Diagnostic performance of rapid antigen testing for SARS-CoV-2: the COVid-19 AntiGen (COVAG) extension study.

Background: This study is the extension of the COVAG study. We compared two RATs, the Panbio COVID-19 Ag Rapid Test (Abbott) and the SD Biosensor Q SARS-CoV-2 Rapid Antigen Test (Roche), against RT-PCR on the foil of new variants. Methods: We included 888 all-comers at a diagnostic center between October 20, 2021, and March 18, 2022. RT-PCR-positive samples with a Ct value ≤32 were examined for SARS-CoV-2 variants. Findings: The sensitivity of the Abbott-RAT and Roche-RAT were 65 and 67%, respectively. For both RATs, lower Ct values were significantly correlated with higher sensitivity. For samples with Ct values ≤25, the sensitivities of the Roche-RAT and of the Abbott-RAT were 96 and 95%, for Ct values 25-30 both were 19%, and for Ct values ≥30 they were 6 and 2%, respectively. The RATs had substantially higher sensitivities in symptomatic than asymptomatic participants (76, 77%, vs. 29, 31%, for Abbott-RAT, Roche-RAT, respectively) and in participants referred to testing by their primary care physician (84, 85%) compared to participants who sought testing due to referral by the health department (55, 58%) or a warning by the Corona-Warn-App (49, 49%). In persons with self-reported previous COVID-19 sensitivities were markedly lower than in patients without previous COVID-19: 27% vs. 75% for Roche-RAT and 27% vs. 73% for Abbott-RAT. We did not find significant correlation between vaccination status and sensitivity. The Omicron variant was detected with a sensitivity of 94 and 92%, the delta variant with a sensitivity of 80 and 80% for Abbott-RAT and Roche-RAT, respectively. This difference is attributable to the lower Ct values of the Omicron samples compared to the Delta samples. When adjusted for the Ct value, a multivariate logistic regression did not show a significant difference between Omicron and Delta. In terms of sensitivity, we found no significant difference between the wild-type and the Omicron and Delta variants, but a significantly lower sensitivity to the alpha variant compared to the other variants.The specificities were > 99% overall.

FOXO3 is a glucocorticoid receptor target and regulates LKB1 and its own expression based on cellular AMP levels via a positive autoregulatory loop.

FOXO3 is a transcription factor involved in the regulation of multiple physiological processes including cell cycle arrest, apoptosis, oxidative stress-response and energy metabolism. Although much is known about its post-translational modification, the transcriptional regulation of FOXO3, as well as the cross-talk between transcription and post-translational events, is still poorly understood. In the present study, we show that FOXO3 is an immediate early glucocorticoid receptor (GR) target, whose transcription is even further enhanced by conditions that mimic metabolic stress. Induction of FOXO3 transcription by GR-binding steroids was reversed by concomitant treatment with the GR antagonist RU-486, but further enhanced by stimuli that activate the AMP-activated protein kinase (AMPK). Analysis of genomic DNA and chromatin immunoprecipitation, as well as luciferase reporter assays, revealed two functional glucocorticoid responsive elements within the FOXO3 promoter. Furthermore, we provide functional evidence for a phosphorylation switch that explains how glucocorticoids induce transcriptional activation of the gene but subsequently inactivate the corresponding protein by site-specific phosphorylation. Only when AMPK is stimulated, pre-existing FOXO3 becomes reverted toward an active form. Energy deprived conditions thus activate FOXO3 on two different levels, namely transcriptional and post-translational. In that way, FOXO3 acts as a metabolic stress sensor that coordinates expression of LKB1, the master upstream kinase involved in metabolic sensing, depending on the energy status of the cell. Additionally, we show that FOXO3 binds and activates its own promoter via a positive autoregulatory feedback loop. In conclusion, our data explain how catabolic glucocorticoid hormones and high intracellular AMP levels cooperate in inducing FOXO3 transcription and in activating the corresponding protein.

Gene expression of the tumour suppressor LKB1 is mediated by Sp1, NF-Y and FOXO transcription factors.

