Transport of Designed Ankyrin Repeat Proteins through reconstituted human bronchial epithelia and protection against SARS-CoV-2.

Clinical studies have proven antiviral effectiveness of treatment with a Designed Ankyrin Repeat Protein (DARPin) specific against the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). More information on transport mechanisms and efficiency to the site of action is desirable. Transepithelial migration through air-liquid interface (ALI) cultures of reconstituted human bronchial epithelia (HBE) was assessed by Enzyme-Linked Immunosorbent Assays and Confocal Laser Scanning Microscopy for different DARPin designs in comparison to a monoclonal antibody. Antiviral efficacy against authentic SARS-CoV-2, applied apically on HBE, was investigated based on viral titers and genome equivalents, after administration of therapeutic candidates on the basal side. Transepithelial translocation of all DARPin candidates and the monoclonal antibody was efficient and dose dependent. Small DARPins and the antibody migrated more efficiently than larger molecules, indicating different transport mechanisms involved. Microscopic analyses support this, demonstrating passive paracellular transport of smaller DARPins and transcellular migration of the larger molecules. All therapeutic candidates applied to the basal side of HBE conferred effective protection against SARS-CoV-2 infection. In summary, we have shown that DARPins specific against SARS-CoV-2 translocate across intact airway epithelia and confer effective protection against infection and viral replication

Temperature dependence of the electrochemical behavior of the 690 Ni-base alloy between 25 and 325 °C

International audienceThe electrochemical behavior and the chemical composition of passive films formed on the Alloy 690 at room temperature in borate buffer solution (pH = 9.0) was studied with different techniques for two surface finishings. XPS and quantum yield measurements showed the presence of Ni and Cr oxides and hydroxides for passive films formed on both the as received and the mechanically polished one, whereas the presence of mixed spinel type Ni (1-x) Fe x Cr 2 O 4 was only observed on the as received material. Additionally, Electrochemical Impedance Spectroscopy (EIS) highlighted a higher corrosion resistance for the as received alloy in comparison with the mechanically polished alloy, which was linked to the chemical composition of the oxide film. Electrochemical measurements were performed before, during, and after oxidation of the Alloy 690 of Steam Generator (SG) tube of Pressurized Water Reactor (PWR) at high temperature and high pressure in the simulated primary circuit of PWR. At high temperature, the interface becomes electrochemically active yielding the precipitation of the corrosion products which form a few tens of nm thick diffusion barrier for the released metal cations. This overlayer is built on the top of a few nm thick, Cr rich inner layer at the alloy/oxide interface which was found to behave similarly to that initially formed at ambient temperature. It is concluded that high temperature oxidation in the static condition of an autoclave at 325°C does not promote a better passivation state than the one already existing initially

Ectopic expression of Kip-related proteins restrains root-knot nematode-feeding site expansion

The development of nematode feeding sites induced by root-knot nematodes involves the synchronized activation of cell cycle processes such as acytokinetic mitoses and DNA amplification. A number of key cell cycle genes are reported to be critical for nematode feeding site development. However, it remains unknown whether plant cyclin-dependent kinase (CDK) inhibitors such as the Arabidopsis interactor/inhibitor of CDK (ICK)/Kip-related protein (KRP) family are involved in nematode feeding site development. This study demonstrates the involvement of Arabidopsis ICK2/KRP2 and ICK1/KRP1 in the control of mitosis to endoreduplication in galls induced by the root-knot nematode Meloidogyne incognita. ! Using ICK/KRP promoter-GUS fusions and mRNA in situ hybridizations, we showed that ICK2/KRP2, ICK3/KRP5 and ICK4/KRP6 are expressed in galls after nematode infection. Loss-of-function mutants have minor effects on gall development and nematode reproduction. Conversely, overexpression of both ICK1/KRP1 and ICK2/KRP2 impaired mitosis in giant cells and blocked neighboring cell proliferation, resulting in a drastic reduction of gall size. ! Studying the dynamics of protein expression demonstrated that protein levels of ICK2/ KRP2 are tightly regulated during giant cell development and reliant on the presence of the nematode. ! This work demonstrates that impeding cell cycle progression by means of ICK1/KRP1 and ICK2/KRP2 overexpression severely restricts gall development, leading to a marked limitation of root-knot nematode development and reduced numbers of offsprin

The small compound inhibitor K22 displays broad antiviral activity against different members of the family Flaviviridae and offers potential as pan-viral inhibitor.

The virus family Flaviviridae encompasses several viruses, including (re)emerging viruses which cause widespread morbidity and mortality throughout the world. Members of this virus family are positive-strand RNA viruses and replicate their genome in close association with reorganized intracellular host cell membrane compartments. This evolutionarily conserved strategy facilitates efficient viral genome replication and contributes to evasion from host cell cytosolic defense mechanisms. We have previously described the identification of a small-compound inhibitor, K22, which exerts a potent antiviral activity against a broad range of coronaviruses by targeting membrane-bound viral RNA replication. To analyze the antiviral spectrum of this inhibitor, we assessed the inhibitory potential of K22 against several members of the Flaviviridae family, including the reemerging Zika virus (ZIKV). We show that ZIKV is strongly affected by K22. Time-of-addition experiments revealed that K22 acts during a postentry phase of the ZIKV life cycle, and combination regimens of K22 together with ribavirin (RBV) or interferon alpha (IFN-α) further increased the extent of viral inhibition. Ultrastructural electron microscopy studies revealed severe alterations of ZIKV-induced intracellular replication compartments upon infection of K22-treated cells. Importantly, the antiviral activity of K22 was demonstrated against several other members of the Flaviviridae family. It is tempting to speculate that K22 exerts its broad antiviral activity against several positive-strand RNA viruses via a similar mechanism and thereby represents an attractive candidate for development as a panviral inhibitor

Screening out irrelevant cell-based models of disease

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell-and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates

lmpact of Steam Generator primary circuit contamination products on PWR primary circuit contamination by corrosion products.

International audienceThis paper analyses the impact of Steam Generator Replacement (SGR) on PWR primary circuit contamination by corrosion products. It presents a comparison of deposited and released activities between three new SG tube materials : alloys 600TT, 690TT and 800. This comparison allows to describe the typical evolutions of 58Co and 60Co contamination due to SGR.The discussion highlights the importance of corrosion product deposits on primary surfaces before SGR and the manufacturing process of SG tubes

The OSCAR code: a simulation tool to assess the PWR contamination for decommissioning

International audienceKnowing the contamination state of the end-of-life nuclear reactor systems by Long-Lived RadioNu-clides (LLRNs) is a key stage for the decommissioning process. Indeed, the initial state is necessary to optimize the decommissioning works and to manage the radioactive waste as well. To address this issue, the contamination state is usually characterized using different types of techniques: in-situ gamma spectrometry, gamma camera scanning, dose rate measurements, , , measurements of samples obtained by smears or scrapings and then chemical separation processes, scaling factor approach… To reduce the amount of these measurements and thus the Occupational Radiation Ex-posure (ORE) and the decommissioning costs, a method is to assess the level of contamination by simulation. Furthermore, at the design stage of a new reactor, its decommissioning has to be taken into account and a simulation tool, such as the OSCAR code, can predict the radioactive source term at the end of life of a future reactor.The OSCAR code (Outil de Simulation de la ContAmination en Réacteur - tOol of Simulation of Con-tAmination in Reactor) has been developed by the CEA in collaboration with EDF and Framatome since the 1970s. The OSCAR code simulates the production and transfer of Activated Corrosion Products (ACPs) and Actinides and Fission Products and (AFPs) in the reactor systems and thus calculates the masses and activities of radionuclides deposited inside piping and heat exchangers of different circuits. As the OSCAR code has been originally devoted to mainly an industrial objective, which is the reduction of the Occupational Radiation Exposure (ORE) for operating PWRs, it deals with the main ACPs, 60Co, 58Co, 54Mn…, which are short-lived radionuclides. Nevertheless, it also cal-culates LLRNs of interest for decommissioning, such as 55Fe, 63Ni, 90Sr, 239Pu…, most of which are difficult to measure. The OSCAR code is validated through on-site measurements, including the EMECC campaigns, an operational experience feedback unique in the world, that consist of measur-ing the gamma surface activities mainly of the PWR primary system but also of auxiliary systems.After a presentation of the features and modeling of the OSCAR code, the paper will present some OSCAR simulation results of long-lived ACPs and AFPs deposited inside PWR circuits and their comparison with measurements. Perspectives of the OSCAR code on the decommissioning pro-grams will also be discussed