Identification of a Novel Aminopeptidase P-Like Gene (OnAPP) Possibly Involved in Bt Toxicity and Resistance in a Major Corn Pest (Ostrinia nubilalis)

Studies to understand the Bacillus thuringiensis (Bt) resistance mechanism in European corn borer (ECB, Ostrinia nubilalis) suggest that resistance may be due to changes in the midgut-specific Bt toxin receptor. In this study, we identified 10 aminopeptidase-like genes, which have previously been identified as putative Bt toxin receptors in other insects and examined their expression in relation to Cry1Ab toxicity and resistance. Expression analysis for the 10 aminopeptidase-like genes revealed that most of these genes were expressed predominantly in the larval midgut, but there was no difference in the expression of these genes in Cry1Ab resistant and susceptible strains. This suggested that altered expression of these genes was unlikely to be responsible for resistance in these ECB strains. However, we found that there were changes in two amino acid residues of the aminopeptidase-P like gene (OnAPP) involving Glu305 to Lys305 and Arg307 to Leu307 in the two Cry1Ab-resistant strains as compared with three Cry1Ab-susceptible strains. The mature OnAPP contains 682 amino acid residues and has a putative signal peptide at the N-terminus, a predicted glycosylphosphatidyl-inositol (GPI)-anchor signal at the C-terminal, three predicted N-glycosylation sites at residues N178, N278 and N417, and an O-glycosylation site at residue T653. We used a feeding based-RNA interference assay to examine the role of the OnAPP gene in Cry1Ab toxicity and resistance. Bioassays of Cry1Ab in larvae fed diet containing OnAPP dsRNA resulted in a 38% reduction in the transcript level of OnAPP and a 25% reduction in the susceptibility to Cry1Ab as compared with larvae fed GFP dsRNA or water. These results strongly suggest that the OnAPP gene could be involved in binding the Cry1Ab toxin in the ECB larval midgut and that mutations in this gene may be associated with Bt resistance in these two ECB strains

An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania

Intra-Organ Variation in Age-Related Mutation Accumulation in the Mouse

Using a transgenic mouse model harboring chromosomally integrated lacZ mutational target genes, we previously demonstrated that mutations accumulate with age much more rapidly in the small intestine than in the brain. Here it is shown that in the small intestine point mutations preferentially accumulate in epithelial cells of the mucosa scraped off the underlying serosa. The mucosal cells are the differentiated villus cells that have undergone multiple cell divisions. A smaller age-related increase, also involving genome rearrangements, was observed in the serosa, which consists mainly of the remaining crypts and non-dividing smooth muscle cells. In the brain we observed an accumulation of only point mutations in no other areas than hypothalamus and hippocampus. To directly test for cell division as the determining factor in the generation of point mutations we compared mutation induction between mitotically active and quiescent embryonic fibroblasts from the same lacZ mice, treated with either UV (a point mutagen) or hydrogen peroxide (a clastogen). The results indicate that while point mutations are highly replication-dependent, genome rearrangements are as easily induced in non-dividing cells as in mitotically active ones. This strongly suggests that the point mutations found to have accumulated in the mucosal part of the small intestine are the consequence of replication errors. The same is likely true for point mutations accumulating in hippocampus and hypothalamus of the brain since neurogenesis in these two areas continues throughout life. The observed intra-organ variation in mutation susceptibility as well as the variation in replication dependency of different types of mutations indicates the need to not only extend observations made on whole organs to their sub-structures but also take the type of mutations and mitotic activity of the cells into consideration. This should help elucidating the impact of genome instability and its consequences on aging and disease

Crowding: risk factor or protective factor for lower respiratory disease in young children?

BACKGROUND: To study the effects of household crowding upon the respiratory health of young children living in the city of São Paulo, Brazil. METHODS: Case-control study with children aged from 2 to 59 months living within the boundaries of the city of São Paulo. Cases were children recruited from 5 public hospitals in central São Paulo with an acute episode of lower respiratory disease. Children were classified into the following diagnostic categories: acute bronchitis, acute bronchiolitis, pneumonia, asthma, post-bronchiolitis wheezing and wheezing of uncertain aetiology. One control, crudely matched to each case with regard to age (<2, 2 years old or more), was selected among healthy children living in the neighborhood of the case. All buildings were surveyed for the presence of environmental contaminants, type of construction and building material. Plans of all homes, including measurements of floor area, height of walls, windows and solar orientation, was performed. Data were analysed using conditional logistic regression. RESULTS: A total of 313 pairs of children were studied. Over 70% of the cases had a primary or an associated diagnosis of a wheezing illness. Compared with controls, cases tended to live in smaller houses with less adequate sewage disposal. Cases and controls were similar with respect to the number of people and the number of children under five living in the household, as well the number of people sharing the child's bedroom. After controlling for potential confounders, no evidence of an association between number of persons sharing the child's bedroom and lower respiratory disease was identified when all cases were compared with their controls. However, when two categories of cases were distinguished (infections, asthma) and each category compared separately with their controls, crowding appeared to be associated with a 60% reduction in the incidence of asthma but with 2 1/2-fold increase in the incidence of lower respiratory tract infections (p = 0.001). CONCLUSION: Our findings suggest that household crowding places young children at risk of acute lower respiratory infection but may protect against asthma. This result is consistent with the hygiene hypothesis

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Administration of M. leprae Hsp65 Interferes with the Murine Lupus Progression

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K409A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F1 mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F1 serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K409A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K409A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process

Imidacloprid-Induced Impairment of Mushroom Bodies and Behavior of the Native Stingless Bee Melipona quadrifasciata anthidioides

Declines in pollinator colonies represent a worldwide concern. The widespread use of agricultural pesticides is recognized as a potential cause of these declines. Previous studies have examined the effects of neonicotinoid insecticides such as imidacloprid on pollinator colonies, but these investigations have mainly focused on adult honey bees. Native stingless bees (Hymenoptera: Apidae: Meliponinae) are key pollinators in neotropical areas and are threatened with extinction due to deforestation and pesticide use. Few studies have directly investigated the effects of pesticides on these pollinators. Furthermore, the existing impact studies did not address the issue of larval ingestion of contaminated pollen and nectar, which could potentially have dire consequences for the colony. Here, we assessed the effects of imidacloprid ingestion by stingless bee larvae on their survival, development, neuromorphology and adult walking behavior. Increasing doses of imidacloprid were added to the diet provided to individual worker larvae of the stingless bee Melipona quadrifasciata anthidioides throughout their development. Survival rates above 50% were only observed at insecticide doses lower than 0.0056 µg active ingredient (a.i.)/bee. No sublethal effect on body mass or developmental time was observed in the surviving insects, but the pesticide treatment negatively affected the development of mushroom bodies in the brain and impaired the walking behavior of newly emerged adult workers. Therefore, stingless bee larvae are particularly susceptible to imidacloprid, as it caused both high mortality and sublethal effects that impaired brain development and compromised mobility at the young adult stage. These findings demonstrate the lethal effects of imidacloprid on native stingless bees and provide evidence of novel serious sublethal effects that may compromise colony survival. The ecological and economic importance of neotropical stingless bees as pollinators, their susceptibility to insecticides and the vulnerability of their larvae to insecticide exposure emphasize the importance of studying these species