Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase-primase

The aminothiazolylphenyl-containing compounds BILS 179 BS and BILS 45 BS are novel inhibitors of the herpes simplex virus helicase-primase with antiviral activity in vitro and in animal models of HSV disease. To verify the mechanism of antiviral action, resistant viruses were selected by serial passage or by single-step plaque selection of HSV-1 KOS in the presence of inhibitors. Three resistant isolates K138r3, K22r5, and K22r1 were found to be 38-, 316-, and 2500-fold resistant to BILS 22 BS, a potent analog of BILS 45 BS. All three viruses had growth properties in vitro similar to wild-type HSV-1 KOS but they were sensitive to acyclovir. Cutaneous and intra-cerebral inoculation of mice with K22r1 or K22r5 resulted in pathogenicity equivalent to that of HSV-1 KOS. Both isolates were fully competent for reactivation from latency following corneal inoculation. Helicase-primase purified from cells infected with resistant viruses showed decreased inhibition in an in vitro DNA-dependent ATPase assay that correlated well with antiviral resistance. Marker transfer experiments and DNA sequence analysis identified single base pair mutations clustered in the N-terminus of the UL5 gene that resulted in single amino acid changes in the UL5 protein. Taken together, the results indicate that helicase-primase inhibitors prevent HSV growth by inhibiting HSV helicase-primase through specific interaction with the UL5 protein.     

Electrochemical determination of Cephalothin antibiotic by adsorptive stripping voltammetric technique

A sensitive and reliable stripping voltammetric method was developed to determine Cephalothin antibiotic drug. This method is based on the adsorptive accumulation of the drug at a hanging mercury drop electrode and then a negative sweep was initiated, which yield a well defined cathodic peak at −625 mV versus Ag/AgCl reference electrode. To achieve high sensitivity, various experimental and instrumental variables were investigated such as supporting electrolyte, pH, accumulation time and potential, drug concentration, scan rate, convection rate and working electrode area. The monitored adsorptive current was directly proportional to the concentration of Cephalothin and it shows a linear response in the range from 4×10⁻⁷ to 1.2×10⁻⁶ mol l⁻¹ (correlation coefficient=0.9995) and the detection limit (S/N=3) is 3.3×10⁻⁹ mol l⁻¹ at an accumulation time of 3 min. The developed AdSV procedure shows a good reproducibility, the relative standard deviation R.S.D.% (n=10) at a concentration level of 5×10⁻⁷ mol l⁻¹ was 0.94%. Possible interferences by other pharmaceutical drugs and surfactants have been also evaluated. The applicability of this approach was illustrated by the determination of Cephalothin in pharmaceutical preparation and biological fluids such as serum and urine.     

Treatment of thyroid associated ophthalmopathy with periocular injections of triamcinolone

AIM: To evaluate the efficacy of periocular triamcinolone acetonide for the treatment of thyroid associated ophthalmopathy (TAO), and the presence of ocular or systemic adverse effects. METHODS: A multicentre prospective pilot study was performed on patients diagnosed with Graves' ophthalmopathy less than 6 months before entry to the study. Patients were admitted to the study and were randomised into two groups: treatment and control. The treatment group received four doses of 20 mg of triamcinolone acetate 40 mg/ml in a peribulbar injection to the inferolateral orbital quadrant. Both groups were evaluated by measuring the area of binocular vision without diplopia on a Goldmann perimeter and the size of the extraocular muscles on computed tomography (CT) scans. Ophthalmological and systemic examinations were done to rule out ocular and systemic adverse effects. Follow up was 6 months for both groups. RESULTS: 50 patients were eligible for the study. 41 patients completed the study. There was an increase in the area of binocular vision without diplopia in the treatment group (Sigma initial: mean 231.1 (SD 99.9) and final absolute change, mean 107.1 (SD 129.0)) compared to the control group (Sigma initial: mean 350.7 (SD 86.5) and final absolute change, mean -4.5 (SD 67.6)). The sizes of the extraocular muscles were reduced in the treatment group (mean (inferior rectus initial values): 1.3 (0.7), final percentage change: -13.2 (25.7), medial rectus initial values: 1.2 (0.6), final percentage change: -8.2 (20.7), superior rectus-levator palpebrae initial values: 1.2 (0.6), final percentage change: -9.5 (29.1), lateral rectus initial values: 1.0 (0.4), final percentage change: -11.5 (20.6)) compared to the control group (inferior rectus initial values: 0.9 (0.3), final percentage change: -4.0 (21.5), medial rectus initial values: 0.9 (0.3), final percentage change: 0.6 (22.4), superior rectus-levator palpebrae initial values: 0.9 (0.3), final percentage change: 12.5 (37.5), lateral rectus initial values: 0.9 (0.4), final percentage change: -0.5 (31.6)). Both measurements (degree of diplopia and muscle thickness) were statistically significant between groups (initial - final). No systemic or ocular adverse effects were found. CONCLUSIONS: Triamcinolone administered as a periocular injection is effective in reducing diplopia and the sizes of extraocular muscles in TAO ophthalmopathy of recent onset. This form of treatment is not associated with systemic or ocular side effects.     

