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.     

Co-segregation analysis and mapping of the anthracnose Co-10 and angular leaf spot Phg-ON disease-resistance genes in the common bean cultivar Ouro Negro

Anthracnose (ANT) and angular leaf spot (ALS) are devastating diseases of common bean (Phaseolus vulgaris L.). Ouro Negro is a highly productive common bean cultivar, which contains the Co-10 and Phg-ON genes for resistance to ANT and ALS, respectively. In this study, we performed a genetic co-segregation analysis of resistance to ANT and ALS using an F₂ population from the Rudá × Ouro Negro cross and the F_2:3 families from the AND 277 × Ouro Negro cross. Ouro Negro is resistant to races 7 and 73 of the ANT and race 63-39 of the ALS pathogens. Conversely, cultivars AND 277 and Rudá are susceptible to races 7 and 73 of ANT, respectively. Both cultivars are susceptible to race 63-39 of ALS. Co-segregation analysis revealed that Co-10 and Phg-ON were inherited together, conferring resistance to races 7 and 73 of ANT and race 63-39 of ALS. The Co-10 and Phg-ON genes were co-segregated and were tightly linked at a distance of 0.0 cM on chromosome Pv04. The molecular marker g2303 was linked to Co-10 and Phg-ON at a distance of 0.0 cM. Because of their physical linkage in a cis configuration, the Co-10 and Phg-ON resistance alleles are inherited together and can be monitored with great efficiency using g2303. The close linkage between the Co-10 and Phg-ON genes and prior evidence are consistent with the existence of a resistance gene cluster at one end of chromosome Pv04, which also contains the Co-3 locus and ANT resistance quantitative trait loci. These results will be very useful for breeding programs aimed at developing bean cultivars with ANT and ALS resistance using marker-assisted selection.     

A Membrane-Translocating Peptide Penetrates into Bilayers without Significant Bilayer Perturbations

Using a high throughput screen, we have identified a family of 12-residue long peptides that spontaneously translocate across membranes. These peptides function by a poorly understood mechanism that is very different from that of the well-known, highly cationic cell penetrating peptides such as the tat peptide from HIV. The newly discovered translocating peptides can carry polar cargoes across synthetic bilayers and across cellular membranes quickly and spontaneously without disrupting the membrane. Here we report on the biophysical characterization of a representative translocating peptide from the selected family, TP2, as well as a negative control peptide, ONEG, from the same library. We measured the binding of the two peptides to lipid bilayers, their secondary structure propensities, their dispositions in bilayers by neutron diffraction, and the response of the bilayer to the peptides. Compared to the negative control, TP2 has a greater propensity for membrane partitioning, although it still binds only weakly, and a higher propensity for secondary structure. Perhaps most revealing, TP2 has the ability to penetrate deep into the bilayer without causing significant bilayer perturbations, a property that may help explain its ability to translocate without bilayer permeabilization.     

Aeromonas molluscorum Av27 is a potential tributyltin (TBT) bioremediator: phenotypic and genotypic characterization indicates its safe application

Aeromonas molluscorum Av27 is an estuarine bacterium highly resistant to tributyltin (TBT). Also, the strain is able to degrade TBT into the less toxic compounds dibutyltin and monobutyltin. Therefore, this bacterium has potential to be employed in bioremediation processes. In this context, defining its biological safety is crucial. With that purpose a number of intrinsic characteristics, usually present/associated with virulent strains, were investigated. Few virulence factors were detected in strain Av27. For instance, a DNase gene is present, but it is not apparently expressed in vitro. Motility, adherence factor and phospholipase activity were also detected. Additionally, cytotoxicity to Vero cells was negative. Resistance to penicillin (10 μg ml^?1), amoxicillin/clavulanic acid (30 μg ml^?1) and cephalothin (30 μg ml^?1) and also to the vibriostatic agent O/129 was observed. Five plasmids (4, 7, 10, 100 kb and one greater than 100 kb) were identified. No Class I and II integrons were detected. Study of the optimal growth conditions showed that Av27 easily adapts to different environmental conditions. Overall, the results suggest that A. molluscorum Av27 can be considered safe to use to bioremediate TBT in contaminated environments.     

