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Searching for molecular mechanisms sustaining tumor formation and progression in Neurofibromatosis type 1
Full text linkSUMMARY
Neurofibromatosis type 1 (NF1, OMIM # 162200), also known as von Recklinghausen, is an autosomal dominant disease caused by mutations of the NF1 gene coding a 2818 amino acid protein, neurofibromin (Nf1). More than 900 different mutations in the NF1 gene have been identified (HGMD, Human Genetic Mutation Database). Mutations of NF1 gene cause a variety of clinical manifestations such as the optic gliomas, neoplasms of the haematopoietic system and learning disabilities. However, the hallmark of NF1 is the development of multiple benign peripheral nerve sheath tumors called neurofibromas. Neurofibromas are complex tumors arising from peripheral nerve sheaths and mainly composed of Schwann Cells (SCs) homozygous for mutated NF1, mast cells (MCs) and fibroblasts (FBRs) both heterozygous for the same mutation. The plexiform variety can progress to highly malignant sarcomas termed Malignant Peripheral Nerve Sheath Tumors (MPNSTs), which are almost invariably lethal.
Up to now, any effective therapy able to either reduces neurofibroma size and its incidence or to counteract its formation, has not been developed yet. The main feature of neurofibroma is a rigid structure due to massive deposition of collagen of different types by activated FBRs. These cells, named myofibroblasts (mFBRs), are massively stimulated by mast cell-secreting Transcription Growth Factor-Beta (TGF-Beta) to produce growth factors such as Platelet Growth Factor (PDGF), Fibroblast Growth Factor (FGF) and collagen. This leads to both potent SCs proliferation and deposition of rigid extracellular matrix (ECM).
Cells’ haploinsufficient for Nf1 display hyper-activation of Rat Sarcoma (Ras), which further increases when Loss of Heterozigozyty (LOH) of NF1 occurs. Thus, the activation of Ras/ Rapidly Accelerated Fibrosarcoma (Raf) /Extracellular signal-regulated Kinase (ERK) signaling in SCs is sufficient to make them more susceptible to proliferative signals provided by a NF1+/- niche. However, the physiological response to Ras hyper-activation is cell-cycle arrest and/or senescence rather than transformation. Ras-mediated transformation of SCs probably relies on a step-wise process that integrates circuits of amplification signals from the local niche. A major component of the niche is the ECM, a complex network of macromolecules whose the elasticity (ranging from soft to stiff and rigid),contributes to development and cancer. ECM elasticity determines how a cell senses and perceives external forces and thus provides a major environmental cue that determines cell behavior. Indeed, the focal adhesion complex, which consists of integrins, multicomplex of adaptors and signaling proteins, can be viewed as a mechanosensor linking the actomyosin cytoskeleton with the ECM. How lack of Nf1 may impact on the complexity of ECM-cell dynamic and how the great rigidity of the ECM in neurofibromas influences SCs’ behavior, is still unknown. Among the three functional domains described in the Nf1 protein, a Focal Adhesion Kinas (FAK) binding domain has been identified and Nf1 has been shown to interact with FAK, paving the way for the enunciation of new hypothesis aimed to explain the route of SCs transformation toward cancer.
Rational: as in other tumors {Lu, 2012 #289}, {Yu, 2011 #292}), also in the plexiform neurofibromas, the tumorigenic phenotype of SCs is fostered by the amplification of integrated signaling pathways triggered by loss of Nf1, Ras hyper activation and deregulated ECM. Changes of the mechanical properties of ECM due to increased collagen secretion by mFBRs might actively contribute to tumor progression by influencing gene expression profile of the cells through the enhancement of Ras signaling pathway triggered by FAK. The deep investigation of these biological changes triggered in SCs by ECM formation is the goal of the present project.
Project Goals: To shed light into this issue, we intend 1) to generate a novel three-dimensional experimental model in vitro reproducing the multicellular complexity of neurofibromas with primary cells. Immortalized cells, indeed, are not suitable for our aims since the molecular oncologists consider the immortalization process as the first hit leading to tumorigenic phenotype, because of the changes which made for cell cycle control in gene expression 2) to assess the requirement ECM for SCs transformation in this new in vitro system identifying the proper ECM composition and stiffness in matrigel (structural and non structural components) required for neurofibroma’s formation.
Results:
Isolation of primary SCs and FBRs from Neurofibromas and their biological characterization:
1) We have already isolated and cultured in 2D our SCs and FBRs NF1+/- according to Serra methodology {Serra, 2000 #210}. These cells have been isolated from plexiform neurofibroma biopsies after informed consent of patients by our Milan and Rome University collaborators.
To get two populations of SCs and FBRs we have cultured cells in selective Medium (according to {Serra, 2000 #210}, and our new unpublished protocol) and characterized them biochemically by: S100B {Tucker, 2011 #321} and p75 markers specifically recognizing SCs and collagen I secretion, alpha smooth muscle actin (α-SMA) expression, Smad2/3 activation, Abl kinase activation characterizing mFBR activity {Kojima, 2010 #150}.
2) We have already obtained colonies of SCs growing in 3D in vitro system as described in step 1 and 2 (in transwell-like chambers to permit autocrine stimulation between mFBRs and SCs). Our preliminary data show that primary SCs generate colonies only when plated in an ECM/reconstituted basement membrane Collagen I-Matrigel of at list 3 mg/ml. However, we have still to set up the culture conditions to keep cells in highly proliferating state.
Preliminary indications in immortalized Mouse Embrionic Fibroblasts (MEFs)
In other cellular models as in mouse FBRs NF1-/-, we have found that the absence of Neurofibromin correlates with deregulation of FAK Y397 and Y925 phosphorylation both in absence of integrin clustering and after ligand stimulation. Further, the tumorigenesis assay showed that MEFNF1-/-ability to form colonies is affected by both MECK inhibitor and FAK inhibitor (Y15) indicating the cooperative role of FAK and PDGFBB growth factor in the tumorigenesis process mediated by Nf1. Consistently, immunoprecipitation experiments showed that in Nf1 null cells, Growth factor receptor-bound protein2 (Grb-2), the RAS pathway initiator, interacts with FAK also in absence of collagen in a PDGFBB ligand-dependent way, thus suggesting that FAK and growth factor receptors can cooperate to increase the Ras activity to a threshold required to induce tumorigenesis.SOMMARIO
Neurofibromatosi tipo 1 (NF1, OMIM # 162200), nota anche come di von Recklinghausen, è una malattia autosomica dominante causata da mutazioni del gene NF1 che codifica una proteina coi 2818 aminoacidi , detta neurofibromina (Nf). Più di 900 diverse mutazioni nel gene NF1 sono state identificate (HGMD, Database di mutazione genetica umana). Mutazioni del gene NF1 causano una varietà di manifestazioni cliniche quali il glioma ottico, neoplasie del sistema ematopoietico e disabilità dell'apprendimento. Tuttavia, il segno distintivo della NF1 è lo sviluppo dei tumori benigni nella guaina dei nervi periferici, chiamati neurofibromi. I neurofibromi sono tumori complessi originati da guaine nervose periferiche e costituiti prevalentemente da cellule di Schwann omozigote mutate per NF1, mastociti e fibroblasti entrambi eterozigoti per la stessa mutazione. I plessiformi possono progredire a sarcomi altamente maligni denominati MPNSTs (schwannomi maligni), che sono quasi sempre letali.
Ad oggi non e’ ancora stata sviluppata alcuna terapia efficace in grado di ridurre la dimensione e incidenza dei neurofibromi, o atta a contrastarne la formazione. La caratteristica principale dei neurofibromi è la loro struttura rigida conseguente alla massiccia deposizione di collagene prodotto dai fibroblasti attivati. Queste cellule, denominate miofibroblasti, sono fortemente stimolate da mastociti che producono fattore di crescita trascrizionale-Beta (TGF-Beta) per produrre poi fattori di crescita, come fattore di crescita piastrinico, fattore di crescita dei fibroblasti e collagene. Ciò comporta sia la potente proliferazione di cellule di Schwann che la deposizione di matrice extracellulare rigida.
Cellule aploinsufficienti per Nf1 comportano iperattivazione di Ras, che aumenta ulteriormente con LOH. L'attivazione di vie di segnale di Ras/Raf/ERK in cellule di Schwann rende le cellule più suscettibili ai segnali proliferativi forniti dalla nicchia NF1+/-. Tuttavia, la risposta fisiologica a Ras iperattivato è l’arresto del ciclo cellulare e/o senescenza piuttosto che trasformazione. La trasformazione Ras-mediata di cellule di Schwann probabilmente si basa su un procedimento che integra diversi segnali dipendenti da circuiti di amplificazione della nicchia stessa. Uno dei più importanti componenti della nicchia è la matrice extracellulare (ECM), una rete complessa di macromolecole con plasticità variabile che contribuisce alla progressione tumorale. L’elasticità di ECM determina la modalità con cui una cellula percepisce le forze esterne e quindi fornisce un importante spunto ambientale che determina il comportamento cellulare. In effetti le adesioni focali, che consistono di integrine, adattatori multicomplesi e proteine di segnale, possono essere visti come meccano-sensori che collegano il citoscheletro con la ECM. Come la mancanza di Nf1 possa avere un impatto significativo sulla complessità di dinamismo di ECM-cellula o come la grande rigidezza dell'ECM in neurofibroma influenzi il comportamento delle cellule di Schwann, è ancora sconosciuto. Tra i tre domini funzionali descritti nella proteina, un dominio di legame, FAK, sulla proteina è stato identificato e Nf1 ha mostrato di interagire con FAK, spianando la strada per l'enunciazione di una nuova ipotesi per spiegare il percorso trasformazionale delle cellule di Schwann verso il cancro.
Razionale: come in altri tumori {Lu, 2012 #289}, {Yu, 2011 #292}), anche nei neurofibromi plessiformi il fenotipo trasformato di SCs è favorito dall'amplificazione della segnalazione di percorsi integrati attivati sia da perdita di Nf1, Ras iperattivazione che deregolamentato di matrice extracellulare (ECM). Le modifiche delle proprietà meccaniche di ECM a causa dell'aumento di secrezione di collagene dai miofibroblasti potrebbe contribuire attivamente alla progressione del tumore, influenzando profili di espressione genica delle cellule attraverso la valorizzazione di segnale di Ras pathway generato dall'adesione focale (FAK). L'indagine in profondità di queste modificazioni biologiche attivate in SCs dalla formazione di ECM è l'obiettivo del presente progetto.
Al fine di far luce su questo argomento, abbiamo intenzione di 1) generare un nuovo modello sperimentale tridimensionale in vitro che riproduce la complessità di neurofibroma pluricellulari con le cellule primarie. Cellule immortalizzate, infatti, non sono adatte per i nostri scopi poiché gli oncologi molecolari consideranno il processo di immortalizzazione come il primo colpo che conduce al fenotipo oncogenico, a causa dei cambiamenti che sono stati fatti per il controllo del ciclo cellulare di espressione genica; 2) valutare l'esigenza di ECM nella trasformazione di cellule di Schwann cell (SCs) in questo nuovo sistema in vitro identificando la corretta composizione dell'ECM e rigidità in matrigel (strutturali e non strutturali) per formazione di neurofibroma.
Risultati:
Isolamento delle cellule di Schwann e Fibroblasti primarie da Neurofibromi e la loro caratterizzazione biologica:
1) Abbiamo già isolato e coltivato in 2D le nostre cellule di Schwann e Fibroblasti NF1+/- secondo la metodologia di Serra {Serra, 2000 #210}. Queste cellule sono state isolate da biopsie di neurofibromi plessiformi dopo aver consenso informato dei pazienti mediante i nostri collaboratori presso Università di Milano e di Roma.
