Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Flight and nutritional status of sylvatic Triatoma sordida and Triatoma guasayana

Nutritional status relating to flight activity was investigated in natural populations of Triatoma sordida and T. guasayana during the dry season in the Bolivian Chaco. The peak flight activity of both species was unimodal and covered the period 61-180 min after sunset. The weight of insects was used as the indicator of nutritional status. Interspecies comparisons employing the same sex and type of capture showed a higher weight for T. guasayana. No significant difference according to weight was observed between flying insects and those collected in natural ecotopes (hollow trees and bromeliads). More than 87% of insects collected from natural ecotopes displayed flight ability under the study conditions, explaining their tendency to invade artificial structures during the dry season.

Entomological and ecological aspects of six sylvatic species of triatomines (Hemiptera, Reduviidae) from the collection of the National Biodiversity Institute of Costa Rica, Central America

A total of 797 specimens of wild adult triatomines, belonging to six species from the entomological collections of the Costa Rican National Biodiversity Institute, was studied from the standpoint of their relative abundance, as reflected by light traps, distribution in the country, seasonal variations and climatic and altitudinal preferences. Triatoma dimidiata was the most abundant species (32.9% of the total specimens), with a very extensive distribution in different ecological zones, being more common between 100 to 400 m above sea level mainly at the end of the dry season. T. dispar was the third in frequency (21.5%), with narrower distribution, more abundant between 600 to 800 m and scarce during the dry season. Panstrongylus geniculatus and P. rufotuberculatus, second and fourth in frequency (22.1% and 15.1%, respectively), were widely distributed on both the Pacific and Caribbean basins, the former being more common between 80 to 270 m all year round and the latter below 800 m mainly during the first semester. Eratyrus cuspidatus which represented only 4.9% of the insects, was also present on both basins mainly below 200 m with a tendency to be scarce during certain months of the year, and was found in all types of ecological zones. Finally, Rhodnius pallescens, the least abundant species (3.6%) was restricted to very humid areas below 20 m, on the north side and Caribbean basin. With the exception of R. pallescens, males were more commonly found than females. Some epidemiological implications related to the six species are discussed.

Vertebrate conserved non coding DNA regions have a high persistence length and a short persistence time.

BACKGROUND: The comparison of complete genomes has revealed surprisingly large numbers of conserved non-protein-coding (CNC) DNA regions. However, the biological function of CNC remains elusive. CNC differ in two aspects from conserved protein-coding regions. They are not conserved across phylum boundaries, and they do not contain readily detectable sub-domains. Here we characterize the persistence length and time of CNC and conserved protein-coding regions in the vertebrate and insect lineages. RESULTS: The persistence length is the length of a genome region over which a certain level of sequence identity is consistently maintained. The persistence time is the evolutionary period during which a conserved region evolves under the same selective constraints.Our main findings are: (i) Insect genomes contain 1.60 times less conserved information than vertebrates; (ii) Vertebrate CNC have a higher persistence length than conserved coding regions or insect CNC; (iii) CNC have shorter persistence times as compared to conserved coding regions in both lineages. CONCLUSION: Higher persistence length of vertebrate CNC indicates that the conserved information in vertebrates and insects is organized in functional elements of different lengths. These findings might be related to the higher morphological complexity of vertebrates and give clues about the structure of active CNC elements.Shorter persistence time might explain the previously puzzling observations of highly conserved CNC within each phylum, and of a lack of conservation between phyla. It suggests that CNC divergence might be a key factor in vertebrate evolution. Further evolutionary studies will help to relate individual CNC to specific developmental processes.

Preretinal partial pressure of oxygen gradients before and after experimental pars plana vitrectomy.

