Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Multifaceted actions of Zeb2 in postnatal neurogenesis from the ventricular-subventricular zone to the olfactory bulb.

The transcription factor Zeb2 controls fate specification and subsequent differentiation and maturation of multiple cell types in various embryonic tissues. It binds many protein partners, including activated Smad proteins and the NuRD co-repressor complex. How Zeb2 subdomains support cell differentiation in various contexts has remained elusive. Here, we studied the role of Zeb2 and its domains in neurogenesis and neural differentiation in the young postnatal ventricular-subventricular zone (V-SVZ), in which neural stem cells generate olfactory bulb-destined interneurons. Conditional Zeb2 knockouts and separate acute loss- and gain-of-function approaches indicated that Zeb2 is essential for controlling apoptosis and neuronal differentiation of V-SVZ progenitors before and after birth, and we identified Sox6 as a potential downstream target gene of Zeb2. Zeb2 genetic inactivation impaired the differentiation potential of the V-SVZ niche in a cell-autonomous fashion. We also provide evidence that its normal function in the V-SVZ also involves non-autonomous mechanisms. Additionally, we demonstrate distinct roles for Zeb2 protein-binding domains, suggesting that Zeb2 partners co-determine neuronal output from the mouse V-SVZ in both quantitative and qualitative ways in early postnatal life.

BMP-SMAD signalling output is highly regionalized in cardiovascular and lymphatic endothelial networks.

BACKGROUND: Bone morphogenetic protein (BMP) signalling has emerged as a fundamental pathway in endothelial cell biology and deregulation of this pathway is implicated in several vascular disorders. BMP signalling output in endothelial cells is highly context- and dose-dependent. Phosphorylation of the BMP intracellular effectors, SMAD1/5/9, is routinely used to monitor BMP signalling activity. To better understand the in vivo context-dependency of BMP-SMAD signalling, we investigated differences in BMP-SMAD transcriptional activity in different vascular beds during mouse embryonic and postnatal stages. For this, we used the BRE::gfp BMP signalling reporter mouse in which the BMP response element (BRE) from the ID1-promotor, a SMAD1/5/9 target gene, drives the expression of GFP. RESULTS: A mosaic pattern of GFP was present in various angiogenic sprouting plexuses and in endocardium of cardiac cushions and trabeculae in the heart. High calibre veins seemed to be more BRE::gfp transcriptionally active than arteries, and ubiquitous activity was present in embryonic lymphatic vasculature. Postnatal lymphatic vessels showed however only discrete micro-domains of transcriptional activity. Dynamic shifts in transcriptional activity were also observed in the endocardium of the developing heart, with a general decrease in activity over time. Surprisingly, proliferative endothelial cells were almost never GFP-positive. Patches of transcriptional activity seemed to correlate with vasculature undergoing hemodynamic alterations. CONCLUSION: The BRE::gfp mouse allows to investigate selective context-dependent aspects of BMP-SMAD signalling. Our data reveals the highly dynamic nature of BMP-SMAD mediated transcriptional regulation in time and space throughout the vascular tree, supporting that BMP-SMAD signalling can be a source of phenotypic diversity in some, but not all, healthy endothelium. This knowledge can provide insight in vascular bed or organ-specific diseases and phenotypic heterogeneity within an endothelial cell population.

BMP-SMAD Signaling Regulates Lineage Priming, but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells.

Naive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases Tet1/2 and low levels of DNA methyltransferases Dnmt3a/b. Moreover, naive mESCs, in which the BMP-SMAD reporter transgene was activated, showed higher resistance to differentiation. Using double Smad1;Smad5 knockout mESCs, we showed that BMP-SMAD signaling is dispensable for self-renewal in both naive and ground state. These mutant mESCs were still pluripotent, but they exhibited higher levels of DNA methylation than their wild-type counterparts and had a higher propensity to differentiate. We showed that BMP-SMAD signaling modulates lineage priming in mESCs, by transiently regulating the enzymatic machinery responsible for DNA methylation.

What tools are available to identify patients with palliative care needs in primary care: a systematic literature review and survey of European practice.

BACKGROUND: It can be difficult to identify when a palliative care approach should be started both in malignant, and particularly, in non-malignant disease, ideally to run alongside disease-modifying care. A structured method or tool may be useful to help general practitioners (GPs) identify patients for early palliative care and trigger assessment and care planning. AIMS: To document what tools for identification of patients with palliative care needs are available in the published literature and to ascertain how GPs in Europe currently identify patients for palliative care. METHODS: A systematic literature search using PubMed and Embase, and a questionnaire survey among key informants in 14 European countries requesting data on methods used to identify patients with palliative care needs. RESULTS: The literature search identified four tools. The questionnaire survey identified a further three in current use and found that in current practice identification is largely based on a GP's own clinical judgement and information received from the hospital: tools are rarely used. CONCLUSIONS: Although several identification tools have been developed, none of these have been validated or widely implemented in Europe. Further collaborative international development, implementation and evaluation of such tools are recommended.

