1,720,955 research outputs found
Vescicole Extracellulari (VE): nuovi biomarcatori per la risposta alla terapia con farmaci immunosopressori in pazienti pediatrici.
No full textLe tiopurine, come la mercaptopurina (MP), sono farmaci immunosoppressori, utilizzati nel trattamento della leucemia linfoblastica acuta (LLA) e delle malattie infiammatorie intestinali croniche (MICI). La Tiopurina S-metiltransferasi (TPMT) è un enzima coinvolto nel metabolismo delle tiopurine, la cui attività è modulata dalla Protein Kinase C e dalla Casein Kinase Substrate in Neurons 2 (PACSIN2). Inoltre, il polimorfismo a singolo nucleotide (SNP) rs2413739 di PACSIN2 è associato alla tossicità gastrointestinale da MP nei bambini affetti da LLA e all'efficacia dell'azatioprina nei pazienti pediatrici affetti da MICI. PACSIN2 è coinvolto nel traffico vescicolare e interagisce con Rac1, una proteina ad attività GTPasica che regola l'organizzazione citoscheletrica e che rappresenta un bersaglio molecolare per la classe dei farmaci tiopurinici. PACSIN2 può influenzare il rilascio e il contenuto delle vescicole extracellulari (VE), che mediano la comunicazione cellulare e il cui carico modifica i fenotipi delle cellule bersaglio. Questo studio valuta i meccanismi che associano il polimorfismo di PACSIN2 alla variabilità interindividuale dell'efficacia delle tiopurine, considerando gli effetti della modulazione di PACSIN2 sulla citotossicità di MP, le proprietà biomeccaniche, il rilascio e il contenuto di VE.
Gli effetti del knock-down (KD) di PACSIN2 sono stati valutati in cellule intestinali LS180. Il saggio di citotossicità MTT è stato utilizzato per verificare la sensibilità al MP in entrambe le linee cellulari, quindi le proprietà biomeccaniche, l'espressione della proteina Rac1 e la sua attività GTPasica sono state studiate rispettivamente con la microscopia a forza atomica (AFM), il saggio di Western Blot (WB) e il saggio G-LISA in cellule non trattate e dopo esposizione al MP per 24, 48 e 72 ore a IC20, IC50 e IC80. Le VE, rilasciate dalle linee cellulari LS180 MOCK e LS180 KD per PACSIN2, sono state isolate mediante ultracentrifuga e caratterizzate mediante analisi di tracciamento delle nanoparticelle (NTA), microscopia elettronica a scansione (SEM), AFM e WB. Il sequenziamento dei miRNA intracellulari e delle VE è stato eseguito con Illumina Hi-seq 2000. Le VE possono alterare la citotossicità dei farmaci, pertanto le cellule LS180 MOCK e LS180 KD per PACSIN2 sono state co-trattate con MP e VE. L'analisi statistica è stata eseguita mediante t-test e ANOVA.
Le LS180 KD per PACSIN2 erano più sensibili al MP rispetto alle cellule di controllo (MOCK) (IC50 LS180 MOCK: 3,23 μM; IC50 LS180 KD: 2,18 μM).
La concentrazione di proteina Rac1 era maggiore nelle cellule LS180 KD per PACSIN2 rispetto alle LS180 MOCK (fold change KD vs MOCK 20,6; p = 0,0009). MP ha causato una riduzione concentrazione e dose-dipendente dell'espressione della proteina Rac1, dell'attività della GTPasi e della rigidità in entrambe le linee cellulari. Non sono state osservate differenze in termini di rilascio di VE tra le cellule intestinali MOCK e KD per PACSIN2 (MOCK: 9,39 * 1010 ± 6,50 * 109 particelle/mL vs KD: 9,91 * 1010 ± 3,41 * 1010; p = 0,8). Il knock-down di PACSIN2 ha alterato l'espressione intracellulare e delle VE rispettivamente di 6 e 23 miRNA. Le VE rilasciate dalla linea cellulare LS180 KD per PACSIN2 (EV-KD) ha ridotto la citotossicità a MP (circa il 10%) e l'espressione della proteina Rac1 nelle cellule LS180 KD per PACSIN2 (MOCK CTRL: 14,0 ± 5,9; VE-MOCK: 22,6 ± 5,0; VE-KD: 51,72 ± 19,8; KD CTRL: 138,9 ± 67,1; VE-MOCK: 103,4 ± 19,0; VE-KD: 63,3 ± 23,9; p < 0,001).
