Tricyclic cyanoguanidines: synthesis, site of action and insecticidal activity of a novel class of reversible acetylcholinesterase inhibitors

Bridged-tricyclic cyanoguanidines 1 were found to be active as insecticides. The preparation and structure-activity relationships of oxacyclic (X=O) and carbocyclic (X=CH(2)) analogues of 1 is described. Compounds 1 were found to inhibit acetylcholinesterase with IC(50) values comparable to the organophosphate Paraoxon. Unlike organophosphates, cyanoguanidines 1 were shown to reversibly bind acetylcholinesterase. This mode of action is shared by the structurally-related natural product Huperzine A.     

Fluorophore labeled kinase detects ligands that bind within the MAPK insert of p38α kinase

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway.     

Infection and transmission of Rift Valley fever viruses lacking the NSs and/or NSm genes in mosquitoes: potential role for NSm in mosquito infection

Background

Rift Valley fever virus is an arthropod-borne human and animal pathogen responsible for large outbreaks of acute and febrile illness throughout Africa and the Arabian Peninsula. Reverse genetics technology has been used to develop deletion mutants of the virus that lack the NSs and/or NSm virulence genes and have been shown to be stable, immunogenic and protective against Rift Valley fever virus infection in animals. We assessed the potential for these deletion mutant viruses to infect and be transmitted by Aedes mosquitoes, which are the principal vectors for maintenance of the virus in nature and emergence of virus initiating disease outbreaks, and by Culex mosquitoes which are important amplification vectors.

Methodology and Principal Findings

Aedes aegypti and Culex quinquefasciatus mosquitoes were fed bloodmeals containing the deletion mutant viruses. Two weeks post-exposure mosquitoes were assayed for infection, dissemination, and transmission. In Ae. aegypti, infection and transmission rates of the NSs deletion virus were similar to wild type virus while dissemination rates were significantly reduced. Infection and dissemination rates for the NSm deletion virus were lower compared to wild type. Virus lacking both NSs and NSm failed to infect Ae. aegypti. In Cx. quinquefasciatus, infection rates for viruses lacking NSm or both NSs and NSm were lower than for wild type virus.

Conclusions/Significance

In both species, deletion of NSm or both NSs and NSm reduced the infection and transmission potential of the virus. Deletion of both NSs and NSm resulted in the highest level of attenuation of virus replication. Deletion of NSm alone was sufficient to nearly abolish infection in Aedes aegypti mosquitoes, indicating an important role for this protein. The double deleted viruses represent an ideal vaccine profile in terms of environmental containment due to lack of ability to efficiently infect and be transmitted by mosquitoes.     

The impact of intermittent umbilical cord occlusions on the inflammatory response in pre-term fetal sheep

Fetal hypoxic episodes may occur antepartum with the potential to induce systemic and cerebral inflammatory responses thereby contributing to brain injury. We hypothesized that intermittent umbilical cord occlusions (UCOs) of sufficient severity but without cumulative acidosis will lead to a fetal inflammatory response. Thirty-one chronically instrumented fetal sheep at ～0.85 of gestation underwent four consecutive days of hourly UCOs from one to three minutes duration for six hours each day. Maternal and fetal blood samples were taken for blood gases/pH and plasma interleukin (IL)-1β and IL-6 levels. Animals were euthanized at the end of experimental study with brain tissue processed for subsequent counting of microglia and mast cells. Intermittent UCOs resulted in transitory fetal hypoxemia with associated acidemia which progressively worsened the longer umbilical blood flow was occluded, but with no cumulative blood gas or pH changes over the four days of study. Fetal arterial IL-1β and IL-6 values showed no significant change regardless of the severity of the UCOs, nor was there any evident impact on the microglia and mast cell counts for any of the brain regions studied. Accordingly, intermittent UCOs of up to three minutes duration with severe, but limited fetal hypoxemia and no cumulative acidemia, do not result in either a systemic or brain inflammatory response in the pre-term ovine fetus. However, fetal IL-1B and IL-6 values were found to be well correlated with corresponding maternal values supporting the placenta as a primary source for these cytokines with related secretion into both circulations. Female fetuses were also found to have higher IL-1β levels than males, indicating that gender may impact on the fetal inflammatory response to various stimuli.     

Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4-FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4-mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.     

Circulating micro-RNAs as potential blood-based markers for early stage breast cancer detection

Introduction

MicroRNAs (miRNAs, miRs) are a class of small, non-coding RNA molecules with relevance as regulators of gene expression thereby affecting crucial processes in cancer development. MiRNAs offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and their characteristic expression in many different diseases. We investigated whether microarray-based miRNA profiling on whole blood could discriminate between early stage breast cancer patients and healthy controls.

Methods

We performed microarray-based miRNA profiling on whole blood of 48 early stage breast cancer patients at diagnosis along with 57 healthy individuals as controls. This was followed by a real-time semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a separate cohort of 24 early stage breast cancer patients from a breast cancer screening unit and 24 age matched controls using two differentially expressed miRNAs (miR-202, miR-718).

Results

Using the significance level of p<0.05, we found that 59 miRNAs were differentially expressed in whole blood of early stage breast cancer patients compared to healthy controls. 13 significantly up-regulated miRNAs and 46 significantly down-regulated miRNAs in our microarray panel of 1100 miRNAs and miRNA star sequences could be detected. A set of 240 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an independent cohort. The relative fold changes of the RT-qPCR validation were in line with the microarray data for both miRNAs, and statistically significant differences in miRNA-expression were found for miR-202.

Conclusions

MiRNA profiling in whole blood has potential as a novel method for early stage breast cancer detection, but there are still challenges that need to be addressed to establish these new biomarkers in clinical use.