Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.     

Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers

Micropropagated plants from two wild-olive species, Olea maderensis and O. europaea ssp. europaea var. sylvestris were screened for genetic stability. O. maderensis shoots were elongated/multiplied on OMG medium with zeatin (9.12 μM), and rooted on 1/2 OMG with NAA (3.22 μM). O. europaea var. sylvestris shoots were elongated/multiplied on OM medium with zeatin, and rooting was optimal after a hormonal shock (IBA 100 μM) followed by transfer to the same medium without growth regulators. In both species, acclimatization was successful and plants looked normal and morphologically identical to the donor field trees. Genetic variability was assessed at several stages of the micropropagation process using flow cytometry (FCM) and nuclear microsatellites (SSR). No changes in ploidy level were found among micropropagated plants, though small deviations, putatively due to the negative effects of cytosolic compounds on propidium iodide staining, between these and field plants were observed. In SSRs analyses, ten SSR markers were able to distinguish between genotypes. No mutations were found in these tested SSR loci among the donor tree and micropropagated plants, suggesting, for the tested markers, genetic uniformity throughout the process. The FCM and SSR results obtained do not exclude the occurrence of other changes in the nuclear genome but, considering the morphological stability of micropropagated plants, indicate that both protocols are suitable and efficient for large scale, true-to-type micropropagation of these two wild olive species.     

Assessing the control/eradication of an invasive species,the American mink,based on field data; how much would it cost?

Biological invasions are an important cause of biodiversity loss, American mink being one of the worst invasive species in Europe. We performed a 13-week control program of the species in the Butron river system (Northern Spain), where a natural population of the European mink is found. Three population estimates were considered: an absolute minimum, an intermediate scenario and a pessimistic one (n = 35, 49 and 70 animals, respectively). After 2,242 cage trap-nights, trapping success varied from 44 to 89% of these estimates. In addition, we evaluated the costs of eradicating the estimated populations; costs ranged between 652.5 and 2,970 € per mink, and would rise up to 83,462 € for the intermediate estimate under an exponential function linking captures and costs, or ca. 172.500 € to capture the highest estimate under a log-function. The implications of these numbers for the design and implementation of future control projects are discussed.     

Growth and mortality patterns in a thinning canopy of post-hurricane regenerating rain forest in eastern Nicaragua (1990-2005)

One of the strongest hypothesis about the maintenance of tree species diversity in tropical areas is disturbance. In order to assess this, the effect of intensive natural disturbances on forest growth and mortality in a thinning canopy was studied after the landfall of hurricane Joan in 1988. We evaluated the growth and mortality rates of the 26 most common tree species of that forest in eastern Nicaragua. Permanent plots were established at two study sites within the damaged area. Growth and mortality rates of all individual trees > or = 3.18cm diameter at breast height were assessed annually from 1990 to 2005. During this period the forest underwent two phases: the building phase (marked by increased number of individuals of tree species present after the hurricane) and the canopy thinning phase (marked by increased competition and mortality). Our results from the thinning phase show that tree survival was independent of species identity and was positively related to the increase in growth rates. The analysis of mortality presented here aims to test the null hypothesis that individual trees die independently of their species identity. These findings were influenced by the mortality observed during the late thinning phase (2003-2005) and provide evidence in favor of a non-niche hypothesis at the thinning phase of forest regeneration.     

Ultracentrifugation of serum samples allows detection of hepatitis C virus RNA in patients with occult hepatitis C

