Genetic detection of cryptic species in the frillfin goby Bathygobius soporator

We compared, through allozyme and cytochrome b sequence analyses, populations of Bathygobius soporator from four localities on the Brazilian coast with those from the Oceanic Islands of the Rocas Atoll (230 km off the Brazilian coast) and the Bahamas (5700 km northwest from Rocas). The population from the Rocas Atoll was genetically more similar to the geographically distant Bahamas (gene identity, I=0.92) than to the supposedly conspecific populations from the Brazilian coast (I<0.55). Five diagnostic allozyme loci and a high level of nucleotide divergence (Kimura 2-parameters=0.21) were found between the two groups, further confirming that they must belong to different biological species. Since the type locality of B. soporator is in the Caribbean, the binomial should be maintained for the Bahamas/Rocas Atoll populations, and the other Brazilian populations of Bathygobius analysed either represent the occurrence of a species of Bathygobius hitherto not cited for the Brazilian coast, or belong to a new species. The coastal populations of this species were also found to be highly structured (F_ST=0.18; p<0.05), indicating that, even along the coast, levels of gene flow of this species are limited. This could be related to their reproductive biology (adhesive benthic eggs with parental care and short length of larval life). The timings of the divergence between the Caribbean and the Southwest Atlantic species found here, estimated from allozymes and cytb sequences, are very similar (around 10 MY bp), and correlate with the start of the Amazon river outflow. The colonization of the Rocas Atoll by populations from the Caribbean seems to have taken place after the Caribbean and the Brazilian coast species had diverged.     

Efficacy of either a single or split treatment of PGF2alpha after a 14 day melengestrol acetate treatment to synchronize estrus and induce luteolysis in Bos indicus x Bos taurus heifers

Two experiments evaluated a modified delivery of prostaglandin F2alpha (PGF2alpha) after a melengestrol acetate (MGA) treatment in Angus and Bos indicus x Bos taurus (BI) heifers. Experiment 1 was replicated three times with yearling BI heifers (n = 695). Heifers received MGA (0.5 mg head(-1) day(-1)) for 14 days. In Replications 1 and 2, heifers received either 25 mg of PGF2alpha im 19 days after MGA (single) or 12.5 mg of PGF2alpha im 19 and 20 days after MGA (split). In Replication 3, heifers received the same treatments, with PGF2alpha initiated either 18 or 19 days after MGA. Estrus was detected for 72 h after PGF2alpha, with AI commencing 8-12 h after a detected estrus. Heifers not observed in estrus by 72 h were timed-AI concomitant with GnRH (100 microg im). Heifers from Replication 2 (n = 146) had blood samples collected at the initial PGF2alpha and at timed-AI to determine corpus luteum (CL) regression by evaluating plasma progesterone concentrations. The interval from MGA withdrawal to PGF2alpha did not have a significant effect on any variable in Replication 3 and there were no treatment by replication effects for any variables, therefore data were pooled. Modifying the PGF2alpha treatment from a single treatment to two treatments on consecutive days increased (P < 0.05) 72 h estrous response (43.2% versus 50.1%), timed-AI (23.9% versus 33.5%) and total-AI pregnancy rates (34.5% versus 42.5%), and CL regression (79.1% versus 92.5%), respectively. In Experiment 2, yearling Angus (n = 66) and 2-year-old BI (n = 68) heifers were synchronized as per Experiment 1 (with the initial PGF2alpha 19 days after MGA). Neither breed nor PGF2alpha treatment effected (P > 0.05) 72 h estrous response, total-AI pregnancy rate, or CL regression rate. In conclusion, treating yearling BI heifers with split treatments of PGF2alpha (given on two consecutive days) improved estrous response and pregnancy rates by increasing PGF2alpha-induced luteolysis.     

Respiratory stimulus in Rhizobium sp. by legume lectins

High molecular weight lectins (> 100 kDa) from seeds of the legumes Canavalia brasiliensis (CnBr), Cratylia floribunda (CFL), Phaseolus vulgaris (PHA) and Vatairea macrocarpa (VML), temporarily stimulate the respiration of Rhizobium tropici-CIAT899 and R. etli-CFN42. These stimulants were significant (P < 0.05) in bacterial suspensions (> 2.85 mg dry biomass ml^–1), having at least 6200 molecules of lectins per bacteria. The VML (20 g ml^–1), induced specific O₂ demand of 2.3–2.5 M O₂ min^–1 mg dry biomass^–1, in CFN42 and CIAT899, respectively. However, CnBr, CFL and PHA induced smaller demands of O₂ (5×), in both strains. The order of affinities of the lectins was approximately VML > PHA > CFL > CnBr, with regard to respiratory stimuli in CIAT899 strain. The co-administration of 10 g VML ml^–1 and 9.8 M galactose, in CIAT899 suspensions, reduced the respiratory stimuli significantly in relation to the treatment with VML alone. These respiratory stimuli, induced by the lectins, increase the significance of the interaction lectin × Rhizobium in terms of bacterial physiology. Its understanding could be important in relation to bacterial symbiotic behaviour.     

Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides

Background

Gastric cancer is the third leading cause of cancer-related mortality worldwide. Recently, it has been demonstrated that gastric cancer cells display a specific miRNA expression profile, with increasing evidence of the role of miRNA-9 in this disease. miRNA-9 upregulation has been shown to influence the expression of E-cadherin-encoding gene, triggering cell motility and invasiveness.

