Salt Tolerant and Sensitive Rice Varieties Display Differential Methylome Flexibility under Salt Stress

DNA methylation has been referred as an important player in plant genomic responses to environmental stresses but correlations between the methylome plasticity and specific traits of interest are still far from being understood. In this study, we inspected global DNA methylation levels in salt tolerant and sensitive rice varieties upon salt stress imposition. Global DNA methylation was quantified using the 5-methylcytosine (5mC) antibody and an ELISA-based technique, which is an affordable and quite pioneer assay in plants, and in situ imaging of methylation sites in interphase nuclei of tissue sections. Variations of global DNA methylation levels in response to salt stress were tissue- and genotype-dependent. We show a connection between a higher ability of DNA methylation adjustment levels and salt stress tolerance. The salt-tolerant rice variety Pokkali was remarkable in its ability to quickly relax DNA methylation in response to salt stress. In spite of the same tendency for reduction of global methylation under salinity, in the salt-sensitive rice variety IR29 such reduction was not statistically supported. In ‘Pokkali’, the salt stress-induced demethylation may be linked to active demethylation due to increased expression of DNA demethylases under salt stress. In ‘IR29’, the induction of both DNA demethylases and methyltransferases may explain the lower plasticity of DNA methylation. We further show that mutations for epigenetic regulators affected specific phenotypic parameters related to salinity tolerance, such as the root length and biomass. This work emphasizes the role of differential methylome flexibility between salt tolerant and salt sensitive rice varieties as an important player in salt stress tolerance, reinforcing the need to better understand the connection between epigenetic networks and plant responses to environmental stresses.     

Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem

Prion-like proteins can undergo conformational rearrangements from an intrinsically disordered to a highly ordered amyloid state. This ability to change conformation is encoded in distinctive domains, termed prion domains (PrDs). Previous work suggests that PrDs change conformation to affect protein function and create phenotypic diversity. More recent work shows that PrDs can also undergo many weak interactions when disordered, allowing them to organize the intracellular space into dynamic compartments. However, mutations within PrDs and altered aggregation properties have also been linked to age-related diseases in humans. Thus, the physiological role of prion-like proteins, the mechanisms regulating their conformational promiscuity and the links to disease are still unclear. Here, we summarize recent work with prion-like proteins in Dictyostelium discoideum. This work was motivated by the finding that D. discoideum has the highest content of prion-like proteins of all organisms investigated to date. Surprisingly, we find that endogenous and exogenous prion-like proteins remain soluble in D. discoideum and do not misfold and aggregate. We provide evidence that this is due to specific adaptations in the protein quality control machinery, which may allow D. discoideum to tolerate its highly aggregation-prone proteome. We predict that D. discoideum will be an important model to study the function of prion-like proteins and their mechanistic links to disease.KEYWORDS: amyloid, Hsp104, prion, molecular chaperone, phase separation, protein aggregate, protein misfolding, ubiquitin/proteasome system     

A New Method to Quantify Ifosfamide Blood Levels Using Dried Blood Spots and UPLC-MS/MS in Paediatric Patients with Embryonic Solid Tumours

Ifosfamide blood concentrations are necessary to monitor its therapeutic response, avoiding any adverse effect. We developed and validated an analytical method by UPLC-MS/MS to quantify ifosfamide in dried blood spots (DBS). Blood samples were collected on Whatman 903® filter paper cards. Five 3 mm disks were punched out from each dried blood spot. Acetonitrile and ethyl acetate were used for drug extraction. Chromatographic separation was carried out in an Acquity UPLC equipment with a BEH-C18 column, 2.1 x 100 mm, 1.7 μm (Waters®). The mobile phase consisted in 5 mM ammonium formate and methanol:acetonitrile (40:48:12 v/v/v) at 0.2 mL/min. LC-MS/MS detection was done by ESI+ and multiple reaction mode monitoring, ionic transitions were m/z¹⁺ 260.99 > 91.63 for ifosfamide and 261.00 > 139.90 for cyclophosphamide (internal standard). This method was linear within a 100–10000 ng/mL range and it was accurate, precise and selective. Ifosfamide samples in DBS were stable for up to 52 days at -80°C. The procedure was tested in 14 patients, ages 1 month to 17 years (9 males and 5 females), with embryonic tumours treated with ifosfamide, alone or combined, at a public tertiary referral hospital. Ifosfamide blood levels ranged from 11.1 to 39.7 μmol/L at 12 hours after the last infusion, while 24-hour levels ranged from 0.7–19.7 μmol/L. The median at 12 hours was 19.5 μmol/L (Q₂₅ 14.4–Q₇₅ 29.0) and 3.8 μmol/L (Q₂₅ 1.5–Q₇₅ 9.9) at 24 hours, p<0.001. This method is feasible to determine ifosfamide plasma levels in paediatric patients.     

The agro-ecological suitability of Atriplex nummularia and A. halimus for biomass production in Argentine saline drylands

The choice of the best species to cultivate in semi-arid and arid climates is of fundamental importance, and is determined by many factors, including temperature and rainfall, soil type, water availability for irrigation and crop purposes. Soil or water salinity represents one of the major causes of crop stress. Species of the genus Atriplex are characterized by high biomass productivity, high tolerance to drought and salinity, and high efficiency in use of solar radiation and water. Based on a search of the international literature, the authors outline an agro-climatic zoning model to determine potential production areas in Argentina for Atriplex halimus and Atriplex numularia. Using the agroclimatic limits presented in this work, this model may be applied to any part of the world. When superimposed on the saline areas map, the agroclimatic map shows the suitability of agro-ecological zoning for both species for energy purposes on land unsuitable for food production. This innovative study was based on the implementation of a geographic information system that can be updated by further incorporation of complementary information, with consequent improvement of the original database.     