Antagonism of Nodal signaling by BMP/Smad5 prevents ectopic primitive streak formation in the mouse amnion.

The strength and spatiotemporal activity of Nodal signaling is tightly controlled in early implantation mouse embryos, including by autoregulation and feedback loops, and involves secreted and intracellular antagonists. These control mechanisms, which are established at the extra-embryonic/embryonic interfaces, are essential for anterior-posterior patterning of the epiblast and correct positioning of the primitive streak. Formation of an ectopic primitive streak, or streak expansion, has previously been reported in mutants lacking antagonists that target Nodal signaling. Here, we demonstrate that loss-of-function of a major bone morphogenetic protein (BMP) effector, Smad5, results in formation of an ectopic primitive streak-like structure in mutant amnion accompanied by ectopic Nodal expression. This suggests that BMP/Smad5 signaling contributes to negative regulation of Nodal. In cultured cells, we find that BMP-activated Smad5 antagonizes Nodal signaling by interfering with the Nodal-Smad2/4-Foxh1 autoregulatory pathway through the formation of an unusual BMP4-induced Smad complex containing Smad2 and Smad5. Quantitative expression analysis supports that ectopic Nodal expression in the Smad5 mutant amnion is induced by the Nodal autoregulatory loop and a slow positive-feedback loop. The latter involves BMP4 signaling and also induction of ectopic Wnt3. Ectopic activation of these Nodal feedback loops in the Smad5 mutant amnion results in the eventual formation of an ectopic primitive streak-like structure. We conclude that antagonism of Nodal signaling by BMP/Smad5 signaling prevents primitive streak formation in the amnion of normal mouse embryos.

Stalk cell phenotype depends on integration of Notch and Smad1/5 signaling cascades.

Gradients of vascular endothelial growth factor (VEGF) induce single endothelial cells to become leading tip cells of emerging angiogenic sprouts. Tip cells then suppress tip-cell features in adjacent stalk cells via Dll4/Notch-mediated lateral inhibition. We report here that Smad1/Smad5-mediated BMP signaling synergizes with Notch signaling during selection of tip and stalk cells. Endothelium-specific inactivation of Smad1/Smad5 in mouse embryos results in impaired Dll4/Notch signaling and increased numbers of tip-cell-like cells at the expense of stalk cells. Smad1/5 downregulation in cultured endothelial cells reduced the expression of several target genes of Notch and of other stalk-cell-enriched transcripts (Hes1, Hey1, Jagged1, VEGFR1, and Id1-3). Moreover, Id proteins act as competence factors for stalk cells and form complexes with Hes1, which augment Hes1 levels in the endothelium. Our findings provide in vivo evidence for a regulatory loop between BMP/TGFß-Smad1/5 and Notch signaling that orchestrates tip- versus stalk-cell selection and vessel plasticity.

Few Smad proteins and many Smad-interacting proteins yield multiple functions and action modes in TGFß/BMP signaling in vivo.

Signaling by the many ligands of the TGFß family strongly converges towards only five receptor-activated, intracellular Smad proteins, which fall into two classes i.e. Smad2/3 and Smad1/5/8, respectively. These Smads bind to a surprisingly high number of Smad-interacting proteins (SIPs), many of which are transcription factors (TFs) that co-operate in Smad-controlled target gene transcription in a cell type and context specific manner. A combination of functional analyses in vivo as well as in cell cultures and biochemical studies has revealed the enormous versatility of the Smad proteins. Smads and their SIPs regulate diverse molecular and cellular processes and are also directly relevant to development and disease. In this survey, we selected appropriate examples on the BMP-Smads, with emphasis on Smad1 and Smad5, and on a number of SIPs, i.e. the CPSF subunit Smicl, Ttrap (Tdp2) and Sip1 (Zeb2, Zfhx1b) from our own research carried out in three different vertebrate models.

Scrapie infection of prion protein-deficient cell line upon ectopic expression of mutant prion proteins.

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.

Prion infection influences murine endogenous retrovirus expression in neuronal cells.

