Palmitoylethanolamide Treatment Reduces Blood Pressure in Spontaneously Hypertensive Rats: Involvement of Cytochrome P450-Derived Eicosanoids and Renin Angiotensin System

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α agonist, has been demonstrated to reduce blood pressure and kidney damage secondary to hypertension in spontaneously hypertensive rat (SHR). Currently, no information is available concerning the putative effect of PEA on modulating vascular tone. Here, we investigate the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids, CYP-dependent arachidonic acid metabolites, as endothelium-derived hyperpolarizing factors (EDHF), and renin angiotensin system (RAS) modulation. To achieve this aim SHR and Wistar-Kyoto rats were treated with PEA (30 mg/kg/day) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF-mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure CYP2C23 and CYP2J2, the key isoenzymes in the formation of epoxyeicosatrienoic acids, and the soluble epoxide hydrolase, which is responsible for their degradation in the corresponding diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme and angiotensin receptor 1 expression. Here, we showed that EDHF-mediated dilation in response to acetylcholine was increased in mesenteric beds of PEA-treated SHR. Western blot analysis revealed that the increase in CYP2C23 and CYP2J2 observed in SHR was significantly attenuated in mesenteric beds of PEA-treated SHR, but unchanged in the carotids. Interestingly, in both vascular tissues, PEA significantly decreased the soluble epoxide hydrolase protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid, implying a reduction in epoxyeicosatrienoic acid hydrolisis. Moreover, PEA treatment down-regulated angiotensin receptor 1 and angiotensin converting enzyme expression, indicating a reduction in angiotensin II-mediated effects. Consistently, a damping of the activation of angiotensin receptor 1 underlying pathways in mesenteric beds was shown in basal conditions in PEA-treated SHR. In conclusion, our data demonstrate the involvement of epoxyeicosatrienoic acids and renin angiotensin system in the blood pressure lowering effect of PEA.     

Effects of Hormone Therapy on Cognition and Mood in Recently Postmenopausal Women: Findings from the Randomized,Controlled KEEPS–Cognitive and Affective Study

Background

Menopausal hormone therapy (MHT) reportedly increases the risk of cognitive decline in women over age 65 y. It is unknown whether similar risks exist for recently postmenopausal women, and whether MHT affects mood in younger women. The ancillary Cognitive and Affective Study (KEEPS-Cog) of the Kronos Early Estrogen Prevention Study (KEEPS) examined the effects of up to 4 y of MHT on cognition and mood in recently postmenopausal women.

Methods and Findings

KEEPS, a randomized, double-blinded, placebo-controlled clinical trial, was conducted at nine US academic centers. Of the 727 women enrolled in KEEPS, 693 (95.3%) participated in the ancillary KEEPS-Cog, with 220 women randomized to receive 4 y of 0.45 mg/d oral conjugated equine estrogens (o-CEE) plus 200 mg/d micronized progesterone (m-P) for the first 12 d of each month, 211 women randomized to receive 50 μg/d transdermal estradiol (t-E2) plus 200 mg/d m-P for the first 12 d of each month, and 262 women randomized to receive placebo pills and patches. Primary outcomes included the Modified Mini-Mental State examination; four cognitive factors: verbal learning/memory, auditory attention/working memory, visual attention/executive function, and speeded language/mental flexibility; and a mood measure, the Profile of Mood States (POMS). MHT effects were analyzed using linear mixed-effects (LME) models, which make full use of all available data from each participant, including those with missing data. Data from those with and without full data were compared to assess for potential biases resulting from missing observations. For statistically significant results, we calculated effect sizes (ESs) to evaluate the magnitude of changes.On average, participants were 52.6 y old, and 1.4 y past their last menstrual period. By month 48, 169 (24.4%) and 158 (22.8%) of the 693 women who consented for ancillary KEEPS-Cog were lost to follow-up for cognitive assessment (3MS and cognitive factors) and mood evaluations (POMS), respectively. However, because LME models make full use all available data, including data from women with missing data, 95.5% of participants were included in the final analysis (n = 662 in cognitive analyses, and n = 661 in mood analyses). To be included in analyses, women must have provided baseline data, and data from at least one post-baseline visit. The mean length of follow-up was 2.85 y (standard deviation [SD] = 0.49) for cognitive outcomes and 2.76 (SD = 0.57) for mood outcomes. No treatment-related benefits were found on cognitive outcomes. For mood, model estimates indicated that women treated with o-CEE showed improvements in depression and anxiety symptoms over the 48 mo of treatment, compared to women on placebo. The model estimate for the depression subscale was −5.36 × 10⁻² (95% CI, −8.27 × 10⁻² to −2.44 × 10^−2; ES = 0.49, p < 0.001) and for the anxiety subscale was −3.01 × 10⁻² (95% CI, −5.09 × 10⁻² to −9.34 × 10⁻³; ES = 0.26, p < 0.001). Mood outcomes for women randomized to t-E2 were similar to those for women on placebo. Importantly, the KEEPS-Cog results cannot be extrapolated to treatment longer than 4 y.

Conclusions

The KEEPS-Cog findings suggest that for recently postmenopausal women, MHT did not alter cognition as hypothesized. However, beneficial mood effects with small to medium ESs were noted with 4 y of o-CEE, but not with 4 y of t-E2. The generalizability of these findings is limited to recently postmenopausal women with low cardiovascular risk profiles.

Trial Registration

ClinicalTrials.gov NCT00154180 and NCT00623311     

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.     

Diatoms and Other Epibionts Associated with Olive Ridley (Lepidochelys olivacea) Sea Turtles from the Pacific Coast of Costa Rica

Although the sea turtles have long been familiar and even iconic to marine biologists, many aspects of their ecology remain unaddressed. The present study is the first of the epizoic diatom community covering the olive ridley turtle’s (Lepidochelys olivacea) carapace and the first describing diatoms living on sea turtles in general, with the primary objective of providing detailed information on turtle epibiotic associations. Samples of turtle carapace including the associated diatom biofilm and epizoic macro-fauna were collected from Ostional beach (9° 59´ 23.7´´ N 85° 41´ 52.6´´ W), Costa Rica, during the arribada event in October 2013. A complex diatom community was present in every sample. In total, 11 macro-faunal and 21 diatom taxa were recorded. Amongst diatoms, the most numerous were erect (Achnanthes spp., Tripterion spp.) and motile (Haslea sp., Navicula spp., Nitzschia spp., Proschkinia sp.) forms, followed by adnate Amphora spp., while the most common macro-faunal species was Stomatolepas elegans (Cirripedia). Diatom densities ranged from 8179 ± 750 to 27685 ± 4885 cells mm^-2. Epizoic microalgae were either partly immersed or entirely encapsulated within an exopolymeric coat. The relatively low diatom species number, stable species composition and low inter-sample dissimilarities (14.4% on average) may indicate a mutualistic relationship between the epibiont and the basibiont. Dispersal of sea turtle diatoms is probably highly restricted and similar studies will help to understand both diatom diversity, evolution and biogeography, and sea turtle ecology and foraging strategies.     

Latitudinal Variation in Termite Species Richness and Abundance along the Brazilian Atlantic Forest Hotspot

In this study, we investigated the termites of the Brazilian Atlantic Forest, one of the most threatened biodiversity hotspots in the world, in regularly spaced sites from 7° S to 27° S latitude. To our knowledge, this is the only report of a latitudinal survey of termites at species level performed with a standardized sampling protocol. We evaluate termite diversity and abundance, and describe patterns of species composition based on feeding groups along the latitudinal gradient. We also describe the relative contribution of environmental variables to explain diversity patterns. Termite assemblages were investigated by standardized surveys at 15 Atlantic Forest sites, on six transects divided into five sections of 10 m², with 30 sections per site (or 300 m²/site), which were investigated by one trained person for one hour. Observed species richness and abundance were negatively correlated with latitude. The influence of latitude was explained mainly by variables related to temperature, precipitation and ambient energy (potential evapotranspiration). Our results also suggest that temperature exerts a greater constraint on Atlantic Forest termites than productivity, because ambient productivity increases with latitude in this forest but termite diversity decreases. Termite species richness in the Atlantic Forest showed a different pattern than those described for other organisms, increasing in diversity where the coastal‐forest strip narrows. Overall, our results indicate comparatively high termite species richness at northeastern sites and a significant impoverishment of termite assemblages in the southeastern and southern regions of the Atlantic Forest.     

An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5′ end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.     

The conserved and divergent roles of carbonic anhydrases in the filamentous fungi Aspergillus fumigatus and Aspergillus nidulans

Carbon dioxide (CO₂) and its hydration product bicarbonate (HCO₃^‐) are essential molecules in various physiological processes of all living organisms. The reversible interconversion between CO₂ and HCO₃^‐ is in equilibrium. This reaction is slow without catalyst, but can be rapidly facilitated by Zn²⁺‐metalloenzymes named carbonic anhydrases (CAs). To gain an insight into the function of multiple clades of fungal CA, we chose to investigate the filamentous fungi Aspergillus fumigatus and A. nidulans. We identified four and two CAs in A. fumigatus and A. nidulans, respectively, named cafA‐D and canA‐B. The cafA and cafB genes are constitutively, strongly expressed whereas cafC and cafD genes are weakly expressed but CO₂‐inducible. Heterologous expression of the A. fumigatus cafB, and A. nidulans canA and canB genes completely rescued the high CO₂‐requiring phenotype of a Saccharomyces cerevisiaeΔnce103 mutant. Only the ΔcafAΔcafB and ΔcanB deletion mutants were unable to grow at 0.033% CO₂, of which growth defects can be restored by high CO₂. Defects in the CAs can affect Aspergilli conidiation. Furthermore, A. fumigatusΔcafA, ΔcafB, ΔcafC, ΔcafD and ΔcafAΔcafB mutant strains are fully virulent in a low‐dose murine infection.