Interference of aging media on the assessment of yeast chronological life span by propidium iodide staining

An increasing number of researchers are using the Saccharomyces cerevisiae chronological aging model to gain insight into the post-mitotic cellular aging. Recently, an alternative approach to the traditional cellular viability assay by colony-forming unit (CFU) counts, based on the propidium iodide (PI) staining combined with flow cytometry (PI-FCM), was proposed for the assessment of yeast chronological aging. Since the chronological aging assessment shows variations particularly concerning the aging media, in this work, the influence of the most common aging media (exhausted media or water) on the assessment of chronological aging by PI staining was studied. Our results show that this methodology is highly affected by the aging media. Indeed, a correlation between CFU counts and the percentage of PI-stained cells is only achieved with the exhausted media. As such, the assessment of yeast chronological aging by PI-FCM water should not be used.     

A melissopalynological study of artisanal honey produced in Catamarca (Argentina)

Spores belonging to 43 taxa of pteridophytes were observed from the ambient air of 11 localities in India. It is suggested that pteridophyte spores could be listed among viable particulate air pollutants, especially in the light of the earlier reports regarding their allergenicity.     

Discovery,synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor antagonists

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1 and orexin 2 receptors along with low cytochrome P450 inhibition potential, good brain penetration and oral bioavailability in rats.     

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio)

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na⁺, K⁺-ATPase, H⁺-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na⁺, K⁺-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na⁺. Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na⁺ import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Influence of seasonal variation on the phenology and liriodenine content of Annona lutescens (Annonaceae)

Annona lutescens Saff. (Annonaceae) grows as a native tree in Chiapas, Mexico in Tropical Dry Forest habitat. Like most Annonaceae, it biosynthesizes benzylisoquinoline alkaloids, mostly liriodenine. To determine the influence of seasonal changes in the accumulation of liriodenine, the monthly variation of liriodenine content in roots, stems and leaves of mature and young trees was observed. These parts of young and mature A. lutescens trees were collected monthly over a 1 year period and the alkaloids were extracted; the liriodenine was quantified by high-resolution liquid chromatography. The phenological stages of the species were also assessed (leaf development, flowering and fruiting) using the Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie (BBCH) scale. The analysis of both young and mature trees showed a significant increase in the liriodenine concentration occurs within roots during the dry season, which coincides with leaf fall. A significant decrease also occurred at the beginning of the rainy season (the period of leaf growth); the liriodenine content for the next rainy season did not reach the levels of the previous dry season. The climatic variation induced phenological and physiological changes in this species.     

Diurnal Variation of Plasmatic Melatonin,Corticosterone and Variation of General Activity in Pigeons under Light-Dark Cycle and Constant Light

This work analyzed the diurnal variation of general activity and plasmatic levels of melatonin and corticosterone in pigeons submitted to a 12:00:12:00 h light-dark cycle (lights on at 6:00 a.m.) or to constant light. In both conditions pigeons were observed in 5-min sessions at times 03:00, 06:00, 09:00, 12:00, 15:00, 18:00, 21:00 and 24:00 h during two successive days. Behavior was video taped in the home cages for posterior categorization and quantification. Radioimmunoassays were used to evaluate plasmatic levels of melatonin and corticosterone. Blood samples were obtained at the times of behavioral observation. In the light-dark condition the results showed day-night variation of general activity (p < 0.001) and a robust diurnal rhythm of plasmatic melatonin (p < 0.001). Both of these variations as well as the oscillatory secretion of corticosterone disappeared under constant light condition. The parallel changes in general activity and blunting of melatonin rhythm secretion in constant light condition agree with previous evidences that melatonin may regulate behavioral oscillations in the pigeon. The present data are related to the proposition that the timing system in pigeons may involve neuroendocrine relations characterized by interactions between blood born signalization by melatonin and corticosterone.     

Circaseptan Rhythms of Semen Characteristics of a Brazilian Breed (“Mangalarga”) Stallion

We describe the semen characteristics of a 21-year old “mangalarga” stallion living under natural temperature and photoperiod conditions in São José do Rio Pardo, São Paulo, Brazil (latitude 21°36' S; longitude 46°53' W). The horse fed on natural pasture and a nutritionally balanced feed twice a day (11:00 and 17:00 h). Water and a mineral supplement were available “ad libitum”. Semen was collected for over 36 months (Oct 89-Dec 92) almost daily between 08:00 and 10:00 h by an artificial vagina and evaluated for volume of ejaculate, spermatozoa motility and concentration by standard procedures. Analysis of data from a total of 128-days was performed according to the Fast Fourier Transform Technique (FFT). Statistically significant periods of 7-day were demonstrated for volume, motility, and spermatozoa concentration. Circaseptan rhythms and, particularly, the circannual rhythm (already described in a previous publication) are probably related to ecological diversity and reproductive strategies.     

Tracking through Life Stages: Adult,Immature and Juvenile Autumn Migration in a Long-Lived Seabird

Seasonal long-distance migration is likely to be experienced in a contrasted manner by juvenile, immature and adult birds, leading to variations in migratory routes, timing and behaviour. We provide the first analysis of late summer movements and autumn migration in these three life stages, which were tracked concurrently using satellite tags, geolocators or GPS recorders in a long-ranging migratory seabird, the Scopoli’s shearwater (formerly named Cory’s shearwater, Calonectris diomedea ) breeding on two French Mediterranean islands. During the late breeding season, immatures foraged around their colony like breeding adults, but they were the only group showing potential prospecting movements around non-natal colonies. Global migration routes were broadly comparable between the two populations and the three life stages, with all individuals heading towards the Atlantic Ocean through the strait of Gibraltar and travelling along the West African coast, up to 8000 km from their colony. However, detailed comparison of timing, trajectory and oceanographic conditions experienced by the birds revealed remarkable age-related differences. Compared to adults and immatures, juveniles made a longer stop-over in the Balearic Sea (10 days vs 4 days in average), showed lower synchrony in crossing the Gibraltar strait, had more sinuous pathways and covered longer daily distances (240 km.d^-1 vs 170 km.d^-1). Analysis of oceanographic habitats along migratory routes revealed funnelling selection of habitat towards coastal and more productive waters with increasing age. Younger birds may have reduced navigational ability and learn progressively fine-scale migration routes towards the more profitable travelling and wintering areas. Our study demonstrates the importance of tracking long-lived species through the stages, to better understand migratory behavior and assess differential exposure to at-sea threats. Shared distribution between life stages and populations make Scopoli’s shearwaters particularly vulnerable to extreme mortality events in autumn and winter. Such knowledge is key for the conservation of critical marine habitats.