Involvement of hnRNP A1 in the matrix metalloprotease-3-dependent regulation of Rac1 pre-mRNA splicing

Rac1b is an alternatively spliced isoform of the small GTPase Rac1 that includes the 57-nucleotide exon 3b. Rac1b was originally identified through its over-expression in breast and colorectal cancer cells, and has subsequently been implicated as a key player in a number of different oncogenic signaling pathways, including tumorigenic transformation of mammary epithelial cells exposed to matrix metalloproteinase-3 (MMP-3). Although many of the cellular consequences of Rac1b activity have been recently described, the molecular mechanism by which MMP-3 treatment leads to Rac1b induction has not been defined. Here we use proteomic methods to identify heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a factor involved in Rac1 splicing regulation. We find that hnRNP A1 binds to Rac1 exon 3b in mouse mammary epithelial cells, repressing its inclusion into mature mRNA. We also find that exposure of cells to MMP-3 leads to release of hnRNP A1 from exon 3b and the consequent generation of Rac1b. Finally, we analyze normal breast tissue and breast cancer biopsies, and identify an inverse correlation between expression of hnRNP A1 and Rac1b, suggesting the existence of this regulatory axis in vivo. These results provide new insights on how extracellular signals regulate alternative splicing, contributing to cellular transformation and development of breast cancer.     

Strategies to select yeast starters cultures for production of flavor compounds in cachaça fermentations

In this work, we have used classical genetics techniques to find improved starter strains to produce cachaça with superior sensorial quality. Our strategy included the selection of yeast strains resistant to 5,5′,5″-trifluor-d,l-leucine (TLF) and cerulenin, since these strains produce higher levels of higher alcohols and esters than parental strains. However, no clear relationship was observed when levels of flavoring compounds were compared with the levels expression of the genes (BAT1, BAT2, ATF2, EEB1 genes) involved with the biosynthesis of flavoring compounds. Furthermore, we determined the stability of phenotypes considered as the best indicators of the quality of the cachaça for a parental strain and its segregants. By applying the principal component analysis, a cluster of segregants, showing a high number of characteristics similar to the parental strain, was recognized. One segregant, that was resistant to TLF and cerulenin, also showed growth stability after six consecutive replications on plates containing high concentrations of sugar and ethanol. “Cachaça” produced at laboratory scale using a parental strain and this segregant showed a higher level of flavoring compounds. Both strains predominated in an open fermentative process through seven cycles, as was shown by mitochondrial restriction fragment length polymorphisms analysis. Based on the physical chemical composition of the obtained products, the results demonstrate the usefulness of the developed strategies for the selection of yeast strains to be used as starters in “cachaça” production.     

Crystalloid body, refractile body and virus-like particles in Apicomplexa: what is in there?

The phylum of Apicomplexa comprises parasitic protozoa that share distinctive features such as the apical complex, the apicoplast, specialized cytoskeletal components and secretory organelles. Other unique cytoplasmic inclusions sharing similar features have been described in some representatives of Apicomplexa, although under different denominations. These are the crystalloid body, present for example in Cryptosporidium, Plasmodium and Cystoisospora; the refractile body in Eimeria and Lankesterella; and virus-like particles, also present in Eimeria and Cryptosporidium. Yet, the specific role of these cytoplasmic inclusions in the cell cycle of these protozoa is still unknown. Here, we discuss their morphology, possible inter-relatedness and speculate upon their function to bring these organelles back to the attention of the scientific community and promote new interest towards original research on these elusive structures.     

Could shading reduce the negative impacts of drought on coffee? A morphophysiological analysis

Based on indirect evidence, it was previously suggested that shading could attenuate the negative impacts of drought on coffee (Coffea arabica), a tropical crop species native to shady environments. A variety (47) of morphological and physiological traits were examined in plants grown in 30-l pots in either full sunlight or 85% shade for 8 months, after which a 4-month water shortage was implemented. Overall, the traits showed weak or negligible responses to the light × water interaction, explaining less than 10% of the total data variation. Only slight variations in biomass allocation were observed in the combined shade and drought treatment. Differences in relative growth rates were mainly associated with physiological and not with morphological adjustments. In high light, drought constrained the photosynthetic rate through stomatal limitations with no sign of apparent photoinhibition; in low light, such constraints were apparently linked to biochemical factors. Sun-grown plants displayed osmotic adjustments, decreased tissue elasticities and improved long-term water use efficiencies, especially under drought. Regardless of the water availability, higher concentrations of lipids, total phenols, total soluble sugars and lignin were found in high light compared to shade conditions, in contrast to the effects on cellulose and hemicellulose concentrations. Proline concentrations increased in water-deprived plants, particularly those grown under full sun. Phenotypic plasticity was much higher in response to the light than to the water supply. Overall, shading did not alleviate the negative impacts of drought on the coffee tree.     

Phytochrome A increases tolerance to high evaporative demand

Stresses resulting from high transpiration demand induce adjustments in plants that lead to reductions of water loss. These adjustments, including changes in water absorption, transport and/or loss by transpiration, are crucial to normal plant development. Tomato wild type (WT) and phytochrome A (phyA)-mutant plants, fri1-1, were exposed to conditions of either low or high transpiration demand and several morphological and physiological changes were measured during stress conditions. Mutant plants rapidly wilted compared to WT plants after exposure to high evaporative demand. Root size and hydraulic conductivity did not show significant differences between genotypes, suggesting that water absorption and transport through this organ could not explain the observed phenotype. Moreover, stomatal density was similar between genotypes, whereas transpiration and stomatal conductance were both lower in mutant than in WT plants. This was accompanied by a lower stem-specific hydraulic conductivity in mutant plants, which was associated to lower xylem vessel number and transversal area in fri1-1 plants, producing a reduction in water supply to the leaves, which rapidly wilted under high evaporative demand. PhyA signaling might facilitate the adjustment to environments differing widely in water evaporative demand in part through the modulation of xylem dimensions.     

High salinity induces dehydrin accumulation in Chenopodium quinoa Willd. cv. Hualhuas embryos

Background and Aims

Chenopodium quinoa can grow at altitudes of 3,600–4,000 masl and is adapted to the highly arid conditions typical of the salty soils in the South American Altiplano, with less than 250?mm of annual rain and temperatures below 0°C. The aim of the study was to investigate the effect of salinity on the dehydrin content of mature embryos harvested from salt-stressed Chenopodium quinoa cv. Hualhuas plants grown at 100 to 500?mM NaCl. To date, no studies exist on the dehydrins of seeds from salt-stressed plants, although dehydrins in the root, stems and leaves have been reported as an adaptation to water deficit produced by salinity.

Methods

Dehydrin-like protein detection was carried out with an antiserum raised against a highly-conserved lysine-rich 15-amino acid sequence known as the K-segment, which is capable of recognizing proteins immunologically related to the dehydrin family.

Results

Dehydrins were analyzed in embryos by both western blot and in situ immunolocalization. Western blot analysis detected at least four dehydrins (55, 50, 34, and 30?kDa) in seeds harvested from quinoa salt-stressed plants treated under a wide range of salinities. The 30?kDa dehydrin increased its accumulation in both 300 and 500?mM NaCl growth conditions as revealed by densitometric analyses. Dehydrin subcellular localization was mostly nuclear at 500?mM of NaCl. A phosphatase treatment of protein extracts caused a mobility shift of the 34 and 30?kDa dehydrin bands suggesting a putative modulation mechanism based on protein phosphorylation.

Conclusions

We propose that these novel observations regarding dehydrin accumulation, subcellular localization and phosphorylation state are related to the high salt stress tolerant phenotype previously reported on this cultivar.     

Pupillometric analysis for assessment of gene therapy in Leber Congenital Amaurosis patients

ABSTRACT: BACKGROUND: Objective techniques to assess the amelioration of vision in patients with impaired visual function are needed to standardize efficacy assessment in gene therapy trials for ocular diseases. Pupillometry has been investigated in several diseases in order to provide objective information about the visual reflex pathway and has been adopted to quantify visual impairment in patients with Leber Congenital Amaurosis (LCA). In this paper, we describe detailed methods of pupillometric analysis and a case study on three Italian patients affected by Leber Congenital Amaurosis (LCA) involved in a gene therapy clinical trial at two follow-up time-points: 1 year and 3 years after therapy administration. METHODS: Pupillary light reflexes (PLR) were measured in patients who had received a unilateral subretinal injection in a clinical gene therapy trial. Pupil images were recorded simultaneously in both eyes with a commercial pupillometer and related software. A program was generated with MATLAB software in order to enable enhanced pupil detection with revision of the acquired images (correcting aberrations due to the inability of these severely visually impaired patients to fixate), and computation of the pupillometric parameters for each stimulus. Pupil detection was performed through Hough Transform and a non-parametric paired statistical test was adopted for comparison. RESULTS: The developed program provided correct pupil detection also for frames in which the pupil is not totally visible. Moreover, it provided an automatic computation of the pupillometric parameters for each stimulus and enabled semi-automatic revision of computerized detection, eliminating the need for the user to manually check frame by frame. With reference to the case study, the amplitude of pupillary constriction and the constriction velocity were increased in the right (treated eye) compared to the left (untreated) eye at both follow-up time-points, showing stability of the improved PLR in the treated eye. CONCLUSIONS: Our method streamlined the pupillometric analyses and allowed rapid statistical analysis of a range of parameters associated with PLR. The results confirm that pupillometry is a useful objective measure for the assessment of therapeutic effect of gene therapy in patients with LCA. Trial registration ClinicalTrials.gov NCT00516477.     

Life tables for the guava psyllid Triozoida limbata in southeastern Brazil

The aim of this study was to identify, quantify, and determine the importance of biological control agents of the psyllid Triozoida limbata (Enderlein) (Hemiptera: Sternorrhyncha: Triozidae). Ecological life tables were developed during four periods to determine the critical stage and key mortality factors of this pest. Predation and parasitism were found to be the two major factors driving the population dynamics of this pest. The major predators of eggs and nymphs of first and second instars were coccinellids, predatory thrips, lacewings, ants, and spiders, while the major predators of third, fourth, and fifth instars were several species of predatory wasps and syrphids. The endoparasitoid Psyllaephagus sp. (Hymenoptera: Encyrtidae) parasitized fifth instar psyllids. The fifth instar was found to be the critical mortality stage of T. limbata, and key mortality factors include the parasitoid Psyllaephagus sp. and predatory wasps.