In conclusione, il knock-down di PACSIN2 aumenta la citotossicità del MP modificando i miRNA delle VE, che potrebbero modulare l'espressione della proteina Rac1. L'associazione dei miRNA candidati con la variabilità interindividuale dell'efficacia delle tiopurine sarà convalidata clinicamente nelle VE isolate dal plasma di pazienti pediatrici affetti da IBD trattati con tiopurine.Thiopurines, such as mercaptopurine (MP), are immunosuppressive agents, used in the treatment of acute lymphoblastic leukemia (ALL) and inflammatory bowel disease (IBD). Thiopurine methyltransferase (TPMT) is an enzyme involved in thiopurines metabolism with activity modulated by Protein Kinase C and Casein Kinase Substrate in Neurons 2 (PACSIN2). Moreover, the PACSIN2 Single Nucleotide Polymorphism (SNP) rs2413739 eQTL is associated with MP-related gastrointestinal toxicity in children with ALL and with azathioprine efficacy in IBD pediatric patients. PACSIN2 is involved in vesicular trafficking and interacts with Rac1, a GTPase regulating cytoskeletal organization, and a molecular target of thiopurines. PACSIN2 may affect the release and content of extracellular vesicles (EVs), which mediate cell communication and whose cargo modifies phenotypes of target cells. This study evaluates mechanisms associating PACSIN2 polymorphism with interindividual variability in efficacy of thiopurines, by considering effects of PACSIN2 modulation on MP cytotoxicity, biomechanical properties and the release and content of EVs.
Effects of stable PACSIN2 knock-down (KD) were evaluated in intestinal LS180 cells. MTT cytotoxicity assay was used to verify MP-sensitivity in both cell lines, then biomechanical properties, Rac1 protein expression and its GTPase activity were investigated respectively by atomic force microscopy (AFM), Western Blot assay (WB) and G-LISA assay in untreated cells and after MP exposure for 24, 48 and 72 hours at IC20, IC50 and IC80. EVs, released by LS180 MOCK and KD were isolated by ultracentrifuge and characterized by nanoparticle tracking analysis (NTA), scanning electron microscopy (SEM), AFM and WB. Intracellular and EVs miRNA-sequencing was performed by Illumina Hi-seq 2000. EVs may alter drug cytotoxicity, therefore LS180 MOCK and KD cells were co-treated with MP and EVs. Statistical analysis was performed using t-test and ANOVA.
LS180 PACSIN2 KD were more sensitive to MP than control cells (MOCK) (IC50 LS180 MOCK 3.23 μM; IC50 LS180 KD 2.18 μM). Rac1 protein concentration was higher in LS180 KD cells compared to LS180 MOCK (fold change KD vs MOCK 20.6; p = 0.0009).
MP caused a concentration- and dose-dependent reduction of Rac1 protein expression, GTPase activity and stiffness in both cell lines.
No differences were observed in terms of release of EVs between MOCK and KD cells (MOCK, 9.39 * 1010 ± 6.50 * 109 particles/mL vs KD 9.91 * 1010± 3.41 * 1010; p = 0.8). PACSIN2 KD altered intracellular and EVs expression of 6 and 23 miRNAs respectively. EV-KD reduced MP cytotoxicity (about 10%): LS180 KD cell lines became more resistent than LS180 MOCK cell line. Moreover, EV-KD reduced Rac1 protein expression in LS180 KD cells (MOCK CTRL: 14.0 ± 5.9; EVs-MOCK: 22.6 ± 5.0; EVs-KD: 51.72 ± 19.8; KD CTRL: 138.9 ± 67.1; EVs-MOCK: 103.4 ± 19.0; EVs-KD: 63.3 ± 23.9; p < 0.001).
In conclusion, PACSIN2 KD increases MP cytotoxicity by modifying EVs miRNAs, which could modulate Rac1 protein expression. The association of candidate miRNAs with interindividual variability in the efficacy of thiopurines will be clinically validated in EVs isolated from plasma of IBD pediatric patients treated with thiopurines
Extracellular Vesicles as Innovative Tools for Assessing Adverse Effects of Immunosuppressant Drugs
No full textExtracellular vesicles (EVs) are a heterogeneous family of small vesicles released by donor cells and absorbed by recipient cells, which represent important mediators with fundamental roles in both physiological and pathological conditions. EVs are present in a large variety of biological fluids and have a great diagnostic and prognostic value. They have gained the interest of the scientific community due to their extreme versatility. In fact, they allow us to hypothesize new therapeutic strategies since, in addition to being cell signal mediators, they play an important role as biomarkers, drug vehicles, and potential new therapeutic agents. They are also involved in immunoregulation, have the ability to transmit resistance to a drug from one cell to a more sensitive one, and can act as drug delivery systems
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
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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