1,721,004 research outputs found
SVILUPPO DI METODI IFENATI ASSOCIATI ALLA SPETTROMETRIA DI MASSA MALDI: APPLICAZIONI PROTEOMICHE E GLICOSFINGOLIPIDOMICHE.
Full text linkL’interesse per lo sviluppo di nuovi metodi e per l’applicazione di tecniche ifenate è
generato dalla necessità di caratterizzare campioni biologici complessi. Questo
lavoro affronta l’applicazione e la messa a punto di tecniche ifenate allo scopo di:
a) identificare un nuovo biomarcatore da associare al PSA (Prostate Specific
Antigen) per la diagnostica del cancro prostatico, b) sviluppare una nuova tecnica
ifenata per l’analisi di glicosfingolipidi.
Negli studi di proteomica clinica, destinati all’identificazione di nuovi biomarcatori, i
maggiori problemi sono legati all’incompatibilità dell’intervallo di sensibilità degli
strumenti di rivelazione con l’intervallo di concentrazione delle specie proteiche e
peptidiche presenti nei fluidi biologici. Pertanto, poiché l’intervallo di sensibilità
degli strumenti di rivelazione non può essere variato, il passaggio cruciale è
rappresentato dalla riduzione dell’intervallo di concentrazione tra le specie più
abbondanti e le specie meno abbondanti nei fluidi biologici in esame. Nel caso del
siero ciò è stato ottenuto grazie all’eliminazione della frazione contenente le
proteine a maggior abbondanza mediante immunodeplezione selettiva. Le frazioni
seriche a bassa abbondanza preparate da 30 pazienti affetti da cancro alla
prostata (ADK) e da 30 soggetti controllo sono state analizzate tramite MALDI
profiling. La metodica applicata ha permesso di individuare delle differenze
statisticamente significative nell’espressione di alcune componenti proteiche,
rivelate dalla presenza di picchi differenzialmente espressi negli spettri di massa
ottenuti. Tuttavia, il punto fondamentale per il trasferimento dei risultati ottenuti in
diagnostica clinica è rappresentato dall’identificazione delle specie proteiche
contenute nel profilo di massa MALDI al fine di poter utilizzare tecniche
immunometriche per la validazione dei dati e sviluppare metodi di dosaggio su
larga scala applicabili in diagnostica clinica. Grazie all’ifenazione tra tecniche
cromatografiche, quali la cromatogafia per gel filtrazione e la cromatografia a fase
inversa, condotte rispettivamente su uno strumento HPLC e su uno strumento
nano LC, e tecniche di spettrometria di massa MALDI, è stato possibile identificare
il picco 2021 m/z, statisticamente variato, che corrisponde al peptide C3f, derivato
dalla proteolisi del fattore del complemento C3, e la sua aumentata espressione in
pazienti ADK+ a bassi valori di PSA, è stata confermata attraverso un esperimento
di immunoblotting. I risultati sono stati validati su un secondo gruppo di pazienti e
controlli. L’aumento di tale peptide indica una deregolazione del fattore del
complemento C3 che viene inattivato a iC3b, il quale inibisce il ciclo della via
alternativa del sistema del complemento impedendo l’associazione del fattore C3b
al fattore B. Il peptide C3f potrebbe pertanto essere usato in associazione con il
valore serico di PSA per ridurre il numero di pazienti “falsi negativi”.
Un altro aspetto importante della ricerca biomedica e indirettamente della
diagnostica clinica è rappresentato da una classe di molecole di segnale che si
sono rivelate importanti in numerose patologie quali malattie cardiovascolari e
neurodegenerative, tumori, malattie d’accumulo lisosomiale e sindrome
metabolica. Nonostante il crescente interesse clinico, l’analisi dei glicosfingolipidi
non gode di tecnologie ad alta automazione che permettano una facile
identificazione e quantizzazione di specie con pesi molecolari spesso molto simili.
Attualmente tale caratterizzazione avviene tramite l’utilizzo di precursori radioattivi,
oppure di coloranti che non permettono una quantizzazione precisa, o di metodi
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LC-MS di grande precisione ma dispendiosi in termini di tempo e di coinvolgimento
dell’operatore.
Attraverso l’ifenazione diretta HPTLC-MALDI, è stato possibile effettuare una fine
analisi qualitativa e quantitativa del profilo glicosfingolipidomico estratto da
mioblasti murini, distinguendo per ogni glicosfingolipide tutte le varianti a diversa
catena di acido grasso presenti. Un aspetto importante è che lo sviluppo
metodologico è stato accoppiato ad una analisi di campioni biologici i cui risultati
sono stati confrontati con quelli precedentemente ottenuti attraverso l’utilizzo di
precursori radioattivi e successiva analisi densitometrica su glicosfingolipidi estratti
da mioblasti wild-type e overesprimenti la sialidasi NEU3. Pertanto, l’ifenazione
proposta si è rivelata di grande applicabilità alla caratterizzazione dei
glicosfingolipidi in quanto riduce i passaggi analitici necessari evitando l’uso di
precursori radioattivi e di procedure di estrazione della banda dal gel di silice, e
potrà essere applicata allo studio di diverse condizioni fisiologiche e patologiche
per identificare potenziali biomarcatori.The interest for the development of new methods and the application of
hyphenated techniques for complex biological samples characterization is growing,
nowadays, more and more. This work faces the application and setup of
hyphenated techniques with two aims: a) to identify a new biomarker that can be
associated to PSA (Prostate Specific Antigen) for prostate cancer (ADK) diagnosis,
b) to develop a hyphenated technique for glycosphingolipids analysis.
In clinical proteomics, the identification of new biomarkers is hampered by the
discrepancy between the quantitative range of detection of instruments and the
concentration range of proteins and peptides in biological fluids. Since the
quantitative range of detection of instruments cannot be changed, the critical point
is to reduce the dynamic range in examined biological fluids. In serum, this can be
obtained by the elimination of high abundant proteins thanks to a selective
immunodepletion. The serum low abundant protein fraction from 30 ADK patients
and 30 controls was analyzed by MALDI profiling. The applied methodology was
able to detect differentially abundant peaks in spectra of ADK vs no ADK controls.
To transfer the obtained results in clinical diagnostic, the identification of species
revealed by MALDI profiles is mandatory for two main reasons: data validation
through the use of immunometric techniques and methodological development for
a large scale test. Thanks to the hyphenation between chromatografic techniques,
such as gel filtration on a HPLC and reverse phase on a nanoLC, and MALDI mass
spectrometry, we were able to identify the highly changed 2021 m/z peak, that
corresponds to C3f peptide and that derives from the proteolyses of Complement
C3b. To confirm its increment in ADK patients with low PSA values an
immunoblotting experiment was conducted. The results were validated on a
second group of patients and controls. The increase of C3f expression could be
related to a deregulation of C3 complement, cleft to iC3b, that inhibits the selffeeding
cycle of the alternative pathway hampering the association of C3b to factor
B. Therefore C3f peptide could be used in association to PSA serum value to
decrease the number of “false negative”.
An important aspect of the biomedical research and, indirectly, of clinical diagnostic
is represented by a class of signal molecules that are important in a number of
diseases, including neurological and cardiovascular disorders, cancer, lipid storage
diseases, and the metabolic syndrome. Despite the increasing clinical interest,
glycosphingolipids (GSLs) analysis cannot benefit by high-throughput techniques
that allow an easy identification and quantitation of these species, characterized by
similar molecular weights. Actually, their characterization is based on radioactive
counts after metabolic radiolabeling, or on staining with proper dyes that doesn’t
allow a precise quantitation. New approaches including LC-MS are largely used but
they are time consuming and operator dependent.
