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This paper presents the findings of a recent reconnaissance survey of the reinforced concrete (RC) construction industry in Australia. The survey involved the review of the structural documentation of a set of case study buildings constructed in Australia. The types of walls and observed methods of detailing are documented. Upper and lower bound values for typical wall attributes have been presented which researchers can utilise to design laboratory test specimens or perform parametric computer studies, to ensure their study accurately represents real-world conditions. A literature review of experimental testing of the lateral in-plane capacity of RC walls is included. The literature review identified 81 experimental studies, in which 501 RC wall test specimens have been tested worldwide. It is shown that very little experimental studies have been performed on RC walls that match what is being constructed in Australia. [ABSTRACT FROM PUBLISHER]

The underwater sound distribution generated by natural sources, shipping and trawling activities has been computed by the Quonops© modelling webservice for the Northern Adriatic Sea (NAS) during 2020, a year characterized by the COVID-19 lockdown restrictions. Modelling has been calibrated by using a year-long time series of field measurements covering the domain of interest. Sound levels (50th percentile) ranged between 75 and 90 dB re 1μPa for all the considered frequencies (63 Hz, 125 Hz, 250 Hz third octave bands). Noisier NAS areas match with the shipping lanes and the distribution of trawling activity. Pressure sound indices based on masking effect were computed for two Ecologically/Biologically Significant Marine Areas (EBSA) located in the NAS. Results indicated a significant contribution of vessel and fishery-generated noise to the local soundscape and provide a basis for addressing NAS underwater noise pollution, with special reference to the Marine Spatial Planning approach., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: This study performs an exploratory analysis of current-future sustainability challenges for ocean planning for the regional seas of Catalonia located in the Western Mediterranean (Spain)., Methods: To address the challenges we develop an Maritime Spatial Planning (MSP)-oriented geodatabase of maritime activities and deploy three spatial models: 1) an analysis of regional contribution to the 30% protection commitment with Biodiversity Strategy 2030; 2) a spatial Maritime Use Conflict (MUC) analysis to address current and future maritime activities interactions and 3) the StressorGenerator QGIS application to locate current and anticipate future sea areas of highest anthropogenic stress., Results & Conclusions: Results show that the i) study area is one of the most protected sea areas in the Mediterranean (44-51% of sea space protected); ii) anthropogenic stressors are highest in 1-4 nautical miles coastal areas, where maritime activities agglomerate, in the Gulf of Roses and Gulf of Saint Jordi. iii) According to the available datasets commercial fishery is causing highest conflict score inside protected areas. Potential new aquaculture sites are causing highest conflict in Internal Waters and the high potential areas for energy cause comparably low to negligible spatial conflicts with other uses. We discuss the added value of performing regional MSP exercises and define five challenges for regional ocean sustainability, namely: Marine protection beyond percentage, offshore wind energy: a new space demand, crowded coastal areas, multi-level governance of the regional sea and MSP knowledge gaps., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Depellegrin D et al.)

Anthropogenic underwater noise is an emergent pollutant. Despite several worldwide monitoring programs, only few data are available for the Mediterranean Sea, one of the global biodiversity hotspots. The results of the first continuous acoustic programme run at a transnational basin scale in the Mediterranean Sea are here presented. Recordings were done from March 2020 to June 2021, including the COVID-19 lockdown, at nine stations in the Northern Adriatic Sea. Spatial-temporal variations of the underwater sound are described, having one third octave band sound pressure levels (SPLs) from 10 Hz to 20 kHz as metrics. Higher and more variable SPLs, mainly related to vessel traffic, were found close to harbours, whereas Natura 2000 stations experienced lower SPLs. Lower values were recorded during the lockdown in five stations. Median yearly SPLs ranged between 64 and 95 as well as 70 and 100 dB re 1 µPa for 63 and 125 Hz bands, respectively. These values are comparable with those previously found in busy shallow EU basins but higher levels are expected during a business-as-usual period. This is a baseline assessment for a highly impacted and environmental valuable area, that needs to be managed in a new sustainable blue growth strategy., (© 2023. The Author(s).)

