Antiprion compounds that reduce PrPSc levels in dividing and stationary-phase cells

During prion diseases, a normally benign, host protein, denoted PrP^C, undergoes alternative folding into the aberrant isoform, PrP^Sc. We used ELISA to identify and confirm hits in order to develop leads that reduce PrP^Sc in prion-infected dividing and stationary-phase mouse neuroblastoma (ScN2a-cl3) cells. We tested 52,830 diverse small molecules in dividing cells and 49,430 in stationary-phase cells. This led to 3100 HTS and 970 single point confirmed (SPC) hits in dividing cells, 331 HTS and 55 confirmed SPC hits in stationary-phase cells as well as 36 confirmed SPC hits active in both. Fourteen chemical leads were identified from confirmed SPC hits in dividing cells and three in stationary-phase cells. From more than 682 compounds tested in concentration–effect relationships in dividing cells to determine potency (EC₅₀), 102 had EC₅₀ values between 1 and 10 μM and 50 had EC₅₀ values of <1 μM; none affected cell viability. We observed an excellent correlation between EC₅₀ values determined by ELISA and Western immunoblotting for 28 representative compounds in dividing cells (R² = 0.75; p <0.0001). Of the 55 confirmed SPC hits in stationary-phase cells, 23 were piperazine, indole, or urea leads. The EC₅₀ values of one indole in stationary-phase and dividing ScN2a-cl3 cells were 7.5 and 1.6 μM, respectively. Unexpectedly, the number of hits in stationary-phase cells was ～10% of that in dividing cells. The explanation for this difference remains to be determined.     

Clomipramine and benznidazole association for the treatment of acute experimental Trypanosoma cruzi infection

Alternative strategies are being designed to identify candidates among drugs already available on the market that could be used in combination to improve the efficacy of Chagas disease treatment. This work evaluates the effect of the association of clomipramine (CLO) with benznidazole (BZN) for the treatment of experimental Chagas disease in the acute stage, in Swiss albino mice infected with Trypanosoma cruzi Tulahuen strain. Infected mice were treated with CLO 5 mg/kg/day and BZN 50 and 100 mg/kg/day, each separately or together. Efficacy of the treatment was evaluated through parasitemia, survival, electrocardiography, histopathological studies, serological and PCR assays at 90 days post-infection (dpi). All treatments significantly (P < 0.05) reduced mortality and decreased parasitemia. Histopathological analysis of liver and kidneys of mice treated with CLO and the drug combination showed less injury than mice treated only with BZN. The lower dose of BZN (50 mg/kg/day) combined with CLO showed the same efficacy as the habitual dose of BZN (100 mg/kg/day) combined with CLO. The therapeutic results from the combination of BZN with CLO presented lesser side effects than the treatment with BZN.     

Using choice experiments to understand public demand for the conservation of nature: A case study in a protected area of Chile

We conducted a choice experiment for the economic valuation of benefits of components of biodiversity that are provided by the natural systems protected in the Peñuelas Lake National Reserve, located in the Mediterranean zone of Chile. The Mediterranean zone of central Chile is one of the world's 34 biodiversity hotpots. Furthermore, we estimated the economic benefits of the water supply provided by the reserve. Unlike most of the previous studies on willingness to pay (WTP) for conservation, part of the data that we produce refers to: (a) public WTP for unpopular species of fauna (an endemic amphibian currently in danger of extinction); and (b) public WTP for flora present at the Reserve (endemic orchid species). Specifically the attributes of the study were the following: existence of endemic orchid species; chances of observing animals with scenic attraction; additional protection for an endemic amphibian; and, availability of drinkable water in the future. A rate of entry to the area was incorporated to estimate WTP for additional protection for the selected attributes. WTP data were obtained from a representative sample of Chilean tourists that visit the area. Factors influencing the visitors’ WTP were also explored.Three hundred and four Chilean visitors of the reserve were randomly selected for interviews. Multinomial Logit and Random Parameter Logit models results show that visitors are willing to pay to protect the selected attributes. Marginal mean WTP/visitor for the single levels of variation of the attributes range from about $1.7 per visitor per visit for securing the existence of five species of endemic orchids to about $8.9 for guaranteeing the availability of drinkable water for 50 years. The analysis of visitors’ WTP for different levels of protection allowed an improved understanding of the sensitivity of the participants to the scope of the information provided.     

