Effects of enod40 overexpression in non legume plants

Il gene ENOD40 \ue8 una nodulina precoce ed \ue8 indotto durante i primi stadi di formazione del nodulo radicale in risposta alle interazioni tra le leguminose ed i batteri simbionti del genere Rhizobia. Omologhi del gene ENOD40 sono stati identificati in diverse specie e la sua espressione, non unicamente correlata alla formazione del nodulo, \ue8 stata osservata in tessuti giovani e meristematici. Una caratteristica che accomuna i geni ENOD40 \ue8 l\u2019assenza di un lunga open reading frame (ORF); al contrario, molte piccole ORF sono generalmente presenti nei trascritti. Il gene contiene due regioni altamente conservate chiamate box1 e box2. Tra le diverse specie \ue8 conservata l\u2019ORF (ORF1) del box1, che sembra codificare per un putativo peptide di 10-13 amminoacidi. Inoltre, il gene contiene regioni corrispondenti a strutture conservate a livello del trascritto. Sei domini sono stati individuati nel mRNA del gene e due di questi domini sono fortemente conservati tra le leguminose e le non leguminose. Nonostante decenni di ricerche, il ruolo del gene ENOD40 non \ue8 stato finora completamente chiarito. La natura biologica del gene \ue8 tuttora in discussione, infatti se l\u2019attivit\ue0 biologica del gene dipenda dall\u2019 RNA o da entrambi \ue8 ancora da chiarire. I due principali obiettivi del mio progetto di ricerca sono: da una parte, indagare la possibile presenza del putativo peptide codificato dal box1 utilizzando cellule BY-2 che overesprimono il gene e dall\u2019altra, studiare il ruolo del gene ENOD40 in piante non leguminose, utilizzando Arabidopsis thaliana. Nella prima parte del lavoro \ue8 stata messa appunto una procedura di purificazione per cercare il putativo peptide in cellule BY-2 che overesprimevano il gene ENOD40 di tabacco. Fin ad ora il putativo peptide non \ue8 mai stato trovato in vivo; \ue8 stato per\uf2 suggerito da diverse osservazioni che il gene potrebbe, almeno in parte, agire attraverso il peptide codificato dall\u2019ORF1. La procedura messa appunto consiste in un cut-off iniziale, seguita da cromatografia a scambio ionico, estrazione di cambio solido, HPLC-DAD e spettrometria di massa (LC-ESI-MS e MALDI-TOF). Purtroppo, nonostante i diversi tentativi per mettere appunto la procedura di purificazione e le diverse tecniche utilizzate per l'analisi delle frazioni putativamente peptide-arricchite, solo l\u2019analisi MALDI-TOF PSD ha dato un primo indizio sulla possibile presenza del peptide in cellule BY-2 che overesprimevano il gene ENOD40. Nella seconda parte del lavoro, il possibile ruolo del gene \ue8 stato indagato mediante l\u2019analisi metabolomica e trascrizionale in piante di Arabidopsis che overesprimevano il gene ENOD40 di soia. I profili metabolici e trascrizionali di tre linee di Arabidopsis trasformate con il gene ENOD40 sono stati acquisiti e confrontati con quelli ottenuti da piante wild type. In seguito, l'analisi dei biomarcatori dei dati ottenuti dalle analisi di metabolomica e trascrittomica \ue8 stata utilizzata per identificare i metaboliti e i trascritti che hanno mostrato un maggiore correlazione con l'overespressione del gene. Dai profili metabolici \ue8 emerso che le tre linee trasformate sono caratterizzate dalla presenza di glucosinolati, mentre i flavonoidi caratterizzano principalmente le piante wild type. Per quanto riguarda i profili trascrizionali, la maggior parte dei geni indotti nelle tre linee trasformate (12 su 23), sono correlati con processi che avvengono nella parete cellulare. Dato che, la parete cellulare determina la forma delle cellule, il gene ENOD40 potrebbe essere coinvolto in un processo che controlla la composizione e le dinamiche della parete. Precedenti studi morfologici condotti sulle stesse linee trasformate di Arabidopsis hanno dimostrato che queste piante presentano organi con dimensioni normali ma formati da celle pi\uf9 piccole; inoltre protoplasti di Arabidopsis trasfettati con il gene ENOD40 sono caratterizzati da una ridotta espansione. Questi dati hanno suggerito che il gene potrebbe avere un ruolo nel mantenere le cellule in uno stadio giovane e poco differenziato. L'osservazione che le linee trasformate di Arabidopsis accumulino glucosinolati, metabolici tipici di tessuti giovani, suggerisce che, anche dal punto di vista metabolico, le cellule trasformate hanno caratteristiche tipiche di cellule pi\uf9 giovani, mentre le cellule wild type accumulano maggiormente i flavonoidi, metaboliti secondari tipici dello stato differenziato. Per quanto riguarda l'analisi trascrizionale, dal momento che le piante trasformate sono morfologicamente caratterizzate da cellule con dimensioni ridotte, i geni indotti in queste linee, potrebbero essere coinvolti nella prevenzione dell\u2019espansione cellulare. Questo ruolo del gene, atto a mantenere le cellule in uno stadio giovanile, \ue8 supportato anche dai profili di espressione del gene riportati in letteratura.ENOD40 is an Early Nodulin gene that it is know to play a key role in nodule formation in response to interaction of legume plants with symbiotic Rhizobium bacteria. Homologues of ENOD40 genes have been identified in several plant species and its expression is observed during the initiation and development of new organs, such as nodules, lateral roots, young leaves and stipule primordia. ENOD40 gene has an unusual structure: it lacks a long open reading frame, but several short ORFs are present. Moreover, at nucleotide level, two regions, named box1 and box2, are highly conserved among all ENOD40 genes. In box 1 region, a highly conserved ORF (ORF 1) is present and it seems to encode a putative peptide of 10-13 amino acids. Furthermore, the gene contains regions corresponding to conserved secondary structures of the transcript. Six domains were identified in ENOD40 mRNA and two of these domains are strongly conserved among legume and non legume species. Despite several researches, the roles of the ENOD40 gene has not been so far completely elucidated. Moreover, whether the biological activity should be ascribed to RNA or peptide, or both, is still unclear. For this reason, the two main goals of the research are: to investigate the possible presence of the putative peptide encoded by box1 of the ENOD40 gene in BY-2 cells and to investigate the role of ENOD40 gene in non legume plants, using Arabidopsis thaliana. That ENOD40 could act, at least in part, through the peptide encode by box1 is suggested by several observations, but no one have revealed biochemically the putative peptide. In the first part of the work a purification procedure consisting of membrane cut-off, ion exchange chromatography, solid exchange extraction, HPLC-DAD and mass spectrometry (LC-ESI-MS and MALDI-TOF) was set up to search for the putative peptide in BY-2 cells overexpressing NtENOD40 gene. Unfortunately, despite several attempts to set up the purification procedure and the different and sensitive techniques used for the analysis of the putatively peptide-enriched fractions, only MALDI-TOF PSD analysis gave an initial clue of the possible presence of the peptide in ENOD40 overexpressing BY2 cells. In the second part of the work, the possible role of the gene has been investigated through the metabolomics and transcriptomics characterization of ENOD40 overexpressing Arabidopsis plants. Metabolite and transcriptional profiles of the three Arabidopsis lines overexpressing soybean ENOD40 gene were acquired and compared to those obtained from wild type plants. Afterward, biomarker analysis of metabolomic and transcriptomic dataset was used in order to identify the metabolites and transcripts that showed the higher correlation with the overexpression of ENOD40 gene. In the metabolite profiles, glucosinolate metabolites characterized all the three transformed lines compared with the wild type, while flavonoids mainly characterized wild type plants. With regard to transcriptional profiling, most of the genes upregulated in the three transformed lines (twelve out of twenty-three), were correlated with processes occurring in the cell wall. Thus, the cell wall is the mechanical determinant of cell shape and size ENOD40 gene could be involved in a process that controls the composition and the dynamics of the cell wall. In conclusion, previous morphological studies on the same Arabidopsis thaliana ENOD40 transformed lines used in this work have been showed that these plants are characterised by normal organs containing smaller cells, and on ENOD40 transfected Arabidopsis protoplasts are characterized by reduced expansion, suggested that the gene could have some role in keeping the cells in a \u201cyoung\u201d state . The observation that ENOD40 transformed Arabidopsis lines accumulate high levels of glucosinolates, that are typical of the young tissues, suggests that, also from the metabolic point of view, the transformed cells have features typical of younger cells, whereas wild type cells use their metabolic resources to accumulate flavonoids, another class of secondary metabolites more typical of differentiated state. With regard to transcriptomic analysis, since transformed plants are morphologically characterized by small cell size, the genes upregulated in the transformed lines, involved in cell wall dynamics and composition, could be involved in the prevention of cell expansion. The role of ENOD40 in maintenance of cells in a \u201cyoung state\u201d is also supported by the expression patterns of ENOD40 genes reported in literature

A multi-model study of extracellular vesicles in bowel cancer invasion and metastasis

Colorectal cancer (CRC) has one of the highest rates of cancer mortality worldwide. Despite progress in improving screening rates, approximately 35% of CRC patients are diagnosed with stage IV (metastatic) disease which has poor prognosis. In vitro 3D models have been developed which provide useful platforms to explore biomolecular processes in pre-clinical studies of cancer and metastasis, enabling the characterisation of mechanisms which can then be applied to the improvement of diagnosis, prognosis and disease treatment. Extracellular vesicles (EVs) are now known to play a critical role in cancer progression. Not only do they support cancer progression towards metastasis, but as carriers of bioactive cargo, they also show promise as potential cancer biomarkers. In this study, organotypic 3D models mimicking cancer invasion and pre-metastatic niche (PMN) in lung were developed to explore the role of EVs in advanced CRC stages and to evaluate their potential as prognostic markers. After validating a transwell system as an effective 3D model, this work establishes that CRC EVs induce the invasion of cancer cells in a stage-dependent manner, with metastatic SW620 EVs driving a more aggressive phenotype than SW480 EVs. Proteomic profiling of the PMN showed differences in protein content upon treatment and a list of potential m/z signals able to distinguish between SW480 and SW620 EV treatment was obtained by multivariate analysis. CRC EVs also induced α-smooth muscle actin (α-SMA) expression in fibroblasts, demonstrating activation of fibroblasts in the 3D model. Expression and activity of TG2, a multifunctional enzyme involved in cancer progression, was increased after CRC EVs treatment in the 3D model of invasion. This led to the exploration of the cell-specific expression of TG2 in a cancer cells/fibroblasts co-culture, which showed an EV-mediated increase of TG2 on the surface of cancer cells and a non-EV mediated decrease of TG2 expression in the fibroblast surface. TG2 activity was further explored in hepatic stellate cells, crucial cellular components of liver metastasis. Finally, a pilot biomarker study on a small cohort of patients was performed, using multivariate analysis of cell responses to plasma-derived EVs and was able to separate patients according to disease stage. The 3D models established and validated provided biomolecular information about the complex milieu of factors involved in CRC progression, including EVs, thus confirming the importance of 3D culture modelling in pre-clinical research. Moreover, this study was able to demonstrate key roles of cancer EVs on cellular activities which could then be translated for the development of alternative screening and prognostic approaches which may have application in improving the early diagnosis of patients with CRC

Survival and prognostic factors of early ovarian cancer.

Survival and prognostic factors were analysed in 150 patients with histologically confirmed epithelial ovarian cancer stage IA-IIA. The relapse-free and overall survival rates were, respectively, 81% and 88% after 3 and 74% and 84% after 5 years. The analysis of various prognostic factors indicates as the main factor the grade differentiation of the tumour

Bone Marrow Endosteal Mesenchymal Progenitors Depend on HIF Factors for Maintenance and Regulation of Hematopoiesis

Summary Maintenance and differentiation of hematopoietic stem cells (HSCs) is regulated through cell-autonomous and non-cell-autonomous mechanisms within specialized bone marrow microenvironments. Recent evidence demonstrates that signaling by HIF-1α contributes to cell-autonomous regulation of HSC maintenance. By investigating the role of HIF factors in bone marrow mesenchymal progenitors, we found that murine endosteal mesenchymal progenitors express high levels of HIF-1α and HIF-2α and proliferate preferentially in hypoxic conditions ex vivo. Inactivation of either HIF-1α or HIF-2α dramatically affects their phenotype, propagation, and differentiation. Also, downregulation of HIF factors provokes an increase in interferon-responsive genes and triggers expansion and differentiation of hematopoietic progenitors by a STAT1-mediated mechanism. Interestingly, in conditions of demand-driven hematopoiesis HIF factors are specifically downregulated in mesenchymal progenitors in vivo. In conclusion, our findings indicate that HIF factors also regulate hematopoiesis non-cell-autonomously by preventing activation of a latent program in mesenchymal progenitors that promotes hematopoiesis

HIF factors cooperate with PML-RARα to promote acute promyelocytic leukemia progression and relapse

Acute promyelocytic leukemia (APL) is epitomized by the chromosomal translocation t(15;17) and the resulting oncogenic fusion protein PML-RARα. Although acting primarily as a transcriptional repressor, PML-RARα can also exert functions of transcriptional co-activation. Here, we find that PML-RARα stimulates transcription driven by HIF factors, which are critical regulators of adaptive responses to hypoxia and stem cell maintenance. Consistently, HIF-related gene signatures are upregulated in leukemic promyelocytes from APL patients compared to normal promyelocytes. Through in vitro and in vivo studies, we find that PML-RARα exploits a number of HIF-1α-regulated pro-leukemogenic functions that include cell migration, bone marrow (BM) neo-angiogenesis and self-renewal of APL blasts. Furthermore, HIF-1α levels increase upon treatment of APL cells with all-trans retinoic acid (ATRA). As a consequence, inhibiting HIF-1α in APL mouse models delays leukemia progression and exquisitely synergizes with ATRA to eliminate leukemia-initiating cells (LICs)

Tratado médico-filosófico de la enagenación del alma o manía

Donación Roberto Escobar Delma

The biological and biomechanical role of transglutaminase-2 in the tumour microenvironment

Transglutaminase-2 (TG2) is the most highly and ubiquitously expressed member of the transglutaminase enzyme family and is primarily involved in protein cross-linking. TG2 has been implicated in the development and progression of numerous cancers, with a direct role in multiple cellular processes and pathways linked to apoptosis, chemoresistance, epithelial-mesenchymal transition, and stem cell phenotype. The tumour microenvironment (TME) is critical in the formation, progression, and eventual metastasis of cancer, and increasing evidence points to a role for TG2 in matrix remodelling, modulation of biomechanical properties, cell adhesion, motility, and invasion. There is growing interest in targeting the TME therapeutically in response to advances in the understanding of its critical role in disease progression, and a number of approaches targeting biophysical properties and biomechanical signalling are beginning to show clinical promise. In this review we aim to highlight the wide array of processes in which TG2 influences the TME, focussing on its potential role in the dynamic tissue remodelling and biomechanical events increasingly linked to invasive and aggressive behaviour. Drug development efforts have yielded a range of TG2 inhibitors, and ongoing clinical trials may inform strategies for targeting the biomolecular and biomechanical function of TG2 in the TME

A Novel Perilla frutescens (L.) Britton Cell-Derived Phytocomplex Regulates Keratinocytes Inflammatory Cascade and Barrier Function and Preserves Vaginal Mucosal Integrity In Vivo

: In the last years, the medicinal plant Perilla frutescens (L.) Britton has gained scientific interest because leaf extracts, due to the presence of rosmarinic acid and other polyphenols, have shown anti-allergic and skin protective potential in pre-clinical studies. Nevertheless, the lack of standardized extracts has limited clinical applications to date. In this work, for the first time, a standardized phytocomplex of P. frutescens, enriched in rosmarinic acid and total polyphenols, was produced through innovative in vitro cell culture biotechnology and tested. The activity of perilla was evaluated in an in vitro inflammatory model of human keratinocytes (HaCaT) by monitoring tight junctions, filaggrin, and loricrin protein levels, the release of pro-inflammatory cytokines and JNK MAPK signaling. In a practical health care application, the perilla biotechnological phytocomplex was tested in a multilayer model of vaginal mucosa, and then, in a preliminary clinical observation to explore its capacity to preserve vaginal mucosal integrity in women in peri-menopause. In keratinocytes cells, perilla phytocomplex demonstrated to exert a marked activity in epidermis barrier maintenance and anti-inflammatory effects, preserving tight junction expression and downregulating cytokines release through targeting JNK activation. Furthermore, perilla showed positive effects in retaining vaginal mucosal integrity in the reconstructed vaginal mucosa model and in vivo tests. Overall, our data suggest that the biotechnological P. frutescens phytocomplex could represent an innovative ingredient for dermatological applications