Prions as causative agents of transmissible spongiform encephalopathies have been well investigated in experimental and modelling work. However, little is known about the molecular pathogenesis of prion-induced encephalopathies, the role of co-factors, and the interaction of prions with cellular components. We investigated the influence of prion infection on expression of murine endogenous retroviruses (ERVs), which compose approximately 10% of the mouse genome. Hypothalamic neuronal cells (GT1) and neuroblastoma cells (N2a) were examined. Both cell lines can be persistently infected with mouse adapted prion strains, i.e., RML. Using a mammalian retrovirus-specific DNA microarray and quantitative PCR methods, we compared the expression profiles of ERVs in prion-infected, uninfected, and anti-prion compound-treated murine neuronal cell lines, including clonal cell populations. The results suggest that prion infection influences ERV expression in neuronal cell lines, that this influence is cell line-specific, ERV-specific, and responsive to anti-prion compound treatment.

Charged bipolar suramin derivatives induce aggregation of the prion protein at the cell surface and inhibit PrPSc replication.

The conversion of the cellular prion protein (PrPc) into a pathogenic isoform (PrP(Sc)) is one of the underlying events in the pathogenesis of the fatal transmissible spongiform encephalopathies (TSEs). Numerous compounds have been described to inhibit prion replication and PrP(Sc) accumulation in cell culture. Among these, the drug suramin induces aggregation and re-targeting of PrPc to endocytic compartments. Plasma membrane and sites of conversion into PrP(Sc) are thereby bypassed. In the present study, a library of suramin analogues was tested as a potential class of new anti-prion compounds and the molecular mechanisms underlying these effects were analysed. Treatment of prion-infected neuroblastoma cells with compounds containing symmetrical aromatic sulfonic acid substitutions inhibited de novo synthesis of PrP(Sc) and induced aggregation and reduction of the half-life of PrPc without downregulating PrPc cell surface expression. Half-molecule compounds lacking the symmetrical bipolar structure or the anionic groups had no effect on PrP(Sc) synthesis or PrPc solubility. Cell surface expression of PrPc was necessary for the activity of effective compounds. Suramin derivatives did not induce aggregation of PrPc when transport along the secretory pathway was compromised, suggesting that their effects occur at a post trans-Golgi network (TGN) site, possibly close to the compartment of conversion into PrP(Sc). In vitro studies with recombinant PrP demonstrated that the inhibitory effect correlated with direct binding to PrP and induction of insoluble PrP aggregates. Our data reveal an anti-prion effect that differs from those characterising other sulphated polyanions and is dependent on the presence of the symmetrical anionic structure of these molecules.

Ultra-sensitive detection of prion protein fibrils by flow cytometry in blood from cattle affected with bovine spongiform encephalopathy.

BACKGROUND: The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the post mortem detection of the pathological form of the prion protein (PrPSc) in brain tissue. Infectivity studies indicate that PrPSc may also be present in body fluids, even at presymptomatic stages of the disease, albeit at concentrations well below the detection limits of currently available analytical methods. RESULTS: We developed a highly sensitive method for detecting prion protein aggregates that takes advantage of kinetic differences between seeded and unseeded polymerization of prion protein monomers. Detection of the aggregates was carried out by flow cytometry. In the presence of prion seeds, the association of labelled recombinant PrP monomers in plasma and serum proceeds much more efficiently than in the absence of seeds. In a diagnostic model system, synthetic PrP aggregates were detected down to a concentration of approximately 10(-8) nM [0.24 fg/ml]. A specific signal was detected in six out of six available serum samples from BSE-positive cattle. CONCLUSION: We have developed a method based on seed-dependent PrP fibril formation that shows promising results in differentiating a small number of BSE-positive serum samples from healthy controls. This method may provide the basis for an ante mortem diagnostic test for prion diseases.

Cell line dependent RNA expression profiles of prion-infected mouse neuronal cells.

The overall impact of prion disease on gene expression is not well characterized. We have carried out a large-scale expression analysis of specific cell types commonly employed in studies of prion disease. Neuroblastoma cells (N2a) and hypothalamic neuronal cells (GT1) can be persistently infected with mouse-adapted scrapie prions, the latter demonstrating cytopathologic effects associated with prion neuropathology. Exploiting a mouse DNA microarray containing approximately 21,000 spotted cDNAs, we have identified several hundred differentially expressed sequences in the two cell lines when infected with prion strain RML. ScN2a and ScGT1 cells demonstrate unique changes in RNA profiles and both differ from the reported changes in human microglia and prion-infected brain studies albeit with some overlap. In addition, several of the identified changes are shared in common with other neurodegenerative diseases such as Alzheimer's disease. The results illustrate that prion infection differs in effect depending on cell type, which could be exploited for diagnostic or therapeutic intervention.