The protection of marine habitats from human-generated underwater noise is an emerging challenge. Baseline information on sound levels, however, is poorly available, especially in the Mediterranean Sea. To bridge this knowledge gap, the SOUNDSCAPE project ran a basin-scale, cross-national, long-term underwater monitoring in the Northern Adriatic Sea. A network of nine monitoring stations, characterized by different natural conditions and anthropogenic pressures, ensured acoustic data collection from March 2020 to June 2021, including the full lockdown period related to the COVID-19 pandemic. Calibrated stationary recorders featured with an omnidirectional Neptune Sonar D60 Hydrophone recorded continuously 24 h a day (48 kHz sampling rate, 16 bit resolution). Data were analysed to Sound Pressure Levels (SPLs) with a specially developed and validated processing app. Here, we release the dataset composed of 20 and 60 seconds averaged SPLs (one-third octave, base 10) output files and a Python script to postprocess them. This dataset represents a benchmark for scientists and policymakers addressing the risk of noise impacts on marine fauna in the Mediterranean Sea and worldwide., (© 2023. The Author(s).)

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99-100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Aquaculture is the fastest growing food production industry in the world yet research and guidance demonstrating strategic multi-objective zoning for sector expansion is scarce. Quantifying and mitigating conflicts and impact on sensitive coastal environments through jointly-optimized objectives for aquaculture and biodiversity simultaneously has not been tested yet. We here develop and evaluate six alternative planning scenarios for one of the European Union's highest priority bivalve shellfish aquaculture areas, the Emilia-Romagna Region in Italy. We i) develop an aquaculture profitability surface as a function of the distance from main ports, and in parallel build a fine-scale aquaculture suitability distribution surface for important commercial species using multi-criteria analysis; ii) prioritize protected areas for biodiversity while testing how different considerations of human impacts influence priorities; iii) simultaneously plan for aquaculture and biodiversity while minimizing impacts on other maritime activities. We compare results from different scenarios according to how well they capture suitable aquaculture habitats and minimize impacts. We introduce a new evaluation method for scenario comparison in spatial optimization using a nearest-neighbor analysis for spatial pattern similarities. Lastly, we test the "value of information" provided by our investment in developing the fine-scale suitability surface to improve efficiencies. We find that an integrated multi-objective zoning approach, which simultaneously optimizes for biodiversity and aquaculture, supports more efficient planning than traditional sector specific growth strategies. We also discovered that the fine-scale suitability model delivered a 5% more efficient solution than the simple distance function, highlighting the role of proxy cost surfaces and diminished returns from investing in comprehensive habitat suitability analysis in regions without much variation in key parameters. We offer evidence of improved efficiency and practical guidance for integrated planning in Blue Growth agendas. Our analysis can be applied in any context where multiple objectives occur for aquaculture sector growth and biodiversity conservation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The North Sea basin is one of the busiest maritime areas globally with a considerable number of anthropogenic pressures impacting the functioning of the marine ecosystem. Due to growing EU ambitions for the deployment of large offshore wind farm projects (OWF), as part of the 2050 renewable energy roadmap, there is a key need for a holistic understanding of OWF potential impacts on the marine ecosystem. We propose a holistic Cumulative Effect Assessment methodology, applied using a geo-spatial open-source software, to assess impacts of OWF related pressures on selected seabed habitats, fish, seabird and mammal species. We take into account pressures specific to the three OWF development phases, spanning 1999-2050, for the entire North Sea basin. Our results underline 2022 as the peak year of cumulative impacts for the approved OWFs, followed by a considerable increase in potential impacts of the planned 212GWs, by 2050. The spatio-temporal analysis of the OWF environmental impacts presents the shift between highly impacted areas over the studied timeline and distinguishes between concentrated areas of high impacts (S-E of UK) and dispersed areas of high impacts (Germany). Our results can inform decision-makers and the OWF industry in a joint effort to mitigate the environmental impacts of future large OWF developments.

Purpose: A growing number of studies on animal models have demonstrated that some ocular diseases are the result of the interaction between hyalocytes and the ocular inflammatory setting. Endogenous and exogenous substances might alter the structure and behavior of hyalocytes that can contribute to the pathogenesis of some ocular diseases. Obtaining primary cultures of human hyalocytes could help understand the role of these cells in response to different treatments., Methods: Hyalocytes were isolated from eyes of 54 patient volunteers subjected to vitrectomy for different clinical reasons. By testing different matrices and growth media, we reproducibly generated primary cultures of hyalocytes that we characterized morphologically and biologically, basally and upon treatment with several agents (basic fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGF-β), platelet-derived growth factor subunit-BB (PDGF-BB), ascorbic acid, dexamethasone, and hydrogen peroxide)., Results: We were able to generate primary cultures from vitreous human samples, growing the cells on collagen-coated plates in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum; primary cells expressed the hyalocyte markers. Specific cytoskeletal modifications were observed upon treatment with bFGF, TGF-β, PDGF-BB, ascorbic acid, dexamethasone, and hydrogen peroxide. Only bFGF was able to promote cell growth and hyaluronic acid production., Conclusions: We describe for the first time the generation and the characterization of primary cultures of human hyalocytes from living donors. Although human hyalocytes share some characteristics with animal hyalocytes, human hyalocytes have their own features typical of the species, confirming how important human experimental models are for investigating human pathologies and their treatments., (Copyright © 2020 Molecular Vision.)

