RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets

MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell lin

Negative transcriptional control of ERBB2 gene by MBP-1 and HDAC1: diagnostic implications in breast cancer

Backgound: The human ERBB2 gene is frequently amplified in breast tumors, and its high expression is associated with poor prognosis. We previously reported a significant inverse correlation between Myc promoter-binding protein-1 (MBP-1) and ERBB2 expression in primary breast invasive ductal carcinoma (IDC). MBP-1 is a transcriptional repressor of the c-MYC gene that acts by binding to the P2 promoter; only one other direct target of MBP-1, the COX2 gene, has been identified so far. Methods: To gain new insights into the functional relationship linking MBP-1 and ERBB2 in breast cancer, we have investigated the effects of MBP-1 expression on endogenous ERBB2 transcript and protein levels, as well as on transcription promoter activity, by transient-transfection of SKBr3 cells. Reporter gene and chromatin immunoprecipitation assays were used to dissect the ERBB2 promoter and identify functional MBP-1 target sequences. We also investigated the relative expression of MBP-1 and HDAC1 in IDC and normal breast tissues by immunoblot analysis and immunohistochemistry. Results: Transfection experiments and chromatin immunoprecipitation assays in SKBr3 cells indicated that MBP-1 negatively regulates the ERBB2 gene by binding to a genomic region between nucleotide -514 and - 262 of the proximal promoter; consistent with this, a concomitant recruitment of HDAC1 and loss of acetylated histone H4 was observed. In addition, we found high expression of MBP-1 and HDAC1 in normal tissues and a statistically significant inverse correlation with ErbB2 expression in the paired tumor samples. Conclusions: Altogether, our in vitro and in vivo data indicate that the ERBB2 gene is a novel MBP-1 target, and immunohistochemistry analysis of primary tumors suggests that the concomitant high expression of MBP-1 and HDAC1 may be considered a diagnostic marker of cancer progression for breast IDC

Biochemical and clinical relevance of alpha lipoic acid: antioxidant and anti-inflammatory activity, molecular pathways and therapeutic potential

The molecular nature of lipoic acid (LA) clarifies its capability of taking part to a variety of biochemical reactions where redox state is meaningful. The pivotal action of LA is the antioxidant activity due to its ability to scavenge and inactivate free radicals. Furthermore, LA has been shown to chelate toxic metals both directly and indirectly by its capability to enhance intracellular glutathione (GSH) levels. This last property is due to its ability to interact with GSH and recycle endogenous GSH. LA exhibits significant antioxidant activity protecting against oxidative damage in several diseases, including neurodegenerative disorders. Interestingly, LA is unique among natural antioxidants for its capability to satisfy a lot of requirements, making it a potentially highly effective therapeutic agent for many conditions related with oxidative damage. In particular, there are evidences showing that LA has therapeutic activity in lowering glucose levels in diabetic conditions. Similarly, LA supplementation has multiple beneficial effects on the regression of the mitochondrial function and on oxidative stress associated with several diseases and aging

Human airway epithelial extracellular vesicle miRNA signature is altered upon asthma development

Background: miRNAs are master regulators of signaling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate whether the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development. Methods: NHBE cells were cultured at air-liquid interface and treated with interleukin (IL)-13 to induce an asthma-like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, and EV-associated miRNAs were identified by a RT-qPCR-based profiling. Significant candidates were confirmed in EVs isolated by size-exclusion chromatography from nasal lavages of children with mild-to-moderate (n = 8) or severe asthma (n = 9), and healthy controls (n = 9). Results: NHBE cells secrete EVs to the apical and basal side. 47 miRNAs were expressed in EVs and 16 thereof were significantly altered in basal EV upon IL-13 treatment. Expression of miRNAs could be confirmed in EVs from human nasal lavages. Of note, levels of miR-92b, miR-210, and miR-34a significantly correlated with lung function parameters in children (FEV1FVC%pred and FEF25-75%pred), thus lower sEV-miRNA levels in nasal lavages associated with airway obstruction. Subsequent ingenuity pathway analysis predicted the miRNAs to regulate Th2 polarization and dendritic cell maturation. Conclusion: Our data indicate that secretion of miRNAs in EVs from the airway epithelium, in particular miR-34a, miR-92b, and miR-210, might be involved in the early development of a Th2 response in the airways and asthma

