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Studio del ruolo dei miRNA nella patogenesi dell'epatocarcinoma hcv-correlato
No full textBACKGROUND:
MicroRNAs (miRNAs) are non-coding RNAs whose role in gene regulation has been discovered and gradually clarified in recent years.
The action of miRNAs, like that of other types of non-coding RNA (commonly called RNA-interference), is based on their physical interaction with gene transcripts (mRNA), and the inhibition or decrease of protein production from them. The biological effect of miRNAs depends on the nature of their targets.
Currently, there is not a precise map of the targets for each of about 700 human miRNAs, but some functional genomics studies have already shown for some of them a direct involvement in the pathogenetic mechanisms of many diseases including cancer.
Hepatocellular carcinoma (HCC) is one of the most aggressive human tumors and both its early detection and even more its therapy remain somewhat problematic. On the other hand, factors that cause and promote its development (hepatitis viruses, alcohol abuse are the most common) are also constantly present and potentially increasing in the population.
Many reasons can then advise the search for new strategies that, starting from scientific analysis, can result in midterm improvements in the health management of the disease. In the case of micro-RNA, several studies have already been carried out showing the existence of a clear pattern of specific miRNA expression in liver affected by HCC, though there currently lacks a clear consensus on the issue, as well as the link with the study of those in clinically distinct but biologically related conditions as chronic hepatitis, liver fibrosis, cirrhosis.
Finally, many scientific evidences advice us to develop and implement quantitative and qualitative analysis on miRNA in tissues from patients with HCC, in order to gather data needed to better understand the pathogenesis of the tumor but also to better study its causes. Moreover, these studies can be exploited in the medium term for the generation of useful biological models for the development of new therapeutic approaches.
OBJECTIVES:
Starting from these assumptions and entering specifically in this study, the objectives are:
- For the methodology point-of-view, the definition of a protocol for the analysis of miRNA in tissue samples, in view of the rapid spread of this kind of research in biomedicine.
- For The scientific point-of-view, the monitoring of the panel of a specific miRNA liver potentially useful for identifying the stages of liver disease progression leading to HCC. This kind of analysis is performed in a comparative context (tumor tissue vs normal tissue) and can provide the basis for future studies focused on molecular and functional interactions between genes and specific miRNAs of interest.
DESIGN of the STUDY
1.Investigation on the expression of a panel of liver-specific and cancer-associated miRNAs in a series of samples from liver cancer patients (mainly HCV-related HCC) by mean of RT-realtime PCR.
2. Analysis of the presence of HCV in tissue samples by PCR and analysis of genotype and subtype of HCV by automatic sequencing, with comparison of the expression profile of miRNAs with virological data.
RESULTS
The results of our studies on HCC, obtained by Real Time PCR with primers specific for a small panel of miRNA, have led us to identify differences in the expression of some of these genes between samples of tumor tissue and samples of adjacent normal tissue. In particular, we confirmed a significant down-regulation of mir-199a, present in almost 70% of the cases studied, while others (including: mir-195, mir-122a, mir-199b) have slightly less frequent but still significant deregulations ( 50-65%). One single miRNA in the selected panel, mir-222, presented a deregulation in the sense of a higher expression in tumor tissue than in normal, in over 40% cases. The other examined micro-RNAs do not show significant differences between the two types of tissue. Slightly different considerations can arise from the comparison of data from HCV positive and negative samples.
These data are consistent with those reported in previous literature, demonstrating the reliability of techniques used.
A parallel activity has focused on the search for HCV viral genomes, and the analysis of viral sequences. Combining the results of the study with the clinical data available, 20 patients out of 29 were HCV-positive. About the viral sequences amplified and analyzed, the alignement with bioinformatic databases gives us the result that 70% of HCV-positive patients belong to the group of subtype 1b, 15% to group 1a, and another 15% to Group 2.
CONCLUSIONS
In conclusion, this study allowed us both to test and develop protocols for analysis to be applied also in other scientific studies, and to collect data on the expression of miRNAs in hepatocellular carcinoma which, although requiring further investigation, appear to be interesting hints of a specific genetic de-regulation of this pathological condition.INTRODUZIONE:
I microRNA (miRNA) sono RNA non codificanti il cui ruolo di regolazione dell’espressione genica è stato scoperto e via via delucidato in anni recenti.
L’azione dei miRNA, non dissimilmente da quella di altre tipologie di RNA non codificanti (comunemente chiamata RNA-interference), si esprime nell’interazione fisica specifica con trascritti genici (mRNA) e nell’impedimento o comunque nella riduzione quantitativa della produzione a partire da questi ultimi di proteine. L’effetto biologico del miRNA dipende pertanto dalla natura del suo bersaglio.
Non è al momento disponibile una mappa precisa dei bersagli di ciascuno dei circa 700 miRNA umani, ma alcuni studi di genomica funzionale hanno già dimostrato per alcuni di essi un coinvolgimento diretto nei meccanismi patogenetici di numerose patologie fra cui il cancro.
L’epatocarcinoma (HCC) è fra i tumori umani uno dei più aggressivi e tanto la sua diagnosi precoce quanto ancor più la sua terapia permangono alquanto problematiche. D’altro canto, i fattori che ne provocano e promuovono l’insorgenza (virus epatitici, abuso di alcool le più comuni) sono a loro volta costantemente presenti nella popolazione e potenzialmente in aumento.
Molte ragioni quindi possono consigliare la ricerca di nuove linee strategiche che, partendo dall’analisi scientifica, possano sfociare nel medio periodo in miglioramenti nella gestione sanitaria della patologia. Nel caso dei micro-RNA, sono già stati effettuati numerosi studi miranti a mostrare l’esistenza di un preciso schema nell’espressione di specifici miRNA nei fegati affetti da HCC, tuttavia manca al momento un chiaro consenso sulla questione, nonché il quanto mai necessario raccordo con lo studio dei medesimi in ambiti clinicamente distinti ma biologicamente attinenti quali epatite cronica , fibrosi epatica, cirrosi.
Concludendo, numerose evidenze scientifiche consigliano di sviluppare e mettere in atto analisi quantitative e qualitative sui miRNA nei tessuti di pazienti affetti da HCC, per poter raccogliere dati necessari ad una miglior comprensione della patogenesi del tumore ma anche per poter studiare meglio le sue cause. Nondimeno, tali studi possono essere sfruttati nel medio periodo per la generazione di modelli biologici utili ai fini dello sviluppo di nuovi approcci terapeutici.
OBIETTIVI:
Partendo da tali presupposti ed entrando nello specifico di questo studio, gli obiettivi sono:
- Sul piano metodologico, la definizione di un protocollo generale per l’analisi dei miRNA in campioni tissutali, nella prospettiva di una rapida diffusione di questo genere di ricerche nell’ambito biomedico.
- Sul piano scientifico, il monitoraggio dell’espressione di un pannello di miRNA specifici per il fegato potenzialmente utili per identificare le tappe della progressione dalla patologia epatica all’HCC. Questo tipo di analisi viene svolta in un contesto comparativo ( tessuto tumorale contro tessuto normale) e può fornire le basi per futuri studi focalizzati su interazioni molecolari e funzionali tra miRNA specifici e geni di interesse.
DISEGNO DELLO STUDIO
1.Indagine sull'espressione di un pannello di miRNA fegato-specifici e associati a tumore in una serie di campioni di pazienti con tumore epatico HCV-associate (principalmente HCC HCV-collegato) tramite metodiche di RT-real time -PCR
2. Analisi della presenza di HCV con PCR nei campioni di tessuto, e analisi del genotipo e sottotipo di HCV mediante sequenziamento automatico; confronto del profilo di espressione di miRNA con i dati virologici.
RISULTATI
I risultati dei nostri studi su HCC, ottenuti mediante Real Time PCR con primers specifici per un ristretto pannello di miRNA, hanno permesso di individuare delle differenze nell’espressione di alcuni di questi geni tra campioni di tessuto tumorale e campioni di tessuto normale adiacente. In particolare abbiamo confermato una significativa sottoespressione di mir-199a, presente in quasi il 70% dei casi analizzati, mentre altri (fra cui: mir-195, mir-122a, mir-199b) presentano deregolazioni leggermente meno frequenti ma pur sempre significative (50-65%). Un solo miRNA del pannello selezionato, mir-222, presenta una deregolazione nel senso di una sovraespressione nel tessuto tumorale rispetto a quello sano, in una percentuale superiore al 40%. Gli altri micro-RNA presi in esame non danno, con questa metodica, differenze significative fra i due tipi di tessuto. Considerazioni leggermente differenti derivano dal confronto dei dati relativi a campioni HCV positivi e negativi.
Questi dati sono compatibili con quelli riferiti nella letteratura pregressa, dimostrando l’attendibilità delle tecniche utilizzate.
L’ attività avviata parallelamente ha riguardato la ricerca di genomi virali di HCV, e successivamente l’analisi delle sequenze virali. Unendo i risultati dell’analisi ai dati clinici disponibili, 20 pazienti su 29 risultano HCV-positivi. Circa le sequenze virali amplificate e analizzate, il confronto coi database bioinformatici ci dà per risultato che il 70% circa dei pazienti HCV-positivi appartiene al gruppo del sottotipo 1b, il 15% al gruppo 1a, e l’altro 15% al gruppo 2.
CONCLUSIONI
In conclusione, questo studio ha permesso sia di testare e mettere a punto protocolli di analisi trasferibili nel breve periodo ad altri studi scientifici, sia di raccogliere dati sull’espressione dei miRNA nell’epatocarcinoma che, pur necessitando di indagini ulteriori, appaiono interessanti indizi di una de-regolazione genetica specifica di questa condizione patologica
In silico approaches to Zika virus drug discovery
No full textAfter the WHO declared Zika virus (ZIKV) as a public health emergency of international concern, intense research for the development of vaccines and drugs has been undertaken, leading to the development of several candidates. Areas covered: This review discusses the developments achieved so far by computational methods in the discovery of candidate compounds targeting ZIKV proteins, i.e. the envelope and capsid structural proteins, the NS3 helicase/protease, and the NS5 methyltransferase/RNA-dependent RNA polymerase. Expert opinion: Research for effective drugs against ZIKV is still in a very early discovery phase. Notwithstanding the intense efforts for the development of new drugs and the identification of several promising candidates by using different approaches, including computational methods, so far only a few candidates have been experimentally tested. An important caveat of anti-flavivirus drug development is represented by the difficult of reproducing the in vivo microenvironment of the replication complex, which may lead to discrepancies between in vitro results and experimental evaluation in vivo. Moreover, anti-ZIKV drugs have the additional requirement of an excellent safety profile in pregnancy and ability to diffuse to different tissues, including the central nervous system, the testis, and the placenta
Zika virus: From pathogenesis to disease control
No full textZika virus is a mosquito-borne flavivirus discovered in Uganda in 1947. The virus has emerged in the recent years and spread in the Pacific Area and the Americas, where it has caused large human outbreaks. Factors involved in virus emergence are still unknown, but probably included the introduction in naïve environments characterized by the presence of high densities of competent Aedes spp. mosquitoes and susceptible human hosts in urban areas. Unique features of Zika virus infection are sexual and transplacental transmission and associated neurological morbidities, i.e. Guillain-Barré syndrome and foetal microcephaly. Diagnosis relies on the detection of viral nucleic acids in biological samples, while detection of a specific antibody response may be inconclusive because of the broad cross-reactivity of antibodies among flaviviruses. Experimental studies have clarified some mechanisms of Zika virus pathogenesis and have identified potential targets for antiviral drugs. In animal models, the virus can infect and efficiently replicate in the placenta and in the brain and induced foetal demise or neural damage, recapitulating human diseases. These animal models have been used to evaluate candidate vaccines and promising results have been achieved
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
Isolation of infectious Zika virus from saliva and prolonged viral RNA shedding in a traveller returning from the Dominican Republic to Italy, January 2016
Full text linkWe report the isolation of infectious Zika virus (ZIKV) in cell culture from the saliva of a patient who developed a febrile illness after returning from the Dominican Republic to Italy, in January 2016. The patient had prolonged shedding of viral RNA in saliva and urine, at higher load than in blood, for up to 29 days after symptom onset. Sequencing of ZIKV genome showed relatedness with strains from Latin America
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
West Nile virus infection in individuals with pre-existing Usutu virus immunity, northern Italy, 2018
Full text linkIn 2018, there was a large West Nile virus (WNV) outbreak in northern Italy. We observed five atypical cases of WNV infection that were characterised by the presence of WNV RNA and WNV IgG at the time of diagnosis, but no IgM response during follow-up. Neutralisation assays demonstrated pre-existing Usutu virus immunity in all patients. Besides challenging diagnosis, the immunological crosstalk between the two viruses warrants further investigation on possible cross-protection or infection enhancement effects
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