The spread of coronavirus (COVID-19) caused an unprecedented implementation of lockdown measures across world's nations. Veneto Region, located in North-Eastern Adriatic Sea was one of the first maritime regions in Italy and Europe subjected to progressive lockdown restrictions. We systematically analyse the effects of national lockdown policies on maritime settings of the region using Automated Identification System (AIS) data from fishing vessels, passenger ships, tanker and cargo vessels collected through the Aqua Alta Oceanographic Tower (AAOT). We derive consequences on vessel activities during the March-April 2020 lockdown, by using a data-driven, comparative spatio-temporal analysis of vessel trajectories. Results show that compared to the same period of 2017, vessel activity were reduced by 69% during the lockdown, fishing activities reduced by 84% and passenger traffic by 78%. We register a restart of fishing activity in the third week of April 2020. We suggest that the presented conceptual and spatial assessment protocol can guide future research on environmental and socio-economic effects of COVID-19 on marine realms and contribute to further interdisciplinary research with other marine scientific fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interests to this manuscript., (Copyright © 2020. Published by Elsevier B.V.)

Gastric cancer is the world's third leading cause of cancer mortality. In spite of significant therapeutic improvements, the clinical outcome for patients with advanced gastric cancer is poor; thus, the identification and validation of novel targets is extremely important from a clinical point of view. We generated a wide, multilevel platform of gastric cancer models, comprising 100 patient-derived xenografts (PDX), primary cell lines, and organoids. Samples were classified according to their histology, microsatellite stability, Epstein-Barr virus status, and molecular profile. This PDX platform is the widest in an academic institution, and it includes all the gastric cancer histologic and molecular types identified by The Cancer Genome Atlas. PDX histopathologic features were consistent with those of patients' primary tumors and were maintained throughout passages in mice. Factors modulating grafting rate were histology, TNM stage, copy number gain of tyrosine kinases/ KRAS genes, and microsatellite stability status. PDX and PDX-derived cells/organoids demonstrated potential usefulness to study targeted therapy response. Finally, PDX transcriptomic analysis identified a cancer cell-intrinsic microsatellite instability (MSI) signature, which was efficiently exported to gastric cancer, allowing the identification, among microsatellite stable (MSS) patients, of a subset of MSI-like tumors with common molecular aspects and significant better prognosis. In conclusion, we generated a wide gastric cancer PDX platform, whose exploitation will help identify and validate novel "druggable" targets and optimize therapeutic strategies. Moreover, transcriptomic analysis of gastric cancer PDXs allowed the identification of a cancer cell-intrinsic MSI signature, recognizing a subset of MSS patients with MSI transcriptional traits, endowed with better prognosis. SIGNIFICANCE: This study reports a multilevel platform of gastric cancer PDXs and identifies a MSI gastric signature that could contribute to the advancement of precision medicine in gastric cancer., (©2019 American Association for Cancer Research.)

Coastal tourism is a growing industry sector in the Mediterranean Basin. This and the other human activities occurring along the coastline share space and resources, leading to conflicts for divergent uses. Moreover, the overexploitation of natural resources degrades and depletes coastal habitats, with negative feedback effects for all human activities. Hence, both tourism and the other human activities have to consider their dependence on coastal ecosystem services, and act at technical and policy level to reach a compromise that preserves natural resources in the long term. Here we provide a conceptual framework illustrating the complex relationships and trade-offs among threats from coastal tourism and from other human activities and coastal ecosystem services, with a focus on cultural ones. We discuss the negative feedbacks on tourism development and provide examples of geospatial analysis on cumulative threats generated by other human activities and affecting tourism itself. The proposed conceptual framework and the threat analysis aim at highlighting the negative feedback effects of human driven threats on the development of Mediterranean coastal tourism, through an ecosystem service perspective. Both tools provide valuable insight for supporting decision makers and planners in achieving integrated coastal management, with a focus on sustainable tourism., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Negative allosteric modulators (NAMs) of the metabotropic glutamate receptor 5 (mGlu 5 ) hold great promise for the treatment of a variety of central nervous system disorders. We have recently reported that prop-2-ynylidenecycloalkylamine derivatives are potent and selective NAMs of the mGlu 5 receptor. In this work, we explored the amide, carbamate, sulfonamide, and urea derivatives of prop-2-ynylidenecycloalkylamine compounds with the aim of improving solubility and metabolic stability. In silico and experimental analyses were performed on the synthesized series of compounds to investigate structure-activity relationships. Compounds 12, 32, and 49 of the carbamate, urea, and amide classes, respectively, showed the most suitable cytochrome inhibition and metabolic stability profiles. Among them, compound 12 showed excellent selectivity, solubility, and stability profiles as well as suitable in vitro and in vivo pharmacokinetic properties. It was highly absorbed in rats and dogs and was active in anxiety, neuropathic pain, and lower urinary tract models.

The Adriatic and Ionian Region is an important area for both strategic maritime development and biodiversity conservation in the European Union (EU). However, given that both EU and non-EU countries border the sea, multiple legal and regulatory frameworks operate at different scales, which can hinder the coordinated long-term sustainable development of the region. Transboundary marine spatial planning can help overcome these challenges by building consensus on planning objectives and making the trade-offs between biodiversity conservation and its influence on economically important sectors more explicit. We address this challenge by developing and testing 4 spatial prioritization strategies with the decision-support tool Marxan, which meets targets for biodiversity conservation while minimizing impacts to users. We evaluated these strategies in terms of how priority areas shift under different scales of target setting (e.g., regional vs. country level). We also examined the trade-off between cost-efficiency and how equally solutions represent countries and maritime industries (n = 14) operating in the region with the protection-equality metric. We found negligible differences in where priority conservation areas were located when we set targets for biodiversity at the regional versus country scale. Conversely, the prospective impacts on industries, when considered as costs to be minimized, were highly divergent across scenarios and biased the placement of protection toward industries located in isolation or where there were few other industries. We recommend underpinning future marine spatial planning efforts in the region through identification of areas of national significance, transboundary areas requiring cooperation between countries, and areas where impacts on maritime industries require careful consideration of the trade-off between biodiversity conservation and socioeconomic objectives., (© 2018 Society for Conservation Biology.)

This paper presents the Tools4MSP software package, a Python-based Free and Open Source Software (FOSS) for geospatial analysis in support of Maritime Spatial Planning (MSP) and marine environmental management. The suite was initially developed within the ADRIPLAN data portal, that has been recently upgraded into the Tools4MSP Geoplatform (data.tools4msp.eu), an integrated web platform that supports MSP through the application of different tools, e.g., collaborative geospatial modelling of cumulative effects assessment (CEA) and marine use conflict (MUC) analysis. The package can be used as stand-alone library or as collaborative webtool, providing user-friendly interfaces appropriate to decision-makers, regional authorities, academics and MSP stakeholders. An effective MSP-oriented integrated system of web-based software, users and services is proposed. It includes four components: the Tools4MSP Geoplatform for interoperable and collaborative sharing of geospatial datasets and for MSP-oriented analysis, the Tools4MSP package as stand-alone library for advanced geospatial and statistical analysis, the desktop applications to simplify data curation and the third party data repositories for multidisciplinary and multilevel geospatial datasets integration. The paper presents an application example of the Tools4MSP GeoNode plugin and an example of Tools4MSP stand-alone library for CEA in the Adriatic Sea. The Tools4MSP and the developed software have been released as FOSS under the GPL 3 license and are currently under further development., Competing Interests: The authors declare there are no competing interests., (©2018 Menegon et al.)

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

This research presents a set of multi-objective spatial tools for sea planning and environmental management in the Adriatic Sea Basin. The tools address four objectives: 1) assessment of cumulative impacts from anthropogenic sea uses on environmental components of marine areas; 2) analysis of sea use conflicts; 3) 3-D hydrodynamic modelling of nutrient dispersion (nitrogen and phosphorus) from riverine sources in the Adriatic Sea Basin and 4) marine ecosystem services capacity assessment from seabed habitats based on an ES matrix approach. Geospatial modelling results were illustrated, analysed and compared on country level and for three biogeographic subdivisions, Northern-Central-Southern Adriatic Sea. The paper discusses model results for their spatial implications, relevance for sea planning, limitations and concludes with an outlook towards the need for more integrated, multi-functional tools development for sea planning., (Copyright © 2017. Published by Elsevier B.V.)

Maritime spatial planning (MSP) is envisaged as a tool to apply an ecosystem-based approach to the marine and coastal realms, aiming at ensuring that the collective pressure of human activities is kept within acceptable limits. Cumulative impacts (CI) assessment can support science-based MSP, in order to understand the existing and potential impacts of human uses on the marine environment. A CI assessment includes several sources of uncertainty that can hinder the correct interpretation of its results if not explicitly incorporated in the decision-making process. This study proposes a three-level methodology to perform a general uncertainty analysis integrated with the CI assessment for MSP, applied to the Adriatic and Ionian Region (AIR). We describe the nature and level of uncertainty with the help of expert judgement and elicitation to include all of the possible sources of uncertainty related to the CI model with assumptions and gaps related to the case-based MSP process in the AIR. Next, we use the results to tailor the global uncertainty analysis to spatially describe the uncertainty distribution and variations of the CI scores dependent on the CI model factors. The results show the variability of the uncertainty in the AIR, with only limited portions robustly identified as the most or the least impacted areas under multiple model factors hypothesis. The results are discussed for the level and type of reliable information and insights they provide to decision-making. The most significant uncertainty factors are identified to facilitate the adaptive MSP process and to establish research priorities to fill knowledge gaps for subsequent planning cycles. The method aims to depict the potential CI effects, as well as the extent and spatial variation of the data and scientific uncertainty; therefore, this method constitutes a suitable tool to inform the potential establishment of the precautionary principle in MSP.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second cause of cancer-related death. Search for genes/proteins whose expression can discriminate between normal and neoplastic liver is fundamental for diagnostic, prognostic and therapeutic purposes. Currently, the most used in vitro hepatocyte models to study molecular alterations underlying transformation include primary hepatocytes and transformed cell lines. However, each of these models presents limitations. Here we describe the isolation and characterization of two rat hepatocyte cell lines as tools to study liver carcinogenesis. Long-term stable cell lines were obtained from a HCC-bearing rat exposed to the Resistant-Hepatocyte protocol (RH cells) and from a rat subjected to the same model in the absence of carcinogenic treatment, thus not developing HCCs (RNT cells). The presence of several markers identified the hepatocytic origin of both cell lines and confirmed their purity. Although morphologically similar to normal primary hepatocytes, RNT cells were able to survive and grow in monolayer culture for months and were not tumorigenic in vivo. On the contrary, RH cells displayed tumor-initiating cell markers, formed numerous colonies in soft agar and spheroids when grown in 3D and were highly tumorigenic and metastatic after injection into syngeneic rats and immunocompromised mice. Moreover, RNT gene expression profile was similar to normal liver, while that of RH resembled HCC. In conclusion, the two cell lines here described represent a useful tool to investigate the molecular changes underlying hepatocyte transformation and to experimentally demonstrate their role in HCC development.

NRF2 has been traditionally considered as a tumor suppressor because its cytoprotective functions are deemed to be the main cellular defense mechanism against exogenous and endogenous insults, including xenobiotics and oxidative stress. However, several recent studies demonstrate that hyperactivation of the NRF2 pathway creates an environment that favors the survival of normal as well as malignant cells, protecting them against oxidative stress, chemotherapeutic agents, and radiotherapy. In a rapidly advancing field, this review summarizes some of the known mechanisms by which NRF2 can exert its oncogenic functions, and describes the current status of NRF2 inhibitors, providing a clear rationale for the consideration of NRF2 as a powerful putative therapeutic target in cancer treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Metabolic changes are associated with cancer, but whether they are just bystander effects of deregulated oncogenic signaling pathways or characterize early phases of tumorigenesis remains unclear. Here we show in a rat model of hepatocarcinogenesis that early preneoplastic foci and nodules that progress towards hepatocellular carcinoma (HCC) are characterized both by inhibition of oxidative phosphorylation (OXPHOS) and by enhanced glucose utilization to fuel the pentose phosphate pathway (PPP). These changes respectively require increased expression of the mitochondrial chaperone TRAP1 and of the transcription factor NRF2 that induces the expression of the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase (G6PD), following miR-1 inhibition. Such metabolic rewiring exclusively identifies a subset of aggressive cytokeratin-19 positive preneoplastic hepatocytes and not slowly growing lesions. No such metabolic changes were observed during non-neoplastic liver regeneration occurring after two/third partial hepatectomy. TRAP1 silencing inhibited the colony forming ability of HCC cells while NRF2 silencing decreased G6PD expression and concomitantly increased miR-1; conversely, transfection with miR-1 mimic abolished G6PD expression. Finally, in human HCC patients increased G6PD expression levels correlates with grading, metastasis and poor prognosis. Our results demonstrate that the metabolic deregulation orchestrated by TRAP1 and NRF2 is an early event restricted to the more aggressive preneoplastic lesions., Competing Interests: No conflict of interest to declare.

Background & Aims: l-2-Hydroxy acid oxidases are flavin mononucleotide-dependent peroxisomal enzymes, responsible for the oxidation of l-2-hydroxy acids to ketoacids, resulting in the formation of hydrogen peroxide. We investigated the role of HAO2, a member of this family, in rat, mouse and human hepatocarcinogenesis., Methods: We evaluated Hao2 expression by qRT-PCR in the following rodent models of hepatocarcinogenesis: the Resistant-Hepatocyte, the CMD and the chronic DENA rat models, and the TCPOBOP/DENA and TCPOBOP only mouse models. Microarray and qRT-PCR analyses were performed on two cohorts of human hepatocellular carcinoma (HCC) patients. Rat HCC cells were transduced by a Hao2 encoding lentiviral vector and grafted in mice., Results: Downregulation of Hao2 was observed in all investigated rodent models of hepatocarcinogenesis. Interestingly, Hao2 mRNA levels were also profoundly downregulated in early preneoplastic lesions. Moreover, HAO2 mRNA levels were strongly downregulated in two distinct series of human HCCs, when compared to both normal and cirrhotic peri-tumoral liver. HAO2 levels were inversely correlated with grading, overall survival and metastatic ability. Finally, exogenous expression of Hao2 in rat cells impaired their tumorigenic ability., Conclusion: Our work identifies for the first time the oncosuppressive role of the metabolic gene Hao2. Indeed, its expression is severely decreased in HCC of different species and etiology, and its reintroduction in HCC cells profoundly impairs tumorigenesis. We also demonstrate that dysregulation of HAO2 is a very early event in the development of HCC and it may represent a useful diagnostic and prognostic marker for human HCC., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Unlabelled: Hepatocellular carcinoma (HCC) develops through a multistage process, but the nature of the molecular changes associated with the different steps, the very early ones in particular, is largely unknown. Recently, dysregulation of the NRF2/KEAP1 pathway and mutations of these genes have been observed in experimental and human tumors, suggesting their possible role in cancer development. To assess whether Nrf2/Keap1 mutations are early or late events in HCC development, we investigated their frequency in the rat Resistant Hepatocyte model, consisting of the administration of diethylnitrosamine followed by a brief exposure to 2-acetylaminofluorene. This model enables the dissection of all stages of hepatocarcinogenesis. We found that Nrf2/Keap1 mutations were present in 71% of early preneoplastic lesions and in 78.6% and 59.3% of early and advanced HCCs, respectively. Mutations of Nrf2 were more frequent, missense, and located in the Nrf2-Keap1 binding region. Mutations of Keap1 occurred at a much lower frequency in both preneoplastic lesions and HCCs and were mutually exclusive with those of Nrf2. Functional in vitro and in vivo studies showed that Nrf2 silencing inhibited the ability of tumorigenic rat cells to grow in soft agar and to form tumors. Unlike Nrf2 mutations, those of Ctnnb1, which are frequent in human HCC, were a later event as they appeared only in fully advanced HCCs (18.5%)., Conclusion: In the Resistant Hepatocyte model of hepatocarcinogenesis the onset of Nrf2 mutations is a very early event, likely essential for the clonal expansion of preneoplastic hepatocytes to HCC, while Ctnnb1 mutations occur only at very late stages. Moreover, functional experiments demonstrate that Nrf2 is an oncogene critical for HCC progression and development., (© 2015 by the American Association for the Study of Liver Diseases.)