Diversity and symbiotic effectiveness of beta-rhizobia isolated from sub-tropical legumes of a Brazilian Araucaria Forest

While the occurrence of Betaproteobacteria occupying the nodules of tropical legumes has been shown, little is known about subtropical areas. Araucaria Forest is a subtropical endangered ecosystem, and a better understanding of the legume-rhizobial symbionts may allow their use in land reclamation. The 16S rRNA gene of bacteria isolated from nine leguminous species was sequenced and their nodulation tested in Mimosa scabrella and Phaseolus vulgaris. 196 isolates were identified as eight genotypes: Pantoea, Pseudomonas, Bradyrhizobium sp1-2, Rhizobium, and Burkholderia sp1-3. The majority of the isolates from native plants (87 %) were taxonomically related to β-rhizobia, namely Burkholderia, however the legumes Galactia crassifolia and Collea speciosa were nodulated by both α and β-rhizobia, and Acacia dealbata, an exotic plant, only by α-rhizobia. The nifH genes of some isolates were sequenced and N-fixing potential shown by the acetylene reduction test. Most of the isolates nodulated the test plants, some were effective in M. scabrella, but all presented low efficiency in the exotic promiscuous legume P. vulgaris. Pantoea and Pseudomonas did not nodulate and probably are endophytic bacteria. The presented data shows diversity of α, β and γ-Proteobacteria in nodules of subtropical legumes, and suggests host specificity with β-rhizobia. Potential isolates were found for M. scabrella, indicating that a high N-fixing strain may be further inoculated in plants for use in reforestation.     

Geographic association and temporal variation of chemical and physical defense and leaf damage in Datura stramonium

The evolution of plant defense traits has traditionally been explained trough the “coevolutionary arms race” between plants and herbivores. According to this, specialist herbivores have evolved to cope effectively with the defensive traits of their host plants and may even use them as a cue for host location. We analyzed the geographic association between leaf trichomes, two tropane alkaloids (putative resistance traits), and leaf damage by herbivores in 28 populations of Datura stramonium in central Mexico. Since the specialist leaf beetles Epitrix parvula and Lema trilineata are the main herbivores of D. stramonium in central Mexico, we predicted a positive association between plant defense and leaf damage across populations. Also, if physical environmental conditions (temperature or precipitation) constrain the expression of plant defense, then the geographic variation in leaf damage should be explained partially by the interaction between defensive traits and environmental factors. Furthermore, we studied the temporal and spatial variation in leaf trichome density and leaf damage in five selected populations of D. stramonium sampled in two periods (1997 vs. 2007). We found a positive association between leaf trichomes density and atropine concentration with leaf damage across populations. The interaction between defensive traits and water availability in each locality had a significant effect on the geographic variation in leaf damage. Differences among populations in leaf trichome density are maintained over time. Our results indicate that local plant–herbivore interaction plays an important role in shaping the geographic and temporal variation in plant defense in D. stramonium.     

Polyethylene glycol‐based low generation dendrimers functionalized with β‐cyclodextrin as cryo‐ and dehydro‐protectant of catalase formulations

Polyethylene glycol (PEG)‐based low generation dendrimers are analyzed as single excipient or combined with trehalose in relation to their structure and efficiency as enzyme stabilizers during freeze‐thawing, freeze‐drying, and thermal treatment. A novel functional dendrimer (DG_o‐CD) based on the known PEG's ability as cryo‐protector and β‐CD as supramolecular stabilizing agent is presented. During freeze‐thawing, PEG and β‐CD failed to prevent catalase denaturation, while dendrimers, and especially DG_o‐CD, offered the better protection to the enzyme. During freeze‐drying, trehalose was the best protective additive but DG_o‐CD provided also an adequate catalase stability showing a synergistic behavior in comparison to the activities recovered employing PEG or β‐CD as unique additives. Although all the studied dendrimers improved the enzyme remaining activity during thermal treatment of freeze‐dried formulations, the presence of amorphous trehalose was critical to enhance enzyme stability. The crystallinity of the protective matrix, either of PEG derivatives or of trehalose, negatively affected catalase stability in the freeze‐dried systems. When humidified at 52% of relative humidity, the dendrimers delayed trehalose crystallization in the combined matrices, allowing extending the protection at those conditions in which normally trehalose fails. The results show how a relatively simple covalent combination of a polymer such as PEG with β‐CD could significantly affect the properties of the individual components. Also, the results provide further insights about the role played by polymer–enzyme supramolecular interactions (host–guest crosslink, hydrogen bonding, and hydrophobic interactions) on enzyme stability in dehydrated models, being the effect on the stabilization also influenced by the physical state of the matrix. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:786–795, 2013     