Myc Promoter-Binding Protein-1 (MBP-1) Is a Novel Potential Prognostic Marker in Invasive Ductal Breast Carcinoma

Background Alpha-enolase is a glycolytic enzyme that catalyses the formation of phosphoenolpyruvate in the cell cytoplasm. \u3b1-Enolase and the predominantly nuclear Myc promoter-binding protein-1 (MBP-1) originate from a single gene through the alternative use of translational starting sites. MBP-1 binds to the P2 c-myc promoter and competes with TATA-box binding protein (TBP) to suppress gene transcription. Although several studies have shown an antiproliferative effect of MBP-1 overexpression on several human cancer cells, to date detailed observations of \u3b1-enolase and MBP-1 relative expression in primary tumors versus normal tissues and their correlation with clinicopathological features have not been undertaken. Methodology and Findings We analyzed \u3b1-enolase and MBP-1 expression in normal breast epithelium and primary invasive ductal breast carcinoma (IDC) from 177 patients by Western blot and immunohistochemical analyses, using highly specific anti-\u3b1-enolase monoclonal antibodies. A significant increase in the expression of cytoplasmic \u3b1-enolase was observed in 98% of the tumors analysed, compared to normal tissues. Nuclear MBP-1 was found in almost all the normal tissues while its expression was retained in only 35% of the tumors. Statistically significant associations were observed among the nuclear expression of MBP-1 and ErbB2 status, Ki-67 expression, node status and tumor grade. Furthermore MBP-1 expression was associated with good survival of patients with IDC. Conclusions MBP-1 functions in repressing c-myc gene expression and the results presented indicate that the loss of nuclear MBP-1 expression in a large number of IDC may be a critical step in the development and progression of breast cancer and a predictor of adverse outcome. Nuclear MBP-1 appears to be a novel and valuable histochemical marker with potential prognostic value in breast cancer

Effect of Lipoic Acid on the Biochemical Mechanisms of Resistance to Bortezomib in SH-SY5Y Neuroblastoma Cells

Neuroblastoma (NB) is an extracranial solid cancer and the most common cancer in infancy. Despite the standard treatment for NB is based on the combination of chemotherapeutic drugs such as doxorubicin, vincristine, cyclophosphamide, and cisplatin, chemoresistance occurs over the time. The aim of the present research was to evaluate the effect of bortezomib (BTZ) (50 nM) on NB cell viability and how lipoic acid (ALA) (100 μM) modifies pharmacological response to this chemotherapeutic agent. Cell viability was assessed by ATP luminescence assay whereas expression of oxidative stress marker (i.e., heme oxygenase-1) and endoplasmic reticulum stress proteins was performed by real-time PCR, western blot, and immunofluorescence. Our data showed that BTZ treatment significantly reduced cell viability when compared to untreated cultures (about 40%). Interestingly, ALA significantly reduced the efficacy of BTZ (about 30%). Furthermore, BTZ significantly induced heme oxygenase-1 as a result of increased oxidative stress and such overexpression was prevented by concomitant treatment with ALA. Similarly, ALA significantly reduced BTZ-mediated endoplasmic reticulum stress as measured by reduction in BiP1 and IRE1α, ERO1α, and PDI expression. In conclusion, our data suggest that BTZ efficacy is dependent on cellular redox status and such mechanisms may be responsible of chemoresistance to this chemotherapeutic agent

Il bivalve più grande del Mediterraneo, Pinna nobilis, nella Laguna di Acquatina: indagini preliminari e prospettive