PURPOSE: To evaluate preretinal partial pressure of oxygen (PO2) gradients before and after experimental pars plana vitrectomy. METHODS: Arteriolar, venous, and intervascular preretinal PO2 gradients were recorded in 7 minipigs during slow withdrawal of oxygen-sensitive microelectrodes (10-μm tip diameter) from the vitreoretinal interface to 2 mm into the vitreous cavity. Recordings were repeated after pars plana vitrectomy and balanced salt solution (BSS) intraocular perfusion. RESULTS: Arteriolar, venous, and intervascular preretinal PO2 at the vitreoretinal interface were 62.3 ± 13.8, 22.5 ± 3.3, and 17.0 ± 7.5 mmHg, respectively, before vitrectomy; 97.7 ± 19.9, 40.0 ± 21.9, and 56.3 ± 28.4 mmHg, respectively, immediately after vitrectomy; and 59.0 ± 27.4, 25.2 ± 3.0, and 21.5 ± 4.5 mmHg, respectively, 2½ hours after interruption of BSS perfusion. PO2 2 mm from the vitreoretinal interface was 28.4 ± 3.6 mmHg before vitrectomy; 151.8 ± 4.5 mmHg immediately after vitrectomy; and 34.8 ± 4.1 mmHg 2½ hours after interruption of BSS perfusion. PO2 gradients were still present after vitrectomy, with the same patterns as before vitrectomy. CONCLUSION: Preretinal PO2 gradients are not eliminated after pars plana vitrectomy. During BSS perfusion, vitreous cavity PO2 is very high. Interruption of BSS perfusion evokes progressive equilibration of vitreous cavity PO2 with concomitant progressive return of preretinal PO2 gradients to their previtrectomy patterns. This indicates that preretinal diffusion of oxygen is not altered after vitrectomy. The beneficial effect of vitrectomy in ischemic retinal diseases or macular edema may be related to other mechanisms, such as increased oxygen convection currents or removal of growth factors and cytokines secreted in the vitreous.

Morphometric comparison of Simulium perflavum larvae (Diptera: Simuliidae) in relation to season and gender in Central Amazônia, Brazil

Number of larval instars, age structure and environmental effects on these parameters represent basic information in the study of insect population biology. When species have economic importance, this information is essential in order to choose the best period to apply different control methods and to determine the stages of the life cycle of the insect that are most susceptible to each treatment. The family Simuliidae has many species of medical/veterinary importance in the world, and some studies in the temperate region have suggested that the number of larval instars and the larval size can vary according to the season, gender and some environmental factors, such as temperature and diet. This study, with the zoophilic species Simulium perflavum Roubaud, is the first in the Neotropics observing some of these factors and will serve as a template for other species of medical importance in the region. S. perflavum larvae were collected in five streams in Central Amazônia (Manaus and Presidente Figueiredo counties, State of Amazonas), in Sept./Oct. 1996 (dry season) and Feb./Mar. 1997 (rainy season). These larvae were measured (lateral length of head capsule and width of cephalic apodema) to determine the number of larval instars (n=3985), to compare the larval size between seasons and genders (last and penultimate larval instars, n=200). Seven larval instars were determined for this species using frequency distributions, t-tests and Crosby´s growth rule. Significant differences were not detected (t-test, p>0.05) in larval size between seasons and genders. Our results differ from some found in temperate regions suggesting that in the Neotropical region the larval size in different seasons and different genders remains constant, although some environmental parameters, such as diet, change depending on the season.

Black flies (Diptera: Simuliidae) of French Guiana: cytotaxonomy and a preliminary list of species

The objectives of the present study were to broaden the survey of simuliid species in French Guiana and to cytologically analyze the species in the Simulium perflavum species group. Twelve species of Simulium were collected from which S. goeldii, S. quadrifidum, S. trombetense, S. near incrustatum, S. metallicum (s.l.) sp1, S. metallicum (s.l.) sp2 and S. ochraceum (s.l.) are reported for the first time for this region. The only species collected in the S. perflavum group was S. rorotaense; 34 larvae of this species were cytologically analyzed, all of which had the standard sequence. S. metallicum (s.l.), S. ochraceum (s.l.), S. guianense (s.l.) and S. oyapockense (s.l.) are involved with transmission of onchocerciasis in Central and South America, however, in French Guiana these species were not found biting humans during the sampling period. With the few collections made during this study, we increase the number of simulid species known in French Guiana from 6 to 13. It is clear that more simuliid species can be expected to be found when more sampling is done, including collections in other ecoregions in French Guiana.