Results

In this study, we designed LNA anti-miRNA oligonucleotides with a complementary sequence to miRNA-9 and tested their properties to both detect and silence the target miRNA. We could identify and visualize the in vitro uptake of low-dosing LNA-based anti-miRNA oligonucleotides without any carrier or transfection agent, as early as 2 h after the addition of the oligonucleotide sequence to the culture medium. Furthermore, we were able to assess the silencing potential of miRNA-9, using different LNA anti-miRNA oligonucleotide designs, and to observe its subsequent effect on E-cadherin expression.

Conclusions

The administration of anti-miRNA sequences even at low-doses, rapidly repressed the target miRNA, and influenced the expression of E-cadherin by significantly increasing its levels.

     

Successful maintenance of a stingless bee population despite a severe genetic bottleneck

Stingless bees play an important ecological role as pollinators of many wild plant species in the tropics and have significant potential for the pollination of agricultural crops. Nevertheless, conservation efforts as well as commercial breeding programmes require better guidelines on the amount of genetic variation that is needed to maintain viable populations. In this context, we carried out a long-term genetic study on the stingless bee Melipona scutellaris to evaluate the population viability consequences of prolonged breeding from a small number of founder colonies. In particular, it was artificially imposed a genetic bottleneck by setting up a population starting from only two founder colonies, and continued breeding from it for a period of over 10?years in a location outside its natural area of occurrence. We show that despite a great reduction in the number of alleles present at both neutral microsatellite loci and the sex-determining locus relative to its natural source population, and an increased frequency in the production of sterile diploid males, the genetically impoverished population could be successfully bred and maintained for at least 10?years. This shows that in stingless bees, breeding from a small stock of colonies may have less severe consequences than previously suspected. In addition, we provide a simulation model to determine the number of colonies that are needed to maintain a certain number of sex alleles in a population, thereby providing useful guidelines for stingless bee breeding and conservation efforts.     

Trypanosoma cruzi and Its Soluble Antigens Induce NET Release by Stimulating Toll-Like Receptors

Neutrophils release fibrous traps of DNA, histones, and granule proteins known as neutrophil extracellular traps (NETs), which contribute to microbicidal killing and have been implicated in autoimmunity. The role of NET formation in the host response to nonbacterial pathogens is not well-understood. In this study, we investigated the release of NETs by human neutrophils upon their interaction with Trypanosoma cruzi (Y strain) parasites. Our results showed that human neutrophils stimulated by T. cruzi generate NETs composed of DNA, histones, and elastase. The release occurred in a dose-, time-, and reactive oxygen species-dependent manner to decrease trypomastigote and increase amastigote numbers of the parasites without affecting their viability. NET release was decreased upon blocking with antibodies against Toll-like receptors 2 and 4. In addition, living parasites were not mandatory in the release of NETs induced by T. cruzi, as the same results were obtained when molecules from its soluble extract were tested. Our results increase the understanding of the stimulation of NETs by parasites, particularly T. cruzi. We suggest that contact of T. cruzi with NETs during Chagas’s disease can limit infection by affecting the infectivity/pathogenicity of the parasite.     

The amplified peptidome: the new treasure chest of candidate biomarkers

Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low-abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement.     

Studies on the effectiveness of diarylheptanoids derivatives against Leishmania amazonensis

In a previous work we demonstrated that diarylheptanoids extracted from Centrolobium sclerophyllum are very active against Leishmania amazonensis promastigotes. In order to continue our studies with these class of compounds, we decided to evaluate the activity of several diarylheptanoids derived from curcumin (diferuloyl methane) against the extracellular form (promastigotes) of L. amazonensis. Furthermore, an experiment against the intracellular form of the parasite (amastigotes) was carried out, comparing the most active compound among the curcumin derivatives (the methylcurcumin) with des-O-methylcentrolobine, the most active diarylheptanoid derived from C. sclerophyllum.     

Seroprevalence of Toxoplasma gondii infection in goats by the indirect haemagglutination, immunofluorescence and immunoenzymatic tests in the region of Uberlândia, Brazil

A comparative study of the indirect haemagglutination (IHA), immunofluorescence (IFAT) and immunoenzymatic (ELISA) tests was carried out to determine the prevalence of Toxoplasma gondii antibodies in goats. One hundred seventy-four serum samples were obtained from four goat herds from the region of Uberlandia, State of Minas Gerais. The distribution of the animals, according to their origin, was as follow: 71 from herd I; 39 from herd II; 37 from herd III; and 27 from herd IV. Serum samples were analyzed by IHA, IFAT and ELISA, considering the reactivity of the serum samples at dilution > or = 1:64 as cut off titer for the three tests. A global seroprevalence of 18.4% was observed, with significantly higher positivity rate in the herd II (66.7%) and older animals (> 36 months). A high and significant positive correlation was found between the titers obtained by the IHA versus IFAT, IHA versus ELISA, and ELISA versus IFAT. Therefore, it can be concluded that the three analyzed tests have shown to be highly concordant and appropriate for epidemiological surveys of Toxoplasma infection in goats. Although the seroprevalence of T. gondii infection in goats is relatively low in this region as compared to other regions of the country, adequate management might be useful and essential to control the infection in the goat herds.     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.