De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus.

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn(2+) than in the presence of Mg(2+). When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>/=50 microM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

Noninvasive detection of Chlamydia trachomatis urethritis in men by a rapid enzyme immunoassay test.

BACKGROUND: The purpose of this investigation was to evaluate the ability of a rapid enzyme immunoassay test to noninvasively detect Chlamydia trachomatis urethritis in men from a urine specimen. METHODS: Urethral samples and urine from 207 patients were evaluated. Urethral and urine sediment Gram stains, leukocyte esterase dipstick tests, and enzyme immunoassay analyses of centrifuged and uncentrifuged urine were compared with urethral C trachomatis culture. RESULTS: The prevalence of infection in this population was 10.3%. Sensitivity and specificity of the enzyme immunoassay on the centrifuged urine specimen were 70% and 96%, respectively. The positive and negative predictive values were 67% and 97%, respectively. The uncentrifuged urine enzyme immunoassay sensitivity was 35.7% and specificity was 98.9%. Leukocyte esterase test sensitivity compared with that of the Neisseria gonorrhoeae and/or C trachomatis cultures was 83.3%, and specificity was 52%. CONCLUSIONS: The rapid enzyme immunoassay clinically complemented the screening urine sediment Gram stain and the leukocyte esterase test. The judicious use of a noninvasive C trachomatis rapid enzyme immunoassay test to identify organism-specific urethritis may improve patient management of sexually transmitted disease.

Pretesting a childbirth handbook.

The development of health education materials for pregnant women involves the work of knowledgeable and experienced health professionals, writers, editors, and artists. Frequently, these materials are also extensively reviewed by various experts and organizations prior to publication. Even with all this expert input and review, however, they can miss the mark if they are not deemed appropriate and acceptable by the audience for whom they were created. In developing a new booklet on childbirth, the New York State Department of Health wanted to ensure that the material would be read and used by pregnant women from various income and educational levels and racial and ethnic groups. Research was conducted by the department to pretest the booklet for its appropriateness for and acceptance by the target audience. Based on the reactions and suggestions from 89 women in eight focus groups, the booklet was revised extensively before being made available to the general public.