Visitare la Laguna di Acquatina, localizzata lungo la costa Adriatica del Salento, è un’esperienza sensoriale straordinaria, ricca di emozioni, che permette agli studiosi e a differenti gruppi di stakeholder di conoscere e apprezzare un patrimonio naturale dal valore inestimabile, di percepire i suoni dell’ambiente circostante e di osservare la biodiversità nelle sue molteplici forme. Fra queste, la biodiversità in specie è l’insieme delle specie animali, vegetali e microorganismi che si trovano in un ecosistema, in un habitat o in un’area predefinita. Le liste di specie, o checklist, sono gli strumenti di indagine con cui i ricercatori analizzano le variazioni della biodiversità in specie nel tempo, nello spazio e in relazione a differenti fattori di perturbazione, naturali e antropici, inclusi i cambiamenti climatici. Nella Laguna di Acquatina si può facilmente osservare anche la biodiversità a un livello di organizzazione ecologica superiore, cioè la biodiversità tra gli habitat. Quest’ultima è definita come l’eterogeneità degli habitat presenti in un ecosistema o in un’area predefinita. Ad Acquatina è facile osservare e individuare distintamente l’habitat acquatico, la macchia mediterranea, il cordone dunale, l’antistante spiaggia e il mare. La biodiversità in specie della Laguna di Acquatina è elevata, caratterizzata dalla presenza di tutti i gruppi tassonomici caratteristici degli ecosistemi acquatici di transizione e costieri dell’Ecoregione Mediterranea. Sono anche numerose le specie di interesse prioritario, cioè quelle specie che a seguito di forti minacce di degrado ed estinzione vengono sottoposte a un regime di tutela straordinario da parte delle istituzioni internazionali, europee e nazionali. A causa della presenza di numerose specie e habitat di interesse prioritario, la Laguna di Acquatina è inserita all’interno di un più ampio sito facente parte della Rete NATURA 2000. La Rete NATURA 2000 è stata istituita dall’Unione Europea e ha la finalità di tutelare e conservare le specie e gli habitat di interesse prioritario. L’ultimo censimento ufficiale relativo al 2015 elenca 33 specie di interesse prioritario per il sito NATURA 2000 denominato Aquatina di Frigole (IT9150003). La maggior parte delle specie riportate nel censimento appartengono all’avifauna, ma Pinna nobilis (Linnaeus, 1758) non era presente nel censimento del 2015. A partire dalla seconda metà del 2017, le attività didattiche, anche di rilievo internazionale, e di ricerca scientifica a carattere ecologico si sono rafforzate. La Laguna di Acquatina si è trasformata in un autentico laboratorio su campo, utile anche per azioni di divulgazione scientifica e di promozione degli sport acquatici ecosostenibili. Alla fine del 2017 sono stati rinvenuti i primi esemplari di Pinna nobilis (L.) durante lo svolgimento di un campionamento scientifico. Si tratta di un mollusco bivalve appartenente alla famiglia “Pinnidae”, comunemente chiamato cozza penna, nacchera o stura. Pinna nobilis è una specie endemica del Mar Mediterraneo, in cui è il più grande bivalve raggiungendo dimensioni anche superiori a un metro di lunghezza. È una specie protetta dall’Unione Europea attraverso le Direttive 92/43/CEE e 2006/105/CE. La pesca e la rimozione sono assolutamente vietate, e sono previste ingenti ammende per chi agisce illegalmente. Usualmente Pinna nobilis viene associata ad ambienti marini, essa vive sepolta per circa un terzo della sua lunghezza nel sedimento, tipicamente in fondi mobili, preferenzialmente associata alle praterie di fanerogame marine (Posidonia oceanica, Cymodocea nodosa e Zostera marina), e anche in zone sabbiose prive di vegetazione. Le pareti esterne delle valve vengono colonizzate da numerose specie incrostanti animali e vegetali, ciò contribuisce a incrementare la biodiversità in specie dell’area in cui si trova P. nobilis. Questa specie può anche colonizzare le porzioni più prossimali e protette di lagune e stagni costieri, in cui può costituire popolazioni ad elevata densità. Gli ambienti lagunari costituiscono, infatti, un “cosmo” singolarmente variegato ed attraente, per la complessità dei fattori ed interazioni ecologiche, e per la varietà di condizioni trofiche e climatiche che, nel complesso, favoriscono la colonizzazione di questi ambienti da parte di specie marine e d’acqua dolce, siano esse indigene o aliene. Per le sue caratteristiche