Development of a simple system for screening anti-hepatitis C virus drugs utilizing mutants capable of vigorous replication.

Replication of infectious hepatitis C virus in Huh7 cells, a human hepatocyte cell line, has become possible due to the unique properties of the JFH1 isolate. Developing reporter virus systems for a simple titration has been attempted by integrating heterologous reporter genes into the JFH1 genome, resulting in a big infectivity reduction that limits the usefulness of such reporter systems. To overcome this problem, JFH1-infected Huh7 cells were cultured continuously for 2 years to obtain Huh7-adapted JFH1 variants capable of yielding up to 1000-fold higher titers. Sequence analysis of variant genome RNA suggested that this adapted population consisted mainly of two variants. By joining the 5'-half of the obtained representative viral complementary DNA (cDNA) fragments of the variants with the 3'-half of the wild-type's, two prototype clones, A/WT and B/WT, were constructed. Replication of A/WT and B/WT viruses in Huh7 cells showed up to 100-1000-fold higher titers than the wild-type. A Renilla luciferase cDNA was inserted into the Nonstructural Protein 5A region of the A/WT and B/WT cDNA to generate A/WT-Rluc and B/WT-Rluc, respectively. Transfection of Huh7 cells with in vitro-transcribed A/WT-Rluc and B/WT-Rluc RNA resulted in production of infectious viruses with approximately 15- and 25-fold higher titers, respectively, than the wild-type RNA. The replication of A/WT-Rluc and B/WT-Rluc viruses was more vigorous than the wild-type even with insertion of the luciferase cDNA showing a good correlation of luciferase activities with infectious titers. Furthermore, interferon-alpha inhibited the replication of A/WT-Rluc and B/WT-Rluc viruses in a dose-dependent manner as determined by a luciferase assay. These results imply that our system is potentially a tool useful for screening anti-hepatitis C virus drugs in a simple and time/cost-saving manner.

Medial profrontal cortex and anterior cingulate cortex in the generation of alpha activity induced by transcendental meditation: a magnetoencephalographic study.

Previous EEG studies have shown that transcendental meditation (TM) increases frontal and central alpha activity. The present study was aimed at identifying the source of this alpha activity using magnetoencephalography (MEG) and electroencephalography (EEG) simultaneously on eight TM practitioners before, during, and after TM. The magnetic field potentials corresponding to TM-induced alpha activities on EEG recordings were extracted, and we attempted to localize the dipole sources using the multiple signal classification (MUSIC) algorithm, equivalent current dipole source analysis, and the multiple spatio-temporal dipole model. Since the dipoles were mapped to both the medial prefrontal cortex (mPFC) and anterior cingulate cortex (ACC), it is suggested that the mPFC and ACC play an important role in brain activity induced by TM.

A novel small molecular weight compound with a carbazole structure that demonstrates potent human immunodeficiency virus type-1 integrase inhibitory activity.

The integration of reverse transcribed proviral DNA into a host genome is an essential event in the human immunodeficiency virus type 1 (HIV-1) replication life cycle. Therefore, the viral enzyme integrase (IN), which plays a crucial role in the integration event, has been an attractive target of anti-retroviral drugs. Several IN inhibitory compounds have been reported previously, yet none has been successful in clinical use. To find a new, more successful IN inhibitor, we screened a diverse library of 12 000 small molecular weight compounds randomly by in vitro strand-transfer assay. We identified a series of substituted carbazoles that exhibit strand-transfer inhibitory activity at low micromolar concentrations. Of these, the most potent compound exhibited an IC50 of 5.00+/-3.31 microM (CA-0). To analyse the structural determinants of strand-transfer inhibitory activity of the carbazole derivatives, we selected 23 such derivatives from our compound library and performed further analyses. Of these 23 compounds, six showed strong strand-transfer inhibition. The inhibition kinetics analyses and ethidium bromide displacement assays indicated that the carbazole derivatives are competitive inhibitors and not intercalators. An HeLa4.5/LTR-nEGFP cell line was employed to evaluate in vitro virus replication inhibition of the carbazole derivatives, and IC50 levels ranged from 0.48-1.52 microM. Thus, it is possible that carbazole derivatives, which possess structures different from previously-reported IN inhibitors, may become novel lead compounds in the development of IN inhibitors.