Physiological and proteomic approaches to address the active role of ozone in kiwifruit post-harvest ripening

Post-harvest ozone application has recently been shown to inhibit the onset of senescence symptoms on fleshy fruit and vegetables; however, the exact mechanism of action is yet unknown. To characterize the impact of ozone on the post-harvest performance of kiwifruit (Actinidia deliciosa cv. ‘Hayward’), fruits were cold stored (0 °C, 95% relative humidity) in a commercial ethylene-free room for 1, 3, or 5 months in the absence (control) or presence of ozone (0.3 μl l−1) and subsequently were allowed to ripen at a higher temperature (20 °C), herein defined as the shelf-life period, for up to 12 days. Ozone blocked ethylene production, delayed ripening, and stimulated antioxidant and anti-radical activities of fruits. Proteomic analysis using 1D-SDS-PAGE and mass spectrometry identified 102 kiwifruit proteins during ripening, which are mainly involved in energy, protein metabolism, defence, and cell structure. Ripening induced protein carbonylation in kiwifruit but this effect was depressed by ozone. A set of candidate kiwifruit proteins that are sensitive to carbonylation was also discovered. Overall, the present data indicate that ozone improved kiwifruit post-harvest behaviour, thus providing a first step towards understanding the active role of this molecule in fruit ripening

The KMT2A recombinome of acute leukemias in 2023

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5’-KMT2A, two patients had a 5’-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.publishedVersionPeer reviewe

Are Small GTPases Signal Hubs in Sugar-Mediated Induction of Fructan Biosynthesis?

External sugar initiates biosynthesis of the reserve carbohydrate fructan, but the molecular processes mediating this response remain obscure. Previously it was shown that a phosphatase and a general kinase inhibitor hamper fructan accumulation. We use various phosphorylation inhibitors both in barley and in Arabidopsis and show that the expression of fructan biosynthetic genes is dependent on PP2A and different kinases such as Tyr-kinases and PI3-kinases. To further characterize the phosphorylation events involved, comprehensive analysis of kinase activities in the cell was performed using a PepChip, an array of >1000 kinase consensus substrate peptide substrates spotted on a chip. Comparison of kinase activities in sugar-stimulated and mock(sorbitol)-treated Arabidopsis demonstrates the altered phosphorylation of many consensus substrates and documents the differences in plant kinase activity upon sucrose feeding. The different phosphorylation profiles obtained are consistent with sugar-mediated alterations in Tyr phosphorylation, cell cycling, and phosphoinositide signaling, and indicate cytoskeletal rearrangements. The results lead us to infer a central role for small GTPases in sugar signaling

Functional Analysis of Arabidopsis Postprenylation CaaX Processing Enzymes and Their Function in Subcellular Protein Targeting1[C][W][OA]

Prenylation is a posttranslational protein modification essential for developmental processes and response to abscisic acid. Following prenylation, the three C-terminal residues are proteoliticaly removed and in turn the free carboxyl group of the isoprenyl cysteine is methylated. The proteolysis and methylation, collectively referred to as CaaX processing, are catalyzed by Ste24 endoprotease or Rce1 endoprotease and by an isoprenyl cysteine methyltransferase (ICMT). Arabidopsis (Arabidopsis thaliana) contains single STE24 and RCE1 and two ICMT homologs. Here we show that in yeast (Saccharomyces cerevisiae) AtRCE1 promoted a-mating factor secretion and membrane localization of a ROP GTPase. Furthermore, green fluorescent protein fusion proteins of AtSTE24, AtRCE1, AtICMTA, and AtICMTB are colocalized in the endoplasmic reticulum, indicating that prenylated proteins reach this compartment and that CaaX processing is likely required for subcellular targeting. AtICMTB can process yeast a-factor more efficiently than AtICMTA. Sequence and mutational analyses revealed that the higher activity AtICMTB is conferred by five residues, which are conserved between yeast Ste14p, human ICMT, and AtICMTB but not in AtICMTA. Quantitative real-time reverse transcription-polymerase chain reaction and microarray data show that AtICMTA expression is significantly lower compared to AtICMTB. AtICMTA null mutants have a wild-type phenotype, indicating that its function is redundant. However, AtICMT RNAi lines had fasciated inflorescence stems, altered phylotaxis, and developed multiple buds without stem elongation. The phenotype of the ICMT RNAi lines is similar to farnesyltransferase β-subunit mutant enhanced response to abscisic acid2 but is more subtle. Collectively, the data suggest that AtICMTB is likely the major ICMT and that methylation modulates activity of prenylated proteins

In Vivo Direct Molecular Imaging of Early Tumorigenesis and Malignant Progression Induced by Transgenic Expression of GFP-Met

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), play an important role in normal developmental processes, as well as in tumorigenicity and metastasis. We constructed a green fluorescent protein (GFP) Met chimeric molecule that functions similarly to the wild-type Met receptor and generated GFP-Met transgenic mice. These mice ubiquitously expressed GFP-Met in specific epithelial and endothelial cells and displayed enhanced GFP-Met fluorescence in sebaceous glands. Thirty-two percent of males spontaneously developed adenomas, adenocarcinomas, and angiosarcomas in their lower abdominal sebaceous glands. Approximately 70% of adenocarcinoma tumors metastasized to the kidneys, lungs, or liver. Quantitative subcellular-resolution intravital imaging revealed very high levels of GFP-Met in tumor lesions and in single isolated cells surrounding them, relative to normal sebaceous glands. These single cells preceded the formation of local and distal metastases. Higher GFP-Met levels correlated with earlier tumor onset and aggressiveness, further demonstrating the role of Met-HGF/SF signaling in cellular transformation and acquisition of invasive and metastatic phenotypes. Our novel mouse model and high-resolution intravital molecular imaging create a powerful tool that enables direct real-time molecular imaging of receptor expression and localization during primary events of tumorigenicity and metastasis at single-cell resolution

MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression