A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: comparison with the in vivo Draize rabbit test

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (DI); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.     

DNA damage-induced cell death： lessons from the central nervous system

DNA damage can, but does not always, induce cell death. While several pathways linking DNA damage signals to mitochondria-dependent and -independent death machineries have been elucidated, the connectivity of these pathways is subject to regulation by multiple other factors that are not well understood. We have proposed two conceptual models to explain the delayed and variable cell death response to DNA damage： integrative surveillance versus autonomous pathways. In this review, we discuss how these two models may explain the in vivo regulation of cell death induced by ionizing radiation （IR） in the developing central nervous system, where the death response is regulated by radiation dose, cell cycle status and neuronal development.     

Dual role for calcium in agrin signaling and acetylcholine receptor clustering.

Agrin is a motoneuron-derived factor that initiates neuromuscular synapse formation; however, the signaling pathway underlying postsynaptic differentiation is not yet understood. We have investigated the role of calcium in agrin signaling through the MuSK receptor tyrosine kinase and in the intracellular signaling cascade that leads to AChR phosphorylation and clustering. We find that agrin- and neuramindase-induced MuSK activation in cultured myotubes is completely blocked by removal of extracellular calcium, but only slightly reduced by clamping of intracellular calcium transients with BAPTA. Following agrin's activation of MuSK, we find that the downstream tyrosine phosphorylation of the AChR beta-subunit was inhibited by BAPTA but not by a slower acting chelator, EGTA. Similarly, agrin-induced clustering of the AChR was blocked by BAPTA but not EGTA. These findings indicate that extracellular calcium is required for the formation of a MuSK signaling complex, and that intracellular calcium regulates phosphorylation and clustering of the AChR in the postsynaptic membrane.     

Rivalry between Stink Bug Females in a Vibrational Communication Network

In the field, male pheromone attracts stink bugs to meet on the same plant and triggers females to call a male by the emission of the calling song. As first among Pentatomidae we describe female rivalry in Chinavia impicticornis, C. ubica and Euschistus heros. Rivalry starts in C. impicticornis by synchronized exchange of the first type of the female calling song pulse trains and proceeds by one of them either to change pulse trains from the first to the second type or to produce readily repeated single pulses. Both reactions either inhibit calling of the rival female or trigger her to respond by alternation with the second type of the calling song pulse trains. Female rivalry in C. ubica differs by the emission of the rival song that replaces alternation with the second type of the calling song typical for C. impicticornis. E. heros females synchronize pulses of the calling song duets and induce emission of the female rival song by one of them that partly inhibits singing of the other. These competitive interactions in Chinavia species reduce the proportion of couples when compared with single couples on a plant. Contrary to both Chinavia species, E. heros female rivalry does not inhibit male response, male signals overlap female emissions and create complex vibrations with modified amplitude modulation pattern caused by interference.     

Conservation of somatic tissue derived from collared peccaries (<Emphasis Type="Italic">Pecari tajacu</Emphasis> Linnaeus, 1758) using direct or solid-surface vitrification techniques

Cryopreservation of somatic tissue can be applied in biodiversity conservation, especially for wild species as collared peccary. We aimed to evaluate the effect of vitrification techniques of ear tissue of collared peccary [direct vitrification in cryovials (DVC) or solid-surface vitrification (SSV)] on the layers of epidermis and dermis by conventional histology and cell ability during the in vitro culture. Thus, both the vitrification methods were able to maintain normal patterns of the epidermis as the cornea and granular layers, furthermore the intercellular space and dermal–epidermal junction of the spinous layer when compared to fresh control. Nevertheless, DVC and SSV percentage of normality decreased in the morphological integrity of cytoplasm (37.5 and 25.0%) of spinous layer, respectively, as compared to the fresh fragments (100%, p < 0.05). Moreover, other differences between the fresh control (100%) and DVC tissues were verified in the intra-epidermal cleavage of the spinous (37.5%) and basal (37.5%) layers. In general, DVC and SSV techniques were efficient for the recovery of the somatic cells according to most of the evaluated parameters for the in vitro culture (p > 0.05). In addition, only at time of 72 h (D3), in the growth curve, DVC fragments showed a reduced cell concentration than fresh control. In conclusion, SSV was found to be a more efficient method for vitrifying collared peccary skin tissue when compared to DVC. These results are relevant for the tissue cryopreservation from collared peccary and could also be useful for mammals with phylogenetic relationships.     

