Quality Control Test for Sequence-Phenotype Assignments

Relating a gene mutation to a phenotype is a common task in different disciplines such as protein biochemistry. In this endeavour, it is common to find false relationships arising from mutations introduced by cells that may be depurated using a phenotypic assay; yet, such phenotypic assays may introduce additional false relationships arising from experimental errors. Here we introduce the use of high-throughput DNA sequencers and statistical analysis aimed to identify incorrect DNA sequence-phenotype assignments and observed that 10–20% of these false assignments are expected in large screenings aimed to identify critical residues for protein function. We further show that this level of incorrect DNA sequence-phenotype assignments may significantly alter our understanding about the structure-function relationship of proteins. We have made available an implementation of our method at http://bis.ifc.unam.mx/en/software/chispas.     

Cytokine,Antibody and Proliferative Cellular Responses Elicited by Taenia solium Calreticulin upon Experimental Infection in Hamsters

Taenia solium causes two diseases in humans, cysticercosis and taeniosis. Tapeworm carriers are the main risk factor for neurocysticercosis. Limited information is available about the immune response elicited by the adult parasite, particularly the induction of Th2 responses, frequently associated to helminth infections. Calreticulin is a ubiquitous, multifunctional protein involved in cellular calcium homeostasis, which has been suggested to play a role in the regulation of immune responses. In this work, we assessed the effect of recombinant T. solium calreticulin (rTsCRT) on the cytokine, humoral and cellular responses upon experimental infection in Syrian Golden hamsters (Mesocricetus auratus). Animals were infected with T. solium cysticerci and euthanized at different times after infection. Specific serum antibodies, proliferative responses in mesenteric lymph nodes and spleen cells, as well as cytokines messenger RNA (mRNA) were analyzed. The results showed that one third of the infected animals elicited anti-rTsCRT IgG antibodies. Interestingly, mesenteric lymph node (MLN) cells from either infected or non-infected animals did not proliferate upon in vitro stimulation with rTsCRT. Additionally, stimulation with a tapeworm crude extract resulted in increased expression of IL-4 and IL-5 mRNA. Upon stimulation, rTsCRT increased the expression levels of IL-10 in spleen and MLN cells from uninfected and infected hamsters. The results showed that rTsCRT favors a Th2-biased immune response characterized by the induction of IL-10 in mucosal and systemic lymphoid organs. Here we provide the first data on the cytokine, antibody and cellular responses to rTsCRT upon in vitro stimulation during taeniasis.     

Proteomic Profiling of the Outer Membrane Fraction of the Obligate Intracellular Bacterial Pathogen Ehrlichia ruminantium

The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed previously, but information is limited about OMPs in this organism and about their role in the protective immune response. Identification of OMPs is also essential for understanding Ehrlichia’s OM architecture, and how the bacterium interacts with the host cell environment. First, we developed an OMP extraction method using the ionic detergent sarkosyl, which enriched the OM fraction. Second, proteins were separated via one-dimensional electrophoresis, and digested peptides were analyzed via nano-liquid chromatographic separation coupled with mass spectrometry (LC-MALDI-TOF/TOF). Of 46 unique proteins identified in the OM fraction, 18 (39%) were OMPs, including 8 proteins involved in cell structure and biogenesis, 4 in transport/virulence, 1 porin, and 5 proteins of unknown function. These experimental data were compared to the predicted subcellular localization of the entire E. ruminantium proteome, using three different algorithms. This work represents the most complete proteome characterization of the OM fraction in Ehrlichia spp. The study indicates that suitable subcellular fractionation experiments combined with straightforward computational analysis approaches are powerful for determining the predominant subcellular localization of the experimentally observed proteins. We identified proteins potentially involved in E. ruminantium pathogenesis, which are good novel targets for candidate vaccines. Thus, combining bioinformatics and proteomics, we discovered new OMPs for E. ruminantium that are valuable data for those investigating new vaccines against this organism. In summary, we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.     

Analysis of Personal and Family Factors in the Persistence of Attention Deficit Hyperactivity Disorder: Results of a Prospective Follow-Up Study in Childhood

Objectives

To study the course of ADHD during childhood and analyze possible personal and family predictor variables of the results.

Method

Sixty-one children with ADHD who were between 6 and 12 years old at the baseline assessment were evaluated 30 months later (mean age at baseline: 8.70 ± 1.97; mean age at follow-up: 10.98 ± 2.19). Status of ADHD in follow-up was identified as persistent (met DSM-IV-TR criteria according to parents’ and teachers’ ratings), contextually persistent (met ADHD criteria according to one informant, and there was functional impairment) and remitted ADHD (with subthreshold clinical symptomatology). Associated psychological disorders of the three groups were analyzed in the follow-up with the Conners'' Rating Scales. The groups were compared on ADHD characteristics (symptoms of ADHD and impairment), child psychopathology, executive functioning (EF; inhibition, working memory) and parenting characteristics (parental stress and discipline styles) at baseline.

Results

At the follow-up, 55.7% of the children continued to meet the DSM-IV-TR criteria for ADHD, 29.5% showed contextual persistence, and 14.8% presented remission of the disorder. The persistent and contextually persistent ADHD groups showed more associated psychological disorders. Inattention, oppositional problems, cognitive problems and impairment at baseline distinguished the remitted ADHD children from the persistent and contextually persistent ADHD children. Moreover, the persistent groups had significantly more emotional liability and higher parental stress than the group in remission, while no differences in EF where found among the groups.

Conclusions

ADHD children continue to present symptoms, as well as comorbid psychological problems, during adolescence and early adulthood. These findings confirm that persistence of ADHD is associated with child psychopathology, parental stress and impairment in childhood.     

Sex-Specific Mediating Role of Insulin Resistance and Inflammation in the Effect of Adiposity on Blood Pressure of Prepubertal Children

Objective

To evaluate the association between obesity indices and blood pressure (BP) at 4 years of age, in each sex, and to quantify to which extent this association is mediated by inflammation and insulin resistance (IR).

Materials and Methods

We studied 1250 4-year-old children selected from the population-based birth cohort Generation XXI. Associations between body mass index (BMI) z-score and waist-to-height ratio (WHtR), office BP, inflammation (high sensitivity C-reactive protein) and IR (HOMA-IR index) were assessed. Path Analysis, a modified multivariate regression approach, was applied to test causal models and quantify direct and indirect effects of predictors of systolic (SBP) and diastolic BP (DBP).

Results

SBP and DBP increased significantly with BMI and WHtR in both sexes. There was a strong direct association (explaining 74.1-93.2% of the total association) of both measures of adiposity with SBP, in both sexes. This association was additionally indirectly mediated by IR, particularly regarding WHtR (20.5% in girls and 9.4% in boys). Mediation by inflammation did not reach statistical significance in either sex. Regarding DBP, the direct effect of adiposity was strong (>95% for BMI and WHtR in boys) and the mediation by IR was much smaller in boys than in girls.

Discussion

The direct association between adiposity and BP in healthy 4-year-old children is strong and IR plays an important mediating role. The strength of effects of IR and inflammation suggests sex differences in the complex interplay between BP, adiposity and inflammation.     

Development and Evaluation of New Microemulsion-Based Hydrogel Formulations for Topical Delivery of Fluconazole

The aim of the present investigation was to develop and evaluate microemulsion-loaded hydrogels (MEHs) for the topical delivery of fluconazole (FZ). The solubility of FZ in oils, surfactants and cosurfactants was evaluated to identify the components of the microemulsion. The pseudo-ternary phase diagrams were constructed using the novel phase diagram by micro-plate dilution method. Carbopol EDT 2020 was used to convert FZ-loaded microemulsions into gel form without affecting their structure. The selected microemulsions were assessed for globule size, zeta potential and polidispersity index. Besides this, the microemulsion-loaded hydrogel (MEH) formulations were evaluated for drug content, pH, rheological properties and in vitro drug release through synthetic membrane and excised pig ear skin in comparison with a conventional hydrogel. The optimised MEH FZ formulations consisting of FZ 2%, Transcutol P 11.5% and 11%, respectively, as oil phase, Lansurf SML 20-propyleneglycol 52% and 50%, respectively, as surfactant–cosurfactant (2:1), Carbopol EDT 2020 1.5% as gelling agent and water 34.5% and 37%, respectively, showed highest flux values and high release rate values, and furthermore, they had low surfactant content. The in vitro FZ permeation through synthetic membrane and excised pig ear skin from the studied MEHs was best described by the zero-order and first-order models. Finally, the optimised MEH FZ formulations showed similar or slightly higher antifungal activity as compared to that of conventional hydrogel and Nizoral® cream, respectively. The results suggest the potential use of developed MEHs as vehicles for topical delivery of FZ, encouraging further in vitro and in vivo evaluation.KEY WORDS: fluconazole, in vitro skin permeation, microemulsion, microemulsion-loaded hydrogel, topical     

Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

Malaria parasites replicating inside red blood cells (RBCs) export a large subset of proteins into the erythrocyte cytoplasm to facilitate parasite growth and survival. PTEX, the parasite-encoded translocon, mediates protein transport across the parasitophorous vacuolar membrane (PVM) in Plasmodium falciparum-infected erythrocytes. Proteins exported into the erythrocyte cytoplasm have been localized to membranous structures, such as Maurer''s clefts, small vesicles, and a tubovesicular network. Comparable studies of protein trafficking in Plasmodium vivax-infected reticulocytes are limited. With Plasmodium yoelii-infected reticulocytes, we identified exported protein 2 (Exp2) in a proteomic screen of proteins putatively transported across the PVM. Immunofluorescence studies showed that P. yoelii Exp2 (PyExp2) was primarily localized to the PVM. Unexpectedly, PyExp2 was also associated with distinct, membrane-bound vesicles in the reticulocyte cytoplasm. This is in contrast to P. falciparum in mature RBCs, where P. falciparum Exp2 (PfExp2) is exclusively localized to the PVM. Two P. yoelii-exported proteins, PY04481 (encoded by a pyst-a gene) and PY06203 (PypAg-1), partially colocalized with these PyExp2-positive vesicles. Further analysis revealed that with P. yoelii, Plasmodium berghei, and P. falciparum, cytoplasmic Exp2-positive vesicles were primarily observed in CD71⁺ reticulocytes versus mature RBCs. In transgenic P. yoelii 17X parasites, the association of hemagglutinin-tagged PyExp2 with the PVM and cytoplasmic vesicles was retained, but the pyexp2 gene was refractory to deletion. These data suggest that the localization of Exp2 in mouse and human RBCs can be influenced by the host cell environment. Exp2 may function at multiple points in the pathway by which parasites traffic proteins into and through the reticulocyte cytoplasm.