Larval ontogeny and morphology of giant trahira Hoplias lacerdae

In the present study, the morphology and behaviour of giant trahira Hoplias lacerdae larvae were investigated, from hatching to complete absorption of the yolk sac, under laboratory conditions. In the first day post‐hatching (dph), the larvae presented a big ovoid‐shaped yolk sac that underwent regression during larval ontogeny. The mouth opened 3 dph, when the pectoral fins were evident. From this day, the larvae were able to perform sudden bursts of activity and appear to be able to swim a few centimetres before sinking again. The branchial apparatus was defined at 5 dph, and by 6 dph the operculum was formed. The internal organs such as intestine, liver, kidney and external sensorial structures were present at 7 dph. The yolk sac remained until 7 dph.     

Major differences in the responses of primary human leukocyte subsets to IFN-beta

Treatment of cell lines with type I IFNs activates the formation of IFN-stimulated gene factor 3 (STAT1/STAT2/IFN regulatory factor-9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-β and used flow cytometry to analyze phosphorylated STAT1, STAT3, and STAT5 in CD4(+) and CD8(+) T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes and CD4(+) and CD8(+) T cells, few B cells activated STAT1 in response to IFN-β, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of suppressor of cytokine signaling 1 or relevant protein tyrosine phosphatases in B cells. Microarray and real-time PCR analyses revealed the induction of STAT1-dependent proapoptotic mRNAs in monocytes but not in B cells. These data show that IFN-stimulated gene factor 3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and, especially in CD4(+) T cells, IFN-β activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-β increases the survival of primary human B cells and CD4(+) T cells but enhances the apoptosis of monocytes, as well as to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections and by type I IFN treatment of patients with multiple sclerosis, hepatitis, or cancer.     

Blessures dans la filiation et infertilité masculine : étude comparative et psychodynamique

The family history of a set of infertile men was the aim of the present study, through a filiation events grid based on a psychoanalytical approach. During the genetic counselling before ICIS procedure, the family chart was established and the filiation grid applied to a group of 30 infertile men requiring assisted reproductive procedures, as well as to a control group of 30 fertile men recruited in a maternity hospital after birth of their child. The filiation events differed significantly between two groups. It can be therefore assumed that difficulties in the past generation may play a part in some cases of infertility. A psychodynamic approach is then developed to highlight the hypothesis of a link between the traumatic impact of these filiation events on these men and its translation as a somatic expression.     

Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults

The present work aims to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers (cholinesterase, glutathione S-transferase and lactate dehydrogenase) as endpoints. Zebrafish eggs showed less sensitivity to Cr (VI) (96 h-LC₅₀ = 145.7 mg/L) than adults (96 h-LC₅₀ = 39.4 mg/L) probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave clear indications about Cr (VI) mode of action: it seems to be neurotoxic (inhibited cholinesterase), to inhibit glutathione S-transferase activity and to interfere with cellular metabolic activity (changes in lactate dehydrogenase activity) in larvae. In adults, only glutathione S-transferase was responsive, showing a clear inhibition. The responsiveness of the analyzed biomarkers in larvae reinforces the idea of the usefulness of early life stage assays in the assessment of chemicals effects. Moreover, early life stage assays also contributed with relevant information regarding anomalies in larvae development and behavior. Further research should focus on the use of biomarkers to assess long term effects which are ecologically more relevant.     

Adaptive Evolution of the Chlamydia trachomatis Dominant Antigen Reveals Distinct Evolutionary Scenarios for B- and T-cell Epitopes: Worldwide Survey

Background

Chlamydia trachomatis is one of the most disseminated human pathogens, for which no vaccine is available yet. Understanding the impact of the host pressure on pathogen antigens is crucial, but so far it was only assessed for highly-restricted geographic areas. We aimed to evaluate the evolutionary picture of the chlamydial key antigen (MOMP), which is one of the leading multi-subunit vaccine candidates, in a worldwide basis.

Methodology/Principal Findings

Using genetics, molecular evolution methods and mathematical modelling, we analyzed all MOMP sequences reported worldwide, composed by 5026 strains from 33 geographic regions of five continents. Overall, 35.9% of variants were detected. The evolutionary pattern of MOMP amino acid gains/losses was found to differ from the remaining chromosome, reflecting the demanding constraints of this porin, adhesin and dominant antigen. Amino acid changes were 4.3-fold more frequent in host-interacting domains (P<10⁻¹²), specifically within B-cell epitopes (P<10⁻⁵), where 25% of them are at fixation (P<10⁻⁵). According to the typical pathogen-host arms race, this rampant B-cell antigenic variation likely represents neutralization escape mutants, as some mutations were previously shown to abrogate neutralization of chlamydial infectivity in vitro. In contrast, T-cell clusters of diverse HLA specificities are under purifying selection, suggesting a strategy that may lead to immune subversion. Moreover, several silent mutations are at fixation, generating preferential codons that may influence expression, and may also reflect recombination-derived ‘hitchhiking-effect’ from favourable nonsilent changes. Interestingly, the most prevalent C. trachomatis genotypes, E and F, showed a mutation rate 22.3-fold lower than that of the remainder (P<10⁻²⁰), suggesting more fitted antigenic profiles.

Conclusions/Significance

Globally, the adaptive evolution of the C. trachomatis dominant antigen is likely driven by its complex pathogenesis-related function and reflects distinct evolutionary antigenic scenarios that may benefit the pathogen, and thus should be taking into account in the development of a MOMP-based vaccine.     

Development and Evaluation of a Sensitive PCR-ELISA System for Detection of Schistosoma Infection in Feces

Background

A PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed to overcome the need for sensitive techniques for the efficient diagnosis of Schistosoma infection in endemic settings with low parasitic burden.

Methodology/Principal Findings

This system amplifies a 121-base pair tandem repeat DNA sequence, immobilizes the resultant 5′ biotinylated product on streptavidin-coated strip-well microplates and uses anti-fluorescein antibodies conjugated to horseradish peroxidase to detect the hybridized fluorescein-labeled oligonucleotide probe. The detection limit of the Schistosoma PCR-ELISA system was determined to be 1.3 fg of S. mansoni genomic DNA (less than the amount found in a single cell) and estimated to be 0.15 S. mansoni eggs per gram of feces (fractions of an egg). The system showed good precision and genus specificity since the DNA target was found in seven Schistosoma DNA samples: S. mansoni, S. haematobium, S. bovis, S. intercalatum, S. japonicum, S. magrebowiei and S. rhodaini. By evaluating 206 patients living in an endemic area in Brazil, the prevalence of S. mansoni infection was determined to be 18% by examining 12 Kato-Katz slides (41.7 mg/smear, 500 mg total) of a single fecal sample from each person, while the Schistosoma PCR-ELISA identified a 30% rate of infection using 500-mg of the same fecal sample. When considering the Kato-Katz method as the reference test, artificial sensitivity and specificity rates of the PCR-ELISA system were 97.4% and 85.1%, respectively. The potential for estimating parasitic load by DNA detection in feces was assessed by comparing absorbance values and eggs per gram of feces, with a Spearman correlation coefficient of 0.700 (P<0.0001).

Conclusions/Significance

This study reports the development and field evaluation of a sensitive Schistosoma PCR-ELISA, a system that may serve as an alternative for diagnosing Schistosoma infection.     

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability

A detailed understanding of the molecular basis of protein folding and stability determinants partly relies on the study of proteins with enhanced conformational stability properties, such as those from thermophilic organisms. In this study, we set up a methodology aiming at identifying the subset of cytosolic hyperstable proteins using Sulfurispharea sp., a hyperthermophilic archaeon, able to grow between 70 and 97 degrees C, as a model organism. We have thermally and chemically perturbed the cytosolic proteome as a function of time (up to 96 h incubation at 90 degrees C), and proceeded with analysis of the remaining proteins by combining one- and two-dimensional gel electrophoresis, liquid chromatography fractionation, and protein identification by N-terminal sequencing and mass spectrometry methods. In total, 14 proteins with enhanced stabilities which are involved in key cellular processes such as detoxification, nucleic acid processing, and energy metabolism were identified including a superoxide dismutase, a peroxiredoxin, and a ferredoxin. We demonstrate that these proteins are biologically active after extensive thermal treatment of the proteome. The relevance of these and other targets is discussed in terms of the organism's ecology. This work thus illustrates an experimental approach aimed at mining a proteome for hyperstable proteins, a valuable tool for target selection in protein stability and structural studies.     

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Colleters in Bathysa nicholsonii K. Schum. (Rubiaceae): ultrastructure, secretion protein composition, and antifungal activity

Colleters are secretory structures well distributed in many organs of Angiosperms. Ultrastructurally, the colleters secretory cell presents an enhanced endoplasmic reticulum, Golgi apparatus, and mitochondria. Secretion synthesis, transportation, and passage through outer cell wall is poorly characterized. This study characterized the anatomy and ultrastructure of BATHYSA NICHOLSONII (Rubiaceae) colleters and evaluated the presence of protein in the secretion and its antifungal property. Samples were collected and prepared according to usual techniques in light and electron microscopy, electrophoresis, and fungal growth inhibition assay. Colleters are of a standard type, cylindrical and elongated, formed by one secretory epidermal palisade layer, and a central axis formed by parenchymatic cells and a vascular trace. Epidermal cells have dense cytoplasm with abundant ribosome, a nucleus, enhanced endoplasmic reticulum and Golgi apparatus. The outer cell wall presented morphologically distinct layers. The presence of secretory cavities was noted in all outer cell wall extents. Secretion preparations analyzed by SDS-PAGE showed that B. NICHOLSONII secretion is a mixture of proteins with molecular masses covering a range of approximately 66 to 24 kDa. This preparation presented an inhibitory effect on the fungi spore growth.