Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

Shadoo (Sho) is a brain glycoprotein with similarities to the unstructured region of PrP^C. Frameshift alleles of the Sho gene, Sprn, are reported in variant Creutzfeldt-Jakob disease (vCJD) patients while Sprn mRNA knockdown in PrP-null (Prnp^0/0) embryos produces lethality, advancing Sho as the hypothetical PrP-like “pi” protein. Also, Sho levels are reduced as misfolded PrP accumulates during prion infections. To penetrate these issues we created Sprn null alleles (Daude et al., Proc. Natl. Acad. Sci USA 2012; 109(23): 9035–40). Results from the challenge of Sprn null and TgSprn transgenic mice with rodent-adapted prions coalesce to define downregulation of Sho as a “tracer” for the formation of misfolded PrP. However, classical BSE and rodent-adapted BSE isolates may behave differently, as they do for other facets of the pathogenic process, and this intriguing variation warrants closer scrutiny. With regards to physiological function, double knockout mice (Sprn^0/0/Prnp^0/0) mice survived to over 600 d of age. This suggests that Sho is not pi, or, given the accumulating data for many activities for PrP^C, that the pi hypothesis invoking a discrete signaling pathway to maintain neuronal viability is no longer tenable.     

Evidence of Phenotypic and Genetic Relationships between Sociality,Emotional Reactivity and Production Traits in Japanese Quail

The social behavior of animals, which is partially controlled by genetics, is one of the factors involved in their adaptation to large breeding groups. To understand better the relationships between different social behaviors, fear behaviors and production traits, we analyzed the phenotypic and genetic correlations of these traits in Japanese quail by a second generation crossing of two lines divergently selected for their social reinstatement behavior. Analyses of results for 900 individuals showed that the phenotypic correlations between behavioral traits were low with the exception of significant correlations between sexual behavior and aggressive pecks both at phenotypic (0.51) and genetic (0.90) levels. Significant positive genetic correlations were observed between emotional reactivity toward a novel object and sexual (0.89) or aggressive (0.63) behaviors. The other genetic correlations were observed mainly between behavioral and production traits. Thus, the level of emotional reactivity, estimated by the duration of tonic immobility, was positively correlated with weight at 17 and 65 days of age (0.76 and 0.79, respectively) and with delayed egg laying onset (0.74). In contrast, a higher level of social reinstatement behavior was associated with an earlier egg laying onset (-0.71). In addition, a strong sexual motivation was correlated with an earlier laying onset (-0.68) and a higher number of eggs laid (0.82). A low level of emotional reactivity toward a novel object and also a higher aggressive behavior were genetically correlated with a higher number of eggs laid (0.61 and 0.58, respectively). These results bring new insights into the complex determinism of social and emotional reactivity behaviors in birds and their relationships with production traits. Furthermore, they highlight the need to combine animal welfare and production traits in selection programs by taking into account traits of sociability and emotional reactivity.     

The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.     

Dot1-Dependent Histone H3K79 Methylation Promotes Activation of the Mek1 Meiotic Checkpoint Effector Kinase by Regulating the Hop1 Adaptor