Metabotropic glutamate receptor 5 (mGlu5) is a biological target implicated in major neurological and psychiatric disorders. In the present study, we have investigated structural determinants of the interaction of negative allosteric modulators (NAMs) with the seven-transmembrane (7TM) domain of mGlu5. A homology model of the 7TM receptor domain built on the crystal structure of the mGlu1 template was obtained, and the binding modes of known NAMs, namely MPEP and fenobam, were investigated by docking and molecular dynamics simulations. The results were validated by comparison with mutagenesis data available in the literature for these two ligands, and subsequently corroborated by the recently described mGlu5 crystal structure. Moreover, a new series of NAMs was synthesized and tested, providing compounds with nanomolar affinity. Several structural modifications were sequentially introduced with the aim of identifying structural features important for receptor binding. The synthesized NAMs were docked in the validated homology model and binding modes were used to interpret and discuss structure-activity relationships within this new series of compounds. Finally, the models of the interaction of NAMs with mGlu5 were extended to include important non-aryl alkyne mGlu5 NAMs taken from the literature. Overall, the results provide useful insights into the molecular interaction of negative allosteric modulators with mGlu5 and may facilitate the design of new modulators for this class of receptors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unlabelled: Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that mediate most of the effects elicited by the thyroid hormone, 3,5,3'-L-triiodothyronine (T3). TRs have been implicated in tumorigenesis, although it is unclear whether they act as oncogenes or tumor suppressors, and at which stage of tumorigenesis their dysregulation occurs. Using the resistant-hepatocyte rat model (R-H model), we found down-regulation of TRβ1 and TRα1 and their target genes in early preneoplastic lesions and hepatocellular carcinoma (HCCs), suggesting that a hypothyroid status favors the onset and progression of preneoplastic lesions to HCC. Notably, TRβ1 and, to a lesser extent, TRα1 down-regulation was observed only in preneoplastic lesions positive for the progenitor cell marker, cytokeratin-19 (Krt-19) and characterized by a higher proliferative activity, compared to the Krt-19 negative ones. TRβ1 down-regulation was observed also in the vast majority of the analyzed human HCCs, compared to the matched peritumorous liver or to normal liver. Hyperthyroidism induced by T3 treatment caused up-regulation of TRβ1 and of its target genes in Krt-19(+) preneoplastic rat lesions and was associated with nodule regression. In HCC, TRβ1 down-regulation was not the result of hypermethylation of its promoter, but was associated with an increased expression of TRβ1-targeting microRNAs ([miR]-27a, -181a, and -204). An inverse correlation between TRβ1 and miR-181a was also found in human cirrhotic peritumoral tissue, compared to normal liver., Conclusion: Down-regulation of TRs, especially TRβ1, is an early and relevant event in liver cancer development and is species and etiology independent. The results also suggest that a hypothyroid status of preneoplastic lesions may contribute to their progression to HCC and that the reversion of this condition may represent a possible therapeutic goal to interfere with the development of this tumor., (© 2014 by the American Association for the Study of Liver Diseases.)

Brugada syndrome is characterised by a typical ECG with ST segment elevation in the right precordial leads. Individuals with this condition are susceptible to ventricular arrhythmias and sudden cardiac death. The principal gene responsible for this syndrome is SCN5A, which encodes the α-subunit of the Nav1.5 voltage-gated sodium channel. Mutations involving other genes have been increasingly reported, but their contribution to Brugada syndrome has been poorly investigated. Here we focused on the SCN1B gene, which encodes the β1-subunit of the voltage-gated sodium channel and its soluble β1b isoform. SCN1B mutations have been associated with Brugada syndrome as well as with other cardiac arrhythmias and familial epilepsy. In this study, we have analysed SCN1B exons (including the alternatively-spliced exon 3A) and 3'UTR in 145 unrelated SCN5A-negative patients from a single centre. We took special care to report all identified variants (including polymorphisms), following the current nomenclature guidelines and considering both isoforms. We found two known and two novel (and likely deleterious) SCN1B variants. We also found two novel changes with low evidence of pathogenicity. Our findings contribute more evidence regarding the occurrence of SCN1B variants in Brugada syndrome, albeit with a low prevalence, which is in agreement with previous reports.

Unlabelled: Studies on gene and/or microRNA (miRNA) dysregulation in the early stages of hepatocarcinogenesis are hampered by the difficulty of diagnosing early lesions in humans. Experimental models recapitulating human hepatocellular carcinoma (HCC) are then used to perform this analysis. We performed miRNA and gene expression profiling to characterize the molecular events involved in the multistep process of hepatocarcinogenesis in the resistant-hepatocyte rat model. A high percentage of dysregulated miRNAs/genes in HCC were similarly altered in early preneoplastic lesions positive for the stem/progenitor cell marker cytokeratin-19, indicating that several HCC-associated alterations occur from the very beginning of the carcinogenic process. Our analysis also identified miRNA/gene-target networks aberrantly activated at the initial stage of hepatocarcinogenesis. Activation of the nuclear factor erythroid related factor 2 (NRF2) pathway and up-regulation of the miR-200 family were among the most prominent changes. The relevance of these alterations in the development of HCC was confirmed by the observation that NRF2 silencing impaired while miR-200a overexpression promoted HCC cell proliferation in vitro. Moreover, T3-induced in vivo inhibition of the NRF2 pathway accompanied the regression of cytokeratin-19-positive nodules, suggesting that activation of this transcription factor contributes to the onset and progression of preneoplastic lesions towards malignancy. The finding that 78% of genes and 57% of dysregulated miRNAs in rat HCC have been previously associated with human HCC as well underlines the translational value of our results., Conclusion: This study indicates that most of the molecular changes found in HCC occur in the very early stages of hepatocarcinogenesis. Among these, the NRF2 pathway plays a relevant role and may represent a new therapeutic target., (© 2013 by the American Association for the Study of Liver Diseases.)

5-HT(1A) receptor and α(1)-adrenoreceptor (α(1)-AR) binding sites recognized by the 1,4-dioxanes 2-4 display reversed stereochemical requirements. (S)-2 proved to be a potent 5-HT(1A) receptor agonist highly selective over α(1)-AR subtypes. Chirality influenced the anticancer activity of 3 and 4 in human prostate cancer cells (PC-3): (R)-4, eutomer at the α(1d)-AR subtype, was the most potent. The decreased effect of 4 and (R)-4 in α(1d)-AR silenced PC-3 cells confirmed that their anticancer activity was α(1d)-AR-dependent.

Twenty X-chromosomal short tandem repeat (STR) loci were typed in 80 families of Italian descent, composed by mother and two or more sons, for a total of 93 meiosis. The analyzed X-STR panel included six clusters of closely linked markers (each spanning<3cM): DXS10135-DXS10148-DXS8378 (Xp22); DXS7132-DXS10074-DXS10079 (Xq12); DXS6801-DXS6809-DXS6789 (Xq21); DXS7424-DXS101 (Xq22); DXS10103-HPRTB-DXS10101 (Xq26); DXS8377-DXS10134-DXS7423-DXS10146 (Xq28). Recombination fractions between pairs of markers calculated by pedigree analysis were compared with those obtained from the second-generation Rutgers combined linkage-physical map of the human genome. The observed differences confirm that recombination is not homogeneous along the X chromosome and that the conventional subdivision of X-STRs in four groups of completely unlinked markers cannot be regarded as true. Significant linkage disequilibrium was found between markers DXS6801 and DXS6809 (p=0.017). The effect on likelihood calculations of inferring haplotype frequencies from allele distributions rather than haplotype count in the relevant population was evaluated., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Twenty-one X-chromosomal short tandem repeat loci, including the six clusters of linked markers DXS10148-DXS10135-DXS8378 (Xp22), DXS7132-DXS10074-DXS10079 (Xq12), DXS6801-DXS6809-DXS6789 (Xq21), DXS7424-DXS101 (Xq22), DXS10103-HPRTB-DXS10101 (Xq26), DXS8377-DXS10146-DXS10134-DXS7423-DXS10011 (Xq28), and the loci DXS6800 and GATA172D05 were typed in a northwestern Algerian population sample (n = 210; 104 men and 106 women). Allele and haplotype frequencies were calculated. No evidence of linkage disequilibrium was observed between pairs of loci within clusters of linked markers. At locus DXS10148, sequence analysis of a subset of alleles displaying unusual amplicon length (>/= 36 repeat units) and anomalous electrophoretic mobility showed that this marker has a complex molecular structure with different repeat variants within alleles of identical amplicon size.

In the frame of the international SOILSAMP project, funded and coordinated by the National Environmental Protection Agency of Italy (APAT), uncertainties due to field soil sampling were assessed. Three different sampling devices were applied in an agricultural area using the same sampling protocol. Cr, Sc and Zn mass fractions in the collected soil samples were measured by k(0)-instrumental neutron activation analysis (k(0)-INAA). For each element-device combination the experimental variograms were calculated using geostatistical tools. The variogram parameters were used to estimate the standard uncertainty arising from sampling. The sampling component represents the dominant contribution of the measurement uncertainty with a sampling uncertainty to measurement uncertainty ratio ranging between 0.6 and 0.9. The approach based on the use of variogram parameters leads to uncertainty values of the sampling component in agreement with those estimated by replicate sampling approach.

Synthesis and biological activity of a novel class of quaternary ammonium salt muscarinic M3 receptor antagonists, showing high selectivity versus the M2 receptor, are described. Selected compounds exhibited potent anticholinergic properties, in isolated guinea-pig trachea, and good functional selectivity for trachea over atria. In vivo, the same compounds potently inhibited acetylcholine-induced bronchoconstriction after intratracheal administration in the guinea pig.