Structural insights into a functional unit from an immunogenic mollusk hemocyanin.

Mollusk hemocyanins, among the largest known proteins, are used as immunostimulants in biomedical and clinical applications. The hemocyanin of the Chilean gastropod Concholepas concholepas (CCH) exhibits unique properties, which makes it safe and effective for human immunotherapy, as observed in animal models of bladder cancer and melanoma, and dendritical cell vaccine trials. Despite its potential, the structure and amino acid sequence of CCH remain unknown. This study reports two sequence fragments of CCH, representing three complete functional units (FUs). We also determined the high-resolution (1.5 Å) X-ray crystal structure of an "FU-g type" from the CCHB subunit. This structure enables in-depth analysis of chemical interactions at the copper-binding center and unveils an unusual, truncated N-glycosylation pattern. These features are linked to eliciting more robust immunological responses in animals, offering insights into CCH's enhanced immunostimulatory properties and opening new avenues for its potential applications in biomedical research and therapies.

Metabolomics Reveals Minor Tambjamines in a Marine Invertebrate Food Chain.

Metabolomics analysis detected tambjamine alkaloids in aqueous and EtOAc extracts of the marine invertebrates Virididentula dentata, Tambja stegosauriformis, Tambja brasiliensis, and Roboastra ernsti. Among several tambjamines, the new amino acid derivatives tambjamines M-O (17-19) were identified by Marfey's advanced analysis, UPLC-MS/MS analyses, and total synthesis. The tambjamine diversity increased from the bryozoan V. dentata to its nudibranch predators T. stegosauriformis and T. brasiliensis and attained a higher diversity in R. ernsti, the nudibranch that preys upon T. stegosauriformis and T. brasiliensis. The total tambjamine content also increases among the trophic levels, probably due to biomagnification. Tambjamines A (1), C (3), and D (4) are the major metabolites in the tissues of V. dentata, T. stegosauriformis, T. brasiliensis, and R. ernsti and are likely the main chemical defenses of these marine invertebrates.

Hemocyanin of the caenogastropod Pomacea canaliculata exhibits evolutionary differences among gastropod clades.

Structural knowledge of gastropod hemocyanins is scarce. To better understand their evolution and diversity we studied the hemocyanin of a caenogastropod, Pomacea canaliculata (PcH). Through a proteomic and genomic approach, we identified 4 PcH subunit isoforms, in contrast with other gastropods that usually have 2 or 3. Each isoform has the typical Keyhole limpet-type hemocyanin architecture, comprising a string of eight globular functional units (FUs). Correspondingly, genes are organized in eight FUs coding regions. All FUs in the 4 genes are encoded by more than one exon, a feature not found in non- caenogastropods. Transmission electron microscopy images of PcH showed a cylindrical structure organized in di, tri and tetra-decamers with an internal collar structure, being the di and tri-decameric cylinders the most abundant ones. PcH is N-glycosylated with high mannose and hybrid-type structures, and complex-type N-linked glycans, with absence of sialic acid. Terminal ß-N-GlcNAc residues and nonreducing terminal α-GalNAc are also present. The molecule lacks O-linked glycosylation but presents the T-antigen (Gal-ß1,3-GalNAc). Using an anti-PcH polyclonal antibody, no cross-immunoreactivity was observed against other gastropod hemocyanins, highlighting the presence of clade-specific structural differences among gastropod hemocyanins. This is, to the best of our knowledge, the first gene structure study of a Caenogastropoda hemocyanin.

Applying the monitoring breakdown structure model to trace metal content in edible biomonitors: An eight-year survey in the Beagle Channel (southern Patagonia).

The purpose of this paper is to investigate the trace metal content in edible biomonitors (i.e., mollusks) in the Beagle Channel (southern Patagonia) and to assess the human health risks associated with their consumption. Rationale: The monitoring breakdown structure (MBS) conceptual model was applied to four sampling campaigns (2005 → 2012) that collected 729 samples of Mytilus chilensis and Nacella magellanica. The composition of trace elements (Cd, Cr, Cu, Ni, Pb and Zn) in the mollusks was determined using graphite furnace (GFAAS) or flame atomic absorption spectrometry (FAAS). We compared the mean obtained values with the maximum levels (MLs) of each element established by international organizations. Then, based on semi-structured interviews, we calculated the estimated daily intake (EDI) of local residents and compared it with safety reference doses, i.e., the provisional tolerable daily intake (PTDI), provisional maximum tolerable daily intake (PMTDI), and tolerable daily intake (TDI), as well as the benchmark dose level lower confidence limit for Pb (BMDL01, a reference point (RP)/point of departure (POD). Moreover, to obtain information about the potential health risks of ingesting heavy metals (HMs) through mollusk consumption, we evaluated the target hazard quotient (THQ) and the hazard index (HI). Findings: For Cd and Pb, 65% and 40% of bivalves exceeded the MLs established by the Mercado Común del Sur (Mercosur), respectively. Except for Cd in N. magellanica (i.e., 1.20 µg/kg/bw/day), EDI values were clearly lower than the safety reference doses. For Cr, Cu, Ni, Pb and Zn, mussels were safe for consumption and did not raise concerns for public health. Likewise, THQ values were well below one for most of the studied metals, indicating that the exposed human population is assumed to be safe. Occasional high consumers of mollusks from the most contaminated sites may be at some health risk. Originality: The food production system and the environment are complex systems; this is crucial to understand when we consider ecosystems as a food source (i.e., marine ecosystems). Here we consider edible biomonitors, that are organisms that can have a dual function. They are food, and at the same time, if properly calibrated, they can act as indicators of environmental quality. This study is the first to investigate relevant essential and non-essential trace metal content in two edible mollusks from the Beagle Channel in a long-term survey (2005 → 2012). The information variety was high; approximately thirteen thousand determinations were conducted to support the risk assessment for mollusk consumption. Other aspects connected with the health risks and the uncertainty factors related to the presence of essential and non-essential minerals in edible mollusks as well as the use of the MBS are also discussed.

Rapid screening fluorescence method applied to detection and quantitation of paralytic shellfish toxins in invertebrate marine vectors.

A rapid screening method is described for the determination of paralytic shellfish toxins (PST), in fresh marine vectors (bivalves and gastropods), at levels ranging from 0.05 to 5.0 mg STX-eq kg-1. PST are extracted from marine vector homogenates with acetic acid according to the Pre-COX-LC-FLD method. At the same time, the obtained extract is oxidised simultaneously in hydrogen peroxide and periodate oxidate to determine PST, non-N-hydroxylated and N-hydroxylated toxins, respectively. Then, they are analysed using a microplate fluorometer (Ex: 335 nm/Em: 405 nm). All the samples were compared with the liquid chromatography post-column oxidation method. Recoveries of PST added to fresh and processed marine vectors averaged 93.9% with a coefficient of variation of 6.1%. Both methods showed a good linear regression (r2 = 0.97). The method shows good intra- and inter-day precisions with a relative coefficient of variation of ≈ 3.8% and 5.7%, respectively. The limit of quantification of the rapid screening fluorescence method was ≈ 0.082 mg STX-eq kg-1, with ≤5% false positives. The established rapid screening fluorescence methods offer highly effective and verifiable pre-analyses of PST contamination in marine vectors and can be used for routine screening of the PST in seafood before formal identification by confirmatory methods (Pre-COX LC-FLD method, Lawrence method).

Valor nutricional de espécies marinhas comercializadas no mercado informal / Nutritional value of marine species comercialized in the informal market

No Brasil, além das espécies marinhas tradicionais de consumo há captura não reportada e comércio informal de espécies não convencionais, com crescente demanda gastronômica. Este estudo analisou o valor nutricional e rendimento do saquaritá Stramonita haemastomae e do pepino-do-mar Holothuria grisea coletados na Baía de Santos, SP, de fevereiro/2018 a junho/2018. Foram coletados indivíduos das duas espécies e a composição centesimal determinada com metodologias oficiais ou reconhecidas. Os resultados indicaram importante aporte nutricional e o rendimento de 29,72% e 22,57% para o pepino-do-mar e saquaritá, respectivamente. Sendo recomendados estudos e ações de gestão ambiental e sanitária, de forma a assegurar a oferta de um produto seguro para consumo humano, em especial dos equinodermas que não são contemplados na regulação para alimentos.