During meiosis, accurate chromosome segregation relies on the proper interaction between homologous chromosomes, including synapsis and recombination. The meiotic recombination checkpoint is a quality control mechanism that monitors those crucial events. In response to defects in synapsis and/or recombination, this checkpoint blocks or delays progression of meiosis, preventing the formation of aberrant gametes. Meiotic recombination occurs in the context of chromatin and histone modifications, which play crucial roles in the maintenance of genomic integrity. Here, we unveil the role of Dot1-dependent histone H3 methylation at lysine 79 (H3K79me) in this meiotic surveillance mechanism. We demonstrate that the meiotic checkpoint function of Dot1 relies on H3K79me because, like the dot1 deletion, H3-K79A or H3-K79R mutations suppress the checkpoint-imposed meiotic delay of a synapsis-defective zip1 mutant. Moreover, by genetically manipulating Dot1 catalytic activity, we find that the status of H3K79me modulates the meiotic checkpoint response. We also define the phosphorylation events involving activation of the meiotic checkpoint effector Mek1 kinase. Dot1 is required for Mek1 autophosphorylation, but not for its Mec1/Tel1-dependent phosphorylation. Dot1-dependent H3K79me also promotes Hop1 activation and its proper distribution along zip1 meiotic chromosomes, at least in part, by regulating Pch2 localization. Furthermore, HOP1 overexpression bypasses the Dot1 requirement for checkpoint activation. We propose that chromatin remodeling resulting from unrepaired meiotic DSBs and/or faulty interhomolog interactions allows Dot1-mediated H3K79-me to exclude Pch2 from the chromosomes, thus driving localization of Hop1 along chromosome axes and enabling Mek1 full activation to trigger downstream responses, such as meiotic arrest.     

Antioxidant therapy: Still in search of the ‘magic bullet’

The therapeutic potential of natural phenolic antioxidants in human diseases associated with oxidative damage has received great attention to date. Appraisal of literature evidences that, in general, antioxidant therapy has enjoyed relative successes in preclinical studies but little benefits in human intervention studies or clinical trials. In fact, despite the huge, largely untapped potential therapeutic benefit of natural phenolic antioxidants, such as vitamins, non-flavonoid and flavonoid compounds, they appear not to be suitable drug candidates. The problem may be related, among others, to their non-drug-likeness properties. Though controversial the results obtained so far confirm the importance of exploring phenolic natural systems as safe templates for the design of new antioxidants. To support the assumption an outlook of the lead structural optimization process to improve ADME properties was given by means of natural hydroxycinnamic acids as a case study. The optimization of drug physicochemical properties and the development of appropriate delivery antioxidant systems can provide in the next future a way out to attain effective therapeutic antioxidant agents.     

Stereochemical features of the envelope protein Domain III of dengue virus reveals putative antigenic site in the five-fold symmetry axis

We bring to attention a characteristic parasitic pattern present in the dengue virus: it undergoes several intensive thermodynamic variations due to host environmental changes, from a vector's digestive tract, through the human bloodstream and intracellular medium. Comparatively, among the known dengue serotypes, we evaluate the effects that these medium variations may induce to the overall structural characteristics of the Domain III of the envelope (E) protein, checking for stereochemical congruences that could lead to the identification of immunologic relevant regions. We used molecular dynamics and principal component analysis to study the protein in solution, for all four dengue serotypes, under distinct pH and temperature. We stated that, while the core of Domain III is remarkably rigid and effectively unaffected by most of the mentioned intensive variations, the loops account for major and distinguishable flexibilities. Therefore, the rigidity of the Domain III core provides a foothold that projects specifically two of these high flexible loop regions towards the inner face of the envelope pores, which are found at every five-fold symmetry axis of the icosahedron-shaped mature virus. These loops bear a remarkable low identity though with high occurrence of ionizable residues, including histidines. Such stereochemical properties can provide very particular serotype-specific electrostatic surface patterns, suggesting a viral fingerprint region, on which other specific molecules and ions can establish chemical interactions in an induced fit mechanism. We assert that the proposed regions share enough relevant features to qualify for further immunologic and pharmacologic essays, such as target peptide synthesis and phage display using dengue patients' sera.     

The Synergistic Effect of Combined Immunization with a DNA Vaccine and Chimeric Yellow Fever/Dengue Virus Leads to Strong Protection against Dengue

The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.     

Trypanosoma cruzi Response to Sterol Biosynthesis Inhibitors: Morphophysiological Alterations Leading to Cell Death

The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs) ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC₅₀/72 h) or killing all cells within 24 hours (EC₁₀₀/24 h). Incubation with inhibitors at the EC₅₀/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC₅₀/72 h. By contrast, treatment with SBIs at the EC₁₀₀/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP), culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the “point of no return” in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite.