"Food Village": An Innovative Alternative Food Network Based on Human Scale Development Economic Model.

Although the different alternative food networks (AFNs) have experienced increases worldwide for the last thirty years, they are still unable to provide an alternative capable of spreading on a large scale. They in fact remain niche experiments due to some limitations on their structure and governance. Thus, this study proposes and applies a design method to build a new sustainable food supply chain model capable of realizing a "jumping scale". Based on the theoretical and value framework of the Civil Economy (CE), the Economy for the Common Good (ECG), and the Development on a Human Scale (H-SD), the proposed design model aims to satisfy the needs of all stakeholders in the supply chain. Max-Neef's Needs Matrix and Design Thinking (DT) tools were used to develop the design model. Applying the design method to the food chain has allowed us to develop the concept of the "Food Village", an innovative food supply network far from the current economic mechanisms and based on the community and eco-sustainability.

Bioaccumulation of algal toxins and changes in physiological parameters in Mediterranean mussels from the North Adriatic Sea (Italy).

The Northwestern Adriatic Sea is a commercially important area in aquaculture, accounting for about 90% of the Italian mussel production, and it was subjected to recurring cases of mussel farm closures due to toxic algae poisoning. A spatial and temporal survey of four sites along the North Adriatic Sea coasts of Emilia Romagna (Italy) was undertaken to study the possible impairments of physiological parameters in Mytilus galloprovincialis naturally exposed to algal toxins. The sites were selected as part of the monitoring network for the assessment of algal toxins bioaccumulation by the competent Authority. Samples positive to paralytic shellfish toxins and to lipophilic toxins were detected through the mouse bioassay. Lipophilic toxins were assessed by HPLC. Decreasing yessotoxins (YTX) levels were observed in mussels from June to December, while homo-YTX contents increased concomitantly. Lysosome membrane stability (LMS), glutathione S-transferase and catalase activities, and multixenobiotic resistance (MXR)-related gene expressions were assessed as parameters related to the mussel health status and widely utilized in environmental biomonitoring. Levels of cAMP were also measured, as possibly involved in the algal toxin mechanisms of action. Low LMS values were observed in hemocytes from mussels positive to the mouse bioassay. MXR-related gene expressions were greatly inhibited in mussels positive to the mouse bioassay. Clear correlations were established between increasing homo-YTX contents (and decreasing YTX) and increasing cAMP levels in the tissues. Similarly, significant correlations were established between the increase of homo-YTX and cAMP levels, and the expressions of three MXR-related genes at submaximal toxin concentrations. In conclusion, YTXs may affect mussel physiological parameters, including hemocyte functionality, gene expression and cell signaling.

Complex toxin profile of Mytilus galloprovincialis from the Adriatic sea revealed by LC-MS.

This paper reports on the determination of toxin profile of mussels (Mytilus galloprovincialis) collected in November-December 2003 along the Emilia Romagna coasts (Italy) when a high concentration of Alexandrium ostenfeldii cells was detected in seawater. Detailed liquid chromatography-mass spectrometry (LC-MS) analyses were performed on the crude extracts in both selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes. They revealed that M. galloprovincialis had accumulated the three major spirolides produced by the alga, namely 13-desMethyl spirolide C, 13,19-didesMethyl spirolide C and 27-hydroxy-13,19-didesMethyl spirolide C, which fully accounted for toxicity of lipophilic extracts shown in mouse bioassay. Interestingly, yessotoxin (YTX) and its analogues were still present in mussel polar extracts but YTX itself was not the major toxin contained in mussels. The presence of pectenotoxin-2 seco acid (PTX-2sa) and its putative epimer was also assessed. The presence of azaspiracids, never reported from the Adriatic sea, as well as of diarrhetic shellfish poisoning toxins (okadaic acid, dinophysistoxins and OA esters) and domoic acid, long known as contaminants of Adriatic mussels, was also investigated.

Protein markers of algal toxin contamination in shellfish.

Filter-feeding bivalve molluscs are often contaminated by algal toxins. We have probed whether proteomic analysis of extracts from the digestive gland (DG) of mussels could be employed to identify biomarkers of contamination due to okadaic acid-group toxins. The protein extracts were obtained from 18 separate mussel samples and were analyzed by two-dimensional gel electrophoresis. When samples were divided into four different classes based on the content of OA-group toxins in the starting material, we found that two proteins varied as a function of OA contamination. By BLAST analysis, the two proteins were identified as a component of photosystem II and a subunit of NADH dehydrogenase. The analysis of peptide homologies showed that the peptide of photosystem II we detected in extracts from the DG of mussels contaminated by OA-group toxins is identical to its counterpart in Dinophysis algae, which are the producers of this group of toxins. We concluded that proteomic analysis can be used for the detection and identification of biomarkers of biotoxin contamination in shellfish, including both proteins expressed by the toxin producers and components that participate to the tissue response to the exogenous bioactive contaminant.

A cytolytic assay for the measurement of palytoxin based on a cultured monolayer cell line.

A cytolytic assay that could detect palytoxin and its congeners has been developed by the use of an established cell line grown as monolayer to replace the current hemolytic method. We used MCF-7 cells and cytolysis was measured by the release of cytosolic lactate dehydrogenase (LDH) in the buffer added to treated cells (culture supernatant). A dose-dependent increase in LDH activity in culture supernatants was detected when MCF-7 cells were exposed to palytoxin and its analogue ostreocin D. The cytolytic response induced by palytoxin and ostreocin D was specific for this group of compounds, acting on Na+/K+-ATPase, as it was prevented when cells were preincubated with ouabain. The specificity of our assay for palytoxin and its congeners was confirmed by the finding that cytolysis was not detected when MCF-7 cells were exposed to unrelated toxins such as maitotoxin, tetrodotoxin, okadaic acid, and yessotoxin, even in the case of compounds that elicit cytotoxic responses under our experimental conditions. Using extracts from biological materials after spiking with the palytoxin standard, we found a good correlation between palytoxin levels measured by our cytolytic assay and the expected values. Our cytolytic assay detected palytoxin in naturally contaminated materials, but estimates were significantly higher than the palytoxin contents determined by LC-MS, indicating that naturally contaminated materials contain biologically active palytoxin congeners. We conclude that our cytolytic assay based on the use of MCF-7 cell monolayers is a viable alternative to animal-based methods for the determination of palytoxin and its congeners in contaminated materials.

Desulfoyessotoxins from Adriatic mussels: a new problem for seafood safety control.

Two new desulfated yessotoxin (YTX) analogues were isolated from a toxic batch of Adriatic mussels collected in October 2004. Their stereostructures were elucidated through extensive NMR and MS-based analysis. The finding of these desulfocarboxyhomoYTXs in shellfish poses additional problems to those institutions entitled to control seafood safety, since desulfated YTXs fail the European Union method currently in force for checking toxicity in mollusks.

A slot blot procedure for the measurement of yessotoxins by a functional assay.

We originally developed a functional assay for the detection of yessotoxins (YTX) based on its capacity to induce dose-dependent changes in cellular levels of two marker proteins, consisting of E-cadherin and an E-cadherin fragment (ECRA100) in epithelial cells. The procedure is time-consuming and we have shortened it by a slot blot format, using antibodies recognizing two different epitopes of E-cadherin (HECD-1 and C20820), thereby discriminating those markers. The best performing membrane under our conditions, in terms of binding capacity and even absorption of proteins, was a positively charged nylon membrane. Treatment of the membrane with 0.5mug of Ab/ml was appropriate for maximal detection of antigens by our slot blot procedure with both HECD-1 and C20820 antibodies. The treatment of cells with YTX, resulting in a relative increase in the cellular levels of ECRA100, led to a dose-dependent increase of the signal detected by Ab HECD-1 without a concomitant increase in the signal detected by Ab C20820 in our slot blot format, and the concentrations of YTX were correlated to both the increase of the signal detected through Ab HECD-1 and to the decrease in the ratio of the signals obtained with the two Abs (C20820 over HECD-1). Upon analyses of extracts from cells treated with shellfish samples, we could detect and quantify YTX in naturally contaminated materials. The slot blot format of our functional assay allows a substantial shortening of its analytical step (about seven hr, as compared to the two working days of the original method), providing YTX measurements that are accurate but show large standard deviations.

Hydrophilic interaction liquid chromatography/mass spectrometry for determination of domoic acid in Adriatic shellfish.

This paper describes a new method for sensitive, specific and direct determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP) syndrome, in shellfish. It is based on combination of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC/MS). The high percentage of organic modifier in the mobile phase and the omission of ion-pairing reagents, both favoured in HILIC, result in enhanced detection limits with MS detection. The new method was set up either on an ionspray ion trap MS instrument operating in MS and MS/MS scanning acquisition modes, or on a turboionspray triple-quadrupole MS system operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes. Positive and negative ion experiments were performed. MRM experiments are recommended for screening contaminated shellfish tissue and for quantitative analyses due to highest sensitivity and selectivity. The minimum detection levels for the toxin in tissue were found to be 63 and 190 ng/g in positive and negative MRM experiments, respectively, which are well below the regulatory limit for DA in tissue (20 microg/g). Application to shellfish samples collected in the Adriatic Sea (Italy) in the period 2000-2004 demonstrated for the first time in Italy the presence of DA as a new toxin that has entered the Adriatic Mytilus galloprovincialis toxin profile.

Structure-activity relationships of yessotoxins in cultured cells.

The structure-activity relationship of yessotoxins (YTX) has been probed by measuring the potency of several YTX analogues to cause the accumulation of a 100 kDa MW fragment of E-cadherin in MCF-7 breast cancer cells. Under our experimental conditions, the EC(50) of YTX, the reference compound, was 0.55 nM. The introduction of a methylene unit adjacent to one of the sulfate groups, as is the case with the homoyessotoxin molecule, did not appear to greatly affect the potency of the analogue, as the measured EC(50) for this compound was 0.62 nM. The EC(50) values we measured for 45-hydroxyhomoyessotoxin and carboxyyessotoxin were about 9.4 and 26 nM, respectively, whereas the EC(50) of noroxoyessotoxin, lacking most of the C(9) chain, was about 50 nM. Thus, significant differences in the potencies of YTX analogues were found when structural changes involved the C(9) terminal chain of these compounds, leading to the conclusion that this portion of the molecule is essential for the activity of YTX in MCF-7 cells. A comparison of our findings with available information regarding the potency of YTX and its analogues in other experimental systems shows that the EC(50)'s we measured for the different compounds are up to 200-fold lower and vary in a wider concentration range. We speculate that YTX effects could involve two separate receptorial systems.

[Risk to human health associated with marine toxic algae]. / Il rischio sanitario associato alle tossine di alghe marine.

Among the about 5000 of marine algal species, some 75 produce algal toxins. These latter mainly belong to the taxa first of dynoflagellates and then diatomes. The so far known human intoxications have been associated with mollusc consumption. The main poisoning syndromes have been termed, on the basis of the observed symptoms, as paralytic, diarrhetic, neurotoxic and amnesic and shorthened as: PSP (paralytic shellfish poisoning), DSP (diarrhetic shellfish poisoning), NSP (neurotoxic shellfish poisoning), ASP (amnesic shellfish poisoning), respectively. This paper is a review of the problem of human health implications associated with marine toxic algae, with particular reference to the situation of the Mediterranean and the Italian coastal areas.

Functional assay to measure yessotoxins in contaminated mussel samples.

Yessotoxin (YTX) treatment of MCF-7 cells results in the accumulation of a 100-kDa fragment of E-cadherin (ECRA(100)) without a parallel loss of the intact protein in cytosoluble extracts. As a consequence, concentration-dependent increases in the total immunoreactivity detectable by anti-E-cadherin antibodies relative to controls (RTI) and in the relative immunoreactivity of ECRA(100) (RI) are observed. These responses have been exploited to develop a functional assay to measure YTX in samples from contaminated mussels by a three-step procedure, consisting of (i) treatment of MCF-7 cells with YTX standard in the concentration range 0-1nM and of unknown samples; (ii) preparation of cellular extracts, fractionation of proteins by polyacrylamide gel electrophoresis under denaturing conditions, and immunoblotting with anti-E-cadherin antibodies, followed by densitometric analyses of autoradiographies and calculation of RI of ECRA(100) and of RTI of the samples; and (iii) interpolation of the YTX concentrations in unknown samples on standard curves, by the RI of ECRA(100) and the RTI of the samples. The procedure has been used to measure yessotoxins in contaminated mussel samples, and the results obtained show that this functional assay is very sensitive (limit of detection of about 100ng equivalent YTX/g of digestive gland), and robust, as (i) it is insensitive to matrix effects in the range of toxin concentrations relevant for risk assessment to protect humans from exposure to YTX, (ii) calculations are based on a molecular parameter (the RI of ECRA(100)) which is not affected by errors in sample preparation, (iii) it can be performed by the use of antibodies commercially available from different companies, and (iv) it does not show an absolute need of calibration by a pure standard within each assay.

Structure and stereochemistry of a new cytotoxic polychlorinated sulfolipid from Adriatic shellfish.

A detailed analysis of the causative toxins contained in the hepatopancreas of toxic mussels from the northern Adriatic sea has been carried out. Along with some DSP (diarrhetic shellfish poisoning) type toxins, such as okadaic acid, yessotoxin, and their derivatives, which are involved in a number of human intoxications throughout the world, we have now isolated a new cytotoxin, a polychlorinated sulfolipid 1, whose gross structure has been elucidated by spectral analysis, including various 2D NMR techniques. The relative stereochemistry of 1 was elucidated by successful application of the J-based configuration analysis developed for acyclic compounds using carbon-proton spin-coupling constants ((2,3)J(C,H)) and proton-proton spin-coupling constants ((3)J(H,H)); its absolute stereochemistry was established by the Mosher method. Compound 1 possesses in vitro cytotoxicity against WEHI 164 and RAW 264.7 cells.

Direct detection of yessotoxin and its analogues by liquid chromatography coupled with electrospray ion trap mass spectrometry.

A liquid chromatography mass spectrometry (LC-MS) method is proposed for the sensitive, specific and direct detection of yessotoxin and its analogues, marine biotoxins which are associated with diarrhetic shellfish poisoning (DSP) and which have been found in the North Adriatic sea since 1995. The LC-MS method provided a detection limit of 70 pg for yessotoxin in full scan mode and was applied to determine the toxic profiles of a number of extracts or partially purified fractions of toxic mussels collected along the Emilia Romagna coasts (Italy) in the period 1995-1999. Detection of a desulfo-yessotoxin derivative from Mytilus galloprovincialis collected in 1998 is also reported.

The detection and identification of 42,43,44,45,46,47,55-heptanor-41-oxoyessotoxin, a new marine toxin from adriatic shellfish, by liquid chromatography-mass spectrometry.

The diarrhetic shellfish toxin composition in the digestive glands of mussels collected in June 2001 from the Northern Adriatic sea was investigated by high-performance liquid chromatography coupled with electrospray ion trap mass spectrometry. Along with known yessotoxins (1, 3-6), identified by comparison of their retention times and mass spectra with those of appropriate standards, a new marine toxin, 42,43,44,45,46,47,55-heptanor-41-oxoyessotoxin, 7, was detected. MS/MS experiments were used to gain structural information. 7 represents a new addition to the class of yessotoxins.

Cytotoxic responses to unfractionated extracts from digestive glands of mussels.

The contamination of bivalve molluscs by lipophylic toxins is mainly detected by the use of unfractionated extracts from contaminated material in mouse bioassays. The development of alternate detection methods based on the use of cultured cells is hampered by difficulties related to the complexity of matrices including toxic compounds obtained from contaminated material. In this paper we have used unfractionated lipid extracts prepared from the digestive gland of mussels, which gave a negative response by the mouse bioassay, and have investigated their effects on functioning of MCF-7 cells. We show that altered growth was induced after addition of lipid extracts corresponding to less than 1mg of digestive gland per ml of culture medium. The cytotoxic effect was also confirmed by the analysis of the effect of the unfractionated extracts on four selected proteins, which were used as markers of general (actin), regulatory (mitogen activated protein kinase isoforms ERK1 and ERK2), as well as differentiative (alpha isoform of estrogen receptor) functions of the MCF-7 human breast cancer cell line.