ecologiche, Pinna nobilis è una specie utilizzata nel biomonitoraggio degli ecosistemi acquatici costieri, inclusi quelli marini, e come specie target nella Strategia Marina (Direttiva MSFD 2008/56/EC). Per quanto riguarda il suo ruolo ecologico, P. nobilis è una specie “filtratrice”- e fornisce numerosi servizi ecosistemici: filtra dalla colonna d’acqua una grande quantità di materia organica e detriti sospesi contribuendo alla rimozione dei nutrienti e alla trasparenza dell’acqua; ospita altre specie determinando un aumento della biodiversità locale; attira subacquei e snorkeler incentivando le attività ricreative e di educazione ambientale. Essendo una specie commestibile, già gli Egizi, i Romani e alcuni popoli Islamici utilizzavano questa specie per scopi alimentari, tessevano il suo bisso per fare dei pregiati ricami e lavoravano le valve per ottenere preziosi ornamenti per le loro vesti. A scala Mediterranea, le informazioni e la letteratura scientifica più recenti su Pinna nobilis (L.) ci raccontano, invece, di una specie esposta a numerose minacce di degrado, di una progressiva riduzione della densità di popolazione e scomparsa dovute alla pesca illegale, ai cambiamenti climatici, agli ancoraggi delle imbarcazioni da turismo e all’azione di un protozoo parassita, denominato Haplosporidium pinnae, che induce la morte degli organismi. La presenza di Pinna nobilis (L.) nella Laguna di Acquatina, Sito di Interesse Comunitario appartenente alla Rete NATURA 2000, sembra essere in controtendenza rispetto alla situazione attuale della specie. Probabilmente la laguna è utilizzata come nursery o habitat rifugio, avendo le condizioni ecologiche ottimali per la specie (Marrocco et al., 2018). L’indagine preliminare a cui si riferisce la ricerca è stata effettuata in un’area limitata della laguna, in vicinanza della foce, e ha permesso di individuare la presenza di undici individui, orientati verso la direzione Nord-Est. I principali parametri biometrici sono stati rilevati in ciascun individuo secondo il seguente schema. La larghezza massima e minima al fondo erano di 15,16 cm ± 0,726 e di 13,81 cm ± 0,611 rispettivamente, e la lunghezza della valva non coperta dal sedimento era di 16,66 cm ± 0,441. Il campionamento è stato “non distruttivo” e nessun individuo è stato rimosso o manipolato. Sono stati anche registrati i parametri abiotici della colonna d’acqua nella zona in cui P. nobilis è stata rilevata (temperatura 12,69 °C ± 0,207; ossigeno disciolto 7,21 mg*l-1 ± 0,278; salinità 24,77 PSU ± 0,963, pH 7,73 ± 0,084) (Marrocco et al., 2018). Benché si tratti di una indagine preliminare, ipotizziamo che la Laguna di Acquatina rappresenti un ambiente protetto per questa specie; qui potrebbe raggiungere dimensioni numeriche tali da permettere il reclutamento e il trans-planting in altri siti idonei, e quindi agevolarne il ripopolamento e la diffusione. La scoperta, effettuata da un team di ricercatori e studenti italiani e stranieri, provenienti da alcuni Paesi asiatici partecipanti al progetto INTER-ASIA, ha suggerito di approfondire l’attività di ricerca, di comunicazione scientifica e di citizen science nell’ambito del progetto IMPRECO “Common strategies and best practices to IMprove the transnational PRotection of ECOsystem integrity and services” – finanziato nell’ambito del Programma di Cooperazione Territoriale Europea “ADRION Adriatic-Ionian Programme INTERREG V-B Transnational 2014-2020, 1st call”. In particolare la ricerca è finalizzata al monitoraggio della specie su una scala spaziale che coinvolge il Mar Adriatico, Ionio ed Egeo. Infatti grazie alla partnership del progetto IMPRECO, distribuita nell’area di riferimento della Strategia Europea per la Regione Adriatico-Ionica (EUSAIR), sarà possibile avviare lo studio della dinamica di popolazione di Pinna nobilis, delle risposte alle perturbazioni e pressioni antropiche, ed ai parassiti mortali. Un protocollo di monitoraggio congiunto e semplificato (Joint Monitoring Protocol of IMPRECO) permetterà di raccogliere dati utili per la conservazione di Pinna nobilis su una ampia scala spaziale, e di coinvolgere gruppi interessati di stakeholder e cittadini nella raccolta dei dati e nella salvaguardia della specie. Per la sua rilevanza scientifica, la ricerca è stata recentemente pubblicata sulla rivista scientifica internazionale Nature Conservation (Marrocco et al., 2018)

In-vitro NET-osis induced by COVID-19 sera is associated to severe clinical course in not vaccinated patients and immune-dysregulation in breakthrough infection

: Since neutrophil extracellular traps formation (NET-osis) can be assessed indirectly by treating healthy neutrophils with blood-derived fluids from patients and then measuring the NETs response, we designed a pilot study to convey high-dimensional cytometry of peripheral blood immune cells and cytokines, combined with clinical features, to understand if NET-osis assessment could be included in the immune risk profiling to early prediction of clinical patterns, disease severity, and viral clearance at 28 days in COVID-19 patients. Immune cells composition of peripheral blood, cytokines concentration and in-vitro NETosis were detected in peripheral blood of 41 consecutive COVID-19 inpatients, including 21 mild breakthrough infections compared to 20 healthy donors, matched for sex and age. Major immune dysregulation in peripheral blood in not-vaccinated COVID-19 patients compared to healthy subjects included: a significant reduction of percentage of unswitched memory B-cells and transitional B-cells; loss of naïve CD3+CD4+CD45RA+ and CD3+CD8+CD45RA+ cells, increase of IL-1β, IL-17A and IFN-γ. Myeloid compartment was affected as well, due to the increase of classical (CD14++CD16-) and intermediate (CD14++CD16+) monocytes, overexpressing the activation marker CD64, negatively associated to the absolute counts of CD8+ CD45R0+ cells, IFN-γ and IL-6, and expansion of monocytic-like myeloid derived suppressor cells. In not-vaccinated patients who achieved viral clearance by 28 days we found at hospital admission lower absolute counts of effector cells, namely CD8+T cells, CD4+ T-cells and CD4+CD45RO+ T cells. Percentage of in-vitro NET-osis induced by patients' sera and NET-osis density were progressively higher in moderate and severe COVID-19 patients than in mild disease and controls. The percentage of in-vitro induced NET-osis was positively associated to circulating cytokines IL-1β, IFN-γ and IL-6. In breakthrough COVID-19 infections, characterized by mild clinical course, we observed increased percentage of in-vitro NET-osis, higher CD4+ CD45RO+ and CD8+ CD45RO+ T cells healthy or mild-COVID-19 not-vaccinated patients, reduced by 24 h of treatment with ACE inhibitor ramipril. Taken together our data highlight the role of NETs in orchestrating the complex immune response to SARS-COV-2, that should be considered in a multi-target approach for COVID-19 treatment

Glomerular autoimmune multicomponents of human lupus nephritis in vivo: α-enolase and annexin AI

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-α-enolase/low anti-annexin AI IgG2 and patients with low anti-α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum

Vaccination with ENO1 DNA Prolongs Survival of Genetically Engineered Mice with Pancreatic Cancer.

BACKGROUND & AIMS:: Pancreatic ductal adenocarcinoma (PDA) is an aggressive tumor, and patients typically present with late-stage disease; rates of 5-year survival after pancreaticoduodenectomy are low. Antibodies against -enolase (ENO1), a glycolytic enzyme, are detected in more than 60% of patients with PDA, and ENO1-specific T cells inhibit the growth of human pancreatic xenograft tumors in mice. We investigated whether an ENO1DNA vaccine elicits anti-tumor immune responses and prolongs survival of mice that spontaneously develop autochthonous, lethal pancreatic carcinomas. METHODS:: We injected and electroporated a plasmid encoding ENO1 (or a control plasmid) into Kras(G12D)/Cre mice (KC) and Kras(G12D)/Trp53 (R172H) /Cre (KPC) mice when they were 4 weeks old (when pancreatic intraepithelial lesions are histologically evident). Anti-tumor humoral and cellular responses were analyzed by histology, immunohistochemistry, ELISAs, flow cytometry, and ELISpot and cytotoxicity assays. Survival was analyzed by Kaplan-Meier analysis. RESULTS:: The ENO1 vaccine induced antibody and a cellular responses and increased survival times by a median 138 days in KC mice and 42 days in KPC mice, compared with mice given the control vector. In histologic analysis, the vaccine appeared to slow tumor progression. The vaccinated mice had increased serum levels of anti-ENO1 immunoglobulin G, which bound the surface of carcinoma cells and induced complement-dependent cytotoxicity. ENO1 vaccination reduced numbers of myeloid-derived suppressor cells and T-regulatory cells, and increased T-helper 1 and 17 responses. CONCLUSIONS:: In a genetic model of pancreatic carcinoma, vaccination with ENO1DNA elicits humoral and cellular immune responses against tumors, delays tumor progression, and significantly extends survival. This vaccination strategy might be developed as a neo-adjuvant therapy for patients with PD