The social wasp Polybia fastidiosuscula Saussure (Hymenoptera: Vespidae) uses herbivore‐induced maize plant volatiles to locate its prey

Social wasps in the Polybia genus are important for use as pest‐control agents in agricultural systems. The objective of this study was to investigate the behavioural responses of Polybia fastidiosuscula Saussure (Hymenoptera: Vespidae) to volatiles from maize, both constitutive volatiles and those induced by the herbivory of Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae). To assess the behavioural response of P. fastidiosuscula to S. frugiperda larvae, undamaged plants, S. frugiperda‐damaged plants, mechanically damaged plants, mechanically damaged plants plus regurgitant from larvae and extracts from various treatments, bioassays were conducted in a Y‐olfactometer. In addition, the volatiles from plants subjected to different treatments were collected via aeration, and they were quantified and identified. The wasps showed a greater preference for plants with damage induced either by larval feeding or by being mechanically damaged plus regurgitant than for undamaged plants or either larvae alone or mechanically damaged plants. Wasps were more attracted to extracts from plants + S. frugiperda larvae and to an extract from mechanically damaged plants + the regurgitant of larvae compared to hexane. The primary compounds induced by herbivory for 5–6 h after the beginning of the damage or regurgitant treatment were identified as α‐pinene, β‐myrcene, (Z)‐3‐hexenyl acetate, limonene, (E)‐ocimene, linalool, DMNT, (E)‐β‐farnesene, TMTT and indole. The results presented here show that the social wasp P. fastidiosuscula uses herbivore‐induced plant volatiles from maize to locate its prey.     

Nest building by a neotropical social wasp using Cecropia trichomes as main construction material (Hymenoptera,Vespidae, Polistinae)

Nest architecture is such a splendid product of social wasp behavior, there being a large diversity of nest types in the Neotropical fauna. Charterginus is a small wasp genus with six species recorded from Honduras through South America, and one species C. fulvus is notable for normally making nests with a very peculiar star shape. Few behavioral traits of this genus have been described, an exception being a record of cleptobiosis between Charterginus species and Azteca ants on Cecropia trees in Amazonia and Central America. Here we describe behavioral traits of C. fulvus related to the construction process of the nest envelope and the materials used, discussing the apparent dependency of this wasp species on resources provided by Cecropia trees. Experimental induction of reconstruction of the envelope resulted in a different oval regular shape, without the characteristic hollow points. Evidence is also presented of the use by this wasp of lichen components to make the white spots typically seen on nests.

     

Efficiency of genome-wide association studies in random cross populations

Genome-wide association studies (GWAS) with plant species have employed inbred lines panels. We evaluated the efficiency of GWAS in non-inbred and inbred populations and assessed factors affecting GWAS. Fifty samples of 800 individuals from populations with linkage disequilibrium were simulated. Individuals were genotyped for 10,000 single nucleotide polymorphisms (SNPs) and phenotyped for traits controlled by ten quantitative trait loci (QTLs) and 90 minor genes, assuming different degrees of dominance and broad sense heritabilities of 40 and 80%. The average SNP density was 0.1 centiMorgan (cM) and the QTL heritabilities ranged from 3.2 to 11.8%. The results for random cross populations evidenced that to increase the QTL detection power, the additive-dominance model must be fitted for traits controlled by dominance effects but must not be fitted for traits showing no dominance. The power of detection was maximized by increasing the sample size to 400 and the false discovery rate (FDR) to 5%. The average power of detection for the low, intermediate, and high heritability QTLs achieved 52.4, 87.0, and 100.0%, respectively. Assuming sample sizes of 400 and 800, the observed FDR was equal to or lower than the specified level of significance. The association mapping was highly precise, since at least 97% of the declared QTLs were detected by the SNP inside it (average bias of 0.4 cM). Besides controlling the FDR, relatedness (and identity by state) efficiently controls the number of significant associations outside the QTL interval (not all false positive associations). The analysis of the inbred random cross population provided essentially the same results as the non-inbred populations.     

An Initial Investigation of an Alternative Model to Study rat Primordial Germ Cell Epigenetic Reprogramming

Background

Primordial germ cells (PGC) are the precursors of the gametes. During pre-natal development, PGC undergo an epigenetic reprogramming when bulk DNA demethylation occurs and is followed by sex-specific de novo methylation. The de novo methylation and the maintenance of the methylation patterns depend on DNA methyltransferases (DNMTs). PGC reprogramming has been widely studied in mice but not in rats. We have previously shown that the rat might be an interesting model to study germ cell development. In face of the difficulties of getting enough PGC for molecular studies, the aim of this study was to propose an alternative method to study rat PGC DNA methylation. Rat embryos were collected at 14, 15 and 19 days post-coitus (dpc) for the analysis of 5mC, 5hmC, DNMT1, DNMT3a and DNMT3b expression or at 16dpc for treatment 5-Aza-CdR, a DNMT inhibitor, in vitro.

Methods

Once collected, the gonads were placed in 24-well plates previously containing 45μm pore membrane and medium with or without 5-Aza-CdR. The culture was kept for five days and medium was changed daily. The gonads were either fixed or submitted to RNA extraction.

Results

5mC and DNMTs labelling suggests that PGC are undergoing epigenetic reprogramming around 14/15dpc. The in vitro treatment of rat embryonic gonads with 1 μM of 5-Aza-CdR lead to a loss of 5mC labelling and to the activation of Pax6 expression in PGC, but not in somatic cells, suggesting that 5-Aza-CdR promoted a PGC-specific global DNA hypomethylation.

Conclusions

This study suggests that the protocol used here can be a potential method to study the wide DNA demethylation that takes place during PGC reprogramming.

     

Consensus strategy in genes prioritization and combined bioinformatics analysis for preeclampsia pathogenesis

Background

Preeclampsia is a multifactorial disease with unknown pathogenesis. Even when recent studies explored this disease using several bioinformatics tools, the main objective was not directed to pathogenesis. Additionally, consensus prioritization was proved to be highly efficient in the recognition of genes-disease association. However, not information is available about the consensus ability to early recognize genes directly involved in pathogenesis. Therefore our aim in this study is to apply several theoretical approaches to explore preeclampsia; specifically those genes directly involved in the pathogenesis.

Methods

We firstly evaluated the consensus between 12 prioritization strategies to early recognize pathogenic genes related to preeclampsia. A communality analysis in the protein-protein interaction network of previously selected genes was done including further enrichment analysis. The enrichment analysis includes metabolic pathways as well as gene ontology. Microarray data was also collected and used in order to confirm our results or as a strategy to weight the previously enriched pathways.

Results

The consensus prioritized gene list was rationally filtered to 476 genes using several criteria. The communality analysis showed an enrichment of communities connected with VEGF-signaling pathway. This pathway is also enriched considering the microarray data. Our result point to VEGF, FLT1 and KDR as relevant pathogenic genes, as well as those connected with NO metabolism.

Conclusion

Our results revealed that consensus strategy improve the detection and initial enrichment of pathogenic genes, at least in preeclampsia condition. Moreover the combination of the first percent of the prioritized genes with protein-protein interaction network followed by communality analysis reduces the gene space. This approach actually identifies well known genes related with pathogenesis. However, genes like HSP90, PAK2, CD247 and others included in the first 1% of the prioritized list need to be further explored in preeclampsia pathogenesis through experimental approaches.