Bacterial structure and characterization of plant growth promoting and oil degrading bacteria from the rhizospheres of mangrove plants

Most oil from oceanic spills converges on coastal ecosystems, such as mangrove forests, which are threatened with worldwide disappearance. Particular bacteria that inhabit the rhizosphere of local plant species can stimulate plant development through various mechanisms; it would be advantageous if these would also be capable of degrading oil. Such bacteria may be important in the preservation or recuperation of mangrove forests impacted by oil spills. This study aimed to compare the bacterial structure, isolate and evaluate bacteria able to degrade oil and stimulate plant growth, from the rhizospheres of three mangrove plant species. These features are particularly important taking into account recent policies for mangrove bioreme-diation, implying that oil degradation as well as plant maintenance and health are key targets. Fifty-seven morphotypes were isolated from the mangrove rhizospheres on Bushneil-Haas (BH) medium supplemented with oil as the sole carbon source and tested for plant growth promotion. Of this strains, 60% potentially fixed nitrogen, 16% showed antimicrobial activity, 84% produced siderophores, 51% had the capacity to solubilize phosphate, and 33% produced the indole acetic acid hormone. Using gas chromatography, we evaluated the oil-degrading potential of ten selected strains that had different morphologies and showed Plant Growth Promoting Rhizobacteria (PGPR) features. The ten tested strains showed a promising degradation profile for at least one compound present in the oil. Among degrader strains, 46% had promising PGPR potential, having at least three of the above capacities. These strains might be used as a consortium, allowing the concomitant degradation of oil and stimulation of mangrove plant survival and maintenance.     

Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches

AIMS: Section Flavi is one of the most significant sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF ICMS) and to evaluate the discriminatory power of the various approaches in species identification. METHODS AND RESULTS: Aspergillus section Flavi isolates obtained from Portuguese almonds were characterized in terms of macro- and micromorphology, mycotoxin pattern, calmodulin gene sequence and MALDI-TOF protein fingerprint spectra. For each approach, dendrograms were created and results were compared. All data sets divided the isolates into three groups, corresponding to taxa closely related to Aspergillus flavus, Aspergillus parasiticus and Aspergillus tamarii. In the A. flavus clade, molecular and spectral analyses were not able to resolve between aflatoxigenic and nonaflatoxigenic isolates. In the A. parasiticus cluster, two well-resolved clades corresponded to unidentified taxa, corresponding to those isolates with mycotoxin profile different from that expected for A. parasiticus.     

Succination of proteins in diabetes

Cysteine is arguably the most reactive amino acid in protein. A wide range of cysteine derivatives is formed in vivo, resulting from oxidation, nitrosation, alkylation and acylation reactions. This review describes succination of proteins, an irreversible chemical modification of cysteine by the Krebs cycle intermediate, fumarate, yielding S-(2-succinyl)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane and develop in concert with mitochondrial and oxidative stress in diabetes. Increased succination of glyceraldehyde-3-phosphate dehydrogenase explains the loss in specific activity of this enzyme in muscle of streptozotocin-diabetic rats and increased succination of adiponectin may explain the decreased secretion of adiponectin from adipose tissue in type 2 diabetes. In addition to GAPDH and adiponectin, other succinated proteins identified in adipocytes include cytoskeletal proteins (tubulin, actin) and chaperone proteins in the endoplasmic reticulum. Succination of adipocyte protein in vitro is inhibited by uncouplers of oxidative phosphorylation and by inhibitors of ER stress. 2SC serves as a biomarker of mitochondrial stress and recent studies suggest that succination is the mechanistic link between mitochondrial and ER stress in diabetes.     