The composition of mineral waters sourced from Europe and North America in respect to bone health: composition of mineral water optimal for bone.

The consumption of mineral waters is increasing in industrialised countries. High intakes of Ca and other alkalising cations as well as a low acid intake are beneficial to bone. We examined which components of mineral waters are conditioning their Ca content and their alkalinising power, in order to define the optimal profile. European mineral waters were randomly selected on the Internet: 100 waters with less than 200 mg Ca/l (9.98 mEq/l) and fifty with more than 200 mg/l, all with complete data for SO4, P, Cl, Na, K, Mg and Ca, and most also for HCO3. For comparison, forty North American mineral waters were randomly chosen. The potential renal acid load (PRAL) was calculated for each mineral water. North American waters did not reveal significant results because of their low mineralisation. We performed correlations between all eight components in order to explore the properties of the mineral waters. In the European waters, twenty-six out of twenty-eight correlations showed a P value of <or= 0.01. In waters with PRAL >0 (acidifying waters), PRAL was positively correlated with SO4, Ca, K and Mg (P < 0.001). In those with PRAL < 0 (alkalinising waters), PRAL was negatively correlated with HCO3, Na, Mg, Ca, K, Cl and SO4 (P < 0.001). SO4 and HCO3 were not found together in high quantities in the same water for geochemical reasons. A high Ca content is associated with either a high SO4 or a high HCO3 content. SO4 theoretically increases Ca excretion, while HCO3 and low PRAL values are associated with positive effects on bone. Therefore, the best waters for bone health are rich in both HCO3 and Ca, and by consequence low in SO4.

Presence of Triatominae (Hemiptera, Reduviidae) and Risk of Transmission of Chagas Disease in Colima, México

With the purpose of evaluating the risk of transmission of the Chagas disease in the State of Colima, México, an entomological survey was performed to obtain triatominae and the rate of infection by Trypanosoma cruzi determined by examination of its dejections. Two hundred eighteen houses located in 16 villages were sampled. In each house the intradomestic and peridomestic habitats were examined by the man-hour-house method, sensor boxes and mouse-baited traps. Also, 12 silvatic places were explored around the same areas using the same techniques as the ones sampled. In total, 456 specimens were captured, of which 139 correspond to Triatoma phyllosoma pallidipennis; 80 to T. p. longipennis; one specimen of T. dimidiata and 236 nymphs of Triatoma sp. Two hundred ninety seven insects were captured in the intradomestic habitat, 132 in the peridomestic and 26 in the silvatic. The index of positive houses was 27%, located in the central area of the state. The rate of natural infection with T. cruzi showed 25.6%. This results confirmed the presence of two important vectors of the Chagas disease in Colima. Its preference for the domestic habitat and its high levels of natural infection with T. cruzi suggested the existence of a significant risk for its transmission in this area of the country.

Eusociality and cooperation

The evolution of eusociality, here defined as the emergence of societies with reproductive division of labour and cooperative brood care, was first seen as a challenge to Darwin's theory of evolution by natural selection. Why should individuals permanently forgo direct reproduction to help other individuals to reproduce? Kin selection, the indirect transmission of genes through relatives, is the key process explaining the evolution of permanently nonreproductive helpers. However, in some taxa helpers delay reproduction until a breeding opportunity becomes available. Overall, eusociality evolved when ecological conditions promote stable associations of related individuals that benefit from jointly exploiting and defending common resources. High levels of cooperation and robust mechanisms of division of labour are found in many animal societies. However, conflicts among individuals are still frequent when group members that are not genetically identical compete over reproduction or resource allocation.

Is pretreatment with Beta-blockers beneficial in patients with acute coronary syndrome?

OBJECTIVES: The role of beta-blockers in the treatment of hypertension is discussed controversially and the data showing a clear benefit in acute coronary syndromes (ACS) were obtained in the thrombolysis era. The goal of this study was to analyze the role of pretreatment with beta-blockers in patients with ACS. METHODS: Using data from the Acute Myocardial Infarction in Switzerland (AMIS Plus) registry, we analyzed outcomes of patients with beta-blocker pretreatment in whom they were continued during hospitalization (group A), those without beta-blocker pretreatment but with administration after admission (group B) and those who never received them (group C). Major adverse cardiac events defined as composed endpoint of re-infarction and stroke (during hospitalization) and/or in-hospital death were compared between the groups. RESULTS: A total of 24,709 patients were included in the study (6,234 in group A, 12,344 in group B, 6,131 in group C). Patients of group B were younger compared to patients of group A and C (62.5, 67.6 and 68.4, respectively). In the multivariate analysis, odds ratio for major adverse cardiac events was 0.59 (CI 0.47-0.74) for group A and 0.66 (CI 0.55-0.83) for group B, while group C was taken as a reference. CONCLUSIONS: beta-Blocker therapy is beneficial in ACS and they should be started in those who are not pretreated and continued in stable patients who had been on chronic beta-blocker therapy before.

Aggregation mediated by faeces and footprints in Triatoma pseudomaculata (Heteroptera: Reduviidae), a Chagas disease vector

The behavioural response of Triatoma pseudomaculata to chemical substances present in their faeces or cuticle (footprints) was analyzed. Groups of larvae were simultaneously exposed to a clean filter paper and to another paper impregnated with a chemical stimulus in a circular arena. In these choice experiments, the insects aggregated significantly around papers impregnated with dry faeces. In addition, the bugs also showed a significant aggregation response to papers impregnated with compounds derived from their cuticle that were deposited by contact on the substrate. These results indicate that chemical compounds that affect the behaviour of T. pseudomaculata are present in the faeces and in the cuticle of this species. Results are discussed in relation to chemical communication in the Triatominae, as well as to the potential use of these substances in traps or sensors for the detection of this species.

The role of energy and nutritional support in the intensive care unit.

Malnutrition is common in critically ill, hospitalized patients and so represents a major problem for intensive care. Nutritional support can be beneficial in such cases and may help preserve vital organ and immune function. Energy requirements, route of delivery and potential complications of nutritional support are discussed in this paper.

Metabolic effects of diets differing in glycaemic index depend on age and endogenous glucose-dependent insulinotrophic polypeptide in mice.

AIMS/HYPOTHESIS: High- vs low-glycaemic index (GI) diets unfavourably affect body fat mass and metabolic markers in rodents. Different effects of these diets could be age-dependent, as well as mediated, in part, by carbohydrate-induced stimulation of glucose-dependent insulinotrophic polypeptide (GIP) signalling. METHODS: Young-adult (16 weeks) and aged (44 weeks) male wild-type (C57BL/6J) and GIP-receptor knockout (Gipr ( -/- )) mice were exposed to otherwise identical high-carbohydrate diets differing only in GI (20-26 weeks of intervention, n = 8-10 per group). Diet-induced changes in body fat distribution, liver fat, locomotor activity, markers of insulin sensitivity and substrate oxidation were investigated, as well as changes in the gene expression of anorexigenic and orexigenic hypothalamic factors related to food intake. RESULTS: Body weight significantly increased in young-adult high- vs low-GI fed mice (two-way ANOVA, p < 0.001), regardless of the Gipr genotype. The high-GI diet in young-adult mice also led to significantly increased fat mass and changes in metabolic markers that indicate reduced insulin sensitivity. Even though body fat mass also slightly increased in high- vs low-GI fed aged wild-type mice (p < 0.05), there were no significant changes in body weight and estimated insulin sensitivity in these animals. However, aged Gipr ( -/- ) vs wild-type mice on high-GI diet showed significantly lower cumulative net energy intake, increased locomotor activity and improved markers of insulin sensitivity. CONCLUSIONS/INTERPRETATION: The metabolic benefits of a low-GI diet appear to be more pronounced in younger animals, regardless of the Gipr genotype. Inactivation of GIP signalling in aged animals on a high-GI diet, however, could be beneficial.