Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.     

Evidence of direct cell-cell fusion in Borrelia by cryogenic electron tomography

Some Borrelia species are the causative agents of tick-borne Lyme disease responsible for different disabilities depending on species and hosts. Borrelia are highly motile bacterial cells, and light microscopy shows that these spirochetes can associate with each other during movement. Using cryo-electron tomography, we observed closely associated Borrelia cells. Some of these showed a single outer membrane surrounding two longitudinally arranged cytoplasmic cylinders. We also observed fusion of two cytoplasmic cylinders and differences in the surface layer density of fused spirochetes. These processes could play a role in the interaction of Borrelia species with the host's immune system.     

Reversed phase liquid chromatography method with fluorescence detection of gemifloxacin in rat plasma and its application to the pharmacokinetic study

A simple, accurate and precise high-performance liquid chromatographic method with fluorescence detection was developed and validated for the determination of gemifloxacin (GEM) in rat plasma using furosemide as internal standard (I.S.). Plasma samples were pretreated by direct deproteinization and all samples and standard solutions were chromatographed at 45°C using triethylamine solution (0.5%, v/v, pH 3.0±0.1), methanol and acetonitrile (63:30:7, v/v/v) as the mobile phase. Chromatographic resolution was achieved using a RP-C(18) column (Atlantis, Waters, 150 mm × 4.6 mm, 5 μm) at a flow rate of 1.0 mL min(-1) and an injection volume of 30 μL. The analytes were measured by fluorescence detection with excitation and emission wavelengths of 344 nm and 399 nm, respectively. The retention times for GEM and I.S. were approximately 7.5 and 12.6 min, respectively. The lower limit of quantitation (LLOQ) was 20 ng mL(-1) and the calibration curves were linear over a concentration range of 20-5000 ng mL(-1). The intra- and inter-day precisions, expressed by relative standard deviation (R.S.D.) were lower than 6.24% and 4.49%, respectively. The accuracy ranged from 91.3% to 112% and from 98.8% to 106% for the lower and upper limit of quantitation of the calibration curve, respectively. Ratio of peak area of analyte to I.S. was used for quantification of plasma samples. No interferences from endogenous substances were found. The recovery of GEM and I.S. from plasma was greater than 90%. Drug stability in plasma was shown at room temperature for 4h, after three freeze-thaw cycles for 24h, in freezer at -80°C for 60 days, and in the autosampler after processing for 12h. The utility of the assay was confirmed by the successful analysis of plasma samples from GEM pharmacokinetics studies in the rats after intravenous administration.     

Structural Insights on Two Hypothetical Secretion Chaperones from <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv<Emphasis Type="Italic">. citri</Emphasis>

Several Gram-negative bacterial pathogens have developed type III secretion systems (T3SSs) to deliver virulence proteins directly into eukaryotic cells in a process essential for many diseases. The type III secretion processes require customized chaperones with high specificity for binding partners, thus providing the secretion to occur. Due to the very low sequence similarities among secretion chaperones, annotation and discrimination of a great majority of them is extremely difficult and a task with low scores even if genes are encountered that codify for small (<20 kDa) proteins with low pI and a tendency to dimerise. Concerning about this, herein, we present structural features on two hypothetical T3SSs chaperones belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest how low resolution models based on Small Angle X-ray Scattering patterns can provide new structural insights that could be very helpful in their analysis and posterior classification.     

Formicidal activity of indole derivatives on Atta opaciceps (Borgmeier): Lethal,behavioural and locomotive effects

The leaf‐cutting ants of the genus Atta are of extreme importance for agriculture and forestry. Few active products can be employed to control these pests and, therefore, the discovery of new insecticidal products represents a fundamental strategy for its management. In this study, we evaluated the mortality, behaviour and locomotion of workers of Atta opaciceps (Borgmeier) exposed to synthesized indole derivatives. The most active compound was 4d [1‐(1H‐indol‐3‐yl)pentan‐1‐one] (LD₅₀ = 0.018 μg/mg), while the 4e [1‐(1H‐indol‐3‐yl)hexan‐1‐one] (LD₅₀ = 3.82 μg/mg) was the least active compound. These two derivatives reduced the survival of A. opaciceps over time and altered the behaviour and locomotion of these ants. This study demonstrates the potential of indole derivatives to produce new formicidal products, since, in addition to being effective, it also affects the ant's behaviour and locomotion.     

Weak relationships between leaf phenology and isohydric and anisohydric behavior in lowland wet tropical forest trees

Wet tropical forest trees display a wide range of leaf phenology dynamics. However, the interrelation between deciduousness, water status, and leaf and stem characteristics have been poorly investigated compared with dry forests. We studied wet forest trees to answer the following questions: (1) do water regulation modes (iso/anisohydric behavior) of evergreen species differ from those found in deciduous species? (2) Does leaf water potential (Ψ_L) influences leaffall and emergence dynamics? (3) Are leaf and stem characteristics consistent across evergreen and deciduous trees? We evaluated vegetative phenology, Ψ_L, and leaf and stem characteristics of six evergreen and three deciduous species monthly for 2 yr. Species exhibited different leaffall and emergence dynamics, as well as different water regulation modes, independent of their deciduousness. Thus, the relationship between leaf phenology and water regulation behaviors appears to be a species‐specific property rather than a functional group attribute. Ψ_L had no direct influence on the dynamics of leaffall and/or emergence, indicating that this process is not modulated by water availability alone. Individual groups of evergreen and deciduous species could not be identified using principal component analysis (PCA), but a decoupling was observed in the leaf and stem economics spectra. The lack of an interrelation between deciduousness and iso/anisohydry, as well as the independence of leaf and stem trade‐offs, emphasizes that more systematic measurements of vegetative phenology and ecophysiological characteristics are necessary to advance our knowledge of leaf habit and water regulation behaviors based on the functional traits of wet forest plants.     

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.