Inhibition of HBV replication by RIG-I stimulation with 5`-triphosphated siRNA in vitro and in vivo

For a proportion of patients, HBV infection results in chronic disease with severe consequences like liver cirrhosis or hepatocellular carcinoma. Approved therapies of chronic hepatitis B include administration of antivirally active IFN-alpha and inhibitors of viral reverse transcription. These drugs control replication, but HBV cccDNA, the episomal transcription template, persists in most cases. Hence, lifelong therapy is required, accompanied by severe side effects and development of drug resistant mutants. An alternative and probably safe immunotherapeutic approach for clearance of HBV may be achieved by the stimulation of pattern-recognition receptors inducing an endogenous type-I IFN response. In this study the cytosolic helicase RIG-I was triggered by in vitro transcribed 5�-triphosphated (3p-) dsRNA. Stimulation induced an RIG-dependent antiviral type-I IFN response and controlled HBV replication in vitro in stable HBV replicating cell lines as well as in HBV infected primary human hepatocytes. In vivo, virus replication in HBV transgenic animals was transiently controlled when Rig I ligands were complexed and i.v. injected. To enhance and prolong the antiviral effect, siRNAs targeting overlapping open reading frames of the HBV genome at the 3´-end of multiple HBV-RNAs were designed and investigated whether they can be in vitro transcribed and act as RIG-I ligands, and thus combine the immune stimulatory potential with HBV specific gene silencing. 3p-siRNAs were transfected into HBV replicating cells, induced INF-I and IFN-stimulated genes. HBV replication markers were significantly reduced and the antiviral effect of 3p-siRNA was superior to siRNA or 3p-RNA alone. Stronger effects of 3p-siRNAs on HBV replication were confirmed in HBV infected primary human hepatocytes, as well as in vivo. Nevertheless, the gene silencing effect of 3p-siRNA seemed to be limited in the mouse model due to insufficient targeting of hepatocytes, the HBV host cells. Application of cholesterol coupled siRNA improved hepatocyte specific targeting. Furthermore, we showed that 3p-siRNA besides a direct antiviral effect additionally induced infiltration of cytotoxic CD8+ T cells to the liver, potentially leading to elimination of infected hepatocytes. The results of this study demonstrate that HBV-specific 5`-triphosphated siRNAs efficiently block HBV replication in vitro and in vivo. Combination of endogenous type-I IFN induction by stimulation of RIG-I with HBV sequence-specific gene silencing by RNAi in one single molecule could be a promising alternative to the actual standard therapeutic approaches against chronic HBV infection

Graph-Based Engineering Systems - A Family of Software Applications and their Underlying Framework

In various engineering disciplines visual modeling techniques are used for the definition as well as representation of complex systems. Besides the pictorial illustration, the included structural information is often used for application-specific procedures. This paper presents a few engineering systems for quite different application fields, but they use a common graph-based model. This model is part of a framework that underlies these applications. Various kinds of applications can be developed on the basis of this framework by means of configurations and extensions. The development of new applications is supported by convenient assemblies of suitable system functions and layout methods as well as by integration of application functionalities. The introduced framework is the basis for a product line of graph-based engineering systems

Highly migratory shark fisheries research by the National Shark Research Consortium (NSRC), 2002-2007

The National Shark Research Consortium (NSRC) includes the Center for Shark Research at Mote Marine Laboratory, the Pacific Shark Research Center at Moss Landing Marine Laboratories, the Shark Research Program at the Virginia Institute of Marine Science, and the Florida Program for Shark Research at the University of Florida. The consortium objectives include shark-related research in the Gulf of Mexico and along the Atlantic and Pacific coasts of the U.S., education and scientific cooperation

The hepatitis B virus pre-core protein p22 activates Wnt sgnaling

An emerging theme for Wnt-addicted cancers is that the pathway is regulated at multiple steps via various mechanisms. Infection with hepatitis B virus (HBV) is a major risk factor for liver cancer, as is deregulated Wnt signaling, however, the interaction between these two causes is poorly understood. To investigate this interaction, we screened the effect of the various HBV proteins for their effect on Wnt/β-catenin signaling and identified the pre-core protein p22 as a novel and potent activator of TCF/β-catenin transcription. The effect of p22 on TCF/β-catenin transcription was dose dependent and inhibited by dominant-negative TCF4. HBV p22 activated synthetic and native Wnt target gene promoter reporters, and TCF/β-catenin target gene expression in vivo. Importantly, HBV p22 activated Wnt signaling on its own and in addition to Wnt or β-catenin induced Wnt signaling. Furthermore, HBV p22 elevated TCF/β-catenin transcription above constitutive activation in colon cancer cells due to mutations in downstream genes of the Wnt pathway, namely APC and CTNNB1. Collectively, our data identifies a previously unappreciated role for the HBV pre-core protein p22 in elevating Wnt signaling. Understanding the molecular mechanisms of p22 activity will provide insight into how Wnt signaling is fine-tuned in cancer

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.Wellcome Trus

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin’s efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections

Priporočila za obravnavo bolnikov s pljučnim rakom

No abstract.Ni abstrakta

Search for the Xb and other hidden-beauty states in the π+π−ϒ(1S) channel at ATLAS

This Letter presents a search for a hidden-beauty counterpart of the X(3872) in the mass ranges of 10.05–10.31 GeV and 10.40–11.00 GeV, in the channel Xb→π+π−ϒ(1S)(→μ+μ−), using 16.2 fb−1 of pp   collision data collected by the ATLAS detector at the LHC. No evidence for new narrow states is found, and upper limits are set on the product of the Xb cross section and branching fraction, relative to those of the ϒ(2S), at the 95% confidence level using the CLS approach. These limits range from 0.8% to 4.0%, depending on mass. For masses above 10.1 GeV, the expected upper limits from this analysis are the most restrictive to date. Searches for production of the ϒ(13DJ), , and states also reveal no significant signals