Effects of bleaching on the pharmacological and toxicological activities elicited by the aqueous extracts prepared from two “fire corals” collected in the Mexican Caribbean

Hydrocorals of the genus Millepora are abundant skeleton-forming inhabitants of coral reefs around the world. These species are popularly known as “fire corals” since contact with them causes severe pain, skin eruptions and blisters as a result of the release of unidentified toxins. Millepora species associate with photosynthetic dinoflagellates of the genus Symbiodinium (“zooxanthellae”), and up to now the role of these symbionts in the toxic effects induced by the “fire corals” is unknown. In this study, we compared the hemolytic, vasoconstrictor, and phospholipase A₂ (PLA₂) activities of the crude aqueous extracts prepared from normal and bleached specimens of two hydrocorals collected in the Mexican Caribbean, Milleporaalcicornis and Millepora complanata. Electrophoretic analysis revealed some differences between the protein profiles of the extracts prepared from normal and bleached specimens. Bleaching decreased, but not abolished, the hemolytic effect induced by the hydrocorals extracts and the phospholipase A₂ activity of M. complanata extract. Furthermore, it did not modify the enzymatic activity of M. alcicornis extract and vasoconstriction elicited by both extracts. Our results suggest that the presence of the symbionts does not importantly influence the pharmacological and toxic effects induced by Millepora ssp. extracts, and indicate that cnidarians are the main source of the bioactive compounds.     

Long signaling cascades tend to attenuate retroactivity

Signaling pathways consisting of phosphorylation/dephosphorylation cycles with no explicit feedback allow signals to propagate not only from upstream to downstream but also from downstream to upstream due to retroactivity at the interconnection between phosphorylation/dephosphorylation cycles. However, the extent to which a downstream perturbation can propagate upstream in a signaling cascade and the parameters that affect this propagation are presently unknown. Here, we determine the downstream-to-upstream steady-state gain at each stage of the signaling cascade as a function of the cascade parameters. This gain can be made smaller than 1 (attenuation) by sufficiently fast kinase rates compared to the phosphatase rates and/or by sufficiently large Michaelis-Menten constants and sufficiently low amounts of total stage protein. Numerical studies performed on sets of biologically relevant parameters indicated that ∼50% of these parameters could give rise to amplification of the downstream perturbation at some stage in a three-stage cascade. In an n-stage cascade, the percentage of parameters that lead to an overall attenuation from the last stage to the first stage monotonically increases with the cascade length n and reaches 100% for cascades of length at least 6.     

Induced pluripotent stem cells' self-renewal and pluripotency is maintained by a bovine granulosa cell line-conditioned medium

Senescence marker protein-30 (SMP30) plays an important role in intracellular Ca²⁺ homeostasis. The aim of the present study was to investigate the effects of estrogens on liver apoptotic damage and changes in SMP30 expression induced by a high saturated fatty acid diet (HSFD). Ovariectomized mice (OVX) and sham-operated mice (SHAM) were randomly divided into five groups: SHAM fed a normal diet (SHAM/ND), SHAM fed HSFD (SHAM/HSFD), OVX fed ND (OVX/ND), OVX fed HSFD (OVX/HSFD) and OVX fed HSFD with 17β-estradiol (E2) supplementation using an implanted slow-release pellet (OVX/HSFD + E2). After 8 weeks, markers of endoplasmic reticulum (ER) stress and apoptosis, and levels of tumor necrosis factor-α (TNFα and SMP30 expression were investigated. Compared with SHAM/ND, OVX/HSFD mice showed significantly increased spliced X-box protein-1 (s-XBP1), phosphorylated eukaryotic initiation factor-2α (p-eIF2α), glucose-regulated protein 78 (GPR78), C/EBP homologous protein (CHOP), cytosolic cytochrome c, caspase-3 activity, and TNFα, and significantly decreased SMP30. These differences in OVX/HSFD mice were restored to the levels of SHAM/ND mice by E2 supplementation. These results suggest that E2 supplementation attenuates HSFD-induced liver apoptotic death in ovariectomized mice by up-regulating SMP30.