Occult hepatitis C virus (HCV) infection of patients with abnormal liver function tests of unknown origin who are anti-HCV and serum HCV RNA negative but who have HCV RNA in the liver has been described. As HCV replicates in the liver cells of these patients, it could be that the amount of circulating viral particles is under the detection limit of the most sensitive techniques. To prove this hypothesis, serum samples from 106 patients with occult HCV infection were analyzed. Two milliliters of serum was ultracentrifuged over a 10% sucrose cushion for 17 h at 100,000 x g(av), where av means average, and HCV RNA detection was performed by strand-specific real-time PCR. Out of the 106 patients, 62 (58.5%) had detectable serum HCV RNA levels after ultracentrifugation, with a median load of 70.5 copies/ml (range, 18 to 192). Iodixanol density gradient studies revealed that HCV RNA was positive at densities of 1.03 to 1.04 and from 1.08 to 1.19 g/ml, which were very similar to those found in the sera of patients with classical chronic HCV infection. Antigenomic HCV RNA was found in the livers of 56 of 62 (90.3%) patients with detectable serum HCV RNA levels after ultracentrifugation, compared to 27 of 44 (61.4%) negative patients (P < 0.001). No differences in the median loads of antigenomic HCV RNA between patients with an those without serum HCV RNA (4.5 x 10(4) [range, 7.9 x 10(2) to 1.0 x 10(6)] versus 2.3 x 10(4) [range, 4.0 x 10(2) to 2.2 x 10(5)]) were found. Alanine aminotransferase and gamma-glutamyl transpeptidase levels, liver necroinflammatory activity, and fibrosis did not differ between both groups. In conclusion, HCV RNA can be detected in the sera of patients with occult HCV infection after circulating viral particles are concentrated by ultracentrifugation.     

Distribution of flower morphs, ploidy level and sexual reproduction of the invasive weed Oxalis pes-caprae in the western area of the Mediterranean region

Background and Aims: Oxalis pes-caprae is a widespread invasive weed in regions witha Mediterranean climate. In its native habitat (southern Africa)this species has been reported as heterostylous with trimorphicflowers and a self- and morph-incompatible reproductive system.In most of the areas invaded, only a pentaploid short-styledmorphotype that reproduces mainly asexually by bulbils is reported,but this has only been confirmed empirically. This study aimsto analyse the floral morph proportions in a wide distributionarea, test the sexual female success, and explain the causesof low sexual reproduction of this species in the western areaof the Mediterranean Basin. Methods: Fifty-five populations of O. pes-caprae were sampled in theIberian Peninsula and Morocco to evaluate the floral morph ratioand individual fruit set. In plants from a dimorphic population,hand-pollination experiments were performed to evaluate theeffect of the pollen source on pollen tube growth through thestyle. The ploidy level and genome size of individuals of eachfloral morph were analysed using flow cytometry. Key Results: From the populations studied 89·1 % were monomorphic,with most of them containing the short-styled (SS) floral morph,and 10·9 % were dimorphic containing long-styled(LS) and SS morphs. In some of these, isoplethy was verifiedbut no fruit production was observed in any population. A sterileform was also recorded in several populations. Hand-pollinationexperiments revealed that pollen grains germinated over recipientstigmas. In intermorph crossings, pollen tubes were able todevelop and fruit initiation was observed in some cases, whilein intramorph pollinations, pollen tube development was sporadicand no fruit initiation was observed. All individuals withineach floral form presented the same DNA ploidy level: SS plantswere pentaploid and LS and the sterile form were tetraploid. Conclusions: The low or null sexual reproduction success of this speciesin the area of invasion studied seems related with the highfrequency of monomorphic populations, the unequal proportionof floral morphs in dimorphic populations and the presence ofdifferent ploidy levels between SS and LS morphs. The discoveryof the occurrence of an LS floral morph and a sterile form,whose invading capacity in these areas is as yet unknown, willbe valuable information for management programmes.     

Path tortuosity of Eurasian badgers (<Emphasis Type="Italic">Meles meles</Emphasis>) in a heterogeneous Mediterranean landscape

Movement is the process by which individual organisms are displaced over time to eat, reproduce and defend resources. Fractal analysis is a technique used to study animal movement that measures spatial complexity of path tortuosity; here, we apply it to characterize the movement patterns of the Eurasian badger (Meles meles) in a Mediterranean landscape. We calculated path tortuosity overall and seasonally, and for individuals of different sexes and social groups. The influence of variables related to badgers’ resources (food, shelter, water), human infra-structures and weather conditions were analysed with respect to the tortuosity of each badger’s path. A total of 55 search paths from six badgers were considered for this study. Although badgers generally displayed convoluted movement, there were two exceptions: (a) males overall and (b) all badgers in summer; for both, movements had a lower fractal value, i.e. were less tortuous. The convoluted movement pattern generally observed is probably adapted to the clumped distribution of food in the study area. Nevertheless, our results suggest that the use of dens and latrines were the principal determinants of tortuosity of badgers’ paths while foraging.