Characterization and use of the total head soluble cholinesterases from mosquitofish (Gambusia holbrooki) for screening of anticholinesterase activity

Inhibition of cholinesterases (ChE) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. Different ChE isoforms may be present in the same tissue and may present distinct sensitivities towards environmental contaminants. The present work characterises the soluble ChE present in mosquitofish (Gambusia holbrooki) total head homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Furthermore, the effects of sodium dodecylsulphate (SDS) on the enzymatic activity were investigated, both in vivo and in vitro. These results showed that acetylcholinesterase (AChE) seemed to be the predominant form present in head homogenates of G. holbrooki, despite the inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) found at high concentrations. SDS was responsible for in vitro, but not in vivo, inhibitory effects. The in vitro AChE inhibitory effects of SDS was partially prevented by the use of increasing amounts of ethanol, suggesting that the inhibition was induced by an emulsion effect, which may explain the lack of effect in vivo.     

Immobilization and stabilization of a bimolecular aggregate of the lipase from Pseudomonas fluorescens by multipoint covalent attachment

The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 °C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol.     

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.     

Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.     

Regulation of growth by the trehalose pathway: Relationship to temperature and sucrose

Carbon signaling can override carbon supply in the regulation of growth. At least some of this regulation is imparted by the sugar signal trehalose 6-phosphate (T6P) through the protein kinase, SnRK1. This signaling pathway regulates biosynthetic processes involved in growth under optimal growing conditions. Recently, using a seedling system we showed that under sub-optimal conditions, such as cold, carbon signaling by T6P/ SnRK1 enables recovery of growth following relief of the stress. The T6P/ SnRK1 mechanism thus could be selected as a means of improving low temperature tolerance. High-throughput automated Fv/Fm measurements provide a potential means to screen for T6P/ SnRK1, and here we confirm through measurements of Fv/Fm in rosettes that T6P promotes low temperature tolerance and recovery during cold to warm transfer. Further, to better understand the coordination between sugars, trehalose pathway, and temperature-dependent growth, we examine the interrelationship between sugars, trehalose phosphate synthase (TPS), and trehalose phosphate phosphatase (TPP) gene expression and T6P content in seedlings. Sucrose, particularly when fed exogenously, correlated well with TPS1 and TPPB gene expression, suggesting that these enzymes are involved in maintaining carbon flux through the pathway in relation to sucrose supply. However, when sucrose accumulated to higher levels under low temperature and low N, TPS1 and TPPB expression were less directly related to sucrose; other factors may also contribute to regulation of TPS1 and TPPB expression under these conditions. TPPA expression was not related to sucrose content and all genes were not well correlated with endogenous glucose. Our work has implications for understanding acclimation to sink-limited growth conditions such as low temperature and for screening cold-tolerant genotypes with altered T6P/ SnRK1 signaling.     

Changing Mortality Profile among HIV-Infected Patients in Rio de Janeiro,Brazil: Shifting from AIDS to Non-AIDS Related Conditions in the HAART Era

Introduction

We describe temporal trends in the mortality rates and factors associated with AIDS and non-AIDS related mortality at the Evandro Chagas Clinical Research Institute (IPEC), Oswaldo Cruz Foundation (FIOCRUZ).

Methods

Adult patients enrolling from 1986 through 2009 with a minimum follow up of 60 days were included. Vital status was exhaustively checked using patients’ medical charts, through active contact with individuals and family members and by linkage with the Rio de Janeiro Mortality database using a previously validated algorithm. The CoDe protocol was used to establish the cause of death. Extended Cox proportional hazards models were used for multivariate modeling.

Results

A total of 3530 individuals met the inclusion criteria, out of which 868 (24.6%) deceased; median follow up per patient was 3.9 years (interquartile range 1.7–9.2 years). The dramatic decrease in the overall mortality rates was driven by AIDS-related causes that decreased from 9.19 deaths/100PYs n 1986–1991 to 1.35/100PYs in 2007–2009. Non-AIDS related mortality rates remained stable overtime, at around 1 death/100PYs. Immunodeficiency significantly increased the hazard of both AIDS-related and non-AIDS-related causes of death, while HAART use was strongly associated with a lower hazard of death from either cause.

Conclusions

Our results confirm the remarkable decrease in AIDS-related mortality as the HIV epidemic evolved and alerts to the conditions not traditionally related to HIV/AIDS which are now becoming more frequent, needing careful monitoring.     

Fish biodiversity of Saint Peter and Saint Paulʼs Archipelago,Mid-Atlantic Ridge,Brazil: new records and a species database

Saint Peter and Saint Paul's Archipelago (SPSPA), one of the smallest and most isolated island groups in the world, is situated on the Mid-Atlantic Ridge, between Brazil and the African continent. SPSPA has low species richness and high endemism; nonetheless, the diversity of fishes from deep habitats (>30 m depth) had not been previously studied in detail. Several expeditions conducted between 2009 and 2018 explored the shallow and deep reefs of SPSPA using scuba, closed-circuit rebreathers, manned submersibles, baited remote underwater stereo-videos (stereo-BRUV) and fishing between 0 and 1050 m depth. These expeditions yielded 41 new records of fishes for SPSPA: 9 in open waters, 9 in shallow waters (0–30 m), 8 in mesophotic ecosystems (30–150 m) and 15 in deeper reefs (>150 m). Combined with literature records of adult pelagic, shallow and deep-reef species, as well as larvae, the database of the fish biodiversity for SPSPA currently comprises 225 species (169 recorded as adult fishes and 79 as larvae, with 23 species found in both stages). Most of them (112) are pelagic, 86 are reef-associated species and 27 are deep-water specialists. Species accumulation curves show that the number of fish species has not yet reached an asymptote. Whereas the number of species recorded in SPSPA is similar to that in other oceanic islands in the Atlantic Ocean, the proportion of shorefishes is relatively lower, and the endemism level is the third highest in the Atlantic. Twenty-nine species are listed as threatened with extinction. Observations confirm the paucity of top predators on shallow rocky reefs of the island, despite the presence of several pelagic shark species around SPSPA. Because all of the endemic species are reef associated, it is argued that the new marine-protected areas created by the Brazilian government do not ensure the protection and recovery of SPSPA's biodiversity because they allow exploitation of the most vulnerable species around the archipelago itself. This study suggests a ban on reef fish exploitation inside an area delimited by the 1000 m isobath around the islands (where all known endemics are concentrated) as the main conservation strategy to be included in the SPSPA management plan being prepared by the Brazilian government.     

A species-level total evidence phylogeny of the microteiid lizard family Alopoglossidae (Squamata: Gymnophthalmoidea)

Alopoglossidae is a family of Neotropical lizards composed of 23 species allocated in two genera (Alopoglossus and Ptychoglossus). There is a lack of knowledge about the phylogenetic relationships and systematics of this family. Published phylogenies that include alopoglossid species have very low taxon coverage within the family, and are usually based on limited character sampling. Considering these shortcomings, we infer the phylogenetic relationships of Alopoglossidae—including all but one species in the family—based on the combined analyses of DNA sequences and morphological characters. We use four loci (the mitochondrial 12S, 16S and ND4; the nuclear C-mos) and a matrix of 143 phenotypic characters from scutellation, tongue morphology, hemipenis morphology, and osteology. The dataset is analyzed with Maximum Parsimony, with four alternative weighting schemes: three under Extended Implied Weighting, and one with equal weighting. The respective resulting topologies are compared in a sensitivity analysis framework. Our analyses support the paraphyly of Ptychoglossus, with Alopoglossus nested within it. We provide an updated classification for the family, where Ptychoglossus Boulenger, 1890 is considered a junior synonym of Alopoglossus Boulenger, 1885.     

Species delimitation based on integrative approach suggests reallocation of genus in Hypostomini catfish (Siluriformes,Loricariidae)

Hydrobiologia - Integrative approaches are particularly useful to resolve taxonomic uncertainties in species-rich groups that have undergone explosive radiation, such as Hypostomini (suckermouth...     

Isoflavone synthase (IFS) gene phylogeny in Trifolium species associated with plant isoflavone contents

This study presents the results of the identification and quantification of 12 isoflavones (prunetin, irilone, pseudobaptigenin, glycitein, daidzin, genistin, daidzein, pratensein, puerarin, biochanin A, formononetin and genistein) in 23 species of Trifolium (T. arvense, T. pratense, T. ligusticum, T. striatum, T. lappaceum, T. angustifolium, T. hirtum, T. subterraneum, T. isthmocarpum, T. stellatum, T. mutabile, T. strictum, T. fragiferum, T. alexandrinum, T. tomentosum. T. nigrescens subsp. petrisavii, T. nigrescens, T. glomeratum, T. subterraneum subsp. brachycalycinum, T. cherleri, T. resupinatum, T. campestre and T. repens). Isoflavones were extracted by an MSPD method and analyzed with HPLC coupled with a diode-array detector. The evaluation of molecular phylogeny of the IFS gene and the relation with isoflavone content was also performed. Five species (T. subterraneum subsp. brachycalycinum, T. alexandrinum, T. pratense, T. subterraneum and T. lappaceum) were identified with high levels of biochanin A (431–83 mg/kg), formononetin (72–365 mg/kg) and genistein (9–509 mg/kg), which could be utilized as alternative sources for the nutraceutical industry. Genetic phylogeny for the IFS gene was found in the species studied, with 20 out of 23 species having been divided into two clades, while the remaining three were genetically distant. Based on our results, we confirm the direct correlation between IFS gene polymorphism and isoflavones content in species of Trifolium particularly noted for formononetin. Therefore, the IFS gene can be utilized for screening Trifolium genotypes for formononetin. The relation of the three isoflavones' contents and the molecular phylogeny of plants determined by the IFS sequences, as a screening marker for plants with high isoflavone contents in Trifolium species, are to the best of our knowledge described for the first time.