Xylella fastidiosa subsp. pauca and fastidiosa Colonize Arabidopsis Systemically and Induce Anthocyanin Accumulation in Infected Leaves.

The bacterium Xylella fastidiosa is a multihost pathogen that affects perennial crops such as grapevine, sweet orange, and olive tree worldwide. It is inherently difficult to study these pathosystems owing to the long-term growth habit of the host plant. Thus, the availability of model plants becomes essential to accelerate discoveries with economic impact. In this study, we uncovered evidence that the model plant Arabidopsis thaliana can be colonized by two different X. fastidiosa subspecies, pauca and fastidiosa. We observed that these bacteria are able to move away from the inoculation point as high bacterial populations were found in distant tissues. In addition, confocal laser scanning microscopy analysis of bacterial movement inside the petiole revealed the ability of the bacterium to move against the net xylem flow during the time course of colonization forming biofilm. These findings provide evidence for the capacity of X. fastidiosa to colonize Arabidopsis. Furthermore, leaves inoculated with X. fastidiosa showed a significant accumulation of anthocyanin. We propose that the X. fastidiosa subsp. pauca or fastidiosa colonization pattern and anthocyanin accumulation in the Arabidopsis ecotype Col-0 can be used as marker phenotypes to facilitate further studies aimed at improving genetic components involved in X. fastidiosa-host interaction.

Analysis of Defense-Related Gene Expression in Citrus Hybrids Infected by Xylella fastidiosa.

Resistance to Xylella fastidiosa was evaluated in 264 hybrids of crosses between Murcott tangor (Citrus reticulata × Citrus sinensis) and Pera sweet orange (C. sinensis) under field conditions. Uninfected hybrids were grafted with buds collected from Pera sweet orange plants infected with X. fastidiosa, forming a plant with two scions (i.e., hybrid branches and Pera sweet orange branches). From these plants, we chose 10 genotypes with three biological replicates. We evaluated gene expression, bacterial multiplication, and citrus variegated chlorosis (CVC) symptom development in both scions. X. fastidiosa was not detected in most hybrid scions and none showed disease symptoms. In contrast, all Pera sweet orange scions were infected with X. fastidiosa and expressed symptoms of CVC. We quantified the expression of 12 defense-related genes by qPCR comparing resistant to susceptible scions. We suggest that some of these genes are involved in resistance of the hybrids to X. fastidiosa, since their expression was significantly higher in the resistant hybrid scions than in tolerant hybrids and scions originated from CVC symptomatic Pera sweet orange buds. However, we note that these data should be interpreted carefully, as the plant genotypes tested are related but necessarily distinct (hybrids of C. reticulata and C. sinensis, in relation to a C. sinensis control). A principal component analysis revealed a relationship between the expression of these genes and hybrid scions, and between scions that originated from infected buds and the presence of the bacteria and plant symptoms. Multiyear field trials are necessary to develop plant resistance to X. fastidiosa. While the experimental design used here had limitations, it allowed us to identify a set of genes potentially involved in Citrus sp. resistance to this pathogen. Future work on the role of these genes in plant defenses to X. fastidiosa infection is necessary to confirm their importance in the displayed resistance phenotype.

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Kinetic study of biodegradation of BTX compounds in mono- and multicomponent systems in reactor with immobilized biomass.

In this study, kinetic parameters were determined for the biodegradation of BTX compounds in a fixed-bed reactor with immobilized biomass, fed with mono- and multicomponent systems. The parameter estimation was achieved through an algorithm using the finite volume method. Different kinetic models were evaluated. The Monod model proved to be suitable to predict the experimental data for the biodegradation individual BTX compound. In multicomponent systems, it was found that the presence of more than one compound tends to cause competitive inhibition. To identify the models that best fit the experimental data, a statistical analysis using the F test was applied. For the two- and three-component systems the presence of more than one compound tends to cause competitive inhibition. In this study, it was possible to predict kinetic parameters in mono- and multicomponent systems as well as different operation conditions for a fixed-bed reactor with immobilized biomass.

