Locating High Affinity Binding Sites of the Carcinogen 4-Nitroquinoline-1-Oxide on PhiX174 DNA Using Restriction Enzyme Activity Assays

Previous NMR and optical titration studies have suggested that the carcinogen 4- nitroquinoline-1-oxide [NQO] exhibits a high degree of selectivity in its binding to DNAs. Restriction enzyme activity assays have been employed to probe the sequence selectivity of a variety of compounds, including the carcinogen acetoxyacetylaminofluorene, to DNAs. To further examine sequence selectivity in the binding of NQO DNA, we have examined the binding of NQO to various locations on the sequenced DNA phiX174. In this study , several restriction enzymes, chosen to have different reaction sequences, have been used to examine the binding of NQO to the differing locations on the DNA phiX174. Samples of the DNA are reacted with varying concentrations of NQO and then digested with one of the restriction enzymes chosen to cleave phiX174 RF DNA once or twice. The digestion products are then separated on electrophoresis agarose gels. From the relative intensities of starting [undigested] DNA gel bands and digestion product gel bands enhancement or inhibition of cleavage by the bound NQO can be observed. The results of the digestions with the different enzymes are compared to detect sequence selectivity. For example, with the enzyme DraI [cleavage sequence TTTAAA], NQO showed enhancement of enzyme cleavage activity. In contrast, NQO produced inhibition at the enzyme XhoI site [CTCGAG]. The results of these experiments show that NQO shows selectivity in its binding activity on phiX174 DNA. When the sequence locations where this carcinogen binds to DNA are located, then we can examine the binding to those specific sequences in detail. These sequences can also be compared to the determined sequences for other carcinogens to provide understanding into how carcinogens, in general, act on the DNA

A New Approach to Dengue Fatal Cases Diagnosis: NS1 Antigen Capture in Tissues

Dengue manifestations may vary from asymptomatic to potentially fatal complications. With an increasing number of Dengue Hemorrhagic fever (DHF) and fatal cases, the availability of new approaches useful for cases confirmation plays an important role for the disease surveillance. The diagnosis of fatal cases in frozen and fixed tissues from autopsies can be determined by techniques such as viral RT-PCR, in situ hybridization, viral proteins detection by immunohistochemistry and NS3 specific immunostaining. We aimed to assess for the first time the usefulness of NS1 capture tests as a diagnostic technique to demonstrate DENV antigens in human tissue specimens. The highest sensitivity was obtained by a rapid ICT which was also the most sensitive in liver, lung, kidney, brain, spleen and thymus. Despite a number of studies demonstrating the usefulness of DENV NS1 antigen detection by different ELISAs in plasma and/or sera of dengue patients, no research has been done previously to demonstrate NS1 presence in tissues of fatal dengue cases. Moreover, the application of NS1 kits to demonstrate the presence of DENV may provide a better understanding of viral tropism in fatal cases and may be useful for studies of pathogenesis in vivo and in experimental animals

Roles of Small GTPase Rac1 in the Regulation of Actin Cytoskeleton during Dengue Virus Infection

An important clinical characteristic of dengue hemorrhagic fever/dengue shock syndrome is increased vascular permeability. Actin cytoskeleton is a significant element of endothelial barrier function regulation. In vitro study showed that dengue virus infection could induce redistributions of actin cytoskeleton. It is not precisely clear the roles of actin and the mechanisms of its reorganization during the infection. Using immunochemical assays, drug inhibition assays and protein interaction profiling methods, we aimed to identify the ways in which dengue virus serotype 2 interacts with actin cytoskeleton. The study showed that dynamic treadmilling of actin is necessary for dengue virus entry, production and release, while small GTPase Rac1 also plays multiple roles during these processes. In addition, we demonstrated the association of viral E protein with actin, indicating a direct effect of viral protein on the structural modifications of actin cytoskeleton. Our results provide evidence for the participation of Rac1 signaling pathways in viral protein-induced actin reorganizations, which may be a mechanism involved in the etiology of dengue hemorrhagic fever

Dengue: a continuing global threat.

Dengue fever and dengue haemorrhagic fever are important arthropod-borne viral diseases. Each year, there are ∼50 million dengue infections and ∼500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas. Illness is produced by any of the four dengue virus serotypes. A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated. Antiviral drugs and vaccines that are currently under development could also make an important contribution to dengue control in the future