Amplification Free Detection of SARS-CoV-2 Using Multi-valent Binding

[Image: see text] We present the development of electrochemical impedance spectroscopy (EIS)-based biosensors for sensitive detection of SARS-CoV-2 RNA using multi-valent binding. By increasing the number of probe–target binding events per target molecule, multi-valent binding is a viable strategy for improving the biosensor performance. As EIS can provide sensitive and label-free measurements of nucleic acid targets during probe–target hybridization, we used multi-valent binding to build EIS biosensors for targeting SARS-CoV-2 RNA. For developing the biosensor, we explored two different approaches including probe combinations that individually bind in a single-valent fashion and the probes that bind in a multi-valent manner on their own. While we found excellent biosensor performance using probe combinations, we also discovered unexpected signal suppression. We explained the signal suppression theoretically using inter- and intra-probe hybridizations which confirmed our experimental findings. With our best probe combination, we achieved a LOD of 182 copies/μL (303 aM) of SARS-CoV-2 RNA and used these for successful evaluation of patient samples for COVID-19 diagnostics. We were also able to show the concept of multi-valent binding with shorter probes in the second approach. Here, a 13-nt-long probe has shown the best performance during SARS-CoV-2 RNA binding. Therefore, multi-valent binding approaches using EIS have high utility for direct detection of nucleic acid targets and for point-of-care diagnostics

Integrating ecosystem services and life cycle assessment: a framework accounting for local and global (socio-)environmental impacts

Purpose: Human activities put pressure on our natural ecosystems in various ways, such as globally through the spread of emissions or locally through the degradation of species-rich landscapes. However, life cycle assessment (LCA) studies that integrate ecosystem services (ES) are still in the minority because of intrinsic differences in data, modelling, and interpretation. This study aims to overcome these challenges by developing and testing a framework that comprehensively evaluates the (socio-)environmental impacts of human activities. Methods: LCA and ecosystem services assessment (ESA) were integrated in two different ways: (1) both methodologies run in parallel and results are combined, and (2) LCA as a driving method where ES are integrated. Because local ESA studies contain the most accurate information but will not be available for all processes in the value chain, it was necessary to advance the life cycle impact assessment method ReCiPe 2016 including three new midpoint impact categories (terrestrial provision, regulation, and cultural ES) and site-generic CFs based on the Ecosystem Services Valuation Database to account for changes in regulating, cultural and provisioning ES due to land use, for the remaining processes in the value chain. Monetary valuation is used to aggregate at the areas of protection (AoP). Results and discussion: A comprehensive LCA$_{+ES}$-ESA sustainability assessment framework is developed to account for local and global impacts due to human activities on three AoPs (natural resources, ecosystem quality, and human health and well-being), of which the results are expressed in monetary terms. The framework is able to visualize all benefits and burdens accounted for through the handprint/footprint approach. A simplified terrestrial case study on Scots pinewood shows the applicability of the proposed framework, resulting in a handprint (€$_{2022}$ 9.81E+02) which is four times larger than the footprint (€$_{2022}$ 2.31E+02) for 1 kg of wood produced. Challenges related to the framework such as data availability and database shortcomings (i.e., beyond land use) and ES interrelations are discussed. Conclusion: While classical LCA studies focus more on burdens, this framework can also take into account benefits, such as the provision of ecosystem services (or the value of the functional unit of the study). Although the integration of both LCA and ESA has been increasingly explored recently, until now no framework has been available that can incorporate results from local ESA, site-specific ESA, and classical LCA studies, which is considered highly relevant to decision-making

D1.4 Critical evaluation of material criticality and product-related circularity approaches

The deliverable presents a critical evaluation of existing approaches addressing material criticality and circularity

Laser ablation of polylactic acid sheets for the rapid prototyping of sustainable, single-use, disposable medical micro-components