Desulfurization and denitrogenation of heavy gas oil by Rhodococcus erythropolis ATCC 4277.

Some of the noxious atmospheric pollutants such as nitrogen and sulfur dioxides come from the fossil fuel combustion. Biodesulfurization and biodenitrogenation are processes which remove those pollutants through the action of microorganisms. The ability of sulfur and nitrogen removal by the strain Rhodococcus erythropolis ATCC 4277 was tested in a biphasic system containing different heavy gas oil concentrations in a batch reactor. Heavy gas oil is an important fraction of petroleum, because after passing through, the vacuum distillation is incorporated into diesel oil. This strain was able to remove about 40% of the nitrogen and sulfur present in the gas heavy oil. Additionally, no growth inhibition occurred even when in the presence of pure heavy gas oil. Results present in this work are considered relevant for the development of biocatalytic processes for nitrogen and sulfur removal toward building feasible industrial applications.

Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.

Expression of Xylella fastidiosa RpfF in citrus disrupts signaling in Xanthomonas citri subsp. citri and thereby its virulence.

Xylella fastidiosa and Xanthomonas citri subsp. citri, that cause citrus variegated chlorosis (CVC) and citrus canker diseases, respectively, utilize diffusible signal factor (DSF) for quorum sensing. DSF, produced by RpfF, are similar fatty acids in both organisms, although a different set of genes is regulated by DSF in each species. Because of this similarity, Xylella fastidiosa DSF might be recognized and affect the biology of Xanthomonas citri. Therefore, transgenic Citrus sinensis and Carrizo citrange plants overexpressing the Xylella fastidiosa rpfF were inoculated with Xanthomonas citri and changes in symptoms of citrus canker were observed. X. citri biofilms formed only at wound sites on transgenic leaves and were thicker; however, bacteria were unable to break through the tissue and form pustules elsewhere. Although abundant growth of X. citri occurred at wound sites on inoculated transgenic leaves, little growth was observed on unwounded tissue. Genes in the DFS-responsive core in X. citri were downregulated in bacteria isolated from transgenic leaves. DSF-dependent expression of engA was suppressed in cells exposed to xylem sap from transgenic plants. Thus, altered symptom development appears to be due to reduced expression of virulence genes because of the presence of antagonists of DSF signaling in X. citri in rpfF-expressing plants.

Dynamic functional impression technique for severely resorbed alveolar ridges.

The process of bone resorption can reduce the volume of the alveolar crest, which makes may make difficult impression taking of the alveolar tissue and the subsequent fit of a new denture. This clinical report describes a fast and simple technique for impressions of edentulous ridges to replace complete dentures, using a temporary tissue conditioner material on the denture base. The existing denture must cover the whole supporting area and should be in harmony with the adjacent oral structures. This technique reduces the number of steps involved and minimizes treatment time and expenses.

Expression of Xylella fastidiosa fimbrial and afimbrial proteins during biofilm formation.

Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in X. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants.

In vivo comparative study of hydroxyapatite labeled with different radioisotopes: evaluation of the scintigraphic images.

Radyosinovectomy (RSV) is a radiotherapeutic modality where a beta-emitting radionuclide is administered locally by intra-articular injection on the form of a colloid or radiolabeled particulate. RSV is a well-accepted therapeutic procedure in inflammatory joint diseases and has been successfully employed for more than 50 years as a viable alternative to surgical and chemical synovectomy. The aim of this work is to compare the in vivo stability of hydroxyapatite labelled with (177)Lu, (90)Y and (153)Sm. All radionuclides were labelled with high yield and were retained in the joint for 7 days, showing stability and usefulness as tools in the RSV treatment. A similar retention of the products in the muscle was observed when the particles were administrated in the muscle. However, the pure form of the radionuclides were rapidly cleared from the blood and accumulated in the liver when injected i.v.. Although (153)Sm-HA is already available for nuclear medicine procedures and clinical studies with (90)Y-HA have been developed, (177)Lu-labeled RSV agents will be economically more viable and has not been studied yet. Its favorable characteristics contribute to follow, to predict and asses the success of RSV by bone scintigraphy studies.

A multitechnique study of structure and dynamics of polyfluorene cast films and the influence on their photoluminescence.

This article describes the microstructure and dynamics in the solid state of polyfluorene-based polymers, poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO), a semicrystalline polymer, and poly[(9,9-dioctyl-2,7-divinylene-fluorenylene)-alt-co-{2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene vinylene}, a copolymer with mesomorphic phase properties. These structures were determined by wide-angle X-ray scattering (WAXS) measurements. Assuming a packing model for the copolymer structure, where the planes of the phenyl rings are stacked and separated by an average distance of approximately 4.5 A and laterally spaced by about approximately 16 A, we followed the evolution of these distances as a function of temperature using WAXS and associated the changes observed to the polymer relaxation processes identified by dynamical mechanical thermal analysis. Specific molecular motions were studied by solid-state nuclear magnetic resonance. The onset of the side-chain motion at about 213 K (beta-relaxation) produced a small increase in the lateral spacing and in the stacking distance of the phenyl rings in the aggregated structures. Besides, at about 383 K (alpha-relaxation) there occurs a significant increase in the amplitude of the torsion motion in the backbone, producing a greater increase in the stacking distance of the phenyl rings. Similar results were observed in the semicrystalline phase of PFO, but in this case the presence of the crystalline structure affects considerably the overall dynamics, which tends to be more hindered. Put together, our data explain many features of the temperature dependence of the photoluminescence of these two polymers.

Perfect 1JCH-resolved HSQC: Efficient measurement of one-bond proton-carbon coupling constants along the indirect dimension.

A versatile 1JCH-resolved HSQC pulse scheme for the speedy, accurate and automated determination of one-bond proton-carbon coupling constants is reported. The implementation of a perfectBIRD element allows a straightforward measurement from the clean doublets obtained along the highly resolved F1 dimension, even for each individual 1JCHa and 1JCHb in diastereotopic HaCHb methylene groups. Real-time homodecoupling during acquisition and other alternatives to minimize accidental signal overlapping in overcrowded spectra are also discussed.

A Bowman-Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition.

Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman-Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment.

Traceability from governmental producers of radiopharmaceuticals in measuring (18)F in Brazil.

Since the inception of its proficiency test program to evaluate radionuclide measurement in hospitals and clinics, the National Metrology Laboratory of Ionizing Radiation-LNMRI, that represents Brazilian National Metrology Institute (NMI) for ionizing radiation has expanded its measurement and calibration capability. Requirements from the National Health Surveillance Agency from Ministry of Health (ANVISA), to producers of radiopharmaceuticals provided an opportunity to improve the full traceability chain to the highest level. Fluorodeoxyglucose (FDG-(18)F) is the only radiopharmaceutical simultaneously produced by all Brazilian radiopharmaceutical production centers (RPCs). By running this proficiency test, LNMRI began to provide them with the required traceability. For evaluation, the ratio of RPC to reference value results and ISO/IEC17043:2010 criteria were used. The reference value established as calibration factor on the secondary standard ionization chamber was obtained from three absolute measurements systems, and routinely confirmed in each round of proficiency test by CIEMAT/NIST liquid scintillation counting. The γ-emitting impurities were checked using a High-Purity Germanium (HPGe) detector. The results show that Brazilian RPCs are in accordance with (accuracy within ±10%) the Brazilian standard for evaluation of measurements with radionuclide calibrators (CNEN NN 3.05., 2013). Nevertheless, the RPCs should improve the methodology of uncertainty estimates, essential when using the statistical criteria of ISO/IEC 17043 standard, in addition to improving accuracy to levels consistent with their position in the national traceability chain.

Amygdalar neuronal activity mediates the cardiovascular responses evoked from the dorsolateral periaqueductal gray in conscious rats.

