Enhancement of the Cytotoxic Effect of Anticancer Agent by Cytochrome c Functionalised Hybrid Nanoparticles in Hepatocellular Cancer Cells

Treatment of hepatocellular cancer with chemotherapeutic agents has limited success in clinical practice and their efficient IC50 concentration would require extremely high doses of drug administration which could not be tolerated due to systemic side effects. In order to potentiate the efficacy of anticancer agents we explored the potential of co-treatment with pro-apoptotic Cytochrome c which activates the apoptotic pathway downstream of p53 that is frequently mutated in cancer. To this end we used hybrid iron oxide-gold nanoparticles as a drug delivery system to facilitate the internalisation of Cytochrome c into cultured HepG2 hepatocellular carcinoma cells. Our results showed that Cytochrome c can be easily conjugated to the gold shell of the nanoparticles which are readily taken up by the cells. We used Cytochrome c in concentration (0.2µgmL-1) below the threshold required to induce apoptosis on its own. When the conjugate was administered to cells treated by doxorubicin, it significantly reduced its IC50 concentration from 9µgmL-1 to 3.5µgmL-1 as detected by cell viability assay, and the efficiency of doxorubicin on decreasing viability of HepG2 cells was significantly enhanced in the lower concentration range between 0.01µgmL-1 to 5µgmL-1. The results demonstrate the potential of the application of therapeutic proteins in activating the apoptotic pathway to complement conventional chemotherapy to increase its efficacy. The application of hybrid iron oxide-gold nanoparticles can also augment the specificity of drug targeting and could serve as a model drug delivery system for pro-apoptotic protein targeting and delivery

Intercalibrating the national classifications of ecological status for Eastern Continental lakes: Biological Quality Element: Benthic invertebrates

The European Water Framework Directive (WFD) requires the national classifications of good ecological status to be harmonised through an intercalibration exercise. In this exercise, significant differences in status classification among Member States are harmonized by comparing and, if necessary, adjusting the good status boundaries of the national assessment methods. Intercalibration is performed for rivers, lakes, coastal and transitional waters, focusing on selected types of water bodies (intercalibration types), anthropogenic pressures and Biological Quality Elements. Intercalibration exercises were carried out in Geographical Intercalibration Groups - larger geographical units including Member States with similar water body types - and followed the procedure described in the WFD Common Implementation Strategy Guidance document on the intercalibration process (European Commission, 2011). The Technical reports are organized in volumes according to the water category (rivers, lakes, coastal and transitional waters), Biological Quality Element and Geographical Intercalibration group. This volume addresses the intercalibration of the Eastern Continental Lake GIG Benthic invertebrate ecological assessment methods. Three countries (Bulgaria, Hungary, Romania) participated in the intercalibration exercise and harmonised their lake benthic invertebrate systems. The results were approved by the WG ECOSTAT and included in the EC Decision on intercalibration (European Commission, 2018).JRC.D.2-Water and Marine Resource

Developments in water quality monitoring and management in large river catchments using the Danube River as an example

Effective management of water quality in large rivers requires information on the influence of activities within the catchment (urban and rural) throughout the whole river basin. However, traditional water quality monitoring programmes undertaken by individual agencies normally relate to specific objectives, such as meeting quality criteria for wastewater discharges, and fail to provide information on basin-scale impacts, especially in transboundary river basins. Ideally, monitoring in large international river basins should be harmonised to provide a basin-scale assessment of sources and impacts of human activities, and the effectiveness of management actions. This paper examines current water quality issues in the Danube River basin and evaluates the approach to water quality monitoring in the context of providing information for a basin-wide management plan. Lessons learned from the monitoring programme in the Danube are used to suggest alternative approaches that could result in more efficient generation of water quality data and provide new insights into causes and impacts of variations in water quality in other large international river basins

Best practice for establishing nutrient concentrations to support good ecological status

The EU Member States, Norway and the European Commission in 2000 have jointly developed a common implementation strategy (CIS) for implementing Directive 2000/60/EC, the Water Framework Directive (WFD) to ensure consistent implementation. The focus is on developing a common understanding of the technical and scientific implications of the WFD. One of the objectives is the development of non-legally binding and practical Guidance Documents on various technical issues of the Directive. These are targeted at experts who are directly or indirectly implementing the WFD in river basins. The structure, presentation and terminology are therefore adapted to their needs and formal, legalistic language is avoided wherever possible. In 2009 CIS Guidance on Eutrophication Assessment (Guidance Document No. 23) was published, providing guidance for evaluating the impacts of nutrient enrichment, a major cause of failure to achieve good status under the WFD. However, an apparently wide range of nutrient boundary values to support good ecological status had been established by the Member States. Water Directors requested that the CIS Working Group ECOSTAT investigate this issue, and the subsequent work has been led by the UK (Freshwaters), Germany (Saline waters) and JRC. The aim of the work was to establish the reasons for differences between Member States in the development and application of nutrient boundaries, leading to the production of this guidance on best practice. This work is an addition to, and not a replacement for, the earlier guidance on eutrophication assessment. In developing this guidance, a number of tasks have been undertaken. The range of nitrogen and phosphorus boundary values in use by Member States, and the methods used to derive those values has been reported separately, for both fresh and saline waters. Further work was undertaken to investigate nutrient pressure-biological response relationships in the different surface water categories. This work was then used to inform the development of this guide and the associated statistical toolkit. During the project a series of workshops were held involving nutrient experts nominated by Member States. These experts contributed to the development and testing of the guidance and toolkit, and provided details of alternative methods of boundary setting in use in some Member States. The purpose of this report is to provide technical guidance to enable Member States to establish new, or review existing, boundaries for phosphorus and nitrogen to support good ecological status. This should facilitate the establishment of comparable and consistent boundaries across all Member States. However it is recognised that alternative methods of arriving at boundary values may be valid, and use of this guidance and the associated statistical toolkit is ultimately a decision for the Member State. The responses of biological elements to nutrient availability are complex, and vary between water categories. This guidance is not therefore a substitute for the application of ecological knowledge and understanding at a local level. Furthermore, responses to nutrients may be confounded by the impact of other pressures acting on a water body, and our understanding of how to account for multiple stressors is still developing. The guidance does not specifically address how the nutrient boundaries are used to derive an overall classification, or to drive action to control nutrients, both of which may be relevant to the level at which the boundaries are set.JRC.D.2-Water and Marine Resource

Intercalibrating the national classifications of ecological status for very large rivers in Europe: Biological Quality Element: Phytoplankton

The European Water Framework Directive (WFD) requires the national classifications of good ecological status to be harmonised through an intercalibration exercise. In this exercise, significant differences in status classification among Member States are harmonized by comparing and, if necessary, adjusting the good status boundaries of the national assessment methods. Intercalibration is performed for rivers, lakes, coastal and transitional waters, focusing on selected types of water bodies (intercalibration types), anthropogenic pressures and Biological Quality Elements. Intercalibration exercises were carried out in Geographical Intercalibration Groups - larger geographical units including Member States with similar water body types - and followed the procedure described in the WFD Common Implementation Strategy Guidance document on the intercalibration process (European Commission, 2011). The Technical reports are organized in volumes according to the water category (rivers, lakes, coastal and transitional waters), Biological Quality Element and Geographical Intercalibration group. This volume addresses the intercalibration of the Very large river Phytoplankton ecological assessment methods. Thirteen countries (Austria, Belgium (Flanders), Bulgaria, Croatia, Czech Republic, Estonia, Germany, Hungary, Latvia, Lithuania, Poland, Romania, Slovakia) participated in the intercalibration exercise and harmonised their benthic invertebrate assessment systems. The results were approved by the WG ECOSTAT and included in the EC Decision on intercalibration (European Commission, 2018). In addition, four countries (Italy, Finland, Norway, Sweden) provided justification for excluding Phytoplankton BQE assessment system.JRC.D.2-Water and Marine Resource

Enhancement of the Cytotoxic Effect of Anticancer Agent by Cytochrome c Functionalised Hybrid Nanoparticles in Hepatocellular Cancer Cells

Treatment of hepatocellular cancer with chemotherapeutic agents has limited success in clinical practice and their efficient IC50 concentration would require extremely high doses of drug administration which could not be tolerated due to systemic side effects. In order to potentiate the efficacy of anticancer agents we explored the potential of co-treatment with pro-apoptotic Cytochrome c which activates the apoptotic pathway downstream of p53 that is frequently mutated in cancer. To this end we used hybrid iron oxide-gold nanoparticles as a drug delivery system to facilitate the internalisation of Cytochrome c into cultured HepG2 hepatocellular carcinoma cells. Our results showed that Cytochrome c can be easily conjugated to the gold shell of the nanoparticles which are readily taken up by the cells. We used Cytochrome c in concentration (0.2µgmL-1) below the threshold required to induce apoptosis on its own. When the conjugate was administered to cells treated by doxorubicin, it significantly reduced its IC50 concentration from 9µgmL-1 to 3.5µgmL-1 as detected by cell viability assay, and the efficiency of doxorubicin on decreasing viability of HepG2 cells was significantly enhanced in the lower concentration range between 0.01µgmL-1 to 5µgmL-1. The results demonstrate the potential of the application of therapeutic proteins in activating the apoptotic pathway to complement conventional chemotherapy to increase its efficacy. The application of hybrid iron oxide-gold nanoparticles can also augment the specificity of drug targeting and could serve as a model drug delivery system for pro-apoptotic protein targeting and delivery

Estimating river nutrient concentrations consistent with good ecological condition: More stringent nutrient thresholds needed

Nutrient pollution remains one of the leading causes of river degradation, making it important to set thresholds that support good ecological condition, which is the main objective of managing Europe's aquatic environment. A wide range of methods has been used by European member states to set river nutrient thresholds in the past, and these vary greatly among countries, even for similar river types. In some countries, thresholds have been set using expert judgement or the statistical distribution of nutrient concentrations. Application of such thresholds creates problems for planning strategies to achieve good ecological status and for managing transboundary river basins. An alternative approach is to examine the statistical relationship between nutrient concentration and one, or more, biological variables. Such relationships can then be used to inform decisions by water managers. We use such 'ecology-based' approaches (univariate regression and mismatch analyses) to derive nutrient thresholds for several river types in Central Europe. Our analysis focused on soluble reactive phosphorus (SRP) and total nitrogen (TN), two variables which were responsible for significant variation (40–55%) in river benthic floras. In this study, for the first time, river nutrient thresholds are estimated using both macrophytes and phytobenthos (EQRs) separately and in combination, calculated as the minimum and the average of the EQRs of the two sub-elements. The resulting thresholds supporting good ecological status range from 21 to 42 µg/L SRP and 0.9–3.5 mg/L TN for the low alkalinity lowland river type, and 32–90 µg/L SRP and 1.0–2.5 mg/L TN for the low alkalinity mid-altitude river type. These targets are compared to the values set by member states. We demonstrate that some national nutrient thresholds fall within the range of predicted values if uncertainty is taken into consideration; however, several threshold values considerably exceed this range. Adopting ecology-based nutrient targets should improve sustainable river management where nutrients are the major pressure preventing the achievement of good ecological status.JRC.D.2-Water and Marine Resource

Application of eDNA method in the detection of Cordulegaster (Insecta: Odonata) species

The aim of this pilot study was to investigate the potential of eDNA techniques to detect the presence of the two dragonfly species Cordulegaster heros and Cordulegaster bidentata. Both species are classified as “near threatened” according to the IUCN Red List and are strictly protected in several countries. Monitoring these species with traditional sampling methods is often difficult, time-consuming and invasive. In this pilot study, we first collected tissue samples from C. heros and C. bidentata to sequence the traditional DNA-barcode gene fragment COI. We then collected further dragonfly COI sequences from BOLD to design species-specific primers. This, however, was impossible given the enormous variability of COI. Therefore, we refrained from species-specific eDNA assays and followed eDNA metabarcoding protocol using universal (BF2/BF2) and a newly designed dragonfly specific primer. For the evaluation of the method, we took water samples from places where Cordulegaster specimens are known to occur. After the extraction, we used two sequential PCR steps for obtaining the desired amplicon (two-step PCR) using universal primers in the first step, and group (dragonfly) specific primers or universal primers. Amplicons were sequenced on an Illumina MiSeq platform and then analysed the data with the JAMP pipeline. With the newly designed primers and we could effectively detect the targeted dragonfly species from tissue samples, and also from filtered environmental samples. The detection of the species with the traditional method is time consuming and involves the destruction of the specimens. In comparison, with the eDNA method we could easily detect these near threatherned odonates and other dragonfly species in a non-invasive way