The binding of synthetic triiodo l-thyronine analogs to human transthyretin: molecular basis of cooperative and non-cooperative ligand recognition

Transthyretin (TTR) is a tetrameric β-sheet-rich transporter protein directly involved in human amyloid diseases. Several classes of small molecules can bind to TTR delaying its amyloid fibril formation, thus being promising drug candidates to treat TTR amyloidoses. In the present study, we characterized the interactions of the synthetic triiodo L-thyronine analogs and thyroid hormone nuclear receptor TRβ-selective agonists GC-1 and GC-24 with the wild type and V30M variant of human transthyretin (TTR). To achieve this aim, we conducted in vitro TTR acid-mediated aggregation and isothermal titration calorimetry experiments and determined the TTR:GC-1 and TTR:GC-24 crystal structures. Our data indicate that both GC-1 and GC-24 bind to TTR in a non-cooperative manner and are good inhibitors of TTR aggregation, with dissociation constants for both hormone binding sites (HBS) in the low micromolar range. Analysis of the crystal structures of TTRwt:GC-1(24) complexes and their comparison with the TTRwt X-ray structure bound to its natural ligand thyroxine (T4) suggests, at the molecular level, the basis for the cooperative process displayed by T4 and the non-cooperative process provoked by both GC-1 and GC-24 during binding to TTR.     

Mucopolysaccharidoses in northern Brazil: Targeted mutation screening and urinary glycosaminoglycan excretion in patients undergoing enzyme replacement therapy

Mucopolysaccharidoses (MPS) are rare lysosomal disorders caused by the deficiency of specific lysosomal enzymes responsible for glycosaminoglycan (GAG) degradation. Enzyme Replacement Therapy (ERT) has been shown to reduce accumulation and urinary excretion of GAG, and to improve some of the patients' clinical signs. We studied biochemical and molecular characteristics of nine MPS patients (two MPS I, four MPS II and three MPS VI) undergoing ERT in northern Brazil. The responsiveness of ERT was evaluated through urinary GAG excretion measurements. Patients were screened for eight common MPS mutations, using PCR, restriction enzyme tests and direct sequencing. Two MPS I patients had the previously reported mutation p.P533R. In the MPS II patients, mutation analysis identified the mutation p.R468W, and in the MPS VI patients, polymorphisms p.V358M and p.V376M were also found. After 48 weeks of ERT, biochemical analysis showed a significantly decreased total urinary GAG excretion in patients with MPS I (p < 0.01) and MPS VI (p < 0.01). Our findings demonstrate the effect of ERT on urinary GAG excretion and suggest the adoption of a screening strategy for genotyping MPS patients living far from the main reference centers.     

A herbivore that manipulates plant defence

Phytopathogens and herbivores induce plant defences. Whereas there is evidence that some pathogens suppress these defences by interfering with signalling pathways involved in the defence, such evidence is scarce for herbivores. We found that the invasive spider mite Tetranychus evansi suppresses the induction of the salicylic acid and jasmonic acid signalling routes involved in induced plant defences in tomato. This was reflected in the levels of inducible defence compounds, such as proteinase inhibitors, which in mite-infested plants were reduced to even lower levels than the constitutive levels in herbivore-free plants. Additionally, the spider mite suppressed the release of inducible volatiles, which are implicated in plant defence. Consequently, the mites performed much better on previously attacked plants than on non-attacked plants. These findings provide a new perspective on plant-herbivore interactions, plant protection and plant resistance to invasive species.     

A morphometric and molecular study of Anastrepha pickeli Lima (Diptera: Tephritidae)

This study investigated the level of morphometric and genetic variability among populations of Anastrepha pickeli Lima from several localities in Brazil, one locality in Bolivia and one in Paraguay. Traditional and geometric morphometric analyses were used, as well as sequencing of a fragment of the cytochrome oxidase gene (COI). Six variables were measured from the aculeus for traditional morphometric analysis and 14 landmarks from the right wing were used for geometric analysis, using 10 specimes/population. The aculeus tip length, aculeus width at the end of the cloaca opening, and the serrate part length contributed with 62.7% for grouping. According to the results from traditional morphometry, there was no significant difference, but the multivariate tests showed that the canonical variables were statistically significant, indicating a difference in the wing conformation among populations. Molecular phylogenetic analysis indicated that the populations clustered into three clades and revealed a high level of genetic variation within A. pickeli populations from various geographic regions. Anastrepha pickeli populations differed among them according to the methods used in this study, showing incongruence among the methods used.     

Enhanced arsenic tolerance of transgenic eastern cottonwood plants expressing gamma-glutamylcysteine synthetase

Arsenic is a metalloid that occurs naturally at parts per million (ppm) levels in the earth's crust. Natural and human activities have contributed to arsenic mobilization and increased concentration in the environment, such that World Health Organization guidelines for arsenic levels in drinking water are exceeded at many locations, worldwide. This translates into an increased risk of arsenic-related illnesses for millions of people. Recent studies demonstrate that increasing thiol-sinks in transgenic plants by overexpressing the bacterial gamma-glutamylcysteine synthetase (ECS) gene results in a higher tolerance and accumulation of metals and metalloids such as cadmium, mercury, and arsenic. We used Agrobacterium-mediated transformation to genetically engineer eastern cottonwood with a bacterial ECS gene. Eastern cottonwood plants expressing ECS had elevated thiol group levels, consistent with increased ECS activity. In addition, these ECS-expressing plants had enhanced growth on levels of arsenate toxic to control plants in vitro. Furthermore, roots of ECS-expressing plants accumulated significantly more arsenic than control roots (approximately twice as much), while shoots accumulated significantly less arsenic than control shoots (approximately two-thirds as much). We discuss potential mechanisms for shifting the balance of plant arsenic distribution from root accumulation to shoot accumulation, as it pertains to arsenic phytoremediation.     

Influence of drying methods over in vitro antitumoral effects of exopolysaccharides produced by Agaricus blazei LPB 03 on submerged fermentation

Agaricus blazei is a mushroom that belongs to the Brazilian biodiversity and is considered as an important producer of bioactive compounds beneficial to human health. Studies have demonstrated that these compounds present immuno-modulatory, antioxidant and antitumor properties. In order to compare the most used method for fungal polysaccharide drying, lyophilization with other industrial-scale methods, the aim of this work was to submit A. blazei LPB 03 polysaccharide extracts to vaucum, spray and freeze drying, and evaluate the maintenance of its antitumoral effects in vitro. Exopolysaccharides produced by A. blazei LPB 03 on submerged fermentation were extracted with ethanol and submitted to drying processes. The efficiency represents the water content that was removed during the drying process. The resultant dried products showed water content around 3% and water activity less than 0.380, preventing therefore the growth of microorganisms and reactions of chemical degradation. Exopolysaccharide extracts dried by vacuum and spray dryer did not showed any significant cytotoxic effect on cell viability of Wistar mice macrophages. Content of total sugars and protein decrease after drying, nevertheless, 20 mg/ml of exopolysaccharides dried by spray dryer reached 33% of inhibition rate over Ehrlich tumor cells in vitro.     

Nuclear insertions of mitochondrial origin: Database updating and usefulness in cancer studies

Nuclear insertions of mitochondrial origin (NUMTs) can be useful tools in evolution and population studies. However, due to their similarity to mitochondrial DNA (mtDNA), NUMTs may also be a source of contamination in mtDNA studies. The main goal of this work is to present a database of NUMTs, based on the latest version of the human genome—GRCh37 draft. A total of 755 insertions were identified. There are 33 paralogous sequences with over 80% sequence similarity and of a greater length than 500 bp. The non-identical positions between paralogous sequences are listed for the first time. As an application example, the described database is used to evaluate the impact of NUMT contamination in cancer studies. The evaluation reveals that 220 positions from 256 with zero hits in the current mtDNA phylogeny could in fact be traced to one or more nuclear insertions of mtDNA. This is due to they are located in non-identical positions between mtDNA and nuclear DNA (nDNA). After in silico primer validation of each revised cancer study, risk of co-amplification between mtDNA and nDNA was detected in some cases, whereas in others no risk of amplification was identified. This approach to cancer studies clearly proves the potential of our NUMT database as a valuable new tool to validate mtDNA mutations described in different contexts. Moreover, due to the amount of information provided for each nuclear insertion, this database should play an important role in designing evolutionary, phylogenetic and epidemiological studies.