Per ottenere due popolazioni delle cellule di Schwann e Fibroblasti abbiamo coltivato le cellule in terreno selettivo (secondo {Serra, 2000 #210}, e il nostro nuovo protocollo inedito) e caratterizzato dal punto di vista biochimico: S100B {Tucker, 2011 #321} e p75 marcatori che riconoscono specificamente le cellule di Schwann e secrezione del collagene di tipo I, espressione dell’actìna alfa del muscolo liscio (α-SMA), attivazione di Smad2/3, attivazione di abl chinasi e caratterizzare l'attività di myo-fibroblasti {Kojima, 2010 #150}.
2) abbiamo già ottenuto le colonie di cellule di Schwann cresciute nel sistema 3D in vitro come descritto nella fase 1 e 2 (in transwell-like chamber per permettere la stimolazione autocrina tra myofibroblasti e le cellule di Schwann). I nostri dati preliminari mostrano che le cellule primarie di Schwann generano delle colonie solo quando piastrate in una ECM/ membrana basale ricostituita del collagene di tipo I di Matrigel di almeno 3 mg/ml. Tuttavia, dobbiamo ancora impostare le condizioni migliori di cultura per mantenere le cellule altamente proliferanti.
Indicazioni preliminari in fibroblasti Embrionali immortalati Murini (MEFs)
In altri modelli cellulari come nel fibroblasti NF1-/- murini (MEFs), abbiamo trovato che l'assenza di neurofibromina scorrela con la deregolata di fosforilazione del FAK in Y397 e Y925 sia in assenza di raggruppamento di integrine che dopo la stimolazione con ligando. Inoltre, il saggio di tumorigenesi mostrava che la capacità di cellule di MEFNf1-/- di formare colonie è influenzata sia da inibitore di MECK che inibitore FAK Y15 indicante il ruolo di cooperatzione di FAK e PDGFBB, fattore di crescita, nel processo di tumorigenesi mediato da NF1. Coerentemente, gli esperimenti di immunoprecipitazione hanno mostrato che in cellule null NF1, il recettore del fattore di crescita di proteina legata2 (Grb-2), l’iniziatore di via di segnale di RAS, interagisce con FAK anche in assenza di collagene in un modo PDGFBB ligando-dipendente, suggerendo così che FAK e recettori di fattori di crescita possono cooperare per aumentare l'attività di Ras con un valore di soglia necessario per indurre la tumorigenesi
New technologies help the functional reconstruction in advanced-stage mandibular medication-related osteonecrosis of the jaw (MRONJ)
No full textExposure to antiresorptive therapy with bisphosphonates does not induce histological changes in human alveolar jawbone
Full text linkAim: The identification of specific alterations in the alveolar jawbone of patients treated with nitrogen-containing bisphosphonates (NBP) but without bisphosphonate-related osteonecrosis of the jaw (BRONJ) may help to identify the early steps of BRONJ and to select patients at risk for it.
Materials and Methods: We performed a case-control study. Cases were 60 individuals treated with NBP without clinical and radiological signs of BRONJ and requiring surgical tooth extraction. Controls were 60 individuals never treated with NBP and requiring tooth extraction. Cases and controls were matched by sex (same) and age (within 5 years). 18 categorical (basophile reversal lines, osteoblasts, osteoblastic lines, osteocytes, empty osteocytic lacunae, osteoclasts, Howship’s lacunae, vessel dilatation, vascular congestion, arteriolar thickening, intravascular fat globules, calcific fat necrosis, fatty bone marrow, ruptured adipocytes, granular cytoplasm of adipocytes, oil cysts, perivascular fibrosis, diffuse fibrous metaplasia) and 2 ordinal histopathological variables (inflammation and bone maturation) were investigated. Exact univariable and multivariable (correction for gender and age) logistic regression was used to test the association between NBP use and the histopathological variables. Because of multiple comparisons, the critical p-value was set to 0.0025 (0.05/20).
Results: Cases and controls did not differ for any study variable except for vascular congestion that was significantly associated with NBP use (multivariable OR = 0.24, exact 95% CI 0.10 to 0.57 for cases vs. controls, p = 0.0006).
Conclusions: Use of NBP does not produce specific histological alveolar bone alterations in the absence of overt BRONJ disease
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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