Sexual behavior and male volatile compounds in wild and mass‐reared strains of the Mexican fruit fly Anastrepha ludens (Diptera: Tephritidae) held under different colony management regimes

We compared the calling and mating behavior and volatile release of wild males Anastrepha ludens (Loew) with males from 4 mass‐reared strains: (i) a standard mass‐reared colony (control), (ii) a genetic sexing strain (Tap‐7), (iii) a colony started from males selected on their survival and mating competitiveness abilities (selected), and (iv) a hybrid colony started by crossing wild males with control females. Selected and wild males were more competitive, achieving more matings under field cage conditions. Mass‐reared strains showed higher percentages of pheromone calling males under field conditions except for Tap‐7 males, which showed the highest percentages of pheromone calling males under laboratory cage conditions. For mature males of all strains, field‐cage calling behavior increased during the last hour before sunset, with almost a 2 fold increase exhibited by wild males during the last half hour. The highest peak mating activity of the 4 mass‐reared strains occurred 30 min earlier than for wild males. By means of solid phase microextraction (SPME) plus gas chromatography‐mass spectrometry (GC‐MS), the composition of volatiles released by males was analyzed and quantified. Wild males emitted significantly less amounts of (E,E)‐α‐farnesene but emitted significantly more amounts of (E,E)‐suspensolide as they aged than mass‐reared males. Within the 4 mass‐reared strains, Tap‐7 released significantly more amounts of (E,E)‐α‐farnesene and hybrid more of (E,E)‐suspensolide. Differences in chemical composition could be explained by the intrinsic characteristics of the strains and the colony management regimes. Characterization of calling behavior and age changes of volatile composition between wild and mass‐reared strains could explain the differences in mating competitiveness and may be useful for optimizing the sterile insect technique in A. ludens.     

QTL mapping for resistance to Ceratocystis wilt in <Emphasis Type="Italic">Eucalyptus</Emphasis>

Ceratocystis wilt caused by the fungus Ceratocystis fimbriata, is currently one of the major diseases in commercial plantations of Eucalyptus trees in Brazil. Deployment of resistant genotypes has been the main strategy for effective disease management. The present study aimed at identifying genomic regions underlying the genetic control of resistance to Ceratocystis wilt in Eucalyptus by quantitative trait loci (QTL) mapping in an outbred hybrid progeny derived from a cross between (Eucalyptus dunnii × Eucalyptus grandis) × (Eucalyptus urophylla × Eucalyptus globulus). A segregating population of 127 individuals was phenotyped for resistance to Ceratocystis wilt using controlled inoculation under a completely randomized design with five clonal replicates per individual plant. The phenotypic resistance response followed a continuous variation, enabling us to analyze the trait in a quantitative manner. The population was genotyped with 114 microsatellite markers and 110 were mapped with an average interval of 12.3 cM. Using a sib-pair interval-mapping approach five QTLs were identified for disease resistance, located on linkage groups 1, 3, 5, 8, and 10, and their estimated individual heritability ranged from 0.096 to 0.342. The QTL on linkage group 3 overlaps with other fungal disease-resistance QTLs mapped earlier and is consistent with the annotation of several disease-resistance genes on this chromosome in the E. grandis genome. This is the first study to identify and attempt to quantify the effects of QTLs associated with resistance to Ceratocystis wilt in Eucalyptus.