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

Antigen Retrieval and Its Effect on the MALDI-MSI of Lipids in Formalin-Fixed Paraffin-Embedded Tissue

Formalin-fixed paraffin-embedded (FFPE) tissue represents the primary source of clinical tissue and is routinely used in MALDI-MSI studies. However, it is not particularly suitable for lipidomics imaging given that many species are depleted during tissue processing. Irrespective, a number of solvent-resistant lipids remain, but their extraction may be hindered by the cross-link between proteins. Therefore, an antigen retrieval step could enable the extraction of a greater number of lipids and may provide information that is complementary to that which can be obtained from other biomolecules, such as proteins. In this short communication, we aim to address the effect of performing antigen retrieval prior to MALDI-MSI of lipids in FFPE tissue. As a result, an increased number of lipid signals could be detected and may have derived from lipid species that are known to be implicated in the lipid-protein cross-linking that is formed as a result of formalin fixation. Human renal cancer tissue was used as a proof of concept to determine whether using these detected lipid signals were also able to highlight the histopathological regions that were present. These preliminary findings may highlight the potential to enhance the clinical relevance of the lipidomic information obtained from FFPE tissue

Biotechnological Phytocomplex of Zanthoxylum piperitum (L.) DC. Enhances Collagen Biosynthesis In Vitro and Improves Skin Elasticity In Vivo

Background: Zanthoxylum piperitum (L.) DC., commonly known as Japanese pepper, is a deciduous shrub native to East Asia. Its berries are widely used as a spice, known for imparting a distinctive, tingly numbing sensation. Biologically, Z. piperitum has antimicrobial, antioxidant, and anti-inflammatory properties, and it is studied for its potential benefits in pain relief and digestive health. This study proposed a novel biotechnological Z. piperitum phytocomplex (ZPP) obtained by plant cell culture for skin health, specifically targeting collagen synthesis, extracellular matrix stability, and resilience against cellular stress. Given the bioactivity of Z. piperitum, we aimed to analyze its efficacy as a sustainable alternative for skin-supportive applications in cosmetics and supplements. Methods: ZPP was produced through stable plant cell cultures, yielding a lignan-rich (3.02% w/w) phytocomplex. Human fibroblasts (HFFs) were treated with varying ZPP concentrations to assess cellular viability, collagen metabolism, and ECM-related enzyme activities, both under normal and cell stress conditions. The in vivo assessment was performed by measuring biophysical skin parameters such as hydration, elasticity, and roughness in female volunteers for a period of six weeks. Results: In vitro, ZPP exhibited non-cytotoxicity at all concentrations tested. Under hyperosmotic stress, ZPP reduced cellular damage, suggesting enhanced resilience. ZPP upregulated lysyl oxidase (LOX) protein levels, critical for collagen cross-linking and ECM stability, with protective effects observed under oxidative/inflammatory conditions. Additionally, ZPP selectively inhibited collagenase, attenuating collagen breakdown, though antioxidant activity was modest. In vivo evaluation highlighted improved skin hydration, elasticity, and roughness. Conclusions: ZPP shows promise as a biotechnological agent for skin health, particularly in supporting collagen integrity, ECM stabilization, and cellular resilience under stress. While further studies are needed to explore its full efficacy, especially for aging and environmentally stressed skin, these findings highlight ZPP's potential as a new ingredient for cosmetic formulations aimed at skin care and the treatment of alterations caused by aging or environmental conditions

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

Detecting Proteomic Indicators to Distinguish Diabetic Nephropathy from Hypertensive Nephrosclerosis by Integrating Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging with High-Mass Accuracy Mass Spectrometry

Introduction: Diabetic nephropathy (DN) and hypertensive nephrosclerosis (HN) represent the most common causes of chronic kidney disease (CKD) and many patients progress to -end-stage renal disease. Patients are treated primarily through the management of cardiovas-cular risk factors and hypertension; however patients with HN have a more favorable outcome. A noninvasive clinical approach to separate these two entities, especially in hypertensive patients who also have diabetes, would allow for targeted treatment and more appropriate resource allocation to those patients at the highest risk of CKD progression. Meth-ods: In this preliminary study, high-spatial-resolution matrix-assisted laser desorption/ion-ization (MALDI) mass spectrometry imaging (MSI) was integrated with high-mass accuracy MALDI-FTICR-MS and nLC-ESI-MS/MS analysis in order to detect tissue proteins within kidney biopsies to discriminate cases of DN (n = 9) from cases of HN (n = 9). Results: Differences in the tryptic peptide profiles of the 2 groups could clearly be detected, with these becoming even more evident in the more severe histological classes, even if this was not evident with routine histology. In particular, 4 putative proteins were detected and had a higher signal intensity within regions of DN tissue with extensive sclerosis or fibrosis. Among these, 2 proteins (PGRMC1 and CO3) had a signal intensity that increased at the latter stages of the disease and may be associated with progression. Discussion/conclusion: This preliminary study represents a valuable starting point for a future study employing a larger cohort of patients to develop sensitive and specific protein biomarkers that could reliably differentiate between diabetic and hypertensive causes of CKD to allow for improved diagnosis, fewer biopsy procedures, and refined treatment approaches for clinicians

MiR-1227 targets SEC23A to regulate the shedding of large extracellular vesicles

Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV

Antigen Retrieval and Its Effect on the MALDI-MSI of Lipids in Formalin-Fixed Paraffin-Embedded Tissue

Formalin-fixed paraffin-embedded (FFPE) tissue represents the primary source of clinical tissue and is routinely used in MALDI-MSI studies. However, it is not particularly suitable for lipidomics imaging given that many species are depleted during tissue processing. Irrespective, a number of solvent-resistant lipids remain, but their extraction may be hindered by the cross-link between proteins. Therefore, an antigen retrieval step could enable the extraction of a greater number of lipids and may provide information that is complementary to that which can be obtained from other biomolecules, such as proteins. In this short communication, we aim to address the effect of performing antigen retrieval prior to MALDI-MSI of lipids in FFPE tissue. As a result, an increased number of lipid signals could be detected and may have derived from lipid species that are known to be implicated in the lipid-protein cross-linking that is formed as a result of formalin fixation. Human renal cancer tissue was used as a proof of concept to determine whether using these detected lipid signals were also able to highlight the histopathological regions that were present. These preliminary findings may highlight the potential to enhance the clinical relevance of the lipidomic information obtained from FFPE tissue

Detecting proteomic indicators to distinguish diabetic nephropathy from hypertensive nephrosclerosis by integrating matrix-assisted laser desorption/ionization mass spectrometry imaging with high-mass accuracy mass spectrometry

Introduction: Diabetic nephropathy (DN) and hypertensive nephrosclerosis (HN) represent the most common causes of chronic kidney disease (CKD) and many patients progress to -end-stage renal disease. Patients are treated primarily through the management of cardiovas-cular risk factors and hypertension; however patients with HN have a more favorable outcome. A noninvasive clinical approach to separate these two entities, especially in hypertensive patients who also have diabetes, would allow for targeted treatment and more appropriate resource allocation to those patients at the highest risk of CKD progression. Meth-ods: In this preliminary study, high-spatial-resolution matrix-assisted laser desorption/ion-ization (MALDI) mass spectrometry imaging (MSI) was integrated with high-mass accuracy MALDI-FTICR-MS and nLC-ESI-MS/MS analysis in order to detect tissue proteins within kidney biopsies to discriminate cases of DN (n = 9) from cases of HN (n = 9). Results: Differences in the tryptic peptide profiles of the 2 groups could clearly be detected, with these becoming even more evident in the more severe histological classes, even if this was not evident with routine histology. In particular, 4 putative proteins were detected and had a higher signal intensity within regions of DN tissue with extensive sclerosis or fibrosis. Among these, 2 proteins (PGRMC1 and CO3) had a signal intensity that increased at the latter stages of the disease and may be associated with progression. Discussion/Conclusion: This preliminary study represents a valuable starting point for a future study employing a larger cohort of patients to develop sensitive and specific protein biomarkers that could reliably differentiate between diabetic and hypertensive causes of CKD to allow for improved diagnosis, fewer biopsy procedures, and refined treatment approaches for clinicians.Proteomic

Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency. Using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but its drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the fusion oncogene PML-RARα and treats APL in cell and animal models and human patients. ATRA-induced Pin1 ablation also inhibits triple negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors