Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions.

Quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful tool used to measure gene expression. However, because of its high sensitivity, the method is strongly influenced by the quality and concentration of the template cDNA and by the amplification efficiency. Relative quantification is an effective strategy for correcting random and systematic errors by using the expression level of reference gene(s) to normalize the expression level of the genes of interest. To identify soybean reference genes for use in studies of flooding stress, we compared 5 candidate reference genes (CRGs) with the NormFinder and GeNorm programs to select the best internal control. The expression stability of the CRGs was evaluated in root tissues from soybean plants subjected to hypoxic conditions. Elongation factor 1-beta and actin-11 were identified as the most appropriate genes for RT-qPCR normalization by both the NormFinder and GeNorm analyses. The expression profiles of the genes for alcohol dehydrogenase 1, sucrose synthase 4, and ascorbate peroxidase 2 were analyzed by comparing different normalizing combinations (including no normalization) of the selected reference genes. Here, we have identified potential genes for use as references for RT-qPCR normalization in experiments with soybean roots growing in O2-depleted environments, such as flooding-stressed plants.

Signal, detection and estimation using a hybrid quantum circuit.

We investigate a hybrid device allowing a photon-phonon coupling of a transmission line radiation (TLR) and a nanoeletromechanical system (NEMS), mediated by a superconducting qubit population imbalance. We demonstrate the derivation of an effective Hamiltonian for the strongly dispersive regime for this system. The qubit works as a quantum switch, allowing a conditioned transfer of excitations between the TLR and NEMS. We show that this regime allows the system to be employed for signal processing and force estimation. Additionally, we explore the ability of the quantum switch to generate non-classical states.

Quantitative assessment of the interplay between DNA elasticity and cooperative binding of ligands.

Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of the total ligand concentration, which we apply to our data of the DNA-cationic ß-cyclodextrin and to the DNA-HU protein data available in the literature, determining the values of the local persistence lengths, the dissociation constant, and the degree of cooperativity for each set of data. In both cases the persistence length behaves nonmonotonically as a function of ligand concentration and based on the results obtained we discuss some physical aspects of the interplay between DNA elasticity and cooperative binding of ligands.

Emergence of the pointer basis through the dynamics of correlations.

We use the classical correlation between a quantum system being measured and its measurement apparatus to analyze the amount of information being retrieved in a quantum measurement process. Accounting for decoherence of the apparatus, we show that these correlations may have a sudden transition from a decay regime to a constant level. This transition characterizes a nonasymptotic emergence of the pointer basis, while the system apparatus can still be quantum correlated. We provide a formalization of the concept of emergence of a pointer basis in an apparatus subject to decoherence. This contrast of the pointer basis emergence to the quantum to classical transition is demonstrated in an experiment with polarization entangled photon pairs.

Biological characteristics of Anticarsia gemmatalis (Lepidoptera: Noctuidae) for three consecutive generations under different temperatures: understanding the possible impact of global warming on a soybean pest.

Climate changes can affect the distribution and intensity of insect infestations through direct effects on their life cycles. Experiments were carried out during three consecutive generations to evaluate the effect of different temperatures (25°C, 28°C, 31°C, 34°C and 37±1°C) on biological traits of the velvetbean caterpillar Anticarsia gemmatalis Hübner, 1818 (Lepidoptera: Noctuidae). The insects were fed on artificial diet and reared in environmental chambers set at 14 h photophase. The developmental cycle slowed with the increase in the temperature, within the 25°C to 34°C range. Male and female longevities were reduced with an increase in temperature from 25°C to 28°C. Egg viability was highest at 25°C, and the sex ratio was not influenced by temperature, in the three generations. There was no interactive effect between development time and temperature on pupal weight. The results suggested that the increase in the temperature negatively impacted A. gemmatalis development inside the studied temperature range, indicating a possible future reduction of its occurrence on soybean crops, as a consequence of global warming, mainly considering its impact on tropical countries where this plant is cropped. A. gemmatalis was not able to adapt to higher temperatures in a three-generation interval for the studied temperature range. However, a gradual increase and a longer adaptation period may favor insect selection and consequently adaptation, and must be considered in future studies in this area. Moreover, it is important to consider that global warming might turn cold areas more suitable to A. gemmatalis outbreaks. Therefore, more than a future reduction of A. gemmatalis occurrence due to global warming, we might expect changes regarding its area of occurrence on a global perspective.

Temperature estimation of a pair of trapped ions.

We apply estimation theory to a system formed by two interacting trapped ions. By using the Fisher matrix formalism, we introduce a simple scheme for estimation of the temperature of the longitudinal vibrational modes of the ions. We use the ions interaction to effectively infer the temperature of the individual ions, by optimising the interaction time evolution and by measuring only over one of the ions. We also investigate the effect of a non-thermal reservoir over the inference approach. The non-classicality of one of the ions vibrational modes, introduced due to a squeezed thermal reservoir, does not directly affect the inference of the individual temperatures, although allowing the modes to be entangled. To check actual experimental conditions, we analyze the temperature inference under heating due to surface-electrode noise.

Molecular, anatomical and physiological properties of a genetically modified soybean line transformed with rd29A:AtDREB1A for the improvement of drought tolerance.

We evaluated the molecular, anatomical and physiological properties of a soybean line transformed to improve drought tolerance with an rd29A:AtDREB1A construct. This construct expressed dehydration- responsive element binding protein DREB1A from the stress-inducible rd29A promoter. The greenhouse growth test included four randomized blocks of soybean plants, with each treatment performed in triplicate. Seeds from the non-transformed soybean cultivar BR16 and from the genetically modified soybean P58 line (T(2) generation) were grown at 15% gravimetric humidity for 31 days. To induce water deficit, the humidity was reduced to 5% gravimetric humidity (moderate stress) for 29 days and then to 2.5% gravimetric humidity (severe stress). AtDREB1A gene expression was higher in the genetically modified P58 plants during water deficit, demonstrating transgene stability in T(2) generations and induction of the rd29A promoter. Drought-response genes, including GmPI-PLC, GmSTP, GmGRP, and GmLEA14, were highly expressed in plants submitted to severe stress. Genetically modified plants had higher stomatal conductance and consequently higher photosynthetic and transpiration rates. In addition, they had more chlorophyll. Overexpression of AtDREB1A may contribute to a decrease in leaf thickness; however, a thicker abaxial epidermis was observed. Overexpression of AtDREB1A in soybean appears to enhance drought tolerance.

Cloning and quantitative expression analysis of drought-induced genes in soybean.

We determined the expression levels of DREB transcription factor (Gmdreb1) and of the genes Gmgols, Gmpip1b, Gmereb, and Gmdefensin in drought-tolerant (MG/BR46-Conquista) and drought-sensitive (BR16) genotypes of soybean, during drought. The trial was carried out in a controlled-environment chamber, set up to provide drought conditions. Sequences of Arabidopsis thaliana DREB-family proteins were used to build a phylogenetic tree through the alignment of the conserved regions near the AP2 domain. We found that Gmdreb1 is similar to Atrap2.1, which is located near the AtDREB1 and AtDREB2 families. The amplified fragment was cloned and sequenced; alignment with the sequence available at Genbank showed total similarity. Expression analysis showed that under drought: a) Gmdreb1 expression increased in leaves and roots of both genotypes and expression level changes occurred that were correlated with the length of the water-deficit period; b) there were increased expression levels of Gmdefensin in roots of MG/BR46; c) expression of Gmgols increased in leaves and roots of the two genotypes; d) Gmpip1b expression generally increased, except in roots of BR16, and e) the same was found for Gmereb, except in roots of MG/BR46.

Unequivocal assignments of flavonoids from Tephrosia sp. (Fabaceae).

(1)H and (13)C NMR chemical shifts of praecansone B, pongaflavone and dehydrorotenone isolated from Tephrosia egregia Sandw and obovatin from T. toxicaria Pers. were unambiguously assigned by 1D and 2D NMR experiments including (1)H, (1)H COSY, gHMQC and gHMBC, allowing the correction of literature assignments.

