Cellular Entry of Ebola Virus Involves Uptake by a Macropinocytosis-Like Mechanism and Subsequent Trafficking through Early and Late Endosomes

Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new therapeutics as well as provide potential insight into the trafficking and entry mechanism of other filoviruses.     

Synthesis of novel Gn-RH analogues using Ugi-4MCR

An efficient method for the synthesis of some Gn-RH analogues based on Ugi reaction has been developed. Four-component reaction of N- and C-terminus peptides, aromatic aldehydes and isocyanides affords novel Gn-RH analogues derived from triptorelin and gonadorelin. All of the products were purified using preparative HPLC and the structures were assigned according to MALDI-mass spectrometry data.     

Preparative purification of Escherichia coli heat-stable enterotoxin

Heat-stable enterotoxin (STa) isolated from bovine Escherichia coli strains was purified to homogeneity by growing the bacterial strains in a chemically defined medium, desalting, and concentrating the culture filtrate by batch adsorption chromatography on Amberlite XAD-2 resin, batch adsorption chromatography on reversed-phase silica, and preparative reversed-phase high-performance liquid chromatography. This rapid preparative purification scheme gave high recovery yields of pure STa which exhibited biochemical homology to STa purified by more complicated procedures.     

Combination vs. disproportionation in dialkyl biradicals. Selectivity reversal in a crystalline solid.

While 1,6-biradicals produced by photodecarbonylation of dimethyl 11-oxodibenzo[c,h]bicyclo[4.4.1]undeca-3,8-diene-1,6-dicarboxylate (1) react exclusively by disproportionation in benzene solution, reactions in crystals lead to radical-radical combination reactions in almost quantitative yield.     

The role of lysolecithin in the relaxation of vascular smooth muscle

The effect of lysolecithin (lysophosphatidylcholine) on the relaxation of rabbit aortic strip closely resembled that produced by acetylcholine (ACh) which releases the endothelium-derived relaxing factor (EDRF). Relaxation induced by lysolecithin depended on the presence of endothelium and was inhibited by hemoglobin and methylene blue. It appeared to be mediated by the second messenger, c-GMP. Lysolecithin induced relaxation was slower but more persistent than that resulting from the endothelium-derived relaxing factor (EDRF) produced by acetylcholine (ACh). Like lysolecithin, Triton X-100, a non-ionic detergent, also preferentially relaxed aortic strips with intact endothelium. The results demonstrate the importance of phospholipids derived from cell membranes in vascular smooth muscle relaxation. Endothelium-derived relaxing factors appear as a group of heterogeneous substances.     

Antibiotics and the post-genome revolution

The emergence of pathogenic bacteria resistant to multiple antimicrobial agents is turning into a major crisis in human and veterinary medicine. This necessitates a serious re-evaluation of our approaches toward antibacterial drug discovery and use. Concurrent advances in genomics including whole-genome sequencing, genotyping, and gene expression profiling have the potential to transform our basic understanding of antimicrobial pathways and lead to the discovery of novel targets and therapeutics.