Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors

The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.     

Rapid and Biosecure Diagnostic Test for Tuberculosis

Early and rapid detection of the causative organism is necessary in tuberculosis. We present here an integrated and dedicated molecular biology system for tuberculosis diagnosis. One hundred and eighty-nine (189) biologic specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl–Neelsen staining, cultivation on a solid medium, and by a balanced heminested fluorometric PCR system (Orange G3TB) that preserves worker safety and produces a rather pure material free of potential inhibitors. DNA amplification was carried out in a low cost using a tuberculosis thermocycler-fluorometer. The double stranded DNA produced is fluorometrically detected. The whole reaction is carried out in one single tube which is never opened after adding the processed sample, thus minimizing the risk of cross contamination with amplicons. The assay is able to detect 30 bacilli/ml of sample having a 99.8 % inter-assay coefficient of variation. PCR was positive in 36 (18.9 %) tested samples (33 of them were smear-negative). In our study, it yields a preliminary overall sensitivity of 97.4 %. In addition, its overall specificity is 98.7 %. The total run time of the test is 4 h with two and a half real working hours. All PCR-positive samples also had a positive result by microbiological culture and clinical criteria. The results obtained showed that it could be a very useful tool to increase efficiency in detecting the tuberculosis disease in low bacillus inoculum samples. Furthermore, its low cost and friendly usage make it feasible to be used in regions with poor development.     

Craniometric sexual dimorphism inLeontopithecus Lesson, 1840 (Callitrichidae, Primates)

Lion tamarins are among the World's most critically endangered primates. Many studies have been produced under guidance of the International Management Committees for the preservation and management of these tamarins. Primates present morphological sexual differences in a wide range of characteristics, including cranial morphology. Studies of sexual dimorphism in the cranial morphology of theLeontopithecus are few in number and contradictory in their results. In order to check for the existence of sexual dimorphism in lion tamarins the present study analyzed 17 craniometric distances on 56 crania of three species of lion tamarins (Leontopithecus): 20L. rosalia (14 females and 6 males); 13L. chrysomelas (6 females and 7 males); and 23L. chrysopygus (8 females and 15 males). All crania are housed in the CPRJ-FEEMA collection (Primatological Center of Rio de Janeiro) and came from animals born in captivity.L. chrysopygus was more sexually dimorphic (10/17 measurements, 59%) thanL. chrysomelas (9/17 measurements, 53%) orL. rosalia (7/17 measurements, 41%). In all three species, male values are greater than the female ones, except for orbital breadth (m7) inL. rosalia. However, this distance is not sexually dimorphic in this species. This study reveals that some cranial distances, especially in the facial region, are sexually dimorphic in lion tamarins.     

Ultrasonographic aspects of the Leontopithecus gestation (Lesson, 1840—Callitrichidae,Primates)

Background

There is a concern about conservation of endangered species today. Among this species, the Leontopithecus (Lesson, 1840) is outstanding. Its population has been whirling reduced. So far the reproductive physiology of Leontopithecus has few studies, it is fundamental requisite to preserve this species. Obstetric sonography has become an essential method in reproductive management of primates.

Method

This method is very helpful to detect early pregnancy and evaluate some deficiency of fetal growth. In this study, 14 pregnancies were monitored using real‐time abdominal sonography. During each evaluation, the number of fetus was recorded, gestational sac and heart beats were observed, and biparietal diameter was measured.

Results

The results showed that abdominal sonography is a reliable method for observation of gross morphological changes during pre‐natal development and to estimate gestational age. No statistically differences were observed between twins and singletons. This study is the first investigation of pre‐natal growth in Leontopithecus.     

Synergistic interaction between ouabain and 8-methoxy-3,9-dihydroxy coumestan, a non-steroidal synthetic inhibitor of Na+,K+-ATPase

The use of combination drugs is very common in therapeutics as in the treatment of infectious diseases, cancer and heart failure but controversies about analysis of these interactions are frequent. The aim of the present work was to characterize the interaction between ouabain and 8-methoxy-3,9-dihydroxy coumestan (LQB93), a non-steroidal synthetic inhibitor of Na⁺,K⁺-ATPase, as well as the interaction between ouabain and ouabagenin, two cardiac glycosides sharing the same binding site. Inhibition of rat kidney Na⁺,K⁺-ATPase with increasing concentrations of the drugs alone or of mixtures of ouabain:ouabagenin and LQB93:ouabain in a fixed 1:4 ratio was performed. In other experiments, increasing concentrations of LQB93 (or ouabain) in the presence of a fixed concentration of ouabain (or ouabagenin) were used for determining the concentration pairs eliciting 50% inhibition in order to construct isobolograms. The mixture (experimental) curve for the ouabain:ouabagenin combination was superimposed on the additive (theoretical) curve indicating additivity, in accordance with the isobolographic analysis. On the other hand, the empirical curve for LQB93:ouabain (IC₅₀ = 10.6 μM) was significantly shifted to the left in relation to the theoretical curve (IC₅₀ = 30.7 μM) indicating synergism, further confirmed by the isobolographic analysis. As a conclusion, we show that the combination of a newly synthesized non-steroidal inhibitor and ouabain have a synergistic effect on Na⁺,K⁺-ATPase, further supporting a mechanism of inhibition different from ouabain. Present data also support the use of both the isobolograms and combination curves for the assessment of drug interactions occurring at the same molecular target, a situation poorly investigated.     

Chromosome phylogeny of the subfamily Pitheciinae (Platyrrhini,Primates) by classic cytogenetics and chromosome painting

Background  

The New World monkey (Platyrrhini) subfamily Pitheciinae is represented by the genera Pithecia, Chiropotes and Cacajao. In this work we studied the karyotypes of Pithecia irrorata (2n = 48) and Cacajao calvus rubicundus (2n = 45 in males and 2n = 46 in females) by G- and C-banding, NOR staining and chromosome painting using human and Saguinus oedipus whole chromosome probes. The karyotypes of both species were compared with each other and with Chiropotes utahicki (2n = 54) from the literature.