Development of topical nanoemulsions containing the isoflavone genistein.

We have recently described the incorporation of genistein into topical nanoemulsions. This study describes the physicochemical properties and the genistein permeation profile from these nanoemulsions. Formulations composed of egg lecithin, medium chain triglycerides (MCT) or octyldodecanol (ODD) and water were prepared by spontaneous emulsification. Irrespective of the oil core employed (MCT or ODD), this procedure yielded monodisperse emulsions with mean droplet sizes in the range of 230-280 nm. The addition of genistein in the oil phase, before emulsification, did not alter the properties of nanoemulsions. The amount of genistein incorporated in both formulations was close to 100% (1 mg/mL). Solubility and DSC experiments suggested that egg-lecithin may play an important role on the incorporation of genistein in nanoemulsions. Genistein permeation from formulations was assessed using pig ear skin in Franz type diffusion cells. The overall results showed a slow permeation profile for genistein from both nanoemulsions.Such results open interesting perspectives for the topical administration of genistein.

Evaluation of the toxicity of oligonucleotide/cationic nanoemulsion complexes on Hep G2 cells through MTT assay.

The purpose of this study was to evaluate the toxicity of the oligonucleotide/cationic nanoemulsion complexes on Hep G2 cells through MTT assay. Complexes exhibit droplet size, zeta potential and viscosity of approximately 270 nm, +50mV, and 1.0 cP. Different parameters which may have an influence on toxicity results obtained by MTT assay, i.e. cells number, concentration of MTT reagent and the addition of Soerensen's glycine buffer were first evaluated. In the optimized conditions (1 x 10(4) cells and 0.5 mg/mL MTT), the overall results showed that the addition of increasing amounts of complexes (or nanoemulsions) lead to a progressive toxicity on cells attributed to the presence of the cationic lipid stearylamine in the formulations, whatever the medias's pH is. The IC50 was approximately 200 microg/ml. Such results open interesting perspectives on the use of these nanoemulsions as oligonucleotide delivery systems for Hep G2 cells.

Associative bacteria influence maize (Zea mays L.) growth, physiology and root anatomy under different nitrogen levels.

Despite the great diversity of plant growth-promoting bacteria (PGPB) with potential to partially replace the use of N fertilisers in agriculture, few PGPB have been explored for the production of commercial inoculants, reinforcing the importance of identifying positive plant-bacteria interactions. Aiming to better understand the influence of PGPB inoculation in plant development, two PGPB species with distant phylogenetic relationship were inoculated in maize. Maize seeds were inoculated with Bacillus sp. or Azospirillum brasilense. After germination, the plants were subjected to two N treatments: full (N+) and limiting (N-) N supply. Then, anatomical, biometric and physiological analyses were performed. Both PGPB species modified the anatomical pattern of roots, as verified by the higher metaxylem vessel element (MVE) number. Bacillus sp. also increased the MVE area in maize roots. Under N+ conditions, both PGPB decreased leaf protein content and led to development of shorter roots; however, Bacillus sp. increased root and shoot dry weight, whereas A. brasilense increased photosynthesis rate and leaf nitrate content. In plants subjected to N limitation (N-), photosynthesis rate and photosystem II efficiency increased in maize inoculated with Bacillus sp., whilst A. brasilense contained higher ammonium, amino acids and total soluble sugars in leaves, compared to the control. Plant developmental and metabolical patterns were switched by the inoculation, regardless of the inoculant bacterium used, producing similar as well as distinct modifications to the parameters studied. These results indicate that even non-diazotrophic inoculant strains can improve the plant N status as result of the morpho-anatomical and physiological modifications produced by the PGPB.

pH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in quasi-anhydrous samples.

pH-sensitive liposomes made of dioleoylphosphatidylethanolamine (DOPE)/oleic acid (OA)/cholesterol (CHOL) mixtures were shown to be very promising carriers for oligonucleotides (ON). However, it appeared necessary to clarify the structural consequence of the interactions of ON with the liposome, and especially on DOPE, the lipid responsible for the pH sensitivity. The present study was carried out by differential scanning calorimetry and X-ray diffraction, at low hydration. In such a case, DOPE generally adopt a hexagonal phase. It could be shown that ON increased DOPE transition temperature and increased v/al, as a result of electrostatic interactions between ON and DOPE headgroups. OA was found to have exactly opposite effects, its presence between DOPE molecules inhibiting the formation of hydrogen bonds. The presence of both ON and OA allowed the system to organize in a lamellar phase below the solid/liquid transition, whereas above this temperature ON preferably interacted with DOPE in a hexagonal phase and led OA to separate.

Pentaoxygenated xanthones and fatty acids from Bredemeyera brevifolia.

The polar portion of an ethanol extract of Bredemeyera brevifolia yielded two pentaoxygenated xanthones whose structures were determined as 1-methoxy-2,3,7,8-dimethylenedioxyxanthone, and 1,7,8-trimethoxy-2,3-methylene-dioxyxanthone. The apolar fraction contained various known fatty acids, structure determinations were accomplished by spectral analysis, mainly NMR, chemical derivatization and comparison to literature values.

Detection of circulating Leishmania chagasi DNA for the non-invasive diagnosis of human infection.

A polymerase chain reaction (PCR) assay for the detection of Leishmania spp. DNA in peripheral blood was optimized and evaluated for the diagnosis of human visceral leishmaniasis (VL) in Brazil during May 2001 to December 2002. Optimization of the technique resulted in a detection limit of 1.65 fg of purified L. (L.) chagasi DNA, equivalent to 1.65 x 10(-2) parasites. Leishmania DNA was detected in the blood of 48 of 53 patients with parasitologically-confirmed VL, which corresponds to a sensitivity of 91%. No DNA was detected in the peripheral blood of 15 healthy, non-exposed volunteers, giving a specificity of 100%. We conclude that detection of parasite DNA in peripheral blood offers a non-invasive, sensitive and rapid method for the detection of VL caused by L. (L.) chagasi.

pH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in excess water.

The cytoplasmic delivery of drugs encapsulated into pH-sensitive liposomes is under the control of a lamellar-to-hexagonal transition. In a previous study, under anhydrous conditions, oligonucleotides (ODN) encapsulated in pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine (DOPE)/oleic acid (OA)/cholesterol (CHOL) were shown to modify the phase behaviour of DOPE. In the present study, the lipid/ODN interactions were evaluated in fully hydrated samples by surface tension measurements, differential scanning calorimetry, X-ray diffraction and turbidimetry. Concerning the lipids, it was shown that OA provoked a disorganisation of DOPE lamellar phases and led to the complete disappearance of hexagonal transition along with heating. The addition of CHOL further decreased the lipid packing in the bilayers. Concerning ODN, these molecules provoked an increase in the surface pressure of a DOPE/OA/CHOL monolayer, indicating the existence of molecular interactions with the lipids. At a supramolecular level, ODN induced a more ordered organisation of DOPE molecules in the lamellar and hexagonal phases, and completely abolished the disorganisational effect of OA and CHOL.

Improvement of in vivo stability of phosphodiester oligonucleotide using anionic liposomes in mice.

Antisense phosphodiester oligonucleotides (ODN) are unstable in biological fluids due to nuclease-mediated degradation and therefore cannot be used in most antisense therapeutic applications. We describe here an in vitro and in vivo stabilization of a 15 mer phosphodiester sequence using anionic liposomes. Two formulations have been studied: DOPC/OA/CHOL and DOPE/OA/CHOL (pH-sensitive liposomes). Our in vitro findings reveal the same stabilization effect in mouse plasma for both anionic liposomes. In vivo investigation showed a great protective effect for both formulations after intravenous administration to mice. By contrast with in vitro results, a higher protection of ODN was observed with DOPC/OA/CHOL liposomes compared to the DOPE/OA/CHOL formulation. The latter was degraded in blood (75% of the injected dose at 5 min) probably due to interactions with blood components, and the remaining (25% at 5 min) was distributed mostly to the liver and spleen. DOPC liposomes were remarkably stable in blood and were distributed more slowly to all studied organs (liver, spleen, kidneys and lungs). Intact ODN was still observed in some organs (liver, spleen, lungs), but not in blood, 24 hours after DOPC liposome administration. These results suggest that this antisense strategy using carrier systems may be applicable to the treatment of diseases involving the reticuloendothelial system.

[Germination of soybeans BR-13 and Paraná: effects on mineral constituents]. / Germinação de soja BR-13 e paraná: efeito sobre os constituintes minerais.

The objective of this work was to evaluate the effect of germination on the minerals constituents (calcium, phosphorus, magnesium, potassium, copper, iron, manganese and zinc) of soybean. For the assays the soybean cultivars-Glycine max (L) Merril-BR-13 and Paraná were used. These were germinated in camera of germination at 25 degrees C and 100% of moisture, from 0 to 72 hours, collecting the samples in intervals of 6 hours. The analysis of variance and comparison among the means indicated that the minerals constituents remained constant up to 72 hours of germination.

Characterization of Injury Caused by Edessa meditabunda (F.), Chinavia impicticornis (Stål), and Piezodorus guildinii (West.) (Hemiptera: Pentatomidae) to Soybean.

Field cage trials were carried out in Ponta Grossa, Paraná, season 2010/2011, aiming to characterize and evaluate the injury caused by Edessa meditabunda (F.), Chinavia impicticornis (Stål), and Piezodorus guildinii (West.) (Hemiptera: Pentatomidae) to soybean. Non-infested plants were compared with infested plants with 2 and 3 adults/m (number of adult specimens per linear meter) at the R5.1-R9, R5.3-R9, R6-R9, and R7-R9 soybean growing stages. The following variables were analyzed: foliar retention, seed germination, and injury levels estimated by the tetrazolium test. Foliar retention index ranged from 2.3 to 4.1 in plants infested with P. guildinii and from 2.6 to 3.3 in plants infested with E. meditabunda and C. impicticornis, respectively. The lowest seed germination ranged from 66.5 to 76.5% and was observed in seeds from plants infested with 2 adults of C. impicticornis at R5.3-R9 and 3 adults of P. guildinii (R5.1-R9) and E. meditabunda (R7-R9). The highest average percentage of injury level for seeds occurred with seeds from plants infested at R5.1 with 2 or 3 adults/m of P. guildinii, ranging from 22.6 to 25.0% and from 63.6 to 68.0% in the first and second trials, respectively. Edessa meditabunda caused the highest damage to the seeds of plants infested at R7 (34.0 and 41.4%), and 2 adults/m of C. impicticornis caused the highest number of damaged seeds (54.0%) in plants infested at R6. Although both species were less harmful than P. guildinii, they could be a constraint factor for grain and seed commercialization.

Resistance of Sugarcane Cultivars to Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae).

The spittlebug Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae) is one of the most important pests of the sugarcane crop in Brazil. Despite of its importance, there is currently a lack of information regarding sugarcane cultivars' resistance to the spittlebug. Therefore, our objective was to evaluate the response of sugarcane genotypes to this species. Two experiments were carried out under laboratory conditions using a random block design with treatments in a factorial arrangement of 2 × 13 (experiment 1) and 2 × 12 (experiment 2), with six replicates. The first factor included two levels of infestation (infested and noninfested plants with spittlebugs), while the second consisted of the cultivars. Nymph survival varied from 47.9 to 84.5%, indicating that there are different levels of antibiosis to M. fimbriolata among the tested cultivars. The highest degree of antibiosis was found in cultivars IACSP96-7586 and IACSP96-2008, in which nymph survival was close to 48%. IACSP96-7586 also presented some degree of tolerance, but IACSP96-7569 and IACSP97-6682 stood out as the most tolerant cultivars to the pest, showing the lowest reduction in weight of aboveground biomass. On average, spittlebug infestations caused a significant reduction in relative leaf chlorophyll content and aboveground biomass weight.