Coordinated adaptations define the ontogenetic shift from worm- to fish-hunting in a venomous cone snail.

Marine cone snails have attracted researchers from all disciplines but early life stages have received limited attention due to difficulties accessing or rearing juvenile specimens. Here, we document the culture of Conus magus from eggs through metamorphosis to reveal dramatic shifts in predatory feeding behaviour between post-metamorphic juveniles and adult specimens. Adult C. magus capture fish using a set of paralytic venom peptides combined with a hooked radular tooth used to tether envenomed fish. In contrast, early juveniles feed exclusively on polychaete worms using a unique "sting-and-stalk" foraging behaviour facilitated by short, unbarbed radular teeth and a distinct venom repertoire that induces hypoactivity in prey. Our results demonstrate how coordinated morphological, behavioural and molecular changes facilitate the shift from worm- to fish-hunting in C. magus, and showcase juvenile cone snails as a rich and unexplored source of novel venom peptides for ecological, evolutionary and biodiscovery studies.

Comparative Venomics of C. flavidus and C. frigidus and Closely Related Vermivorous Cone Snails.

Cone snail venom biodiversity reflects dietary preference and predatory and defensive envenomation strategies across the ≈900 species of Conidae. To better understand the mechanisms of adaptive radiations in closely related species, we investigated the venom of two phylogenetically and spatially related species, C. flavidus and C. frigidus of the Virgiconus clade. Transcriptomic analysis revealed that the major superfamily profiles were conserved between the two species, including 68 shared conotoxin transcripts. These shared transcripts contributed 90% of the conotoxin expression in C. frigidus and only 49% in C. flavidus, which showed greater toxin diversification in the dominant O1, I2, A, O2, O3, and M superfamilies compared to C. frigidus. On the basis of morphology, two additional sub-groups closely resembling C. flavidus were also identified from One Tree Island Reef. Despite the morphological resemblance, the venom duct proteomes of these cryptic sub-groups were distinct from C. flavidus. We suggest rapid conotoxin sequence divergence may have facilitated adaptive radiation and the establishment of new species and the regulatory mechanisms facilitating species-specific venom evolution.

Venom duct origins of prey capture and defensive conotoxins in piscivorous Conus striatus.

The venom duct origins of predatory and defensive venoms has not been studied for hook-and-line fish hunting cone snails despite the pharmacological importance of their venoms. To better understand the biochemistry and evolution of injected predatory and defensive venoms, we compared distal, central and proximal venom duct sections across three specimens of C. striatus (Pionoconus) using proteomic and transcriptomic approaches. A total of 370 conotoxin precursors were identified from the whole venom duct transcriptome. Milked defensive venom was enriched with a potent cocktail of proximally expressed inhibitory α-, ω- and µ-conotoxins compared to milked predatory venom. In contrast, excitatory κA-conotoxins dominated both the predatory and defensive venoms despite their distal expression, suggesting this class of conotoxin can be selectively expressed from the same duct segment in response to either a predatory or defensive stimuli. Given the high abundance of κA-conotoxins in the Pionoconus clade, we hypothesise that the κA-conotoxins have evolved through adaptive evolution following their repurposing from ancestral inhibitory A superfamily conotoxins to facilitate the dietary shift to fish hunting and species radiation in this clade.

Characterisation of d-Conotoxin TxVIA as a Mammalian T-Type Calcium Channel Modulator.

The 27-amino acid (aa)-long d-conotoxin TxVIA, originally isolated from the mollusc-hunting cone snail Conus textile, slows voltage-gated sodium (NaV) channel inactivation in molluscan neurons, but its mammalian ion channel targets remain undetermined. In this study, we confirmed that TxVIA was inactive on mammalian NaV1.2 and NaV1.7 even at high concentrations (10 µM). Given the fact that invertebrate NaV channel and T-type calcium channels (CaV3.x) are evolutionarily related, we examined the possibility that TxVIA may act on CaV3.x. Electrophysiological characterisation of the native TxVIA on CaV3.1, 3.2 and 3.3 revealed that TxVIA preferentially inhibits CaV3.2 current (IC50 = 0.24 mM) and enhances CaV3.1 current at higher concentrations. In fish bioassays TxVIA showed little effect on zebrafish behaviours when injected intramuscular at 250 ng/100 mg fish. The binding sites for TxVIA at NaV1.7 and CaV3.1 revealed that their channel binding sites contained a common epitope.

Conotoxins: Chemistry and Biology.

The venom of the marine predatory cone snails (genus Conus) has evolved for prey capture and defense, providing the basis for survival and rapid diversification of the now estimated 750+ species. A typical Conus venom contains hundreds to thousands of bioactive peptides known as conotoxins. These mostly disulfide-rich and well-structured peptides act on a wide range of targets such as ion channels, G protein-coupled receptors, transporters, and enzymes. Conotoxins are of interest to neuroscientists as well as drug developers due to their exquisite potency and selectivity, not just against prey but also mammalian targets, thereby providing a rich source of molecular probes and therapeutic leads. The rise of integrated venomics has accelerated conotoxin discovery with now well over 10,000 conotoxin sequences published. However, their structural and pharmacological characterization lags considerably behind. In this review, we highlight the diversity of new conotoxins uncovered since 2014, their three-dimensional structures and folds, novel chemical approaches to their syntheses, and their value as pharmacological tools to unravel complex biology. Additionally, we discuss challenges and future directions for the field.

Venomics-Accelerated Cone Snail Venom Peptide Discovery.

Cone snail venoms are considered a treasure trove of bioactive peptides. Despite over 800 species of cone snails being known, each producing over 1000 venom peptides, only about 150 unique venom peptides are structurally and functionally characterized. To overcome the limitations of the traditional low-throughput bio-discovery approaches, multi-omics systems approaches have been introduced to accelerate venom peptide discovery and characterisation. This "venomic" approach is starting to unravel the full complexity of cone snail venoms and to provide new insights into their biology and evolution. The main challenge for venomics is the effective integration of transcriptomics, proteomics, and pharmacological data and the efficient analysis of big datasets. Novel database search tools and visualisation techniques are now being introduced that facilitate data exploration, with ongoing advances in related omics fields being expected to further enhance venomics studies. Despite these challenges and future opportunities, cone snail venomics has already exponentially expanded the number of novel venom peptide sequences identified from the species investigated, although most novel conotoxins remain to be pharmacologically characterised. Therefore, efficient high-throughput peptide production systems and/or banks of miniaturized discovery assays are required to overcome this bottleneck and thus enhance cone snail venom bioprospecting and accelerate the identification of novel drug leads.

Accelerated proteomic visualization of individual predatory venoms of Conus purpurascens reveals separately evolved predation-evoked venom cabals.

Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens. High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.

Toxicology of Gambierdiscus spp. (Dinophyceae) from Tropical and Temperate Australian Waters.

Ciguatera Fish Poisoning (CFP) is a human illness caused by the consumption of marine fish contaminated with ciguatoxins (CTX) and possibly maitotoxins (MTX), produced by species from the benthic dinoflagellate genus Gambierdiscus. Here, we describe the identity and toxicology of Gambierdiscus spp. isolated from the tropical and temperate waters of eastern Australia. Based on newly cultured strains, we found that four Gambierdiscus species were present at the tropical location, including G. carpenteri, G. lapillus and two others which were not genetically identical to other currently described species within the genus, and may represent new species. Only G. carpenteri was identified from the temperate location. Using LC-MS/MS analysis we did not find any characterized microalgal CTXs (P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) or MTX-1; however, putative maitotoxin-3 (MTX-3) was detected in all species except for the temperate population of G. carpenteri. Using the Ca2+ influx SH-SY5Y cell Fluorescent Imaging Plate Reader (FLIPR) bioassay we found CTX-like activity in extracts of the unidentified Gambierdiscus strains and trace level activity in strains of G. lapillus. While no detectable CTX-like activity was observed in tropical or temperate strains of G. carpenteri, all species showed strong maitotoxin-like activity. This study, which represents the most comprehensive analyses of the toxicology of Gambierdiscus strains isolated from Australia to date, suggests that CFP in this region may be caused by currently undescribed ciguatoxins and maitotoxins.

Comparative Venomics Reveals the Complex Prey Capture Strategy of the Piscivorous Cone Snail Conus catus.

Venomous marine cone snails produce a unique and remarkably diverse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides.

Ribosomal protein S15A augments human osteosarcoma cell proliferation in vitro.

As a highly conserved housekeeping gene, the biological implications of ribosomal protein S15A (RPS15A) during various processes, including carcinogenesis, remain elusive. Herein, the authors reported that knockdown of RPS15A expression significantly inhibited human osteosarcoma U2OS cell proliferation and colony formation in vitro by using a lentivirus-mediated RNA interference (RNAi) system. Moreover, an excess accumulation of cells in the G0/G1 phase was observed in U2OS cells transduced with lentivirus targeting RPS15A, suggesting that the growth inhibition mediated by RPS15A knockdown in osteosarcoma cells was probably due to the induction of cell cycle arrest. Taken together, this study highlights the crucial role of RPS15A in promoting osteosarcoma cell proliferation, and provides a foundation for further study into the clinical potential of inhibition of RPS15A for the treatment of osteosarcoma.

Fumigaclavine C from a marine-derived fungus Aspergillus fumigatus induces apoptosis in MCF-7 breast cancer cells.

Recently, much attention has been given to discovering natural compounds as potent anti-cancer candidates. In the present study, the anti-cancer effects of fumigaclavine C, isolated from a marine-derived fungus, Aspergillus fumigatus, was evaluated in vitro. In order to investigate the impact of fumigaclavine C on inhibition of proliferation and induction of apoptosis in breast cancer, MCF-7 cells were treated with various concentrations of fumigaclavine C, and fumigaclavine C showed significant cytotoxicity towards MCF-7 cells. Anti-proliferation was analyzed via cell mobility and mitogen-activated protein kinase (MAPK) signaling pathway. In addition, fumigaclavine C showed potent inhibition on the protein and gene level expressions of MMP-2, -9 in MCF-7 cells which were manifested in Western blot and reverse transcription polymerase chain reaction (RT-PCR) results. The apoptosis induction abilities of the fumigaclvine C was studied by analyzing the expression of apoptosis related proteins, cell cycle analysis, DNA fragmentation and molecular docking studies. It was found that fumigaclavine C fragmented the MCF-7 cell DNA and arrested the cell cycle by modulating the apoptotic protein expressions. Moreover, fumigaclavine C significantly down-regulated the NF-kappa-B cell survival pathway. Collectively, data suggest that fumigaclavine C has a potential to be developed as a therapeutic candidate for breast cancer.

Triterpenoids of marine origin as anti-cancer agents.

Triterpenoids are the most abundant secondary metabolites present in marine organisms, such as marine sponges, sea cucumbers, marine algae and marine-derived fungi. A large number of triterpenoids are known to exhibit cytotoxicity against a variety of tumor cells, as well as anticancer efficacy in preclinical animal models. In this review efforts have been taken to review the structural features and the potential use of triterpenoids of marine origin to be used in the pharmaceutical industry as potential anti-cancer drug leads.

Tyrosol exerts a protective effect against dopaminergic neuronal cell death in in vitro model of Parkinson's disease.

Experimental evidence suggests that tyrosol [2-(4-hydroxyphenyl)ethanol] exhibits potent protective activities against several pathogeneses. In this study, we evaluated the protective effect of tyrosol against 1-methyl-4-phenylpyridinium (MPP(+))-induced CATH.a neuron cell death. Tyrosol dose-dependently protected CATH.a cells from MPP(+)-induced cell death and the protection was more apparent after prolong incubation (48h). The data showed that tyrosol treatment suppressed the reduction of phospho-tyrosine hydroxylase level in CATH.a cells. Further, the compound repressed MPP(+)-induced depletion of mitochondrial membrane potential (Δψm) and thereby maintained intracellular ATP production in the cell. The cellular signalling pathway studies revealed that tyrosol protected CATH.a cells from MPP(+)-induced apoptotic signalling, most likely via activation of PI3K/Akt signalling pathway along with up-regulation of anti-oxidative enzymes (SOD-1 and SOD-2) and DJ-1 protein in the cell. Collectively, present study demonstrates that tyrosol significantly protects dopaminergic neurons from MPP(+)-induced degradation, and reveals potential neuroprotective mechanism of tyrosol.

EGFR tyrosine kinase inhibitory peptide attenuates Helicobacter pylori-mediated hyper-proliferation in AGS enteric epithelial cells.

Helicobacter pylori infection is one of the most critical causes of stomach cancer. The current study was conducted to explore the protective effects of an isolated active peptide H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser) from microbial hydrolysates of Chlamydomonas sp. against H. pylori-induced carcinogenesis. The peptide H-P-6 has effectively suppressed H. pylori-induced hyper-proliferation and migration of gastric epithelial cells (AGS). However, the peptide did not inhibit the viability of the bacteria or invasion into AGS cells. Therefore, the effect of the peptide on regulating H. pylori-induced molecular signaling was investigated. The results indicated that H. pylori activates the EGFR tyrosine kinase signaling and nuclear translocation of the ß-catenin. The EGFR activation has led to the up-regulation of PI3K/Akt signaling pathway. Moreover, the nuclear translocation levels of ß-catenin were significantly increased as a result of Akt mediated down-regulation of GSK3/ß protein levels in the cytoplasm. Both of these consequences have resulted in increased expression of cell survival and migration related genes such as c-Myc, cyclin-D, MMP-2 and matrilysin. Interestingly, the isolated peptide potently inhibited H. pylori-mediated EGFR activation and thereby down-regulated the subsequent P13K/Akt signaling leading to ß-catenin nuclear translocation. The effect of the peptide was confirmed with the use of EGFR tyrosine kinase inhibitor AG1487 and molecular docking studies. Collectively this study identifies a potent peptide which regulates the H. pylori-induced hyper-proliferation and migration of AGS cells at molecular level.

Neoechinulin A suppresses amyloid-ß oligomer-induced microglia activation and thereby protects PC-12 cells from inflammation-mediated toxicity.

A pathological hallmark of Alzheimer's disease (AD), aggregation and deposition of amyloid-ß peptides, has been recognized as a potent activator of microglia-mediated neuroinflammation and neuronal dysfunction. Therefore, downregulation of microglial activation has a significant therapeutic demand. In this study, focus was given to evaluate the ability of neoechinulin A, an indole alkaloid isolated from marine-derived Microsporum sp., to attenuate microglial activation by oligomeric amyloid-ß 1-42 (Aß42). Neoechinulin A treatment significantly inhibited the generation of reactive oxygen and nitrogen species in Aß42-activated BV-2 microglia cells. In addition, we found that neoechinulin A significantly suppressed the production of neurotoxic inflammatory mediator tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) in activated BV-2 cells. Moreover, the treatment downregulated the protein and gene expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1ß and IL-6. Further, activated microglia-mediated apoptosis of PC-12 pheochromocytoma cells was significantly repressed by neoechinulin A. The molecular mechanism studies suggested that neoechinulin A may block the phosphorylation of mitogen-activated protein kinase (MAPK) molecule p38, apoptosis signal-regulating kinase 1 (ASK-1) and nuclear translocation of nuclear factor-κB (NF-κB) p65 and p50 subunits. Regulation of these signalling pathways have most probably contributed to the anti-inflammatory activity of neoechinulin A. Collectively, these results suggest that with further studies neoechinulin A have a potential to be developed as a modulator of neuroinflammatory process in AD.

Peptide isolated from Japanese flounder skin gelatin protects against cellular oxidative damage.

Gelatin was extracted from the skin of Japanese flounder ( Palatichtys olivaceus ) and was subjected to enzymatic hydrolysis. The peptic hydrolysate resulted in a potent antioxidative peptide Gly-Gly-Phe-Asp-Met-Gly (582 Da), which bears +12.61 kcal/mol hydrophobicity. The antioxidative potential of the peptide was characterized by analyzing the protective effect of the peptide on reactive oxygen species (ROS)-mediated intracellular macromolecule damage. It was found that the peptide is a potent scavenger of intracellular ROS, thereby protecting the radical-mediated damage of membrane lipids, proteins, and DNA. Moreover, the peptide is capable of upregulating the expression of inherent antioxidative enzymes, superoxide dismutase-1, glutathione, and catalase. Collectively, it can be concluded that Japanese flounder skin, a processing byproduct of filleting, can be effectively used to produce a bioactive peptide with potent antioxidant capacity.

Paeonol from Hippocampus kuda Bleeler suppressed the neuro-inflammatory responses in vitro via NF-κB and MAPK signaling pathways.

Inflammation has recently been implicated as a critical mechanism responsible for neurodegenerative diseases. In this study, paeonol (1-(2-hydroxy-4-methoxyphenyl)ethanone) isolated from the sea horse Hippocampus kuda Bleeler was studied as an agent to suppress LPS induced activation of BV-2 microglial and RAW264.7 macrophage cells. The results obtained showed that paeonol significantly suppressed LPS induced release of pro-inflammatory products such as nitric oxide (NO), prostaglandin E2 (PGE(2)), and cytokines; tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Furthermore, the compound down regulated the protein and gene expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1ß and IL-6 in both cell lines. Molecular signaling pathway studies showed that paeonol inhibited the translocation of nuclear factor-κB (NF-κB) p65 and p50 subunits to the nucleus by blocking IKKα/ß (IκB kinase α/ß) mediated degradation of IκBα. Moreover, it suppressed the phosphorylation of mitogen activated protein kinase (MAPK) pathway molecules; c-Jun N-terminal kinases (JNK) and p38 in both cell lines. Collectively these results indicate that paeonol blocked the LPS stimulated inflammatory responses in BV-2 and RAW264.7 cells via modulating MAPK and NF-κB signaling pathways. Therefore, paeonol could be a promising candidate to be used in neuro-inflammatory therapy.

Triterpene glycosides from sea cucumbers and their biological activities.

Triterpenoid glycosides are abundantly present in sea cucumbers, which are responsible for the toxicity of these echinoderms. More than 100 triterpenoid glycosides have been isolated in the past 20 years and those are grouped into four main structural categories considering their aglycone structure: 3ß-hydroxyholost-9(ll)-ene aglycone skeleton, 3ß-hydroxyholost-7-ene skeleton, other holostane type aglycones and nonholostane aglycone. Most of the triterpenoid glycosides are found to be possessing potential biological activities. Among the biological activities, anticancer activity and antiviral activity are the most widely studied areas. In this communication, we have presented a general view of the structural characteristics of triterpenoid glycosides and their major biological activities. The structural significance and the application limitations of triterpene glycosides are also discussed.

Sulfated chitooligosaccharide II (SCOS II) suppress collagen degradation in TNF-induced chondrosarcoma cells via NF-κB pathway.

Chitooligosaccharides (COS), the hydrolyzed product of chitosan and its derivatives, are known to have interesting pharmaceutical and medicinal applications due to its high solubility, non-toxicity, and increased functionality. Among them sulfated chitooligosaccharides (SCOSs) have been identified to possess enhanced biological activities. This study reports the effects of SCOSs with different molecular weights on the degradation of articular cartilage through unregulated collagenase expression. The results indicated that the SCOS II (3-5kDa) effectively inhibited the expressions of collagenases 1 and 3 and thereby prevented TNF-α induced degradation of collagen in human chondrosarcoma cells (SW-1353). Moreover, the signaling cascade responsible for this effect was found as SCOS II mediated suppression of NF-κB activation. Based on these data, it can be concluded that SCOS II prevented collagen degradation by inhibiting collagenases 1 and 3 via suppressing TNF-α induced NF-κB signaling. We suggest that SCOS II can be further studied as a potential candidate for the treatment of arthritis.

Chitooligosaccharides attenuate UVB-induced damages in human dermal fibroblasts.

As a continuation of our research on chitooligosaccharides (COS), this study focused on the protective effect of COS of various molecular weights (1-3, 3-5, and 5-10 kDa) on cellular damage caused by ultraviolet B (UVB)-induced oxidative stress in human dermal fibroblast cells. The results show that the protective effect of COS on UVB-stressed human fibroblasts was dependent on molecular weight. COS suppressed UVB irradiation-induced reactive oxygen species generation and DNA damage, accompanied by the downregulation of matrix metalloproteinase (MMP)-1 and MMP-13. In a comparative analysis, COS (3-5 kDa) exhibited the most potent protective effect on UVB-stressed fibroblasts. The presence of COS (3-5 kDa) attenuated UVB-induced collagenolytic MMP production and collagen degradation. The photoprotective activity of COS (3-5 kDa) was confirmed by transcriptional phosphorylation of mitogen-activated protein kinase-responsive signaling pathways.