Phosphodiesterase and thymidine phosphorylase-inhibiting salirepin derivatives from Symplocos racemosa

A re-investigation of the chemical constituents of the stem bark of Symplocos racemosa Roxb. led to the isolation of four new glycosides, symplocomoside (1), symponoside (2), symplososide (3) and symploveroside (4). Benzoylsalireposide (5) and salireposide (6) were re-isolated from this plant. The structures of the new compounds were determined by 1D and 2D-homonuclear and heteronuclear NMR spectroscopy, chemical evidence, and by comparison with the published data of the closely related compounds. The glycosides 1-4 displayed in vitro inhibitory activity against phosphodiesterase I with IC50 values of 122 +/- 0.017, 698 +/- 0.06, 722 +/- 0.03, 909 +/- 0.09 microM, respectively. The compounds 1-6 also showed in vitro inhibitory activity against thymidine phosphorylase with IC50 values of 189.96 +/- 1.02, 195.56 +/- 2.36, 207.61 +/- 1.06, 488.89 +/- 4.10, 427.20 +/- 5.36, 354.2 +/- 5.69 microM, respectively while 1 was also found to be a urease inhibitor with an IC50 value of 54.13 +/- 0.71 microM.     

Capping of radiation-induced DNA breaks in mouse early embryos

The aim of the study was to investigate the spectrum and frequencies of chromosome aberrations induced by the exposure of different mouse spermatogenic germ cell stages to ionizing radiation. Male mice were exposed in vivo to X-rays. Chromosome aberrations were analyzed in first- and second-embryonic cleavages obtained from mating irradiated males with nonirradiated females at different periods after radiation exposure. A combination of telomeric and centromeric labeling as well as whole Y chromosome painting was used to characterize the rejoining pattern and the telomere status of the radiation-induced DNA breaks. The frequency of chromosome aberrations observed in eggs fertilized with sperm irradiated at the early spermatid stage was markedly higher than the frequency in eggs fertilized with sperm irradiated at the other spermatogenic stages when reference was made to the chromosome aberrations recovered in early embryos. At the first division postirradiation, distal rejoining of broken chromosome ends (in regard to the position of the centromere) was more frequent than proximal rejoining; thus compound acentric fragments were more frequently observed than dicentric chromosomes. The presence of additional telomere signals at the broken chromosome ends in mouse germ cells and early embryos, compatible with de novo formation of telomeres, was not frequent.     

Repeated-measures modeling improved comparison of diagnostic tests in meta-analysis of dependent studies

OBJECTIVE: Current methods for meta-analysis of diagnostic tests do not allow utilizing all the information from papers in which several tests have been studied on the same patient sample. We demonstrate how to combine several studies of diagnostic tests, where each study reports on more than one test and some tests (but not necessarily all of them) are shared with other papers selected for the meta-analysis. We adopt statistical methodology for repeated measurements for the purpose of meta-analysis of diagnostic tests. STUDY DESIGN AND SETTING: The method allows for missing values of some tests for some papers, takes into account different sample sizes of papers, adjusts for background and confounding factors including test-specific covariates and paper-specific covariates, and accounts for correlations of the repeated measurements within each paper. It does not need individual-level data, although it can be modified to use them, and uses the two-by-two table of test results vs. gold standard. RESULTS: The results are translated from diagnostic odds ratios (DOR) to more clinically useful measures such as predictive values, post-test probabilities, and likelihood ratios. Models to capture between-study variation are introduced. The fit and influence of specific studies on the regression can be evaluated. Furthermore, model-based tests for homogeneity of DORs across papers are presented. CONCLUSION: The use of this new method is illustrated using a recent meta-analysis of the D-dimer test for the diagnosis of deep venous thrombosis.     

Computer-assisted visualization and quantitation of experimental gastric lesions in rats

INTRODUCTION: Photography is commonly used to evaluate macroscopic morphology of experimental gastric lesions. The conventional methods for quantitation of these lesions tend to be highly subjective, error-prone, and tedious. This study reports a simple and novel method for visualization of experimental gastric lesions by direct scanning of stomach samples and their quantitation by using computer-assisted image analysis. METHODS: Gastric mucosal lesions were produced by indomethacin or ethanol in male Wistar rats. The stomachs were removed, opened along the greater curvature, and washed. The flattened stomach samples were sandwiched within a transparent plastic folder and scanned. The captured image was saved and subjected to lesion quantitation using a freely accessible image analysis program, Scion Image. A macro subroutine was optimized for automation of the entire procedure for ease in frequent applications. RESULTS: The images obtained by direct scanning of stomach samples showed excellent morphologic visualization of gastric lesions. The standard spots having variable areas of same density, or same areas of variable densities, showed a direct relation with signal intensity (pixels). A linear correlation was observed between measured and actual values of the known standard areas. The representative samples of indomethacin- and ethanol-induced gastric ulcers showed the total lesion areas of 4.62+/-0.74 and 169.26+/-13.23 mm(2), respectively. DISCUSSION: Direct scanning of stomach samples is a simple, cost-effective, rapid, and efficient procedure for morphologic evaluation of experimental gastric lesions. The output images of scanned stomach samples are superior to scanned photographs, and can be instantly used for quantitative assessment of mucosal injury using the image analysis program.