Valor nutricional de espécies marinhas comercializadas no mercado informal / Nutritional value of marine species comercialized in the informal market

No Brasil, além das espécies marinhas tradicionais de consumo há captura não reportada e comércio informal de espécies não convencionais, com crescente demanda gastronômica. Este estudo analisou o valor nutricional e rendimento do saquaritá Stramonita haemastomae e do pepino-do-mar Holothuria grisea coletados na Baía de Santos, SP, de fevereiro/2018 a junho/2018. Foram coletados indivíduos das duas espécies e a composição centesimal determinada com metodologias oficiais ou reconhecidas. Os resultados indicaram importante aporte nutricional e o rendimento de 29,72% e 22,57% para o pepino-do-mar e saquaritá, respectivamente. Sendo recomendados estudos e ações de gestão ambiental e sanitária, de forma a assegurar a oferta de um produto seguro para consumo humano, em especial dos equinodermas que não são contemplados na regulação para alimentos.(AU)

Inter-species variability of okadaic acid group toxicity in relation to the content of fatty acids detected in different marine vectors.

Okadaic acid group (OA-group) is a set of lipophilic toxins which are characterised by being produced by species associated with the genera Dinophysis and Prorocentrum. OA-group has been regularly detected in endemic shellfish species from the southern zone of Chile only through the mouse bioassay. The purpose of this work was to determine the variability of OA-group toxins in endemic aquatic organisms (bivalves, crabs, gastropods and fish) and to establish the relationship with the concentration of fatty acids (FAs) detected in the evaluated species. The toxicity of OA-group and the FA profiles were determined using LC-MS/MS and gas chromatography with flame-ionisation detection, respectively. In the study area, the dinoflagellate Dinophysis acuta was detected in densities ≈2000 cells ml-1 with a toxicity ≈18.3 pg OA equiv cel-1. The analysis identified OA and dinophysistoxin-1 in shellfish in a range of ≈90 to ≈225 µg OA eq kg-1, where no toxins in fish were detected. A positive relationship between the FA level and the concentration of OA-group toxins in the digestive glands of bivalves and gastropods was established, noted for high levels of saturated FAs (C14:0 and C16:0). The toxic variability of OA-group toxins determined in the different species allowed us to establish that the consumption of these vectors, regulated by non-analytical methods, can be harmful when consumed by humans, thus suggesting that the sanitary regulations for the control of OA-group in Chile should be updated.

Effect of the earthquake-tsunami (Chile, 2010) on toxic metal content in the Chilean abalone mollusc Concholepas concholepas.

Toxic metal content were measured in samples of mollusc Concholepas concholepas obtained from the Chilean coast. Samples were collected during two periods, one before and one after the earthquake-tsunami which occurred in the Maule Region, Chile, February 27th, 2010 as a result of an earthquake with a magnitude of 8.8. Quantification of toxic metals in samples of hepatopancreas and muscle tissue was performed using flame atomic absorption spectroscopy. The analytical methodology was validated with certified reference material. The content means measured in muscle tissue for January 2010 were Cu: 10.3; Cr: 0.7; Cd: < 0.1; Mn: 3.6 and Zn: 38.6 µg g-1 (dry weight). For October 2014, the means were Cu: 8; Cr: 2.4; Cd: < 0.1; Mn: 5.6 and Zn: 45.4 µg g-1 (dry weight). In hepatopancreas tissue, the content means were Cu: 14.8; Cr: 2.4; Cd: 246.2; Mn: 4.4 and Zn: 1552.9 µg g-1 (dry weight). For October 2014, the means were Cu: 53.7; Cr: 3.5; Cd: 118; Mn: 13.4 and Zn: 1352.3 µg g-1 (dry weight). Cd, Cr, Cu, Mn and Zn content in the samples of hepatopancreas were generally higher than those recorded in muscle tissue however they were not always statistically significant. Statistical analysis showed that Cu and Mn content in the post-tsunami period increased in the hepatopancreas tissue. The concentrations of Cd, Cr, Cu, Mn, and Zn measured in the muscular tissue (edible part) of the species Conchalepas concholepas, were lower than the maximum contents allowed by the current legislation (FAO/WHO, 2004; EU, 2001) and its consumption is not a risk to human health.

PAHs contamination in edible gastropods from north Patagonian harbor areas.

PAHs are persistent pollutants released into the environment by fossil fuels burning and leak during petroleum operations. Associated with suspended particles upon entering marine ecosystem are accumulated by benthic fauna. Human exposure occurs mainly from ingestion such as gastropods consumption. The objective was to determine PAHs in sediments and in the marine gastropod Buccinanops globulosus in sites with different maritime and urban influences. In sampling sites located 20 km from the harbor, PAHs were non-detected; while in harbor gastropods, the level of PAH4 was exceeded according to international normative. Level of dibenzo[a,h]anthracene in sediments was between the ISQG and PEL. Since these are the first results of PAHs in edible gastropods in South America, we concluded that PAHs can be dangerous for consumers according to ingestion frequency. Integrative studies are necessary to evaluate the interaction among pollutants in maritime areas and the incidence in human health due to shellfish consumption.

Analysis of classical neurotransmitter markers in tapeworms: Evidence for extensive loss of neurotransmitter pathways.

Parasitic flatworms have complex neuromuscular systems that serve important functions in their life cycles. However, our understanding of neurotransmission in parasitic flatworms is limited. Pioneering studies have suggested the presence of several classical neurotransmitter systems, but their molecular components have not been characterized in most cases. Because these components are conserved in bilaterian animals, we searched the genomes of parasitic flatworms for orthologs of genes required for neurotransmitter synthesis, vesicular transport, reuptake, and reception. Our results indicate that tapeworms have lost the genes that are specifically required in other animals for synaptic signaling using the classical neurotransmitters dopamine, tyramine, octopamine, histamine and gamma-aminobutyric acid (GABA). These results imply that these signaling pathways are either absent in these parasites, or that they require completely different molecular components in comparison with other animals. The orthologs of genes related to histaminergic and GABA signaling are also missing in trematodes (although Schistosoma-specific histaminergic receptors have been previously described). In contrast, conserved genes required for glutamatergic, serotonergic and cholinergic signaling could be found in all analyzed flatworms. We analyzed the expression of selected markers of each pathway in the tapeworm Hymenolepis microstoma by whole-mount in situ hybridization. Each marker was specifically expressed in the nervous system, although with different patterns. In addition, we analyzed the expression of proprotein convertase 2 as a marker of peptidergic cells. This gene showed the widest expression in the nervous system, but was also expressed in other tissues, suggesting additional roles of peptidergic signaling in tapeworm development and reproduction.

Polyunsaturated fatty acids from Phyllocaulis boraceiensis mucus block the replication of influenza virus.

Influenza viruses cause worldwide outbreaks and pandemics in humans and animals every year with considerable morbidity and mortality. The molecular diversity of secondary metabolites extracted from mollusks is a good alternative for the discovery of novel bioactive compounds with unique structures and diverse biological activities. Phyllocaulis boraceiensis is a hermaphroditic slug that exudes mucus, in which was detected hydroxy polyunsaturated fatty acids that exhibited potent antiviral activity against measles virus. The objective of this study was to evaluate this property against Influenza viruses. Cell viability and toxicity of the mucus were evaluated on Madin-Darby canine kidney (MDCK) cells by MTT assay. Antiviral activity from mucus against influenza viruses was carried out by determination of the virus infection dose and by immunofluorescence assays. The crude mucus and its fractions exhibited low cytotoxicity on MDCK cells. A significant inhibition of viral replication, reduced by the order of eight times, was observed in influenza-induced cytopathic effect. In immunofluorescence assay was observed a decrease of more than 80% of the viral load on infected MDCK cell treated with mucus and its fractions. The viral glycoproteins hemagglutinin and neuraminidase located on the surface of the virus are crucial for the replications and infectivity of the influenza virus. Some authors demonstrated that lipids, such as, polyunsaturated fatty acids exhibited multiple roles in antiviral innate and adaptive responses, control of inflammation, and in the development of antiviral therapeutics. As corroborated by other studies, hydroxy polyunsaturated fatty acids interfered with the binding of influenza virus on host cell receptor and reduced viral titers. The results obtained indicated that polyunsaturated fatty acids from P. boraceiensis crude mucus and fractions 39 exerted antiviral activity against influenza virus.

Antitumor activity and carrier properties of novel hemocyanins coupled to a mimotope of GD2 ganglioside.

Conjugation to carrier proteins is a way to improve the immunogenicity of peptides. Such is the case for peptides mimicking carbohydrate tumor-associated antigens in cancer vaccine development. The most used protein for this purpose is the keyhole limpet hemocyanin (KLH) from Megathura crenulata. Its limited bioavailability has prompted interest in finding new candidates; nevertheless, it is not known whether other hemocyanins might be equally efficient as carrier of carbohydrate peptide mimotopes to promotes anti-tumor responses. Here, we evaluated the carrier and antitumor activity of novel hemocyanins with documented immunogenicity obtained from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH), coupled through sulfo-SMCC to P10, a mimetic peptide of GD2, the major ganglioside constituent of neuroectodermal tumors, and incorporating AddaVax as an adjuvant. The humoral immune responses of mice showed that CCH-P10 and FLH-P10 conjugates elicited specific IgM and IgG antibodies against P10 mimotope, similar to those obtained with KLH-P10, which was used as a positive control. The CCH-P10 and FLH-P10 antisera, exhibited cross-reactivity with murine and human melanoma cells, like anti-CCH and anti-FLH sera suggesting a cross-reaction of CCH and FLH glycosylations with carbohydrate epitopes on the tumor cell surfaces, similar to the KLH antisera. When mice were primed with each hemocyanin-P10 and challenged with melanoma cells, better antitumor effects were observed for FLH-P10 than for CCH-P10 and, as for KLH-P10, irrespective of conjugation. These data demonstrate that CCH and FLH are useful carriers of carbohydrate mimotopes; however, the best antitumor activity of FLH preparations, indicate that is a suitable candidate for further cancer vaccines research.

The impact of pesticides on the macroinvertebrate community in the water channels of the Río Negro and Neuquén Valley, North Patagonia (Argentina).

Agriculture represents the second most important economic activity in the North Patagonian Region of Argentina and non-selective insecticides are still being used with significant implications to the quality of the environment. The range of concentrations (µg/L) determined for azinphosmethyl, chlorpyrifos, and carbaryl in drainage channels were from non-detected to 1.02, 1.45, and 11.21, respectively. Macroinvertebrate abundance and taxon richness in drainage channels were significantly lower in November compared to the other sampling months (October, February). The decrease in taxon richness observed in November was associated with chlorpyrifos and azinphosmethyl peak concentrations. The most remarkable changes were the decrease in sensitive taxa such as Baetidae and the increase in some tolerant taxa such as Chironomidae and Gastropoda.For all three pesticides, the acute hazard quotient exceeded the risk criteria for invertebrates. The effects of the three pesticides on aquatic organisms, characterized by joint probability curves, showed that the LC50 of 10% of the species were exceeded five and three times by the concentrations of azinphosmethyl and chlorpyrifos during the study period, respectively. However, the correlation between the pesticide concentrations and both taxon richness and abundance of macroinvertebrates at each site (irrigation and drainage channels) was indicative that only chlorpyrifos was negatively correlated with both parameters (Spearman r2 - 0.61, p = 0.0051 and Spearman r2 - 0.59, p = 0.0068 for taxon richness and abundance correlation, respectively). We conclude that macroinvertebrate assemblages in drainage channels were highly affected by chlorpyrifos levels.

Assessment of organotins and imposex in two estuaries of the northeastern Brazilian coast.

Butyltin compounds (BTs) were used worldwide, especially because of their properties as biocides. Due to its high toxicity, the use of tributyltin (TBT) in antifouling paints has been prohibited in most countries. The occurrence and impact of BTs were assessed in surface sediments and in Stramonita rustica populations of two tropical estuaries that host major ports in northeastern Brazil. ΣBT concentrations ranged from

Methanolic extract of Euchelus asper exhibits in-ovo anti-angiogenic and in vitro anti-proliferative activities.

BACKGROUND: The marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME). METHODS: The anti-angiogenic activity of EAME (50-800 µg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histological investigations. In vitro testing of EAME (5-20 µg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student's t test for two unpaired groups. RESULTS: Analysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9). CONCLUSION: Overall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.

Rearranged Terpenoids from the Marine Sponge Darwinella cf. oxeata and Its Predator, the Nudibranch Felimida grahami.

