RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

Urban distribution of Phlebotominae in a cutaneous leishmaniasis focus, Argentina

Urbanization and vector domestication are currently proposed as factors that contributed to the recent increase of American cutaneous leishmaniasis (ACL). Is likely also urban transmission? Oran is the main city in the Argentinean hyper-endemic area of ACL, and human cases in urban residences are usually reported. In order to assess the spatial distribution of risk, phlebotomine traps were located in different environments of Oran. A total of 7,787 sand flies were captured: Lutzomyia neivai (98.1%), Lutzomyia migonei (1.2%), Lutzomyia cortelezzii (0.7%), and one Lutzomyia shannoni. During the season of transmission (April-May) a single sand fly was obtained in one out of five urban sites, while a trap in a peri-urban pigsty captured up to 2,985 Lu. neivai/night. Captures performed in the other season of vector activity (September-October) revealed that small-scale changes in the pigsty environment resulted in noticeable changes in the abundance of Lu. neivai. In addition, in a new neighbourhood, on the fringe of the city, 1,073 Lu. neivai/site were captured in the forested edge but one in the yard of the houses. Therefore, in this urban ACL focus the human-vector effective contact risk is still associated with peri-urban vegetation and ecotone modifications despite the urban residence of the cases.

Ecological control of Triatoma dimidiata (Latreille, 1811): five years after a Costa Rican pilot project

An ecological pilot project for the control of Triatoma dimidiata allowed a new evaluation four and five years after environmental modifications in the peridomestic areas of 20 households. It was verified that the two groups of houses, 10 case-houses and 10 control-houses, were free of insects after those periods of time. In the first group, the owners started a chicken coop in the backyard and a colony of bugs was found there without infesting the house. In the second group, the inhabitants of one house once again facilitated the conditions for the bugs to thrive in the same store room, reaffirming that man-made ecotopes facilitates colonization. This ecological control method was revealed to be reliable and sustainable and it is recommended to be applied to those situations where the vectors of Chagas disease can colonize houses and are frequent in wild ecotopes.

Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

Understanding mechanisms and the role of differentiation in pathogenesis of Toxoplasma gondii: a review

Parasite differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission of the intracellular protozoan pathogen Toxoplasma gondii. The presence of bradyzoite-containing cysts in human hosts and their subsequent rupture can cause life-threatening recrudescence of acute infection in the immunocompromised and cyst formation in other animals contributes to zoonotic transmission and widespread dissemination of the parasite. In this review, we discuss the evidence showing how the clinically relevant process of bradyzoite differentiation is regulated at both transcriptional and post-transcriptional levels. Specific regulatory factors implicated in modulating bradyzoite differentiation include promoter-based cis-elements, epigenetic modifications and protein translation control through eukaryotic initiation factor -2 (eIF2). In addition to a summary of the current state of knowledge in these areas we discuss the pharmacological ramifications and pose some questions for future research.

Host genetic and epigenetic factors in toxoplasmosis

Analysing human genetic variation provides a powerful tool in understanding risk factors for disease. Toxoplasma gondii acquired by the mother can be transmitted to the fetus. Infants with the most severe clinical signs in brain and eye are those infected early in pregnancy when fetal immunity is least well developed. Genetic analysis could provide unique insight into events in utero that are otherwise difficult to determine. We tested the hypothesis that propensity for T. gondii to cause eye disease is associated with genes previously implicated in congenital or juvenile onset ocular disease. Using mother-child pairs from Europe (EMSCOT) and child/parent trios from North America (NCCCTS), we demonstrated that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4 previously associated with juvenile onset retinal dystrophies including Stargardt's disease. Polymorphisms at COL2A1 encoding type II collagen, previously associated with Stickler syndrome, associated only with ocular disease in congenital toxoplasmosis. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting, which provided an explanation for the patterns of inheritance observed. These genetic and epigenetic risk factors provide unique insight into molecular pathways in the pathogenesis of disease.

Laboratorial atopy markers in children with human immunodeficiency virus

Changes in immune system functions are one of the most important consequences of human immunodeficiency virus (HIV) infection. Studies have reported a higher prevalence of disease mediated by immunological hypersensitivity mechanisms in HIV-positive patients. This study aims to observe how immunological changes in HIV-infected children interfere in atopy determinants. Fifty-seven HIV-positive children were studied between June 2004-August 2005 to evaluate the possible modifications in atopy diagnosis from prick test environmental allergen reactivity. Patients were subjected to two evaluations: on both occasions, atopic and non-atopic groups were correlated with immunological (CD4+ and CD8+ lymphocyte concentrations and serum levels of IgA, IgM, IgG and IgE) and viral parameters (HIV viral load). The percent atopy was 20.05 in the first and 29.82 in the second evaluation and atopy was diagnosed in patients without immunosuppression or with moderate immunosuppression. Six patients changed from a negative to a positive atopy profile. One patient with a decreased CD4+ T lymphocyte concentration failed to demonstrate prick test positivity between evaluations. Multivariate analysis showed that the variables associated with atopy diagnosis included a personal history of allergic diseases as well as elevated IgE for age and elevated IgE levels. Atopy development in HIV-infected children seems to be modulated by genetic and environmental factors as well as immunological condition.

Short-term therapy with simvastatin reduces inflammatory mediators and heart inflammation during the acute phase of experimental Chagas disease

Trypanosoma cruzi infection induces progressive cardiac inflammation that leads to fibrosis and modifications in the heart architecture and functionality. Statins, such as 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, have been studied due to their pleiotropic roles in modulating the inflammatory response. Our goal was to evaluate the effects of simvastatin on the cardiac inflammatory process using a cardiotropic strain of T. cruzi in a murine model of Chagas cardiomyopathy. C57BL/6 mice were infected with 500 trypomastigotes of the Colombian strain of T. cruzi and treated with an oral dose of simvastatin (20 mg/Kg/day) for one month and inflammatory and morphometric parameters were subsequently evaluated in the serum and in the heart, respectively. Simvastatin reduced the total cholesterol and inflammatory mediators (interferon-gamma, tumour necrosis factor-alpha, CCL2 and CCL5) in the serum and in the heart tissue at 30 days post-infection. Additionally, a proportional reduction in heart weight and inflammatory infiltration was observed. Simvastatin also reduced epimastigote proliferation in a dose-dependent manner in vitro and was able to reduce blood trypomastigotes and heart amastigote nests during the acute phase of Chagas disease in vivo. Based on these data, we conclude that simvastatin exerts a modulatory effect on the inflammatory mediators that are elicited by the Colombian strain of T. cruzi and ameliorates the heart damage that is observed in a murine model of Chagas disease.

New approaches in antimalarial drug discovery and development: a review

Malaria remains a major world health problem following the emergence and spread of Plasmodium falciparum that is resistant to the majority of antimalarial drugs. This problem has since been aggravated by a decreased sensitivity of Plasmodium vivax to chloroquine. This review discusses strategies for evaluating the antimalarial activity of new compounds in vitro and in animal models ranging from conventional tests to the latest high-throughput screening technologies. Antimalarial discovery approaches include the following: the discovery of antimalarials from natural sources, chemical modifications of existing antimalarials, the development of hybrid compounds, testing of commercially available drugs that have been approved for human use for other diseases and molecular modelling using virtual screening technology and docking. Using these approaches, thousands of new drugs with known molecular specificity and active against P. falciparum have been selected. The inhibition of haemozoin formation in vitro, an indirect test that does not require P. falciparum cultures, has been described and this test is believed to improve antimalarial drug discovery. Clinical trials conducted with new funds from international agencies and the participation of several industries committed to the eradication of malaria should accelerate the discovery of drugs that are as effective as artemisinin derivatives, thus providing new hope for the control of malaria.

Cardiomyocyte dysfunction during the chronic phase of Chagas disease

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Anopheles darlingi polytene chromosomes: revised maps including newly described inversions and evidence for population structure in Manaus

Salivary gland polytene chromosomes of 4th instar Anopheles darlingi Root were examined from multiple locations in the Brazilian Amazon. Minor modifications were made to existing polytene photomaps. These included changes to the breakpoint positions of several previously described paracentric inversions and descriptions of four new paracentric inversions, two on the right arm of chromosome 3 and two on the left arm of chromosome 3 that were found in multiple locations. A total of 18 inversions on the X (n = 1) chromosome, chromosome 2 (n = 7) and 3 (n = 11) were scored for 83 individuals from Manaus, Macapá and Porto Velho municipalities. The frequency of 2Ra inversion karyotypes in Manaus shows significant deficiency of heterozygotes (p < 0.0009). No significant linkage disequilibrium was found between inversions on chromosome 2 and 3. We hypothesize that at least two sympatric subpopulations exist within the An. darlingi population at Manaus based on inversion frequencies.

Content validation of the nursing diagnosis Nausea

This study aimed to evaluate the content validity of the nursing diagnosis of nausea in the immediate post-operative period, considering Fehring’s model. Descriptive study with 52 nurses experts who responded an instrument containing identification and validation of nausea diagnosis data. Most experts considered the domain 12 (Comfort), Class 1 (Physical Comfort) and the statement (Nausea) adequate to the diagnosis. Modifications were suggested in the current definition of this nursing diagnosis. Four defining characteristics were considered primary (reported nausea, increased salivation, aversion to food and vomiting sensation) and eight secondary (increased swallowing, sour taste in the mouth, pallor, tachycardia, diaphoresis, sensation of hot and cold, changes in blood pressure and pupil dilation). The total score for the diagnosis of nausea was 0.79. Reports of nausea, vomiting sensation, increased salivation and aversion to food are strong predictors of nursing diagnosis of nausea.

Scientific Literature on the Reliability and Validity of the Manchester Triage System (MTS) Protocol: A Integrative Literature Review

OBJECTIVE To analyze the scientific production about the validity and reliability of the Manchester Triage System (MTS) protocol. METHOD A descriptive study of an integrative literature review. Articles on the validity and reliability of the MTS developed with children and adults published between 1999 and 2013 were included. RESULTS 14 articles were selected from a total of 8438, nine of validity and five of reliability. The reliability of the MTS ranged from moderate to almost perfect, with higher intra-evaluation. Regarding validity, the results seem to point to equivalent and satisfactory sensibility and specificity levels of the MTS. The instrument proved to be a good predictor of the need for hospitalization and of hospital mortality. CONCLUSION The reliability and validity of the MTS obtained in the studies is varied. It is recommended that new studies indicate necessary modifications to the MTS so that it is more safely used by nurses.

Adult sex ratio effects on male survivorship of Drosophila melanogaster Meigen (Diptera, Drosophilidae)

The behavioral biology has a central role in evolutionary biology mainly because the antagonistic relations that occur in the sexual reproduction. One involves the effect of reproduction on the future life expectation. In this scenario, changes in male operational sex ratio could lead to an increase in mortality due to costs associated with excessive courtship and mating displays. Thus, this work experimentally altered the male sex ratio of Drosophila melanogaster Meigen, 1830, to determine its impact on mortality. The results indicated that mortality increases as the sex ratio changes, including modifications in the survivorship curve type and in the curve concavity, measured by entropy.

Phylogeny and revision of the bee genus Rhinocorynura Schrottky (Hymenoptera, Apidae, Augochlorini), with comments on its female cephalic polymorphism

Phylogeny and revision of the bee genus Rhinocorynura Schrottky (Hymenoptera, Apidae, Augochlorini), with comments on its female cephalic polymorphism. A taxonomic revision and a phylogeny for the species of Rhinocorynura are provided. Six species are recognized: R. briseis, R. crotonis, R. inflaticeps and R. vernoniae stat. nov., the latter removed from synonymy with R. inflaticeps, in addition to two newly described species, R. brunnea sp. nov. and R. viridis sp. nov. Lectotypes for Halictus crotonis Ducke, 1906 and Halictus inflaticeps Ducke, 1906 are hereby designated. Another available name included in Rhinocorynura, Corynuropsis ashmeadi Schrottky, 1909, is removed from the genus and treated as species inquerenda in Augochlorini. Rhinocorynura is monophyletic in the phylogenetic analysis and the following relationships were found among its species: (R. crotonis (R. briseis ((R. brunnea sp. nov. + R. viridis sp. nov.) (R. inflaticeps + R. vernoniae)))). Biogeographic relationships within the genus and comparisons with related taxa are presented. Females of all species exhibit pronounced variation in body size, in two of them, R. inflaticeps and R. vernoniae, with structural modifications possibly linked to division of labor. Identification key and illustrations for the species are provided.