Local Approach of the Charpy Test at Low Temperature

Charpy V-notch impact testing is widely used in the toughness assessment of large forged components, e.g. the pressure vessel for pressurised water reactors (PWR). At low temperature, A508 Cl.3 nuclear pressure vessel steel fails by cleavage fracture. The results reported here are part of both an experimental program and numerical investigations which aim at the establishment of a non-empirical relationship between the lower shelf Charpy V-notch energy, CVN, and the fracture toughness, KIc, of this material. Here, the applicability of the Beremin cleavage fracture model to the Charpy specimen is demonstrated

Identification of Zantedeschia aethiopica Cat1 and Cat2 catalase genes and their expression analysis during spathe senescence and regreening

Plants encode catalase (CAT; EC 1.11.1.6) as multigene families, which may reflect the multiple and diverse roles played by this enzyme. Catalases from higher plants can be subdivided into three distinct types, according to their phylogenetic relationship. However, there is not a specific correlation of phylogeny and function within these groups, as catalases from the same type can play different specific roles. We report on the characterization of two monocot catalase genes from Zantedeschia aethiopica (a C_3-type Araceae) and its expression analysis during spathe senescence and regreening. Z. aethiopica CAT2 appears to play an exclusive role in scavenging photorespiratory H_2O_2, but is more phylogenetically related to the unspecific Zea mays CAT3 and Oryza sativa CATA than to the photorespiratory-related Zea mays CAT2 and Oryza sativa CATC. Z. aethiopica CAT1 is more phylogenetically related to Zea mays CAT1 and Oryza sativa CATB and appears to have a dual role in scavenging glyoxysomal and peroxisomal H_2O_2.Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XXI/BD/16280/98 , SFRH/BD/12081/2003

Diurnal changes of polysome loading track sucrose content in the rosette of wildtype Arabidopsis and the starchless pgm mutant

Growth is driven by newly fixed carbon in the light, but depends at night on reserves, like starch, that are laid down in the light. Unless plants coordinate their growth with diurnal changes in the carbon supply, they will experience acute carbon starvation during the night. Protein synthesis represents a major component of cellular growth. Polysome loading was investigated during the diurnal cycle, an extended night and low CO2 in Arabidopsis Col-0 and in the starchless pgm mutant. In Col-0, polysome loading was 60-70% in the light, 40-45% for much of the night and <20% in an extended night, whilst in pgm it fell to <25% early in the night. Quantification of rRNA species using qRT-PCR revealed that polysome loading remained high for much of the night in the cytosol, was strongly light-dependent in the plastid, and was always high in mitochondria. The rosette sucrose content correlated with overall and with cytosolic polysome loading. Ribosome abundance did not show significant diurnal changes. However, compared to Col-0, pgm had decreased and increased abundance of plastidic and mitochondrial ribosomes, respectively. Incorporation of label from 13CO2 into protein confirmed that protein synthesis continues at a diminished rate in the dark. Modelling revealed that a decrease in polysome loading at night is required to balance protein synthesis with the availability of carbon from starch breakdown. Costs are also reduced by using amino acids that accumulated in previous light period. These results uncover a tight coordination of protein synthesis with the momentary supply of carbon

Metabolism and Growth in Arabidopsis Depend on the Daytime Temperature but Are Temperature-Compensated against Cool Nights

Diurnal cycles provide a tractable system to study the response of metabolism and growth to fluctuating temperatures. We reasoned that the response to daytime and night temperature may vary; while daytime temperature affects photosynthesis, night temperature affects use of carbon that was accumulated in the light. Three Arabidopsis thaliana accessions were grown in thermocycles under carbon-limiting conditions with different daytime or night temperatures (12 to 24 degrees C) and analyzed for biomass, photosynthesis, respiration, enzyme activities, protein levels, and metabolite levels. The data were used to model carbon allocation and growth rates in the light and dark. Low daytime temperature led to an inhibition of photosynthesis and an even larger inhibition of growth. The inhibition of photosynthesis was partly ameliorated by a general increase in protein content. Low night temperature had no effect on protein content, starch turnover, or growth. In a warm night, there is excess capacity for carbon use. We propose that use of this capacity is restricted by feedback inhibition, which is relaxed at lower night temperature, thus buffering growth against fluctuations in night temperature. As examples, the rate of starch degradation is completely temperature compensated against even sudden changes in temperature, and polysome loading increases when the night temperature is decreased

Inflammatory response in hematopoietic stem and progenitor cells triggered by activating SHP2 mutations evokes blood defects

Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with SHP2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2 D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies, and in vivo transplantability of HSPCs. Single-cell mRNA sequencing of HSPCs from Shp2 D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2 D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies

Differential Phosphorylation of Ribosomal Proteins in Arabidopsis thaliana Plants during Day and Night

Protein synthesis in plants is characterized by increase in the translation rates for numerous proteins and central metabolic enzymes during the day phase of the photoperiod. The detailed molecular mechanisms of this diurnal regulation are unknown, while eukaryotic protein translation is mainly controlled at the level of ribosomal initiation complexes, which also involves multiple events of protein phosphorylation. We characterized the extent of protein phosphorylation in cytosolic ribosomes isolated from leaves of the model plant Arabidopsis thaliana harvested during day or night. Proteomic analyses of preparations corresponding to both phases of the photoperiod detected phosphorylation at eight serine residues in the C-termini of six ribosomal proteins: S2-3, S6-1, S6-2, P0-2, P1 and L29-1. This included previously unknown phosphorylation of the 40S ribosomal protein S6 at Ser-231. Relative quantification of the phosphorylated peptides using stable isotope labeling and mass spectrometry revealed a 2.2 times increase in the day/night phosphorylation ratio at this site. Phosphorylation of the S6-1 and S6-2 variants of the same protein at Ser-240 increased by the factors of 4.2 and 1.8, respectively. The 1.6 increase in phosphorylation during the day was also found at Ser-58 of the 60S ribosomal protein L29-1. It is suggested that differential phosphorylation of the ribosomal proteins S6-1, S6-2 and L29-1 may contribute to modulation of the diurnal protein synthesis in plants

Translatome and metabolome effects triggered by gibberellins during rosette growth in Arabidopsis

Although gibberellins (GAs) are well known for their growth control function, little is known about their effects on primary metabolism. Here the modulation of gene expression and metabolic adjustment in response to changes in plant (Arabidopsis thaliana) growth imposed on varying the gibberellin regime were evaluated. Polysomal mRNA populations were profiled following treatment of plants with paclobutrazol (PAC), an inhibitor of GA biosynthesis, and gibberellic acid (GA3) to monitor translational regulation of mRNAs globally. Gibberellin levels did not affect levels of carbohydrates in plants treated with PAC and/or GA3. However, the tricarboxylic acid cycle intermediates malate and fumarate, two alternative carbon storage molecules, accumulated upon PAC treatment. Moreover, an increase in nitrate and in the levels of the amino acids was observed in plants grown under a low GA regime. Only minor changes in amino acid levels were detected in plants treated with GA3 alone, or PAC plus GA3. Comparison of the molecular changes at the transcript and metabolite levels demonstrated that a low GA level mainly affects growth by uncoupling growth from carbon availability. These observations, together with the translatome changes, reveal an interaction between energy metabolism and GA-mediated control of growth to coordinate cell wall extension, secondary metabolism, and lipid metabolism