Attenuation of SARS-CoV-2 replication and associated inflammation by concomitant targeting of viral and host cap 2'-O-ribose methyltransferases.

The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.

Rapid and sensitive identification of omicron by variant-specific PCR and nanopore sequencing: paradigm for diagnostics of emerging SARS-CoV-2 variants.

On November 26, 2021, the World Health Organization classified B.1.1.529 as a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VoC), named omicron. Spike-gene dropouts in conventional SARS-CoV-2 PCR systems have been reported over the last weeks as indirect diagnostic evidence for the identification of omicron. Here, we report the combination of PCRs specific for heavily mutated sites in the spike gene and nanopore-based full-length genome sequencing for the rapid and sensitive identification of the first four COVID-19 patients diagnosed in Germany to be infected with omicron on November 28, 2021. This study will assist the unambiguous laboratory-based diagnosis and global surveillance for this highly contagious VoC with an unprecedented degree of humoral immune escape. Moreover, we propose that specialized diagnostic laboratories should continuously update their assays for variant-specific PCRs in the spike gene of SARS-CoV-2 to readily detect and diagnose emerging variants of interest and VoCs. The combination with established nanopore sequencing procedures allows both the rapid confirmation by whole genome sequencing as well as the sensitive identification of newly emerging variants of this pandemic ß-coronavirus in years to come.

Intermittent versus continuous sciatic block combined with femoral block for patients undergoing knee arthroplasty. A randomized controlled trial.

PURPOSE: Many institutions perform peripheral femoral and/or sciatic nerve blocks for analgesia after total knee arthroplasty. The aim of the present investigation was to compare the analgesic effect of an intermittent sciatic block (boluses on demand only) with a continuous infusion. METHODS: One hundred and forty patients receiving a femoral and a sciatic nerve block (SNB) by catheter were assessed. The femoral catheter was infused continuously in both groups. In the continuous group (n = 70), the sciatic catheter was used continuously as well. In the intermittent group (n = 70), after a single injection of 20 ml ropivacaine 0.75 %, injections into the sciatic catheter were performed on demand only. Frequency of injections, ventral and dorsal numerical pain scores (NRSs) of the knee, functional outcome (degree of flexion/extension) and additional opioid requirements were assessed during the post-operative period. RESULTS: In both groups, dorsal and ventral NRSs were below 3 at rest and below 5 during mobilisation over the complete period of assessment. Differences between the groups could not be observed. The cumulative number of supplemental injections into the sciatic catheter was higher in the intermittent group (52) compared with the continuous group (24; p < 0.05). Groups did not differ from each other with regard to functional outcome and opioid consumption. CONCLUSIONS: The use of a sciatic block performed as intermittent bolus injection on demand only did not affect post-operative outcome parameters with regard to pain scores, functional outcome or opioid requirements. This approach might therefore be considered as an alternative to a continuous infusion concept. ClinicalTrials.gov Identifier: NCT01843153.

Comprehensive analysis of ß-catenin target genes in colorectal carcinoma cell lines with deregulated Wnt/ß-catenin signaling.

BACKGROUND: Deregulation of Wnt/ß-catenin signaling is a hallmark of the majority of sporadic forms of colorectal cancer and results in increased stability of the protein ß-catenin. ß-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto-oncogenes MYC and CCND1 as well as the genes encoding the basic helix-loop-helix (bHLH) proteins ASCL2 and ITF-2B. To identify genes commonly regulated by ß-catenin in colorectal cancer cell lines, we analyzed ß-catenin target gene expression in two non-isogenic cell lines, DLD1 and SW480, using DNA microarrays and compared these genes to ß-catenin target genes published in the PubMed database and DNA microarray data presented in the Gene Expression Omnibus (GEO) database. RESULTS: Treatment of DLD1 and SW480 cells with ß-catenin siRNA resulted in differential expression of 1501 and 2389 genes, respectively. 335 of these genes were regulated in the same direction in both cell lines. Comparison of these data with published ß-catenin target genes for the colon carcinoma cell line LS174T revealed 193 genes that are regulated similarly in all three cell lines. The overlapping gene set includes confirmed ß-catenin target genes like AXIN2, MYC, and ASCL2. We also identified 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are regulated similarly in DLD1 and SW480 cells and one pathway - the steroid biosynthesis pathway - was regulated in all three cell lines. CONCLUSIONS: Based on the large number of potential ß-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T cells as well as the large overlap with confirmed ß-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study Wnt/ß-catenin signaling and associated colorectal carcinogenesis. Furthermore, the confirmed and the newly identified potential ß-catenin target genes are useful starting points for further studies.

