Functional Improvement of Human Cardiotrophin 1 Produced in Tobacco Chloroplasts by Co-Expression with Plastid Thioredoxin m

Human cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant protein’s overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts

TP53 abnormalities are underlying the poor outcome associated with chromothripsis in chronic lymphocytic leukemia patients with complex karyotype

Simple Summary Chromothripsis, a genomic event that generates massive chromosomal rearrangements, has been described in 1-3% of CLL patients and is associated with poor prognostic factors (e.g., TP53 abnormalities and genomic complexity). However, previous studies have not assessed its role in CLL patients with complex karyotypes. Herein, we aimed to describe the genetic characteristics of 33 CLL patients with high genomic complexity and chromothripsis. Moreover, we analyzed the clinical impact of chromothripsis, comparing these patients against a cohort of 129 patients with complex karyotypes not presenting this catastrophic event. Nine cases were also assessed via the novel cytogenomic methodology known as optical genome mapping. We confirmed that this phenomenon is heterogeneous and associated with a shorter time to first treatment. Nonetheless, our findings suggested that TP53 abnormalities, rather than chromothripsis itself, underlie the dismal outcome. Chromothripsis (cth) has been associated with a dismal outcome and poor prognosis factors in patients with chronic lymphocytic leukemia (CLL). Despite being correlated with high genome instability, previous studies have not assessed the role of cth in the context of genomic complexity. Herein, we analyzed a cohort of 33 CLL patients with cth and compared them against a cohort of 129 non-cth cases with complex karyotypes. Nine cth cases were analyzed using optical genome mapping (OGM). Patterns detected by genomic ..

Functional Improvement of Human Cardiotrophin 1 Produced in Tobacco Chloroplasts by Co-Expression with Plastid Thioredoxin m

Human cardiotrophin 1 (CT1), a cytokine with excellent therapeutic potential, was previously expressed in tobacco chloroplasts. However, the growth conditions required to reach the highest expression levels resulted in an impairment of its bioactivity. In the present study, we have examined new strategies to modulate the expression of this recombinant protein in chloroplasts so as to enhance its production and bioactivity. In particular, we assessed the effect of both the fusion and co-expression of Trx m with CT1 on the production of a functional CT1 by using plastid transformation. Our data revealed that the Trx m fusion strategy was useful to increase the expression levels of CT1 inside the chloroplasts, although CT1 bioactivity was significantly impaired, and this was likely due to steric hindrance between both proteins. By contrast, the expression of functional CT1 was increased when co-expressed with Trx m, because we demonstrated that recombinant CT1 was functionally active during an in vitro signaling assay. While Trx m/CT1 co-expression did not increase the amount of CT1 in young leaves, our results revealed an increase in CT1 protein stability as the leaves aged in this genotype, which also improved the recombinant protein’s overall production. This strategy might be useful to produce other functional biopharmaceuticals in chloroplasts

Effects of water-deficit irrigation on hormonal content and nitrogen compounds in developing berries of Vitis vinifera L. cv. Tempranillo

13 Págs., 3 Figs., 4 Tabls.Water-deficit irrigation to grapevines reduces plant growth, yield, and berry growth, altering the ripening process, all of which may influence fruit composition and wine quality. Therefore, the goals of this study were (1) to investigate the influence of the main endogenous berry hormones, abscisic acid (ABA), indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acid (JA), on berry growth and ripening under water-deficit conditions and (2) to analyze changes in fruit composition, specifically N compounds, under water deprivation. The study was carried out using container-grown Tempranillo grapevines grown under controlled conditions in a greenhouse. Two irrigation treatments were imposed: control (well-watered) and sustained deficit irrigation (SDI). Water deficit decreased leaf area and the source-to-sink ratio, reduced yield and berry size, and decreased concentrations of the main phenolic compounds. SDI also modified berry hormonal status. At the pea-size stage, SDI berries had lower IAA and higher JA and SA than nonstressed berries. At veraison (onset of ripening), accumulation of ABA was less accentuated in SDI than in control berries. At harvest, the content of amino acids and free ammonium was low in both treatments but SDI-treated berries showed a significant accumulation of amines. Results suggest that water restrictions to grapevines might be playing a physiological role in reducing berry growth through affecting hormone dynamics, phenolic synthesis, and the berry amino acid content and composition, which could compromise fruit quality. Possible roles of endogenous IAA controlling berry size and endogenous ABA and SA controlling levels of anthocyanins and flavonols at harvest are discussed. © 2013 Springer Science+Business Media New York.This work was supported by Fundación Universitaria de Navarra (2011) and Ministerio de Ciencia e Innovación (MCINN BFU2011-26989), Spain. F. Morales thanks Gobierno de Aragón (A03 research group) for financial support. M. Niculcea was the recipient of a grant from Asociación de Amigos de la Universidad de Navarra.Peer reviewe