynthesis occur primarily in the roots of F. picrosperma. This study provides a resource for future gene-related biodiscovery investigations in F. picrosperma and diterpene biosynthesis, in particular for tigilanol tiglate and related macrocyclic diterpenes.

Drug Seller Provision Practices and Knowledge of Misoprostol in Bangladesh.

CONTEXT: In Bangladesh, prior to the availability of the approved combination regimen of mifepristone and misoprostol for menstrual regulation (MR), drug seller provision of misoprostol-only regimens for MR without a prescription was widespread but service quality was poor. Examining provider practices relating to misoprostol-only provision in Bangladesh may increase understanding of misoprostol use and provision in other low-resource, legally restrictive settings. METHODS: In 2013-2014, a countrywide cross-sectional knowledge, attitudes and practice survey was conducted among 777 randomly selected drug sellers; data were analyzed descriptively. Logistic regression was used to test the associations between exposure to three interventions designed to improve drug seller practice (nongovernmental organization [NGO]-led training, a call center and in-shop training from pharmaceutical company representatives) and correct knowledge of the misoprostol-only MR regimen. RESULTS: Almost all (97%) of the drug sellers reported providing medications intended for MR; misoprostol-only was more commonly sold than the combination regimen (96% vs. 26%). Nine percent had received NGO-led training, 62% had received in-shop training from a pharmaceutical company representative and 27% had used the call center. Overall, 19% of drug sellers knew the correct misoprostol-only MR regimen, and 74% wanted more information about this regimen. Correct regimen knowledge was positively associated with receipt of NGO training and call center utilization (odds ratios, 2.0 and 1.9, respectively). CONCLUSIONS: NGO-led training and call centers should be considered in other settings in which misoprostol alone is provided off-label for pregnancy termination.

RESUMEN Contexto: En Bangladesh, antes de que el régimen combinado de mifepristona y misoprostol fuera aprobado para la regulación menstrual (RM), la provisión sin receta de regímenes de misoprostol solo para RM por parte de vendedores de medicamentos estuvo muy generalizada, pero la calidad de servicio era deficiente. Examinar las prácticas de los proveedores relacionadas con la provisión de misoprostol solo en Bangladesh podría aumentar la comprensión sobre el uso y la provisión de misoprostol en otros entornos de bajos recursos restringidos legalmente. Métodos: Entre 2013 y 2014, se realizó una encuesta transversal de conocimientos, actitudes y prácticas en todo el país entre 777 vendedores de medicamentos seleccionados al azar; los datos fueron analizados descriptivamente. Se utilizó regresión logística para evaluar las asociaciones entre la exposición a tres intervenciones diseñadas para mejorar las prácticas de los vendedores de medicamentos (capacitación conducida por una organización no gubernamental [ONG], un centro de atención telefónica y capacitación en el negocio por parte de representantes de las compañías farmacéuticas), así como el conocimiento correcto del régimen de misoprostol solo usado para RM. Resultados: Casi la totalidad (97%) de los vendedores de medicamentos informaron que estaban vendiendo medicamentos para RM; que la venta de misoprostol solo era más común que el régimen combinado (96% vs 26%). El nueve por ciento había recibido capacitación impartida por ONG, el 62% había recibido capacitación en su negocio de un representante de una compañía farmacéutica y el 27% había utilizado el centro de llamadas. En general, el 19% de los vendedores de medicamentos conocía el régimen correcto de RM basado en misoprostol solo y el 74% quería más información sobre ese régimen. El conocimiento correcto del régimen se asoció positivamente con la recepción de capacitación de las ONG y la utilización del centro de atención telefónica (razón de probabilidades, 2.0 y 1.9, respectivamente). Conclusiones: La capacitación conducida por ONG y el uso del centro de atención telefónica deberían considerarse en otros entornos restringidos en los que el misoprostol solo se proporciona sin autorización para la interrupción del embarazo.

RÉSUMÉ Contexte: Au Bangladesh, avant la disponibilité du traitement homologué au mifépristone associé au misoprostol pour la régulation menstruelle (RM), la prestation par les vendeurs de médicaments des traitements de RM au misoprostol seul sans ordonnance était répandue, mais la qualité du service était faible. L'examen des pratiques de prestation relatives à la fourniture de misoprostol seul au Bangladesh peut aider à mieux cerner l'usage et l'offre de ce médicament dans d'autres contextes à faibles ressources soumis à des lois restrictives. Méthodes: En 2013­2014, une étude transversale sur les connaissances, les attitudes et les pratiques à l'échelle du pays a été menée auprès de 777 vendeurs de médicaments sélectionnés aléatoirement, pour analyse descriptive des données. La régression logistique a servi au test des associations entre l'exposition à trois interventions conçues pour améliorer la pratique des vendeurs de médicaments (formation sous la conduite d'organisations non gouvernementales [ONG], établissement d'un centre d'appels et formation par des représentants de laboratoires pharmaceutiques) et la connaissance correcte du traitement de RM à base de misoprostol seul. Résultats: Presque tous les vendeurs de médicaments (97%) ont déclaré vendre des médicaments destinés à la RM. Le misoprostol seul était vendu plus fréquemment que le traitement d'association (96% vs 26%). Neuf pour cent avaient bénéficié d'une formation par une ONG, 62% d'une formation locale assurée par un représentant de laboratoire pharmaceutique et 27% avaient eu recours au centre d'appels. Globalement, 19% des vendeurs avaient une connaissance correcte du traitement de RM à base de misoprostol seul et 74% désiraient plus d'information à ce sujet. La connaissance correcte du traitement était associée positivement à l'obtention d'une formation par une ONG et au recours au centre d'appels (RC, 2,0 et 1,9, respectivement). Conclusions: La formation sous la conduite d'une ONG et l'établissement d'un centre d'appels doivent être envisagés dans d'autres contextes sujets à une législation restrictive dans lesquels le misoprostol seul est proposé hors indication pour l'interruption d'une grossesse.

Evidence for a Saponin Biosynthesis Pathway in the Body Wall of the Commercially Significant Sea Cucumber Holothuria scabra.

The sea cucumber (phylum Echinodermata) body wall is the first line of defense and is well known for its production of secondary metabolites; including vitamins and triterpenoid glycoside saponins that have important ecological functions and potential benefits to human health. The genes involved in the various biosynthetic pathways are unknown. To gain insight into these pathways in an echinoderm, we performed a comparative transcriptome analysis and functional annotation of the body wall and the radial nerve of the sea cucumber Holothuria scabra; to define genes associated with body wall metabolic functioning and secondary metabolite biosynthesis. We show that genes related to signal transduction mechanisms were more highly represented in the H. scabra body wall, including genes encoding enzymes involved in energy production. Eight of the core triterpenoid biosynthesis enzymes were found, however, the identity of the saponin specific biosynthetic pathway enzymes remains unknown. We confirm the body wall release of at least three different triterpenoid saponins using solid phase extraction followed by ultra-high-pressure liquid chromatography-quadrupole time of flight-mass spectrometry. The resource we have established will help to guide future research to explore secondary metabolite biosynthesis in the sea cucumber.

Biomolecular changes that occur in the antennal gland of the giant freshwater prawn (Machrobrachium rosenbergii).

In decapod crustaceans, the antennal gland (AnG) is a major primary source of externally secreted biomolecules, and some may act as pheromones that play a major role in aquatic animal communication. In aquatic crustaceans, sex pheromones regulate reproductive behaviours, yet they remain largely unidentified besides the N-acetylglucosamine-1,5-lactone (NAGL) that stimulates male to female attraction. In this study, we used an AnG transcriptome of the female giant freshwater prawn (Macrobrachium rosenbergii) to predict the secretion of 226 proteins, including the most abundantly expressed transcripts encoding the Spaetzle protein, a serine protease inhibitor, and an arthropodial cuticle protein AMP 8.1. A quantitative proteome analysis of the female AnG at intermolt, premolt and postmolt, identified numerous proteins of different abundances, such as the hemocyanin subunit 1 that is most abundant at intermolt. We also show that hemocyanin subunit 1 is present within water surrounding females. Of those metabolites identified, we demonstrate that the NAGL and N-acetylglucosamine (NAG) can bind with high affinity to hemocyanin subunit 1. In summary, this study has revealed components of the female giant freshwater prawn AnG that are released and contribute to further research towards understanding crustacean conspecific signalling.

Attenuation of UV-B exposure-induced inflammation by abalone hypobranchial gland and gill extracts.

Exposure to solar ultraviolet B (UV-B) is a known causative factor for many skin complications such as wrinkles, black spots, shedding and inflammation. Within the wavelengths 280­320 nm, UV-B can penetrate to the epidermal level. This investigation aimed to test whether extracts from the tropical abalone [Haliotis asinina (H. asinina)] mucus-secreting tissues, the hypobranchial gland (HBG) and gills, were able to attenuate the inflammatory process, using the human keratinocyte HaCaT cell line. Cytotoxicity of abalone tissue extracts was determined using an AlamarBlue viability assay. Results showed that HaCaT cells could survive when incubated in crude HBG and gill extracts at concentrations between <11.8 and <16.9 µg/ml, respectively. Subsequently, cell viability was compared between cultured HaCaT cells exposed to serial doses of UV-B from 1 to 11 (x10) mJ/cm2 and containing 4 different concentrations of abalone extract from both the HBG and gill (0, 0.1, 2.5, 5 µg/ml). A significant increase in cell viability was observed (P<0.001) following treatment with 2.5 and 5 µg/ml extract. Without extract, cell viability was significantly reduced upon exposure to UV-B at 4 mJ/cm2. Three morphological changes were observed in HaCaT cells following UV-B exposure, including i) condensation of cytoplasm; ii) shrunken cells and plasma membrane bubbling; and iii) condensation of chromatin material. A calcein AM­propidium iodide live­dead assay showed that cells could survive cytoplasmic condensation, yet cell death occurred when damage also included membrane bubbling and chromatin changes. Western blot analysis of HaCaT cell COX­2, p38, phospho­p38, SPK/JNK and phospho­SPK/JNK following exposure to >2.5 µg/ml extract showed a significant decrease in intensity for COX­2, phospho­p38 and phospho­SPK/JNK. The present study demonstrated that abalone extracts from the HGB and gill can attenuate inflammatory proteins triggered by UV-B. Hence, the contents of abalone extract, including cellmetabolites and peptides, may provide new agents for skin anti­inflammation, preventing damage due to UV-B.

Comparative analysis of serum zinc, copper, manganese, iron, calcium, and magnesium level and complexity of interelement relations in generalized anxiety disorder patients.

The purpose of the study was to determine the concentration of serum trace and other essential elements of generalized anxiety disorder patients and to find out the relationship between element levels and nutritional status or socioeconomic factors. The study was conducted among 50 generalized anxiety disorder patients and 51 healthy volunteers. Patients were selected and recruited in the study with the help of a clinical psychologist by random sampling. The concentrations of serum trace elements (Zn, Cu, Mn, and Fe) and other two essential elements (Ca and Mg) were determined by graphite furnace and flame atomic absorption spectroscopy. Data were analyzed by independent t test, Pearson's correlation analysis, regression analysis, and analysis of variance. The serum concentrations of Zn, Cu, Mn, Fe, Ca, and Mg in generalized anxiety disorder patients were 1.069 ± 0.40, 1.738 ± 0.544, 1.374 ± 0.750, 3.203 ± 2.065, 108.65 ± 54.455, and 21 ± 4.055 mg/L, while those were 1.292 ± 0.621, 0.972 ± 0.427, 0.704 ± 0.527, 1.605 ± 1.1855, 101.849 ± 17.713, and 21.521 ± 3.659 mg/L in control subjects. Significantly decreased (p < 0.05) serum Zn concentration was found in the patient group compared to the control group while serum level of Cu, Mn, and Fe was significantly (p < 0.05) higher, but the differences of the concentration of Ca and Mg between the patient and control groups were not significant (p > 0.05). Socioeconomic data revealed that most of the patients were in the lower middle class group and middle-aged. Mean BMI of the control group (23.63 ± 3.91 kg/m(2)) and the patient group (23.62 ± 3.77 kg/m(2)) was within the normal range (18.5-25.0 kg/m(2)). The data obtained from different interelement relations in the generalized anxiety disorder patients and control group strongly suggest that there is a disturbance in the element homeostasis. So changes in the serum trace element level in generalized anxiety disorder patients occur independently and they may provide a prognostic tool for the diagnosis and treatment of this disease.