Modification of composition of a nanoemulsion with different cholesteryl ester molecular species: Effects on stability, peroxidation, and cell uptake

Purpose: Use of lipid nanoemulsions as carriers of drugs for therapeutic or diagnostic purposes has been increasingly studied. Here, it was tested whether modifications of core particle constitution could affect the characteristics and biologic properties of lipid nanoemulsions. Methods: Three nanoemulsions were prepared using cholesteryl oleate, cholesteryl stearate, or cholesteryl linoleate as main core constituents. Particle size, stability, pH, peroxidation of the nanoemulsions, and cell survival and uptake by different cell lines were evaluated. Results: It was shown that cholesteryl stearate nanoemulsions had the greatest particle size and all three nanoemulsions were stable during the 237-day observation period. The pH of the three nanoemulsion preparations tended to decrease over time, but the decrease in pH of cholesteryl stearate was smaller than that of cholesteryl oleate and cholesteryl linoleate. Lipoperoxidation was greater in cholesteryl linoleate than in cholesteryl oleate and cholesteryl stearate. After four hours' incubation of human umbilical vein endothelial cells (HUVEC) with nanoemulsions, peroxidation was minimal in the presence of cholesteryl oleate and more pronounced with cholesteryl linoleate and cholesteryl stearate. In contrast, macrophage incubates showed the highest peroxidation rates with cholesteryl oleate. Cholesteryl linoleate induced the highest cell peroxidation rates, except in macrophages. Uptake of cholesteryl oleate nanoemulsion by HUVEC and fibroblasts was greater than that of cholesteryl linoleate and cholesteryl stearate. Uptake of the three nanoemulsions by monocytes was equal. Uptake of cholesteryl oleate and cholesteryl linoleate by macrophages was negligible, but macrophage uptake of cholesteryl stearate was higher. In H292 tumor cells, cholesteryl oleate showed the highest uptakes. HUVEC showed higher survival rates when incubated with cholesteryl stearate and smaller survival with cholesteryl linoleate. H292 survival was greater with cholesteryl stearate. Conclusion: Although all three nanoemulsion types were stable for a long period, considerable differences were observed in size, oxidation status, and cell survival and nanoemulsion uptake in all tested cell lines. Those differences may be helpful in protocol planning and interpretation of data from experiments with lipid nanoemulsions.

The four hexamerin genes in the honey bee: structure, molecular evolution and function deduced from expression patterns in queens, workers and drones

Background: Hexamerins are hemocyanin-derived proteins that have lost the ability to bind copper ions and transport oxygen; instead, they became storage proteins. The current study aimed to broaden our knowledge on the hexamerin genes found in the honey bee genome by exploring their structural characteristics, expression profiles, evolution, and functions in the life cycle of workers, drones and queens. Results: The hexamerin genes of the honey bee (hex 70a, hex 70b, hex 70c and hex 110) diverge considerably in structure, so that the overall amino acid identity shared among their deduced protein subunits varies from 30 to 42%. Bioinformatics search for motifs in the respective upstream control regions (UCRs) revealed six overrepresented motifs including a potential binding site for Ultraspiracle (Usp), a target of juvenile hormone (JH). The expression of these genes was induced by topical application of JH on worker larvae. The four genes are highly transcribed by the larval fat body, although with significant differences in transcript levels, but only hex 110 and hex 70a are re-induced in the adult fat body in a caste-and sex-specific fashion, workers showing the highest expression. Transcripts for hex 110, hex 70a and hex70b were detected in developing ovaries and testes, and hex 110 was highly transcribed in the ovaries of egg-laying queens. A phylogenetic analysis revealed that HEX 110 is located at the most basal position among the holometabola hexamerins, and like HEX 70a and HEX 70c, it shares potential orthology relationship with hexamerins from other hymenopteran species. Conclusions: Striking differences were found in the structure and developmental expression of the four hexamerin genes in the honey bee. The presence of a potential binding site for Usp in the respective 5' UCRs, and the results of experiments on JH level manipulation in vivo support the hypothesis of regulation by JH. Transcript levels and patterns in the fat body and gonads suggest that, in addition to their primary role in supplying amino acids for metamorphosis, hexamerins serve as storage proteins for gonad development, egg production, and to support foraging activity. A phylogenetic analysis including the four deduced hexamerins and related proteins revealed a complex pattern of evolution, with independent radiation in insect orders.

A molecular phylogeny and the evolution of nest architecture and behavior in Trigona s.s. (Hymenoptera : Apidae : Meliponini)

Stingless bees exhibit extraordinary variation in nest architecture within and among species. To test for phylogenetic association of behavioral traits for species of the Neotropical stingless bee genus Trigona s.s., a phylogenetic hypothesis was generated by combining sequence data of 24 taxa from one mitochondrial (16S rRNA) and four nuclear gene fragments (long-wavelength rhodopsin copy 1 (opsin), elongation factor-1 alpha copy F2, arginine kinase, and 28S rRNA). Fifteen characteristics of the nest architecture were coded and tested for phylogenetic association. Several characters have significant phylogenetic signal, including type of nesting substrate, nest construction material, and hemipterophily, the tending of hemipteroid insects in exchange for sugar excretions. Phylogenetic independent habits encountered in Trigona s.s. include coprophily and necrophagy.

The role of disulfide bridges in the 3-D structures of the antimicrobial peptides gomesin and protegrin-1: a molecular dynamics study

Some antimicrobial peptides have a broad spectrum of action against many different kinds of microorganisms. Gomesin and protegrin-1 are examples of such antimicrobial peptides, and they were studied by molecular dynamics in this research. Both have a beta-hairpin conformation stabilized by two disulfide bridges and are active against Gram-positive and Gram-negative bacteria, as well as fungi. In this study, the role of the disulfide bridge in the maintenance of the tertiary peptide structure of protegrin-1 and gomesin is analyzed by the structural characteristics of these peptides and two of their respective variants, gomy4 and proty4, in which the four cysteines are replaced by four tyrosine residues. The absence of disulfide bridges in gomy4 and proty4 is compensated by overall reinforcement of the original hydrogen bonds and extra attractive interactions between the aromatic rings of the tyrosine residues. The net effects on the variants with respect to the corresponding natural peptides are: i) maintenance of the original beta-hairpin conformation, with great structural similarities between the mutant and the corresponding natural peptide; ii) combination of positive F and. Ramachandran angles within the hairpin head region with a qualitative change to a combination of positive (F) and negative (.) angles, and iii) significant increase in structural flexibility. Experimental facts about the antimicrobial activity of the gomesin and protegrin-1 variants have also been established here, in the hope that the detailed data provided in the present study may be useful for understanding the mechanism of action of these peptides.

Study of the antimicrobial peptide indolicidin and a mutant in micelle medium by molecular dynamics simulation

The antimicrobial peptide indolicidin (IND) and the mutant CP10A in hydrated micelles were studied using molecular dynamics simulations in order to observe whether the molecular dynamics and experimental data could be sufficiently correlated and a detailed description of the interaction of the antimicrobial peptides with a model of the membrane provided by a hydrated micelle system could be obtained. In agreement with the experiments, the simulations showed that the peptides are located near the surface of the micelles. Peptide insertions agree with available experimental data, showing deeper insertion of the mutant compared with the peptide IND. Major insertion into the hydrophobic core of the micelle by all tryptophan and mutated residues of CP10A in relation to IND was observed. The charged residues of the terminus regions of both peptides present similar behavior, indicating that the major differences in the interactions with the micelles of the peptides IND and CP10A occur in the case of the hydrophobic residues.

Beam-energy and system-size dependence of dynamical net charge fluctuations

We present measurements of net charge fluctuations in Au+Au collisions at s(NN)=19.6, 62.4, 130, and 200 GeV, Cu+Cu collisions at s(NN)=62.4 and 200 GeV, and p+p collisions at s=200 GeV using the dynamical net charge fluctuations measure nu(+-,dyn). We observe that the dynamical fluctuations are nonzero at all energies and exhibit a modest dependence on beam energy. A weak system size dependence is also observed. We examine the collision centrality dependence of the net charge fluctuations and find that dynamical net charge fluctuations violate 1/N(ch) scaling but display approximate 1/N(part) scaling. We also study the azimuthal and rapidity dependence of the net charge correlation strength and observe strong dependence on the azimuthal angular range and pseudorapidity widths integrated to measure the correlation.

First Report of the Isolation and Molecular Characterization of Rickettsia amblyommii and Rickettsia felis in Central America

During 2010, 15 adult ticks, identified as Amblyomma cajennense, were collected from horses in Cahuita and Turrialba districts, whereas 7 fleas, identified as Ctenocephalides felis, were collected from a dog in San Jose city, Costa Rica. In the laboratory, three A. cajennense specimens, two from Cahuita and one from Turrialba, were individually processed for rickettsial isolation in cell culture, as was a pool of seven fleas. Rickettsiae were successfully isolated and established in Vero cell culture from the three ticks and from a pool of seven fleas in C6/36 cell culture. The three tick isolates were genotypically identified as Rickettsia amblyommii, and the flea isolate was identified as Rickettsia felis through DNA sequencing of portions of the rickettsial genes gltA, ompA, and ompB of each isolate. In addition, other seven ticks were shown to contain rickettsial DNA. Polymerase chain reaction products of at least two of these ticks were sequenced and also showed to correspond to R. amblyommii. Overall, 66.7% (10/15) of the A. cajennense adult ticks were found to be infected with rickettsiae. This is the first report of a successful isolation in cell culture of R. amblyommii and R. felis from Central America.

Molecular and geographic analyses of vampire bat-transmitted cattle rabies in central Brazil

Background: Vampire bats are important rabies virus vectors, causing critical problems in both the livestock industry and public health sector in Latin America. In order to assess the epidemiological characteristics of vampire bat-transmitted rabies, the authors conducted phylogenetic and geographical analyses using sequence data of a large number of cattle rabies isolates collected from a wide geographical area in Brazil. Methods: Partial nucleoprotein genes of rabies viruses isolated from 666 cattle and 18 vampire bats between 1987 and 2006 were sequenced and used for phylogenetic analysis. The genetic variants were plotted on topographical maps of Brazil. Results: In this study, 593 samples consisting of 24 genetic variants were analyzed. Regional localization of variants was observed, with the distribution of several variants found to be delimited by mountain ranges which served as geographic boundaries. The geographical distributions of vampire-bat and cattle isolates that were classified as the identical phylogenetic group were found to overlap with high certainty. Most of the samples analyzed in this study were isolated from adjacent areas linked by rivers. Conclusion: This study revealed the existence of several dozen regional variants associated with vampire bats in Brazil, with the distribution patterns of these variants found to be affected by mountain ranges and rivers. These results suggest that epidemiological characteristics of vampire bat-related rabies appear to be associated with the topographical and geographical characteristics of areas where cattle are maintained, and the factors affecting vampire bat ecology.

Determination and Molecular Analysis of the Complete Genome Sequence of Two Wild-Type Rabies Viruses Isolated from a Haematophagous Bat and a Frugivorous Bat in Brazil

The complete genome sequences of two Brazilian wild-type rabies viruses (RABV), a BR-DR1 isolate from a haematophagous bat (Desmodus rotundus) and a BR-AL1 isolate from a frugivorous bat (Artibeus lituratus), were determined. The genomes of the BR-DR1 and RR-AL1 had 11,923 and 11,922 nt, respectively, and both encoded the five standard genes of rhabdoviruses. The complete nucleotide sequence identity between the BR-DR1 and BR-AL1 isolates was 97%. The BR-DR1 and BR-AL1 isolates had some conserved functional sites revealed by the fixed isolates, whereas both isolates had unique amino acid substitutions in the antigenic region IV of the nucleocapsid gene. Therefore, it is speculated that both isolates were nearly identical in virologic character. According to our phylogenetic analysis based on the complete genomes, both isolates belonged to genotype 1, and to the previously defined ""vampire bat-related RABV lineage"" which consisted of mainly D. rotundus- and A. lituratus- isolates; however, a branch pattern with high bootstrap values suggested that BR-DR1 was more closely related to the 9001FRA isolate, which was collected from a dog bitten by a bat in French Guiana, than to BR-AL1. This result suggests that the vampire bat-related RABV lineage includes Brazilian vampire bat and Brazilian frugivorous bat RABV and is further divided into Brazilian vampire bat and Brazilian frugivorous bat RABV sub-lineages. The phylogenetic analysis based on the complete genomes was valuable in discriminating among very closely related isolates.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results: 8489 transcripts were detected across the two oocyte groups, of which similar to 25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of a-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

PRACTICAL USE OF MOLECULAR MARKERS IN BEEF AND DAIRY ZEBU CATTLE IN BRAZIL

Selection of zebu (Bos indicus) beef and dairy cattle in Brazil and the validation process of genetic markers for growth, carcass and meat quality traits and also for milk production, fat and protein milk content are discussed as concerned to the concepts and details of their use as auxiliary tools in selection processes. It is highlighted, also, the importance of right selection of ova donor cows for production of embryos to be transferred.

The young, tight, and low-mass binary TWA22AB: a new calibrator for evolutionary models? Orbit, spectral types, and temperature

Context. Tight binaries discovered in young, nearby associations are ideal targets for providing dynamical mass measurements to test the physics of evolutionary models at young ages and very low masses. Aims. We report the binarity of TWA22 for the first time. We aim at monitoring the orbit of this young and tight system to determine its total dynamical mass using an accurate distance determination. We also intend to characterize the physical properties (luminosity, effective temperature, and surface gravity) of each component based on near-infrared photometric and spectroscopic observations. Methods. We used the adaptive-optics assisted imager NACO to resolve the components, to monitor the complete orbit and to obtain the relative near-infrared photometry of TWA22 AB. The adaptive-optics assisted integral field spectrometer SINFONI was also used to obtain medium-resolution (R(lambda) = 1500-2000) spectra in JHK bands. Comparison with empirical and synthetic librairies were necessary for deriving the spectral type, the effective temperature, and the surface gravity for each component of the system. Results. Based on an accurate trigonometric distance (17.5 +/- 0.2 pc) determination, we infer a total dynamical mass of 220 +/- 21 M(Jup) for the system. From the complete set of spectra, we find an effective temperature T(eff) = 2900(-200)(+200) K for TWA22A and T(eff) = 2900(-100)(+200) for TWA22 B and surface gravities between 4.0 and 5.5 dex. From our photometry and an M6 +/- 1 spectral type for both components, we find luminosities of log(L/L(circle dot)) = -2.11 +/- 0.13 dex and log(L/L(circle dot)) = -2.30 +/- 0.16 dex for TWA22 A and B, respectively. By comparing these parameters with evolutionary models, we question the age and the multiplicity of this system. We also discuss a possible underestimation of the mass predicted by evolutionary models for young stars close to the substellar boundary.

Mn, Cu, and Zn abundances in barium stars and their correlations with neutron capture elements

Barium stars are optimal sites for studying the correlations between the neutron-capture elements and other species that may be depleted or enhanced, because they act as neutron seeds or poisons during the operation of the s-process. These data are necessary to help constrain the modeling of the neutron-capture paths and explain the s-process abundance curve of the solar system. Chemical abundances for a large number of barium stars with different degrees of s-process excesses, masses, metallicities, and evolutionary states are a crucial step towards this goal. We present abundances of Mn, Cu, Zn, and various light and heavy elements for a sample of barium and normal giant stars, and present correlations between abundances contributed to different degrees by the weak-s, mains, and r-processes of neutron capture, between Fe-peak elements and heavy elements. Data from the literature are also considered in order to better study the abundance pattern of peculiar stars. The stellar spectra were observed with FEROS/ESO. The stellar atmospheric parameters of the eight barium giant stars and six normal giants that we analyzed lie in the range 4300 < T(eff)/K < 5300, -0.7 < [Fe/H] <= 0.12 and 1.5 <= log g < 2.9. Carbon and nitrogen abundances were derived by spectral synthesis of the molecular bands of C(2), CH, and CN. For all other elements we used the atomic lines to perform the spectral synthesis. A very large scatter was found mainly for the Mn abundances when data from the literature were considered. We found that [Zn/Fe] correlates well with the heavy element excesses, its abundance clearly increasing as the heavy element excesses increase, a trend not shown by the [Cu/Fe] and [Mn/Fe] ratios. Also, the ratios involving Mn, Cu, and Zn and heavy elements usually show an increasing trend toward higher metallicities. Our results suggest that a larger fraction of the Zn synthesis than of Cu is owed to massive stars, and that the contribution of the main-s process to the synthesis of both elements is small. We also conclude that Mn is mostly synthesized by SN Ia, and that a non-negligible fraction of the synthesis of Mn, Cu, and Zn is owed to the weak s-process.

The taxonomic status of the endangered thin-spined porcupine, Chaetomys subspinosus (Olfers, 1818), based on molecular and karyologic data

Background: The thin-spined porcupine, also known as the bristle-spined rat, Chaetomys subspinosus (Olfers, 1818), the only member of its genus, figures among Brazilian endangered species. In addition to being threatened, it is poorly known, and even its taxonomic status at the family level has long been controversial. The genus Chaetomys was originally regarded as a porcupine in the family Erethizontidae, but some authors classified it as a spiny-rat in the family Echimyidae. Although the dispute seems to be settled in favor of the erethizontid advocates, further discussion of its affinities should be based on a phylogenetic framework. In the present study, we used nucleotide-sequence data from the complete mitochondrial cytochrome b gene and karyotypic information to address this issue. Our molecular analyses included one individual of Chaetomys subspinosus from the state of Bahia in northeastern Brazil, and other hystricognaths. Results: All topologies recovered in our molecular phylogenetic analyses strongly supported Chaetomys subspinosus as a sister clade of the erethizontids. Cytogenetically, Chaetomys subspinosus showed 2n = 52 and FN = 76. Although the sexual pair could not be identified, we assumed that the X chromosome is biarmed. The karyotype included 13 large to medium metacentric and submetacentric chromosome pairs, one small subtelocentric pair, and 12 small acrocentric pairs. The subtelocentric pair 14 had a terminal secondary constriction in the short arm, corresponding to the nucleolar organizer region (Ag-NOR), similar to the erethizontid Sphiggurus villosus, 2n = 42 and FN = 76, and different from the echimyids, in which the secondary constriction is interstitial. Conclusion: Both molecular phylogenies and karyotypical evidence indicated that Chaetomys is closely related to the Erethizontidae rather than to the Echimyidae, although in a basal position relative to the rest of the Erethizontidae. The high levels of molecular and morphological divergence suggest that Chaetomys belongs to an early radiation of the Erethizontidae that may have occurred in the Early Miocene, and should be assigned to its own subfamily, the Chaetomyinae.

Design and fabrication of microcavity-array superhydrophobic surfaces

We have modeled, fabricated, and characterized superhydrophobic surfaces with a morphology formed of periodic microstructures which are cavities. This surface morphology is the inverse of that generally reported in the literature when the surface is formed of pillars or protrusions, and has the advantage that when immersed in water the confined air inside the cavities tends to expel the invading water. This differs from the case of a surface morphology formed of pillars or protrusions, for which water can penetrate irreversibly among the microstructures, necessitating complete drying of the surface in order to again recover its superhydrophobic character. We have developed a theoretical model that allows calculation of the microcavity dimensions needed to obtain superhydrophobic surfaces composed of patterns of such microcavities, and that provides estimates of the advancing and receding contact angle as a function of microcavity parameters. The model predicts that the cavity aspect ratio (depth-to-diameter ratio) can be much less than unity, indicating that the microcavities do not need to be deep in order to obtain a surface with enhanced superhydrophobic character. Specific microcavity patterns have been fabricated in polydimethylsiloxane and characterized by scanning electron microscopy, atomic force microscopy, and contact angle measurements. The measured advancing and receding contact angles are in good agreement with the predictions of the model. (C) 2010 American Institute of Physics. [doi:10.1063/1.3466979]