Workshop on Regulatory Preparedness for Innovation in Nanotechnology

This report summarises the presentations and discussions at the first NanoReg2 Workshop on Regulatory Preparedness for Innovation in Nanotechnology held in Ispra, Italy 5 to 6 October 2017 and attended by approximately 60 regulators, industry representatives and other stakeholders. NanoReg2 is a European Union (EU) Horizon 2020 project. At the workshop, Regulatory Preparedness was defined as the regulators' timely awareness of innovations and the regulator's actions to check whether present legislation covers all safety aspects of each innovation, including initiating revision of the legislation as appropriate. Regulatory Preparedness, and Safe-by-Design (SbD) jointly constitute the NanoReg2 Safe Innovation Approach (SIA) for developing innovative products based on nanotechnology. The workshop aimed to gather views and identify current practices in regulatory work on safety of innovative products, tools already in use or needed, and potential difficulties in implementing Regulatory Preparedness in the EU. Presentations addressed the current state of the safety of nanotechnology innovation. The viewpoints included the regulatory framework, the principles behind it and the agencies and authorities enforcing it; nanosafety research projects and their support system (e.g. the current EU Horizon 2020 Framework Programme); national nanosafety initiatives; and the development of tools, such as foresight tools and harmonised test guidelines by the OECD for data generation. The workshop served to generate ideas for achieving Regulatory Preparedness. The participants recognised that while regulators deal with the safety of innovations, only few systematic approaches to this work exist. Some innovative products may reach the market before their safety has been appropriately assessed, as illustrated by RAPEX, the Rapid Exchange of Information System. A continuous and proactive combination of interconnected activities was considered to be required for ensuring Regulatory Preparedness. Thus, anticipation, e.g. horizon scanning, was seen as important, as was communication between regulators, innovators (industry) and other stakeholders. Regulators need to become aware of innovative products under development to ensure that the legislation and methods for safety assessment are available and adequate. Innovators must be aware of regulatory requirements and their likely development. This mutual awareness helps to develop safe products and to avoid delays or other problems in obtaining market approval. Awareness can be achieved through communication, which requires trust, e.g. promoted via "trusted environments" for confidential inquiries and information sharing. Furthermore, regulators need early access to the existing information and data relevant to safety assessment of innovative products to provide timely guidance and advice to Industry as well as to develop strategies for dealing with uncertainty, e.g. by applying the precautionary principle. Regulatory Preparedness was discussed as part of the SIA, and a "road map" of actions was suggested and outlined. The workshop has thus contributed towards acceptance of implementing Regulatory Preparedness for innovation in nanotechnology through the participation of a variety of stakeholders. This paves the way for a better dialogue among stakeholders in a fast economic development cycle, where it is even more important to quickly identify emerging needs for new approaches to regulatory issues for innovationJRC.F.2-Consumer Products Safet

Modeling Multi-organ Infection by SARS-CoV-2 Using Stem Cell Technology.

SARS-CoV-2, the virus causing the current COVID-19 pandemic, primarily targets the airway epithelium and in lungs can lead to acute respiratory distress syndrome. Clinical studies in recent months have revealed that COVID-19 is a multi-organ disease causing characteristic complications. Stem cell models of various organ systems-most prominently, lung, gut, heart, and brain-are at the forefront of studies aimed at understanding the role of direct infection in COVID-19 multi-organ dysfunction

Modeling Multi-organ Infection by SARS-CoV-2 Using Stem Cell Technology.

SARS-CoV-2, the virus causing the current COVID-19 pandemic, primarily targets the airway epithelium and in lungs can lead to acute respiratory distress syndrome. Clinical studies in recent months have revealed that COVID-19 is a multi-organ disease causing characteristic complications. Stem cell models of various organ systems-most prominently, lung, gut, heart, and brain-are at the forefront of studies aimed at understanding the role of direct infection in COVID-19 multi-organ dysfunction

Data from: Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola

Background: Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Results: Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were ‘orphans’. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Conclusions: Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs

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fasta file of the sequences used for the phylogenetic analysi

Process intensification for high yield production of influenza H1N1 Gag virus-like particles using an inducible HEK-293 stable cell line

Influenza virus dominant antigens presentation using virus like particle (VLP) approach is attractive for the development of new generation of influenza vaccines. Mammalian cell platform offers many advantages for VLP production. However, limited attention has been paid to the processing of mammalian cell produced VLPs. Better understanding of the production system could contribute to increasing the yields and making large-scale VLP vaccine manufacturing feasible. In a previous study, we have generated a human embryonic kidney HEK-293 inducible cell line expressing Hemagglutinin (HA) and Neuraminidase (NA), which was used to produce VLPs upon transient transfection with a plasmid containing HIV-1 Gag. In this work, to streamline the production process, we have developed a new HEK-293 inducible cell line adapted to suspension growth expressing the three proteins HA, NA (H1N1 A/PR/8/1934) and the Gag fused to GFP for monitoring the VLP production. The process was optimized to reach higher volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. A 5-fold improvement was achieved by doing media evaluation at small scale. Furthermore, a 3-L perfusion bioreactor mirrored the performance of small-scale shake flask cultures with sequential medium replacement. The cell density was increased to 14 7 106 cells/ml at the time of induction which augmented by 60-fold the volumetric yield to 1.54 7 1010 Gag-GFP fluorescent events/ml, as measured by flow cytometry. The 9.5-L harvest from the perfusion bioreactor was concentrated by tangential flow filtration at low shear rate. The electron micrographs revealed the presence of VLPs of 100\u2013150 nm with the characteristic dense core of HIV-1 particles. The developed process shows the feasibility of producing high quantity of influenza VLPs from an inducible mammalian stable cell line aiming at large scale vaccine manufacturing.Peer reviewed: YesNRC publication: Ye

Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate

Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150\u2013200 nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.Peer reviewed: YesNRC publication: Ye

Modelling T-cell immunity against hepatitis C virus with liver organoids in a microfluidic coculture system.

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here, we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV non-structural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells

SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients.

Although COVID-19 causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human iPSC-derived heart cells to SARS-CoV-2 revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural proteins corroborated adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and numerous iPSC-cardiomyocytes lacking nuclear DNA. Human autopsy specimens from COVID-19 patients displayed similar sarcomeric disruption, as well as cardiomyocytes without DNA staining. These striking cytopathic features provide new insights into SARS-CoV-2 induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise serious concerns about the long-term consequences of COVID-19

SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients.

Although COVID-19 causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human iPSC-derived heart cells to SARS-CoV-2 revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural proteins corroborated adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and numerous iPSC-cardiomyocytes lacking nuclear DNA. Human autopsy specimens from COVID-19 patients displayed similar sarcomeric disruption, as well as cardiomyocytes without DNA staining. These striking cytopathic features provide new insights into SARS-CoV-2 induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise serious concerns about the long-term consequences of COVID-19