Palmiro Poltronieri

Jasmonate signaling in plant development and defense response to multiple (a)biotic stresses

Plants frequently live in environments characterized by the presence of simultaneous and different stresses. The intricate and finely tuned molecular mechanisms activated by plants in response to abiotic and biotic environmental factors are not well understood, and less is known about the integrative signals and convergence points activated by plants in response to multiple (a)biotic stresses. Phytohormones play a key role in plant development and response to (a)biotic stresses. Among these, one of the most important signaling molecules is an oxylipin, the plant hormone jasmonic acid. Oxylipins are derived from oxygenation of polyunsaturated fatty acids. Jasmonic acid and its volatile derivative methyl jasmonate have been considered for a long time to be the bioactive forms due to their physiological effects and abundance in the plant. However, more recent studies showed unambiguously that they are only precursors of the active forms represented by some amino acid conjugates. Upon developmental or environmental stimuli, jasmonates are synthesized and accumulate transiently. Upon perception, jasmonate signal transduction process is finely tuned by a complex mechanism comprising specific repressor proteins which in turn control a number of transcription factors regulating the expression of jasmonate responsive genes. We discuss the latest discoveries about the role of jasmonates in plants resistance mechanism against biotic and abiotic stresses. Finally, the deep interplay of different phytohormones in stresses signaling will be also discussed.

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples.

We developed a novel filtration-based method that can eliminate dead or severely damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p<0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R(2)>0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.

Non-protein coding RNA biomarkers and differential expression in cancers: a review.

BackgroundIn these years a huge number of human transcripts has been found that do not code for proteins, named non-protein coding RNAs. In most cases, small (miRNAs, snoRNAs) and long RNAs (antisense RNA, dsRNA, and long RNA species) have many roles, functioning as regulators of other mRNAs, at transcriptional and post-transcriptional level, and controlling protein ubiquitination and degradation. Various species of npcRNAs have been found differentially expressed in different types of cancer. This review discusses the published data and new results on the expression of a subset of npcRNAs.ConclusionThese results underscore the complexity of the RNA world and provide further evidence on the involvement of functional RNAs in cancer cell growth control.

Differential expression of poly(ADP-ribose) polymerase and DNA polymerase β in rat tissues

The activities of two DNA repair-related enzymes, poly(ADP-ribose) polymerase and DNA polymerase beta, and their mRNA levels were measured in 17 tissues of Wistar rats. A large variety in enzyme activity values could be detected in the tissues examined; the highest levels of activity for both enzymes were found in the testis. A good correlation between poly(ADP-ribose) polymerase activity and the level of the transcript of the gene coding for the enzyme was observed in many tissues. A less satisfactory correlation could be evidenced for DNA polymerase beta. The almost parallel amounts of the mRNAs for poly(ADP-ribose) polymerase and DNA polymerase beta in the tissues examined suggest a possible coexpression of the genes coding for these enzymes. Additional studies have been carried out in testis and liver by immunohistochemical techniques and by in situ hybridization analyses. While in the testis the spermatocytes were shown to contain both enzymes and their transcripts, in other types of cells this could not be observed. In the liver mRNAs and enzymes were only found in 20% of the hepatocytes. This may in part explain both the low levels of the mRNAs and the modest activities of the two enzymes in that tissue.

Biosensors for the Detection of Food Pathogens

Food pathogens frequently cause foodborne diseases. There is a need to rapidly identify the source of the bacteria in order to contain their spread and epidemics. A pre-enrichment culture or a direct culture on agar plate are standard microbiological methods. In this review, we present an update on alternative molecular methods to nucleic acid-based detection for species identification. Biosensor-based methods rely on the recognition of antigen targets or receptors by antibodies, aptamers or high-affinity ligands. The captured antigens may be then directly or indirectly detected through an antibody or high-affinity and high-specificity recognition molecule. Various different detection methods are discussed, from label-free sensors and immunosensors to fluorescence-based ones. Each method shows advantages and disadvantages in terms of equipment, sensitivity, simplicity and cost-effectiveness. Finally, lab-on-a-chip (LOC) devices are introduced briefly, with the potential to be fast, sensitive and useful for on-site bacteria detection in food processing laboratories to check potential contamination by sample monitoring combined with a rapid pre-enrichment step.

Transcriptomic analysis of oxylipin biosynthesis genes and chemical profiling reveal an early induction of jasmonates in chickpea roots under drought stress

Drought is one of the major constraints in subtropical agriculture. Therefore improving water stress tolerance is of great importance to breed for drought tolerance in future. The first plant organ sensing dehydration is the root. Aim of the present work was to clarify the potential impact of the phyto-oxylipins pathway on drought tolerance of chickpea (Cicer arietinum), the third important legume crop worldwide. Therefore, we measured the expression of key genes involved in oxylipins metabolism by qPCR on samples from stressed and non-stressed roots of a drought-tolerant and a drought-sensitive chickpea variety using commercially available TaqMan assays. We demonstrate that the drought tolerant variety reacts to drought with sustained and earlier activation of a specific lipoxygenase (Mt-LOX 1) gene, two hydroperoxide lyases (Mt-HPL 1 and Mt-HPL 2), an allene oxide synthase (Mt-AOS), and an oxo-phytodienoate reductase (Mt-OPR). We further show that gene over-expression positively correlates with the levels of major oxylipin metabolites from the AOS branch of the pathway, which finally leads to the synthesis of jasmonates. Higher levels of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA) and the active form JA-isoleucine (JA-Ile) were especially detected in the root tissues of the tolerant variety, prompting us to assume a role of jasmonates in the early signalling of drought stress in chickpea and its involvement in the tolerance mechanism of the drought-tolerant variety.

Potential of Anti-Cancer Therapy Based on Anti-miR-155 Oligonucleotides in Glioma and Brain Tumours

MicroRNAs are aberrantly expressed in many cancers and can exert tumour‐suppressive or oncogenic functions. As oncomirs promote growth of cancer cells and support survival during chemotherapy, thus microRNA‐silencing therapies could be a valuable approach to be associated with anticancer drugs and chemotherapy treatments. miR‐155 microRNA was found overexpressed in different types of cancer, such as leukaemias (PML, B‐cell lymphomas), lung cancer and glioblastoma. GABA‐A receptor downregulation was found correlated with glioma grading, with decreasing levels associated with higher grade of malignancies. A relationship between knock‐down of miR‐155 and re‐expression of GABRA 1 protein in vivo was recently individuated. This finding has implication on the effectiveness of RNA‐silencing approaches against miR‐155 with the scope to control proliferation and signalling pathways regulated by GABA‐A receptor. Applying microRNAs for treatment of brain tumours poses several problems, and fields to be solved are mainly the passage of the brain–blood barrier and the targeted delivery to specific cell types. Glioblastoma multiforme cells bud off microvesicles that deliver cytoplasmic contents to nearby cells. Thus, the exploitation of these mechanisms to deliver antagomir therapeutics targeting microvescicles in the brain could take the lead in the near future in the treatment for brain cancers in substitution of invasive surgical intervention.

Non-coding RNAs change their expression profile after Retinoid induced differentiation of the promyelocytic cell line NB4

BackgroundThe importance of non-coding RNAs (ncRNAs) as fine regulators of eukaryotic gene expression has emerged by several studies focusing on microRNAs (miRNAs). miRNAs represent a newly discovered family of non coding-RNAs. They are thought to be crucial players of human hematopoiesis and related tumorigenesis and to represent a potential tool to detect the early stages of cancer. More recently, the expression regulation of numerous long ncRNAs has been linked to cell growth, differentiation and cancer although the molecular mechanism of their function is still unknown.NB4 cells are promyelocytic cells that can be induced to differentiation upon retinoic acid (ATRA) treatment and represent a feasible model to study changes of non coding RNAs expression between cancer cells and their terminally differentiated counterpart.Findingswe screened, by microarray analysis, the expression of 243 miRNAs and 492 human genes transcribing for putative long ncRNAs different from miRNAs in NB4 cells before and after ATRA induced differentiation. Our data show that 8 miRNAs, and 58 long ncRNAs were deregulated by ATRA induced NB4 differentiation.Conclusionour data suggest that ATRA-induced differentiation lead to deregulation of a large number of the ncRNAs that can play regulatory roles in both tumorigenesis and differentiation.

Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets.

Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets (LD), whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum (ER). Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin-green fluorescent protein (GFP) peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein (BiP), were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.

In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed.