Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni

Schistosomiasis, one of the world's greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs. METHODOLOGY/PRINCIPAL FINDINGS: The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery. CONCLUSIONS: The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies

Investigating the antiparasitic potential of the marine sesquiterpene avarone, its reduced form avarol, and the novel semisynthetic thiazinoquinone analogue thiazoavarone

The chemical analysis of the sponge Dysidea avara afforded the known sesquiterpene quinone avarone, along with its reduced form avarol. To further explore the role of the thiazinoquinone scaffold as an antiplasmodial, antileishmanial and antischistosomal agent, we converted the quinone avarone into the thiazinoquinone derivative thiazoavarone. The semisynthetic compound, as well as the natural metabolites avarone and avarol, were pharmacologically investigated in order to assess their antiparasitic properties against sexual and asexual stages of Plasmodium falciparum, larval and adult developmental stages of Schistosomamansoni (eggs included), and also against promastigotes and amastigotes of Leishmania infantum and Leishmania tropica. Furthermore, in depth computational studies including density functional theory (DFT) calculations were performed. A toxic semiquinone radical species which can be produced starting both from quinone- and hydroquinone-based compounds could mediate the anti-parasitic effects of the tested compounds

Italian Mutual Banks: Performance, Efficiency and Mergers and Acquisitions

1) Are Italian mutual banks efficient? Evidence from two different cost frontier techniques, by Juan Sergio Lopez, Alessandra Appenini, and Stefania P.S. Rossi. The aim of the first paper is to analyze the efficiency of co-operative banks in Italy. The increasing competition induced by the ongoing process of liberalization in Europe has been affecting also these small financial institutions that used to operate in a more protected environment. Based on a panel of about 450 banks covering the period 1995-99, two different techniques were employed: non-parametric frontier analysis, and parametric frontier analysis. By means of this analysis it is possible to compare the results obtained using these two methodologies and analyze the determinants of bank inefficiency 2) Mergers and acquisitions between mutual banks in Italy: an analysis of the effects on performance and productive efficiency, by Roberto Di Salvo, Maria Carmela Mazzilis, and Andrea Guidi. The second paper is aimed at testing the hypothesis that the M&A wave over the past ten years has increased the level of efficiency of co-operative credit banks (CCBs), both in terms of overall performance and productive efficiency. The logical development is hinged on two steps: 1) an explorative analysis which is based on the observation of balance sheet ratios by quantiles, 2) a DEA application for estimating productive efficiency scores. The analysis refers to 94 CCBs which have been involved in M&As over the period 1995-98 and is carried out on both merged and non-merged banks, either before concentration or in the subsequent years. The explorative analysis mainly shows a higher level of fee-based income for merged banks, which is consistent with the hypothesis that concentration strategies enhance diversification. It also detects some degree of cost reduction just after merging. The DEA application models (CRS and VRS) tends to confirm the results of the previous analysis and estimates higher efficiency for merged banks, a lower efficiency degree for pre-merger banks, and a significant degree of scale econonomies.

Valutazione della tossicità acuta sul tessuto retinico dopo somministrazione di aminoglicosidi e fluorochinoloni in un modello sperimentale animale su ratto albino.

Titolo della tesi: Valutazione della tossicità acuta sul tessuto retinico dopo somministrazione di aminoglicosidi e fluorochinoloni in un modello sperimentale animale su ratto albino. Introduzione – L'occhio è un organo sferoidale, posto nella cavità orbitaria, direttamente a contatto con l’ambiente esterno e pur essendo protetto dalle palpebre e da altri annessi oculari è soggetto all’attacco di numerosi batteri gram-positivi e gram-negativi. Le infezioni oculari più frequenti sono la congiuntivite, la cheratite e l’endoftalmite. La terapia ad oggi più utilizzata nelle patologie oftalmiche batteriche è costituita da due classi di antibiotici: aminoglicosidi e fluorochinoloni, in particolare da Tobramicina, Netilmicina (aminoglicosidi) e Norfloxacina (fluorochinolone). In letteratura sono noti alcuni aspetti circa la tossicità oculare a carico di alcuni aminoglicosidi mentre mancano studi dettagliati circa la possibile tossicità oculare di fluorochinoloni. Scopo della ricerca – Nel nostro studio abbiamo voluto valutare le eventuali alterazioni retiniche causate dalla Tobramicina, dalla Netilmicina e dalla Norfloxacina dopo somministrazione topica in acuto, in un modello sperimentale su ratto. Abbiamo quindi valutato l'attività elettrica della retina in risposta a particolari stimoli luminosi, il numero delle file di fotorecettori presenti nello strato nucleare esterno della retina e l’intera morfologia retinica. Materiali e metodi – Sono stati utilizzati 18 ratti maschi Wistar (Harlan, Italy) adulti sani, con un peso di circa 250g ± 25g che sono stati randomizzati nei seguenti gruppi sperimentali:1.Gruppo trattato con Norfloxacina; 2.Gruppo trattato con Tobramicina; 3.Gruppo trattato con Netilmicina. Gli animali di ciascun gruppo hanno ricevuto il rispettivo farmaco topicamente nell’occhio destro, 2 gocce applicate 6 volte al giorno per 5 giorni, mentre l’occhio sinistro, usato come controllo, è stato trattato con soluzione fisiologica con identica modalità di somministrazione. Prima dell’inizio del trattamento, dopo la prima applicazione e a fine trattamento è stato effettuato l’ERG da flash per andare a valutare l’attività elettrica della retina. Subito dopo l’ultima registrazione elettroretinografica gli animali sono stati anestetizzati, operati per l’asportazione di entrambi gli occhi e sacrificati. Abbiamo poi isolato il tessuto retinico per l’allestimento dei preparati istologici. Le retine sono state fissate, incluse e criosezionate seguendo procedure standard. Parte delle sezioni sono state colorate con ioduro di propidio per valutare la morfologia dello strato esterno plessiforme di cellule nervose della retina, andando a contare il numero di file di nuclei presenti in tale strato. Le sezioni rimanenti sono state colorate con ematossilina-eosina in modo da valutare la morfologia dell’intero tessuto retinico. Risultati – Le registrazioni elettroretinografiche prima del trattamento, dopo 2h, e dopo 5gg di somministrazione topica di Norfloxacina, di Netilmicina e di Tobramicina, non evidenziano alterazioni significative dell’ampiezza dell’onda b, se confrontati con il controllo. Limitando l’osservazione al tracciato dell’occhio che ha ricevuto Tobramicina si osserva una riduzione dell’ampiezza dell’onda b. Tale riduzione è tuttavia non significativa e costituisce, al momento, solo un trend. Dalla valutazione isto-patologica dello strato nucleare esterno della retina non si osservano diminuzioni, in numero, delle file di cellule nervose successivamente a somministrazione dei tre antibiotici. La morfologia retinica non evidenzia alterazioni patologiche successivamente a somministrazione dei tre antibiotici. Conclusioni – Dai risultati sperimentali ottenuti possiamo concludere che la somministrazione topica di Netilmicina e Norfloxacina risulta priva di effetti collaterali sulla visione, infatti non si evidenziano variazioni delle ampiezze dell’onda b dopo 5gg; mentre è necessario utilizzare con cautela la Tobramicina; dal momento che è stato rilevato un trend negativo nell’andamento delle ampiezze dell’onda b dopo 5gg, Tuttavia dato che da un punto di vista morfologico, per nessuno dei tre farmaci, non sono state rilevate alterazioni, per escludere il verificarsi di degenerazione del tessuto retinico si rendono necessarie ulteriori future analisi con tempi di somministrazione più prolungati

Towards zero marine plastic pollution: a multidimensional measurement model for evaluating waste management solutions informed by SDGs 12 and 14

Purpose: This paper explores how marine plastic waste management solutions can be holistically evaluated in alignment with Sustainable Development Goals (SDGs) 12 and 14. It aims to address the fragmentation in the existing literature by developing an integrated model for sustainability assessment that combines cost-benefit analysis (CBA) and multi-criteria decision analysis (MCDA). Design/methodology/approach: Presenting an interventionist case study, the research examines a project aimed at collecting plastic waste from the sea and constructing a pyrolysis plant to convert non-recyclable plastics into energy in a major Italian port. A multidimensional evaluation model was developed through iterative stakeholder engagement, integrating financial and non-financial criteria across economic, environmental, and social dimensions to assess the investment. Data were gathered through semi-structured interviews, document analysis and field observations. Findings: The study introduces an integrated CBA–MCDA framework tailored to the complexities of sustainability-oriented investments. The model enables a comprehensive assessment of infrastructure projects by combining economic valuation with participatory evaluation of qualitative impacts. It also strengthens alignment with the targets of SDGs 12 and SDG 14 by incorporating criteria related to waste recovery, emissions reduction and social outcomes. Originality/value: This research contributes to the literature by operationalising the interdependence between SDGs 12 and 14 within a unified decision-making framework. It advances existing approaches by demonstrating how integrated methodologies can support investment decisions in contexts where environmental, economic, and social objectives converge, offering a replicable model for sustainability evaluation in waste management

New provisions for the labelling of fishery and aquaculture products: Difficulties in the implementation of Regulation (EU) n. 1379/2013

The European Union (EU), within the renewal plan of the Common Fisheries Policy and the Common Market Organization, with the Cape IV of Reg. (EU) n. 1379/2013 have introduced new requirements for the labelling of fisheries and aquaculture products. These, as well as providing consumers with more complete information, integrate the provisions of Reg. (EU) n. 1169/2011 and acts as a tool to prevent frauds and illegal fishing. In this work the new seafood labelling provisions were evaluated, starting from the analysis of the art. 35 of the Chapter IV and comparing it with the previous EU dispositions (Reg. (EC) no. 104/2000 and no. 2065/2001). The exclusion of prepared and processed products and aquatic invertebrates from the application of the mandatory seafood labelling provisions and the role of the mass caterer operators with respect to the labelling requirements were identified as the two major shortcomings that still need to be better addressed by the legislator. Overall, what emerged from this work is that, if on the one hand the European legislation on seafood labelling has achieved important goals, evolving and improving itself, on the other it is still controversial and plagued by the same problems as 15 years ago. Therefore, the authors suggest that the regulation is modified at least extending its scope to all products and to at all stages of the fishery logistic chai

Occurrence and viability of Anisakis spp. larvae in ready to eat products made of herring (Clupea harengus) sold in Italy

The study aimed to assess the occurrence and viability of Anisakis spp. larvae in ready to eat products made of herring, a common host of Anisakis spp. and the third most commercialized fish species in the EU. A total of 135 products consisting of 50 smoked whole specimens and 85 filleted products (25 smoked, 30 marinated, 30 canned) were sampled from 2016 to 2018. Viscera and muscle of whole herrings were visually inspected and separately digested. Filleted products were also visually inspected and digested. Larvae viability was assessed, then they were counted and microscopically identified to genus level. A subsample was molecularly identified. At least one Anisakis spp. larva was found in 56 products (41.5%), for a total of 1715 larvae collected (0-172 larvae/product). Most of the larvae (91%) were found in the viscera of 49 of the 50 whole herrings (98%). A highly significant difference was observed between the positivity rate and larval density at muscle level, as 149 larvae were found in the muscle of 31 whole herrings (positivity rate 62%, 0.022 larval density/g), while only 7 larvae were found in the 85 filleted products (positivity rate 7%, 0.001 larval density/g). Larvae were molecularly identified as A. simplex. The study showed that obviously contaminated products are commercialized. All the larvae were dead, proving a negligible risk of developing anisakiasis. However, their allergenic potential is debated and the significant difference between muscle infection levels in whole and filleted herrings may result in a different risk of exposure to antigens

A Conventional Multiplex PCR Assay for the Detection of Toxic Gemfish Species (Ruvettus pretiosus and Lepidocybium flavobrunneum): A Simple Method to Combat Health Frauds

The meat of Ruvettus pretiosus and Lepidocybium flavobrunneum (gemfishes) contains high amounts of indigestible wax esters that provoke gastrointestinal disorders. Although some countries have banned the sale of these species, mislabeling cases have been reported in sushi catering. This work developed a simple conventional multiplex PCR, which discriminates the two toxic gemfishes from other potentially replaced species, such as tunas, cod, and sablefish. A common degenerate forward primer and three species-specific reverse primers were designed to amplify cytochrome oxidase subunit I (COI) gene regions of different lengths (479, 403, and 291 bp) of gemfishes, tunas, and sablefish, respectively. A primer pair was designed to amplify a fragment (193 bp) of the cytb gene of cod species. Furthermore, a primer pair targeting the 16S rRNA gene was intended as common positive control (115 bp). The method developed in this study, by producing the expected amplicon for all of the DNA samples tested (reference and commercial), provides a rapid and reliable response in identifying the two toxic species to combat health frauds

Fish species identification in canned pet food by BLAST and Forensically Informative Nucleotide Sequencing (FINS) analysis of short fragments of the mitochondrial 16s ribosomal RNA gene (16S rRNA)

Nowadays, pet food claiming high-valued fish among ingredients is largely available on the market. Unfortunately, the modifications induced by processing make species identification by visual inspection difficult and hinder the enforcement of the legislation on traceability. In this work, after aligning 819 sequences of Clupeidae, Engraulidae, Salangidae and Scombridae families, we developed new universal primers for the amplification and sequencing of 2 short fragments (±118 and ±213) of the mitochondrial 16s ribosomal RNA (16S rRNA) gene. Once tested on 130 DNA reference samples, these primers were used in the analysis of highly degraded DNA extracted from 43 canned cat food containing whole minnows (whitebait) (M) and tuna, or bonito or mackerel fillets (F). Three M and 2 F samples were analyzed for each can. A BLAST and a FINS analysis, the latter performed only on the 118 bp fragment, were performed separately on the sequences obtained from M and F samples. All the M samples were identified at the species or genus level by both BLAST and FINS analysis. This allowed to highlight an impressive rate of mislabeling (100%). F samples, for which FINS was less performing in species identification, resulted mislabeled in 40% of the products

Multiple DNA BARCODING for fish species identification in sushi products

The aim of this work was to perform a molecular survey based on DNA barcoding to identify the seafood species used in the preparation of ethnic products (sushi). Twenty-one raw products (each composed of 3 to 8 pieces, for a total of 88 samples) were purchased in ethnic restaurants in the provinces of Pisa (11), Lucca (2), Livorno (3) and Florence (5). The total DNA extracted (1) was evaluated by gel electrophoresis and amplified using universal primers for mitochondrial (COI, 16SrRNA) or nuclear genes (PEPCK) depending on the species (fish, mollusk or crustacean) and the level of DNA degradation. Different primers (2,3,4,5,6,7) for the amplification of a long (~700 bp) or a short (~139-200 bp) fragment were used. Ninety-five PCR products were obtained (for some products two genes were analyzed). Of these, 30 have already been sequenced (Experimental Zooprophylactic Institute of Latium and Tuscany (Rome)). The sequences were elaborated with Clustal W in Bioedit 7.0.9.0, and analyzed by a BLAST analysis on GenBank and by using the Identification System on BOLD. A top match with a sequence similarity of at least 98% was used to designate potential species identification (8). DNA was degraded in almost one third of the samples. This was probably due to rice acidification, to repeated cycles of freezing/thawing or to prolonged storage. The degradation was confirmed by PCR amplification. In fact, we obtained long amplicons in 72.6% of the cases (n=69) and short amplicons for 27.3% of the samples (n=26). The average length of the long sequences was 595 bp for the COI FDB and 490 bp for the PEPCK gene, while the length of the short sequences was ~210bp for the 16S rRNA and 139bp for the COI MDB. All the samples were identified at least at the genus level, with identity values ranging from 99 to 100%. Although for some samples it was impossible to achieve a specific identification, the results were informative enough to verify the information given by the producers. No samples were found mislabeled. Even though the COI gene represents the most exploited target for seafood species identification, issues were found during amplification and comparison with the databases. Thus, in order to increase the PCR output, new universal primers, able to amplify a wide range of taxa, would be desirable. Finally, in case of degraded DNA samples, where the number of diagnostic mutation is limited, a multiple gene analysis is advisable