We develop the direct hyphenation HPTLC-MALDI, to obtain a precise qualitative
and quantitative analysis of the glycosphingolipids profile from murine myoblasts,
and to distinguish every fatty acid chain variant for each GSL. An important aspect
is that the methodological setup was coupled to biological samples analysis and
results were compared to those obtained through the use of radioactive precursors
and densitometric analysis on GSLs extracted from wild type and NEU3
overexpressing murine myoblasts. The proposed hyphenation is of great
applicability to the glycosphingolipids characterization because it reduces the
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analytical steps, it avoids the use of radioactive precursors and silica scraping, and
it could be applied to the study of different physiological and pathological
conditions to identify potential biomarkers
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
La tecnica ELISA applicata alla diagnosi delle malattie infettive di interesse veterinario
No full textHPTLC-MALDI MS for (glyco)sphingolipid multiplexing in tissues and blood: A promising strategy for biomarker discovery and clinical applications
No full textSphingolipids have hydrophilic and hydrophobic properties, different saturation and combination of the oligosaccharide chains and mass homology of species located in a narrow m/z region hampering their recognition. To target sphingolipids for diagnostic purposes, standardized methods for lipid extraction, quali- and quantitative assessments are required. In this study, HPTLC-MALDI MS was adopted to establish sphingolipid and glycosphingolipid profiles in muscle, brain and serum to create a database of molecules to be searched in the preclinical and clinical investigations. Specific protocols for lipid extraction were set up based on the characteristics of the tissue or/and fluids; this approach maximizes the HPTLC-MALDI MS analytical throughput both for lipids extracted in organic and aqueous phase. This study indicates that alkaline hydrolysis is necessary for the detection of low abundant species such as Gb3Cer and ceramides in serum and Gb4Cer, CerP and HexCer in muscle tissue. The high hydrophobicity of ceramides has been overcome by the development of HPTLC plate in chloroform:methanol/50:3.5, which increases the number and the intensity of low abundant Cer species. MS/MS analysis has been conducted directly on HPTLC plate allowing the molecular recognition; furthermore a dataset of spectra was acquired to create a database for future profiling of these molecules
Set-up for human sera MALDI profiling: the case of rhEPO treatment
No full textThe implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology even though the serum high dynamic range imposes some limitations, preventing detection and identification of less abundant species. Efforts to increase the MALDI profiling detection ability are needed. A set-up has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (α-cyano-4-hydroxycinnamic acid and 2’,4’-dihydroxyacetophenone) for spectra quality improvement.
Immunodepletion skills of both columns were determined by 2D-DIGE, which precisely revealed the efficacy of Hu14 in protein removal and in serum dynamic range decrement.
After optimization of the type of matrix and sample dilution, these new efficient conditions were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that combination of Hu14 column and 2’,4’-dihydroxyacetophenone matrix increases data quality allowing to discriminate between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO
Set-up for MALDI profiling of human sera: the case of rhEPO teatment
No full textThe implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. To this purpose, MALDI profiling appears a robust and sensitive technology even though the high dynamic range imposes a major limitation a hampering the identification of less abundant species and decreasing the quality of MALDI profiling for clinical implementations. A number of methodologies have been developed to overcome this issue and in this study the efficacy and reproducibility of immunodepletion for human serum profiling was investigated. Two commercially available columns (MARS Hu7 and Hu14) were tested. The effects of both on serum were assessed by 2D-DIGE: the comparison of eluted fraction from Hu14 versus Hu7 revealed the presence of a different protein distribution and of 128 differentially changed spots (p-value<0.01), out of them 114 were identified by mass spectrometry. In particular, 34 spots strongly decreased in Hu14 sample correspond to proteins and their isoforms expected to be significantly reduced by this column. In addition, the type of matrix and the sample dilution, representing crucial elements for MALDI profiling improvement, were determined. Finally, the selected experimental conditions were applied to sera profiling of 8 subjects before and after erythropoietin treatment. The results indicate that a combination of Hu14 column immunodepletion and DHAP matrix increases data quality and appears an appropriate approach for human serum profiling by MALDI
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
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