Phosphate-solubilizing peanut associated bacteria: screening for plant growth-promoting activities

Carbon and nitrogen are supplied by a variety of sources in the desert food web; both vascular and non-vascular plants and cyanobacteria supply carbon, and cyanobacteria and plant-associated rhizosphere bacteria are sources of biological nitrogen fixation. The objective of this study was to compare the relative influence of vascular plants and biological soil crusts on desert soil nematode and protozoan abundance and community composition. In the first experiment, biological soil crusts were removed by physical trampling. Treatments with crust removed had fewer nematodes and a greater relative ratio of bacterivores to microphytophages than treatments with intact crust. However, protozoa composition was similar with or without the presence of crusts. In a second experiment, nematode community composition was characterized along a spatial gradient away from stems of grasses or shrubs. Although nematodes generally occurred in increasing abundance nearer to plant stems, some genera (such as the enrichment-type Panagrolaimus) increased disproportionately more than others (such as the stress-tolerant Acromoldavicus). We propose that the impact of biological soil crusts and desert plants on soil microfauna, as reflected in the community composition of microbivorous nematodes, is a combination of carbon input, microclimate amelioration, and altered soil hydrology.     

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity

Background

Eosinophils are involved in various inflammatory processes including allergic inflammation during which angiogenesis has been documented. Angiogenesis is most likely connected to the hypoxia which characterizes inflamed tissues. Eosinophils produce VEGF and are pro-angiogenic. However, to the best of our knowledge no study has been performed to verify the existence of a direct link between eosinophils, hypoxia and angiogenesis in allergic inflammation.

Objective

To characterize eosinophil function and angiogenic potential under hypoxic conditions.

Methods

Human peripheral blood eosinophils were cultured in normoxic or hypoxic conditions with or without cytokines. Viability and apoptosis were assessed by Annexin V/PI staining. Anti- or pro-apoptotic protein levels, HIF-1α levels and MAPK phosphorylation were analyzed by immunoblot analysis. Angiogenic mediator release was evaluated by ELISA.

Results

Hypoxic eosinophils were more viable than normoxic ones after up to three days. In addition in hypoxia, anti-apoptotic Bcl-XL protein levels increased more than pro-apoptotic Bax levels. Hypoxia increased VEGF and IL-8 release. In hypoxic eosinophils high levels of HIF-1α were observed, particularly in the presence of GM-CSF. MAPK, particularly ERK1/2 inhibitors, decreased hypoxia-mediated VEGF release and HIF-1α expression.

Conclusion

Eosinophils respond to hypoxia by up-regulation of survival and of some of their pro-angiogenic functions indicating a correlation between eosinophilic inflammation and angiogenesis.     

Structural and functional studies of the HAMP domain of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli

The HAMP domain plays an essential role in signal transduction not only in histidine kinase but also in a number of other signal-transducing receptor proteins. Here we expressed the EnvZ HAMP domain (Arg(180)-Thr(235)) with the R218K mutation (termed L(RK)) or with L(RK) connected with domain A (Arg(180)-Arg(289)) (termed LA(RK)) of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli, by fusing it with protein S. The L(RK) and LA(RK) proteins were purified after removing protein S. The CD analysis of the isolated L protein revealed that it consists of a random structure or is unstructured. This suggests that the EnvZ HAMP domain by itself is unable to form a stable structure and that this structural fragility may be important for its role in signal transduction. Interestingly the substitution of Ala(193) in the EnvZ HAMP domain with valine or leucine in Tez1A1, a chimeric protein of Tar and EnvZ, caused a constitutive OmpC phenotype. The CD analysis of LA(RK)(A193L) revealed that this mutated HAMP domain possesses considerable secondary structures and that the thermostability of this entire LA(RK)(A193L) became substantially lower than that of LA(RK) or just domain A, indicating that the structure of the HAMP domain with the A193L mutation affects the stability of downstream domain A. This results in cooperative thermodenaturation of domain A with the mutated HAMP domain. These results are discussed in light of the recently solved NMR structure of the HAMP domain from a thermophilic bacterium (Hulko, M., Berndt, F., Gruber, M., Linder, J. U., Truffault, V., Schultz, A., Martin, J., Schultz, J. E., Lupas, A. N., and Coles, M. (2006) Cell 126, 929-940).     

Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease

The human LINE-1 endonuclease (L1-EN) is the targeting endonuclease encoded by the human LINE-1 (L1) retrotransposon. L1-EN guides the genomic integration of new L1 and Alu elements that presently account for ~28% of the human genome. L1-EN bears considerable technological interest, because its target selectivity may ultimately be engineered to allow the site-specific integration of DNA into defined genomic locations. Based on the crystal structure, we generated L1-EN mutants to analyze and manipulate DNA target site recognition. Crystal structures and their dynamic and functional analysis show entire loop grafts to be feasible, resulting in altered specificity, while individual point mutations do not change the nicking pattern of L1-EN. Structural parameters of the DNA target seem more important for recognition than the nucleotide sequence, and nicking profiles on DNA oligonucleotides in vitro are less well defined than the respective integration site consensus in vivo. This suggests that additional factors other than the DNA nicking specificity of L1-EN contribute to the targeted integration of non-LTR retrotransposons.