There is ample evidence that both lateral/dorsolateral periaqueductal gray (l/dlPAG) and basolateral amygdala (BLA) are essential for the regulation of the autonomic responses evoked during innate reactions to threatening stimuli. However, it is not well established to what extent the BLA regulates the upstream functional connection from the l/dlPAG. Here we evaluated the role of the BLA and its glutamatergic receptors in the cardiovascular responses induced by l/dlPAG stimulation in rats. We examined the influence of acute inhibition of the BLA, unilaterally, by injecting muscimol on the cardiovascular responses evoked by the injection of N-methyl D-aspartate (NMDA) into the l/dlPAG. We also evaluated the role of BLA ionotropic glutamate receptors in these responses by injecting antagonists of NMDA and AMPA/kainate receptor subtypes into the BLA. Our results show that the microinjection of NMDA in the BLA increased the mean arterial pressure (MAP) and heart rate (HR). Injection of NMDA into the l/dlPAG caused similar increases in these variables, which was prevented by the prior injection of muscimol, a GABAA agonist, into the BLA. Moreover, injection of glutamatergic antagonists (2-amino-5-phosphonopentanoate (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)) into the BLA reduced the increase in MAP and HR induced by l/dlPAG activation. Finally, the inhibition of the central amygdala neurons failed to reduce the cardiovascular changes induced by l/dlPAG activation. These results indicate that physiological responses elicited by l/dlPAG activation require the neuronal activity in the BLA. This ascending excitatory pathway from the l/dlPAG to the BLA might ensure the expression of the autonomic component of the defense reaction.

An Evaluation of Kinetic Models in the Biodesulfurization of Synthetic Oil by Rhodococcus erythropolis ATCC 4277.

Biodesulfurization is an eco-friendly technology applied in the removal of sulfur from fossil fuels. This technology is based on the use of microorganisms as biocatalysts to convert the recalcitrant sulfur compounds into others easily treatable, as sulfides. Despite it has been studied during the last decades, there are some unsolved questions, as per example the kinetic model which appropriately describes the biodesulfurization globally. In this work, different kinetic models were tested to a batch desulfurization process using dibenzothiophene (DBT) as a model compound, n-dodecane as organic solvent, and Rhodococcus erythropolis ATCC 4277 as biocatalyst. The models were solved by ODE45 function in the MATLAB. Monod model was capable to describe the biodesulfurization process predicting all experimental data with a very good fitting. The coefficients of determination achieved to organic phase concentrations of 20, 80, and 100 % (v/v) were 0.988, 0.995, and 0.990, respectively. R. erythropolis ATCC 4277 presented a good affinity with the substrate (DBT) since the coefficients of saturation obtained to reaction medium containing 20, 80, and 100 % (v/v) were 0.034, 0.07, and 0.116, respectively. This kinetic evaluation provides an improvement in the development of biodesulfurization technology because it showed that a simple model is capable to describe the throughout process.

Vertebrate hosts and vectors of Trypanosoma rangeli in the Amazon Basin of Brazil.

A total of 46 Trypanosoma rangeli stocks were isolated from naturally infected mammals and triatomine vectors. Twenty-two stocks were from the common opossum (Didelphis marsupialis), one from the brown "4-eyed" opossum (Metachirus nudicaudatus), one from the anteater (Tamandua tetradactyla), one from the coati (Nasua nasua), seven from Rhodnius pictipes and 14 from Rhodnius robustus. Two stocks were also isolated from recently fed sandflies (Lutzomyia sp., Shannoni group). The stocks were identified as T. rangeli on the basis of natural or experimental salivary gland infections in Rhodnius, inoculative (anterior station) transmission to mice, morphological parameters in parasitemic mice and comparisons of isozyme profiles with a known stock of T. rangeli isolated from man. Three other trypanosome stocks from D. marsupialis, T. tetradactyla and the three-toed sloth (Bradypus tridactylus) were morphologically similar to T. rangeli in culture but had quite different isozyme profiles and were not identified. It is concluded that T. rangeli is widely distributed in Amazonas, Pará and Rondonia States of Brazil, and probably extends into other regions where R. pictipes and R. robustus are known to occur. R. pictipes is light-attracted into houses and occasionally transmits Chagas' disease to man. It is likely that T. rangeli is also occasionally transmitted to man in the Amazon basin.

Breeding structure of the sand fly Lutzomyia longipalpis (Lutz & Neiva) in Brazil.

Eleven populations of Lutzomyia longipalpis (Lutz & Neiva), the sand fly vector of Leishmania chagasi, from different areas of Brazil were analyzed for genetic variation at 16 enzyme loci. In this region, the prevalence of visceral leishmaniasis (VL) caused by L. chagasi is spotty and reproductive isolation among populations of Lu. longipalpis has been reported. It is thought that morphologically similar cryptic species with varying vectorial capacity may be responsible for the discontinuous distribution of VL. The aim was to study the genetic structure of populations within this region and to identify demes that may represent sibling species. Genotypic frequencies within populations were in close compliance to Hardy-Weinberg expectations, suggesting there are no sympatric species among these 11 populations. Levels of genetic distance between pairs of populations were very low (< 0.03), consistent with local populations within a single sand fly species. When genotypic frequency data for all populations were pooled, 9 of the 13 polymorphic loci deviated from Hardy-Weinberg expectations, indicating some degree of genetic substructuring. Estimates of effective migration rates (N(e)m) among all populations were low, 2.73, suggesting that gene flow is restricted among populations, which is probably the reason for the observed genetic substructuring.

Some methods for the enzymic characterization of Latin-American Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Leishmania hertigi.

30 Brazilian stocks of Leishmania mexicana amazonensis and 13 stocks of subspecies of Leishmania hertigi were characterized by starch-gel electrophoresis, using 18 enzymes selected from a total of 36 investigated. L. m. amazonensis was separable from subspecies of L. hertigi by enzymic profiles of 11 enzymes. The L. m. amazonensis stocks, which were from a wide range of hosts in a large geographical area, were enzymically extremely homogeneous, and could only be subdivided on two enzymes; sub-groups did not relate to each other or to any differences in epidemiological characters, including the clinical form of the human disease. 12 stocks regarded as L. hertigi deanei, that were isolated from Coendou prehensilis prehensilis and Coendou sp. in Pará State, Brazil, were separable into two sub-groups by three enzymes. A single stock of L. hertigi hertigi from Panama was separable from both enzymic sub-groups of L. h. deanei, in each case by three enzymes. The significance of these and other characters of diversity is discussed, together with the use of enzymes for the identification of the leishmaniae.

Leishmaniasis in Brazil: XV. Biochemical distinction of Leishmania mexicana amazonensis, L. braziliensis braziliensis and L. braziliensis guyanensis--aetiological agents of cutaneous leishmaniasis in the Amazon Basin of Brazil.

Enzymic profiles of the three known agents of human cutaneous leishmaniasis in the lower Amazon region are compared. Of 14 enzymes, 10 (ASAT, ALAT, GPI, G5PD, MDH, ACON, PEP, HK, MPI and ACP) differentiate Leishmania mexicana amazonensis from L. braziliensis braziliensis or L. braziliensis guyanensis: this supports their taxonomic status as distinct species. In contrast, only slight mobility differences of four enzymes (ASAT, ALAT, PGM, MPI) separate L. b. braziliensis and L. b. guyanensis, which are distinguished biochemically for the first time: this indicates that they are closely related. Four stocks of L. b. panamensis correspond with L. b. guyanensis on mobilities of 10 enzymes (ASAT, ALAT, PGM, GPI, G6PD, MDH, PK, HK, MPI, ACP), although these two subspecies are known to be separable by kinetoplast DNA buoyancies and the enzyme 6PGDH. The generation of practical, regional biochemical keys to the medically important leishmanias is discussed.