The employment of single-use, disposable medical equipment has increased the amount of medical waste produced and the advent of point-of-care diagnostics in lab-on-chip format is likely to add further volume. Current materials used for the manufacture of these devices are derived from petroleum sources and are, therefore, unsustainable. In addition, disposal of these plastics necessitates combustion to reduce infection risk, which has, depending on material composition, an undesirable environmental impact. To address these issues, we have developed a general approach for the rapid prototyping of single-use point-of-care cartridges prepared from poly­(lactic acid), a sustainable material which can be milled and laser-cut as well as molded for translation to mass-market products. Here, the laser workability of poly­(lactic acid) sheets is reported together with examples of microfluidic components. Furthermore, the low molecular adsorption in laser-ablated poly­(lactic acid) channels and the compatibility of poly­(lactic acid) for common on-chip bioassays, such as polymerase chain reaction (PCR), are demonstrated. This innovative prototyping technique can be easily translated to high volume manufacturing and presents exciting opportunities for future sustainable microfluidic laboratories as well as potential for sustainable disposable single-use microcomponents for clinical applications

IL-17A/F-Signaling Does Not Contribute to the Initial Phase of Mucosal Inflammation Triggered by S. Typhimurium

Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium) causes diarrhea and acute inflammation of the intestinal mucosa. The pro-inflammatory cytokines IL-17A and IL-17F are strongly induced in the infected mucosa but their contribution in driving the tissue inflammation is not understood. We have used the streptomycin mouse model to analyze the role of IL-17A and IL-17F and their cognate receptor IL-17RA in S. Typhimurium enterocolitis. Neutralization of IL-17A and IL-17F did not affect mucosal inflammation triggered by infection or spread of S. Typhimurium to systemic sites by 48 h p.i. Similarly, Il17ra−/− mice did not display any reduction in infection or inflammation by 12 h p.i. The same results were obtained using S. Typhimurium variants infecting via the TTSS1 type III secretion system, the TTSS1 effector SipA or the TTSS1 effector SopE. Moreover, the expression pattern of 45 genes encoding chemokines/cytokines (including CXCL1, CXCL2, IL-17A, IL-17F, IL-1α, IL-1β, IFNγ, CXCL-10, CXCL-9, IL-6, CCL3, CCL4) and antibacterial molecules was not affected by Il17ra deficiency by 12 h p.i. Thus, in spite of the strong increase in Il17a/Il17f mRNA in the infected mucosa, IL-17RA signaling seems to be dispensable for eliciting the acute disease. Future work will have to address whether this is attributable to redundancy in the cytokine signaling network

Ex Vivo Expansion of Human CD8+ T Cells Using Autologous CD4+ T Cell Help

Background: Using in vivo mouse models, the mechanisms of CD4+ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4+ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4+ T cell help of CD8+ T cell proliferation using a novel in vitro model. Methods/Principal Findings: We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3+ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4+ T cells was associated with significantly improved CD8+ T cell expansion in vitro. The CD4+ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4+ T cell help of CD8+ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8+ T cells. Conclusions: We have developed an in vitro model that advances our understanding of the immunobiology of human CD4+ T cell help of CD8+ T cells. Our data suggests that human CD4+ T cell help can be leveraged to expand CD8+ T cells in vitro

Developmental roadmap for antimicrobial susceptibility testing systems

Antimicrobial susceptibility testing (AST) technologies help to accelerate the initiation of targeted antimicrobial therapy for patients with infections and could potentially extend the lifespan of current narrow-spectrum antimicrobials. Although conceptually new and rapid AST technologies have been described, including new phenotyping methods, digital imaging and genomic approaches, there is no single major, or broadly accepted, technological breakthrough that leads the field of rapid AST platform development. This might be owing to several barriers that prevent the timely development and implementation of novel and rapid AST platforms in health-care settings. In this Consensus Statement, we explore such barriers, which include the utility of new methods, the complex process of validating new technology against reference methods beyond the proof-of-concept phase, the legal and regulatory landscapes, costs, the uptake of new tools, reagent stability, optimization of target product profiles, difficulties conducting clinical trials and issues relating to quality and quality control, and present possible solutions

Hazardous substances and human health : exposure impact and external cost assessment at the European scale /

There is widespread public concern about hazardous chemicals that are contained in air, soil, water and food. Policy has therefore adopted a series of laws and regulations concerning emissions into and concentration levels in different media including food. As policy makers do not only have to consider the protection of the environment but also need to ensure a well-functioning economy at the same time, these limit or target values need to be set in a balanced way. The main problem, however, is to compare the costs for achieving these targets with the benefits to society by having a smaller exposure to hazardous substances (cost-benefit analysis). This book sets out to improve the reliability of cost-benefit analyses particularly of hazardous substances present in air, water, soil and food. It suggests that the human health risk assessment of chemicals is performed in a bottom-up analysis, i.e., following a spatially resolved multimedia modelling approach. In order to support cost-benefit analyses, the approach is accompanied by monetary valuation of human health impacts, yielding so-called external costs. Results for selected priority metals show that these external costs are small compared to those by the classical air pollutants and involve rather long time horizons touching on the aspect of intergenerational equity within sustainable development. When including further hazardous substances, the total external costs attributable to contaminants are expected to be more substantial.There is widespread public concern about hazardous chemicals that are contained in air, soil, water and food. Policy has therefore adopted a series of laws and regulations concerning emissions into and concentration levels in different media including food. As policy makers do not only have to consider the protection of the environment but also need to ensure a well-functioning economy at the same time, these limit or target values need to be set in a balanced way. The main problem, however, is to compare the costs for achieving these targets with the benefits to society by having a smaller exposure to hazardous substances (cost-benefit analysis). This book sets out to improve the reliability of cost-benefit analyses particularly of hazardous substances present in air, water, soil and food. It suggests that the human health risk assessment of chemicals is performed in a bottom-up analysis, i.e., following a spatially resolved multimedia modelling approach. In order to support cost-benefit analyses, the approach is accompanied by monetary valuation of human health impacts, yielding so-called external costs. Results for selected priority metals show that these external costs are small compared to those by the classical air pollutants and involve rather long time horizons touching on the aspect of intergenerational equity within sustainable development. When including further hazardous substances, the total external costs attributable to contaminants are expected to be more substantial.1. Introduction. 2. Assessment of human health impacts and the approach followed. 3. Multimedia environmental fate and/or exposure assessment of prioritised contaminants. 4. Multimedia environmental fate assessment framework: outline, atmospheric modelling and spatial differentiation. 5. Modelling the environmental fate in the terrestrial environment. 6. Modelling the environmental fate in the aquatic environment. 7. Expose and impact assessment. 8. Valuation. 9. Evaluation of results. 10. Case studies on emissions from signle facilities. 11. Whole economy case study. 12. Concluding remarks. Appendix A.A model formulation. Appendix B. Substance-independent data. Appendix C. Substance-dependent data. Appendix D. Symbols, indices and compartment acronyms used for parameter and process description.Includes bibliographical references and index.Print version record.Elsevie

Health-Related External Cost Assessment in Europe: Methodological Developments from ExternE to the 2013 Clean Air Policy Package

“Getting the prices right” through internalizing external costs is a guiding principle of environmental policy making, one recent example being the EU Clean Air Policy Package released at the end of 2013. It is supported by impact assessments, including monetary valuation of environmental and health damages. For over 20 years, related methodologies have been developed in Europe in the Externalities of Energy (ExternE) project series and follow-up activities. In this study, we aim at analyzing the main methodological developments over time from the 1990s until today with a focus on classical air pollution-induced human health damage costs. An up-to-date assessment including the latest European recommendations is also applied. Using a case from the energy sector, we identify major influencing parameters: differences in exposure modeling and related data lead to variations in damage costs of up to 21%; concerning risk assessment and monetary valuation, differences in assessing long-term exposure mortality risks together with assumptions on particle toxicity explain most of the observed changes in damage costs. These still debated influencing parameters deserve particular attention when damage costs are used to support environmental policy making