EFEMP1 expression promotes in vivo tumor growth in human pancreatic adenocarcinoma.

The progression of pancreatic cancer is dependent on local tumor growth, angiogenesis, and metastasis. EFEMP1, a recently discovered member of the fibulin family, was characterized with regard to these key elements of pancreatic cancer progression. Differential gene expression was assessed by mRNA microarray hybridization in FG human pancreatic adenocarcinoma cells and L3.6pl cells, a highly metastatic variant of FG. In vivo orthotopic tumor growth of EFEMP1-transfected FG cells was examined in nude mice. To assess the angiogenic properties of EFEMP1, vascular endothelial growth factor (VEGF) production of tumor cells, endothelial cell proliferation and migration, and tumor microvessel density were analyzed in response to EFEMP1. Further, tumor cell apoptosis, cell cycle progression, and resistance to cytotoxic agents were quantitated by propidium iodide staining and flow cytometry. In microarray hybridization, EFEMP1 was shown to be significantly up-regulated in L3.6pl cells compared with FG cells. Concordantly, EFEMP1 transfection of FG cells stimulated orthotopic and metastatic tumor growth in vivo. EFEMP1 expression resulted in a stimulation of VEGF production by tumor cells and an increased number of CD31-positive microvessels. Endothelial cell proliferation and migration were not altered by EFEMP1, indicating an indirect angiogenic effect. Further, EFEMP1 expression decreased apoptosis and promoted cell cycle progression in response to serum starvation or exposure to gemcitabine, 5-fluorouracil, and irinotecan. EFEMP1 has protumorigenic effects on pancreatic cancer in vivo and in vitro mediated by VEGF-driven angiogenesis and antiapoptotic mechanisms. Hence, EFEMP1 is a promising candidate for assessing prognosis and individualizing therapy in a clinical tumor setting.

OPG is regulated by beta-catenin and mediates resistance to TRAIL-induced apoptosis in colon cancer.

PURPOSE: Resistance to apoptosis is a hallmark of cancer and correlates with aggressiveness of tumors and poor prognosis. The Wnt/beta-catenin pathway plays a pivotal role in the genesis of colorectal cancer by mechanisms not fully elucidated yet. Previous studies have linked regulation of osteoprotegerin (OPG) in bone to Wnt/beta-catenin signaling. As OPG also serves as a decoy receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we hypothesized that OPG might play a role in mediating resistance to apoptosis in colorectal cancer cells. EXPERIMENTAL DESIGN: Expression analysis and functional studies in human colorectal cancer cell lines and determination of expression in primary tumors and sera from patients with colorectal cancer. RESULTS: We found production of OPG in colorectal cancer cells to be regulated by beta-catenin/Tcf-4. Addition of exogenous OPG to colorectal cancer cells caused resistance to TRAIL. Similarly, accumulation of OPG in medium of cultivated cells caused resistance to TRAIL, and this could be reverted by removal of OPG. Furthermore, OPG levels were significantly increased in serum of patients with advanced disease. CONCLUSIONS: We conclude that the Wnt/beta-catenin pathway contributes to carcinogenesis and cancer cell survival by driving expression of OPG. Expression of the survival factor OPG might provide colorectal cancer cells with an essential growth advantage and contribute to cell invasion and metastasis. Inhibition of OPG expression might offer a new therapeutic approach for the treatment of patients with colorectal tumors overexpressing OPG and make these tumors sensitive to TRAIL-induced apoptosis.

Dickkopf-4 is frequently down-regulated and inhibits growth of colorectal cancer cells.

Like Dickkopf-1 (DKK1), DKK4 is a target of beta-catenin/Tcf-4 in colorectal cancer. However, as a negative regulator of Wnt signalling its function in colorectal cancer cells is not well understood. We report that DKK4 is frequently down-regulated in colorectal cancer cell lines with deregulated beta-catenin/Tcf-4 and in primary colorectal cancers. Exposure of cancer cells to DKK4 strongly inhibits basal beta-catenin/Tcf-4 signalling activity, cancer cell growth and cell cycle progression. Therefore, loss of this negative feed-back loop provides Wnt factor expressing cancer cells with a growth advantage. Our data demonstrate that DKK4 is an important negative regulator of colon cancer cell growth.