Characterization of young and aged ferrets as animal models for SARS-CoV-2 infection with focus on neutrophil extracellular traps

Neutrophil extracellular traps (NETs) are net-like structures released by activated neutrophils upon infection [e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] as part of the innate immune response that have protective effects by pathogen entrapment and immobilization or result in detrimental consequences for the host due to the massive release of NETs and their impaired degradation by nucleases like DNase-1. Higher amounts of NETs are associated with coronavirus disease 2019 (COVID-19) severity and are a risk factor for severe disease outcome. The objective of our study was to investigate NET formation in young versus aged ferrets to evaluate their value as translational model for SARS-CoV-2-infection and to correlate different NET markers and virological parameters. In each of the two groups (young and aged), nine female ferrets were intratracheally infected with 1 mL of 106 TCID50/mL SARS-CoV-2 (BavPat1/2020) and euthanized at 4, 7, or 21 days post-infection. Three animals per group served as negative controls. Significantly more infectious virus and viral RNA was found in the upper respiratory tract of aged ferrets. Interestingly, cell-free DNA and DNase-1 activity was generally higher in bronchoalveolar lavage fluid (BALF) but significantly lower in serum of aged compared to young ferrets. In accordance with these data, immunofluorescence microscopy revealed significantly more NETs in lungs of aged compared to young infected ferrets. The association of SARS-CoV-2-antigen in the respiratory mucosa and NET markers in the nasal conchae, but the absence of virus antigen in the lungs, confirms the nasal epithelium as the major location for virus replication as described for young ferrets. Furthermore, a strong positive correlation was found between virus shedding and cell-free DNA or the level of DNAse-1 activity in aged ferrets. Despite the increased NET formation in infected lungs of aged ferrets, the animals did not show a strong NET phenotype and correlation among tested NET markers. Therefore, ferrets are of limited use to study SARS-CoV-2 pathogenesis associated with NET formation. Nevertheless, the mild to moderate clinical signs, virus shedding pattern, and the lung pathology of aged ferrets confirm those animals as a relevant model to study age-dependent COVID-19 pathogenesis

FasL is more frequently expressed in liver metastases of colorectal cancer than in matched primary carcinomas

Colorectal carcinoma cells have recently been shown to express Fas ligand (FasL). This ligand could allow the tumour cells to evade activated tumour-infiltrating lymphocytes (TILs) by inducing their apoptosis and would thus promote tumour survival and possibly metastasis formation. To test this hypothesis in vivo we analysed the expression of FasL mRNA and protein in paired tissue samples of normal colonic mucosa (N), primary colorectal carcinomas (T) and their metastases (M) from a total of 21 patients by four different methods. Additionally, the presence and activation status of infiltrating lymphocytes, which might contribute to the total amount of FasL in the tissue, was determined by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) in the same samples. The frequency of FasL detection was 30–40% in T and was 60–100% in M, depending on the sensitivity of the method. Simultaneously, the amount of CD25 mRNA, used as a measure of the number of activated TILs, was in 90% of patients lower in M than in T. The increased frequency of FasL detection in liver metastases was therefore not due to the presence of activated TILs. We conclude that metastasizing subpopulations of colorectal tumour cells express FasL more frequently than the primary carcinomas and may be able to eliminate activated TILs in vivo via Fas/FasL-induced apoptosis or other hitherto unknown mechanisms. © 1999 Cancer Research Campaig

Sleep, vigilance, and thermosensitivity

The regulation of sleep and wakefulness is well modeled with two underlying processes: a circadian and a homeostatic one. So far, the parameters and mechanisms of additional sleep-permissive and wake-promoting conditions have been largely overlooked. The present overview focuses on one of these conditions: the effect of skin temperature on the onset and maintenance of sleep, and alertness. Skin temperature is quite well suited to provide the brain with information on sleep-permissive and wake-promoting conditions because it changes with most if not all of them. Skin temperature changes with environmental heat and cold, but also with posture, environmental light, danger, nutritional status, pain, and stress. Its effect on the brain may thus moderate the efficacy by which the clock and homeostat manage to initiate or maintain sleep or wakefulness. The review provides a brief overview of the neuroanatomical pathways and physiological mechanisms by which skin temperature can affect the regulation of sleep and vigilance. In addition, current pitfalls and possibilities of practical applications for sleep enhancement are discussed, including the recent finding of impaired thermal comfort perception in insomniacs

Escherichia coli genome-wide promoter analysis: Identification of additional AtoC binding target elements

<p>Abstract</p> <p>Background</p> <p>Studies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of <it>E. coli </it>activates the expression of <it>atoDAEB </it>operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that <it>atoSC </it>is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the <it>atoDAEB </it>promoter as the single, <it>cis</it>-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the <it>E. coli </it>genome.</p> <p>Results</p> <p>Through the implementation of a computational <it>de novo </it>motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the <it>E. coli </it>genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the <it>in vivo </it>binding of AtoC to the predicted sites. This process verified twenty-two additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences.</p> <p>Conclusions</p> <p>This study, by tracing a number of putative AtoC binding sites, has indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation.</p

Rebuffing Royals? Afrikaners and the royal visit to South Africa in 1947’

This article traces the responses of Afrikaners to the symbolism and political purposes of the 1947 royal visit to Southern Africa, the first post-war royal tour and the first visit of a reigning sovereign to the Union of South Africa. Taking place in the aftermath of a war that had caused bitter political divisions within Afrikaner ranks and stimulated radical populist nationalism, a royal tour intended to express the crown's gratitude for South Africa's participation in that war was bound to be contentious. Drawing on press accounts, biographies, autobiographies and archival sources, this article argues that the layered reactions of Afrikaners demonstrate that, even on the eve of the National Party's electoral victory on a republican and apartheid platform, attitudes towards monarchy and the British connection were more fluid and ambiguous than either contemporary propaganda or recent accounts have allowed. The diverse meanings attributed to this iconic royal tour reveal a process of intense contestation and reflection about South Africa's place in an empire that was in the throes of post-war redefinition and transformation, and confirm recent characterisations of the 1940s as one of manifold possibilities such that outcomes, like the electoral victory of the National Party in the following year, was far from pre-determined