The serine/threonine kinase LKB1 is a tumour suppressor that regulates multiple biological pathways, including cell cycle control, cell polarity and energy metabolism by direct phosphorylation of 14 different AMP-activated protein kinase (AMPK) family members. Although many downstream targets have been described, the regulation of LKB1 gene expression is still poorly understood. In this study, we performed a functional analysis of the human LKB1 upstream regulatory region. We used 200 base pair deletion constructs of the 5'-flanking region fused to a luciferase reporter to identify the core promoter. It encompasses nucleotides -345 to +52 relative to the transcription start site and coincides with a DNase I hypersensitive site. Based on extensive deletion and substitution mutant analysis of the LKB1 promoter, we identified four cis-acting elements which are critical for transcriptional activation. Using electrophoretic mobility shift assays as well as chromatin immunoprecipitations, we demonstrate that the transcription factors Sp1, NF-Y and two forkhead box O (FOXO) family members FOXO3 and FOXO4 bind to these elements. Overexpression of these factors significantly increased the LKB1 promoter activity. Conversely, small interfering RNAs directed against NF-Y alpha and the two FOXO proteins greatly reduced endogenous LKB1 expression and phosphorylation of LKB1's main substrate AMPK in three different cell lines. Taken together, these results demonstrate that Sp1, NF-Y and FOXO transcription factors are involved in the regulation of LKB1 transcription.

Development of a novel fluorescent substrate for Autolysin E, a bacterial type II amidase.

The bifunctional Autolysin E from Staphylococcus epidermidis, contains a Zn(2+)-dependent N-acetylmuramoyl-L-alanine amidase AmiE (EC 3.5.1.28). This enzyme hydrolyzes the amide bond between the carbohydrate chain and the peptide stem of bacterial peptidoglycan. Since peptidoglycan is the mayor component of bacterial cell walls, type II amidases like Autolysin E play an essential role in the bacterial life cycle. Therefore bacterial amidases are appropriate drug targets in the development of antibiotics. The drug discovery process relies on sensitive enzyme assay systems to test possible lead candidates for enzyme inhibition. However, specific determination of bacterial amidase activity is complicated because a simple and accurate enzyme assay is currently unavailable. In this study we developed a sensitive fluorescent substrate for the type II amidase Autolysin E from S. epidermidis, which is suitable for quantifying amidase activity in a high throughput compatible fashion. Using derivatives of the substrate Mca-Ala-D-isoGln-Lys(Dnp)-D-Ala-Arg-OH, we were further able to characterize the amidase substrate specificity of Autolysin E.

Quantifying cathepsin S activity in antigen presenting cells using a novel specific substrate.

Cathepsin S (CatS) is a lysosomal cysteine protease belonging to the papain superfamily. Because of the relatively broad substrate specificity of this family, a specific substrate for CatS is not yet known. Based on a detailed study of the CatS endopeptidase specificity, using six series of internally quenched fluorescent peptides, we were able to design a specific substrate for CatS. The peptide series was based on the sequence GRWHTVGLRWE-Lys(Dnp)-DArg-NH2, which shows only one single cleavage site between Gly and Leu and where every substrate position between P-3 and P-3' was substituted with up to 15 different amino acids. The endopeptidase specificity of CatS was mainly determined by the P-2, P-1', and the P-3' substrate positions. Based on this result, systematically modified substrates were synthesized. Two of these modified substrates, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2 and Mca-GRWHPMGAPWE-Lys(Dnp)-DArg-NH2, did not react with the purified cysteine proteases cathepsin B (CatB) and cathepsin L (CatL). Using a specific CatS inhibitor, we could further show that these two peptides were not cleaved by endosomal fractions of antigen presenting cells (APCs), when CatS was inhibited and related cysteine proteases cathepsin B, H, L and X were still active. Although aspartic proteases like cathepsin E and cathepsin D were also present, our substrates were suitable to quantify cathepsin S activity specifically in APCs, including B cells, macrophages, and dendritic cells without the use of any protease inhibitor. We find that CatS activity differs significantly not only between the three types of professional APCs but also between endosomal and lysosomal compartments.