Marine sponges are a rich source of terpenoids with rearranged spongian carbon skeletons. Investigation of extracts from the sponge Darwinella cf. oxeata yielded four new rearranged diterpenoids, oxeatine (2) and oxeatamides H-J (3-5), as well as the known metabolites oxeatamide A (6), oxeatamide A methyl ester (7), and membranolide (1). Oxeatine (2) has a new heterocyclic skeleton, while oxeatamide J (5) has an N-methyl urea group included in a γ-lactam moiety. UPLC-QTOF analysis of the extract obtained from the mantle of the nudibranch Felimida grahami indicated the presence of 1 and 4.

Time course of lead induced proteomic changes in gill of the Antarctic limpet Nacella Concinna (Gastropoda: Patellidae).

The effect of increasing levels of metals from anthropogenic sources on Antarctic invertebrates is poorly understood. Here we exposed limpets (Nacella concinna) to 0, 0.12 and 0.25 µg L− 1 lead for 12, 24, 48 and 168 h. We subsequently quantified the changes in protein abundance from gill, using 2D gel electrophoresis and mass spectrometry. We identified several antioxidant proteins, including the metal binding Mn-superoxide dismutase and ferritin, increasing abundances early on. Chaperones involved in the redox-dependent maturation of proteins in the endoplasmic reticulum (ER) showed higher abundance with lead at 48 h. Lead also increased the abundance of Zn-binding carbonic anhydrase at 12 h, suggesting a challenge to acid-base balance. Metabolic proteins increased abundance at 168 h, suggesting a greater ATP demand during prolonged exposure. Changes in abundance of the small G-protein cdc42, a signaling protein modifying cytoskeleton, increased early and subsequently reversed during prolonged exposure, possibly leading to the modification of thick filament structure and function. We hypothesize that the replacement of metals initially affected antioxidant proteins and increased the production of reactive oxygen species. This disrupted the redox-sensitive maturation of proteins in the ER and caused increased ATP demand later on, accompanied by changes in cytoskeleton. SIGNIFICANCE: Proteomic analysis of gill tissue in Antarctic limpets exposed to different concentrations of lead (Pb) over a 168 h time period showed that proteomic changes vary with time. These changes included an increase in the demand of scavenging reactive oxygen species, acid-base balance and a challenge to protein homeostasis in the endoplasmic reticulum early on and subsequently an increase in energy metabolism, cellular signaling, and cytoskeletal modifications. Based on this time course, we hypothesize that the main mode of action of lead is a replacement of metal-cofactors of key enzymes involved in the scavenging of reactive oxygen species and the regulation of acid-base balance.

Methanolic extract of Euchelus asper exhibits in-ovo anti-angiogenic and in vitro anti-proliferative activities

BACKGROUND: The marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME). METHODS: The anti-angiogenic activity of EAME (50-800 µg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histo-logical investigations. In vitro testing of EAME (5-20 µg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student's t test for two unpaired groups. RESULTS: Analysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9). CONCLUSION: Overall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.

Determination of the toxic variability of lipophilic biotoxins in marine bivalve and gastropod tissues treated with an industrial canning process.

Contamination of shellfish with lipophilic marine biotoxins (LMB), pectenotoxins (PTXs), yessotoxins (YTXs) and okadaic acid (OA) toxin groups in southern Chile is a constant challenge for the development of miticulture considering the high incidence of toxic episodes that tend to occur. This research is focused on using methodologies for assessing the decrease in toxins of natural resources in Chile with high value, without altering the organoleptic properties of the shellfish. The species were processed through steaming (1 min at 121°C) and subsequent canning (5 min at 121°C). Changes in the profiles of toxins and total toxicity levels of LMB in endemic bivalves and gastropods were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total reduction of toxicity (≈ 15%) was not related to the destruction of the toxin, but rather to the loss of LMB on removing the shells and packing media of canned products (***p < 0.001). Industrial processing of shellfish reduces LMB contents by up to 15% of the total initial contents, concomitant only with the interconversion of PTX-group toxins into PTX-2sa. In soft bottom-dwelling species with toxicities beyond the standard for safe human consumption (≥ 160 µg OA-eq kg-1), toxicity can be reduced to safe levels through industrial preparation procedures.