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Laura Giusti,
Pietro Iacconi,
Ylenia Da Valle,
Federica Ciregia,
Tiziana Ventroni, Elena Donadio,
Gino Giannaccini,
Massimo Chiarugi,
Liborio Torregrossa,
Agnese Proietti,
Fulvio Basolo,
Antonio Lucacchini
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ABSTRACT: Washing fluid (WF) from the colon rectal tract after surgical resection might represent a first step in obtaining a mixture of proteins derived from the secretion of tumoral epithelial cells potentially involved in the pathological progression of tissue. In this study, we performed a proteomic analysis of colorectal WF to search for potential biomarkers of colon cancer. The outcome of this approach might open the possibility of using WF to screen for the precancerous and early stages of colorectal cancer (CRC). Samples of WFs were obtained during surgery from 35 patients submitted to colon resection for suspicious adenocarcinoma or carcinoma, while the respective controls were obtained by washing the healthy sections. WFs were immediately centrifuged, concentrated and trichloroacetic acid (TCA) was added to obtain protein pellets. After two-dimensional gel electrophoresis (2DE), the protein patterns of malignant samples were compared with respective normal samples. Forty-one protein spots were found to be differentially expressed exhibiting ≥2 fold-change of mean value spot intensities. After mass spectrometry, these protein spots collapsed into 38 different proteins. Interestingly, 19 of the differentially expressed proteins identified in the study corresponded to those suggested as being potential biomarkers of CRC. In accordance with the literature, these proteins showed the same direction of change (up or down for all proteins). Our results suggest that WF has the potential of being a method for the exploration of clinical samples for biomarker and drug target discovery.
Molecular BioSystems 04/2012; 8(4):1088-99. · 3.53 Impact Factor
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ABSTRACT: The aim of this study was to compare two methods used to measure serum cystatin C (Cys) and their accuracy to predict glomerular filtration rate (GFR).
Three hundred and sixty-seven adult chronic kidney disease (CKD) patients with different functional impairments participated in this study. GFR was determined as the renal clearance of 99mTc-DTPA. Serum concentrations of cystatin C (SCys) were determined with an immunonephelometric method and with an immunoturbidimetric method.
A very high linear correlation was found between the two measurements of SCys (r=0.929). The mean difference of SCysTurb-SCysNeph was 0.02±0.43 mg/L (not significant). A high logarithmic correlation was also found between SCys and GFR (r was 0.919 for SCysNeph and 0.937 for SCysTurb). By means of multiple regression analysis, we developed formulae to predict GFR from SCysNeph, SCysTurb and SCr. For comparison, GFR was predicted using published formulae. A good agreement was found between predicted GFR and measured GFR. The results showed that the accuracy of SCysNeph, SCysTurb and SCr and of the different prediction formulae were quite similar.
The immunoturbidimetric method seems adequate to measure SCys and to predict GFR and its impairment in CKD, at least similar to the immunonephelometric method. The accuracy of SCys and of derived formulae was not higher than that of SCr and SCr-based formulae.
Nephrology Dialysis Transplantation 03/2012; 27(7):2826-38. · 3.40 Impact Factor
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Chiara Baldini,
Laura Giusti,
Federica Ciregia,
Ylenia Da Valle,
Camillo Giacomelli, Elena Donadio,
Francesca Sernissi,
Laura Bazzichi,
Gino Giannaccini,
Stefano Bombardieri,
Antonio Lucacchini
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ABSTRACT: A growing interest has arisen in salivary proteomics as a tool for the identification of biomarkers for primary Sjögren's syndrome (pSS). Nonetheless, only a limited number of preclinical validation studies have been performed, limiting the possibility of translating proteomic results into clinical practice. The primary aim of this study was to refine the diagnostic power of a panel of candidate salivary biomarkers described in pSS with respect to both healthy volunteers and pathological controls. We also explored the pathogenetic function of the detected putative biomarkers both in the local exocrinopathy and in the systemic inflammatory processes of SS.
One hundred and eighty patients were included in the study overall. In the first "exploratory phase", we enrolled 40 females with pSS, 40 sex- and age-matched healthy volunteers, 10 patients with sicca non-SS and 15 secondary SS (sSS) patients. The testing cohort of the second "challenge phase" of the study was represented by 75 unselected, consecutive subjects: 19 pSS, 21 healthy volunteers, 10 sicca non-SS and 25 sSS patients. Salivary proteomic analysis was performed combining two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Western blot (WB) analysis and enzyme-linked immunosorbent assay (ELISA) were employed to validate 2DE results. Ingenuity Pathway Analysis (IPA) Knowledge base was adopted to associate candidate biomarkers in a signalling pathogenetic network.
A total of 28, 6, 7 and 12 protein spots were found to be significantly different in pSS samples with respect to healthy volunteers, non-SS sicca syndrome, SSc-sSS and rheumatoid arthritis-sSS, leading to the identification of 15 differently expressed proteins. Among them, α-amylases precursor, carbonic anhydrase VI, β-2 microglobulin, glyceraldehydes-3-phosphate dehydrogenase (G3PDH), epidermal fatty acid binding protein (E-FABP) and immunoglobulin k light chain (IGK-light chain) apparently showed the most significant differences in pSS when compared to healthy volunteers and non-SS pathological controls. On the other hand, as expected, pSS and sSS salivary profiles shared a great number of similarities.
This study demonstrated that salivary fluid might represent a novel ideal milieu for the detection of a diagnostic panel of candidate biomarkers for pSS, and to gain an insight into the pathogenetic processes underlying glandular and systemic autoimmune disorders.
Arthritis research & therapy 11/2011; 13(6):R194. · 4.27 Impact Factor
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ABSTRACT: Albumin is the most important antioxidant substance in plasma and performs many physiological functions. Furthermore, albumin is the major carrier of endogenous molecules and exogenous ligands. This paper reviews the importance of post-translational modifications of albumin and fragments thereof in patients with renal disease. First, current views and controversies on renal handling of proteins, mainly albumin, will be discussed. Post-translational modifications, namely the fragmentation of albumin found with proteomic techniques in nephrotic patients, diabetics, and ESRD patients will be presented and discussed. It is reasonable to hypothesize that proteolytic fragmentation of serum albumin is due to a higher susceptibility to proteases, induced by oxidative stress. The clinical relevance of the fragmentation of albumin has not yet been established. These modifications could affect some physiological functions of albumin and have a patho-physiological role in uremic syndrome. Proteomic analysis of serum allows the identification of over-expressed proteins and can detect post-translational modifications of serum proteins, hitherto hidden, using standard laboratory techniques.
Clinica chimica acta; international journal of clinical chemistry 11/2011; 413(3-4):391-5. · 2.54 Impact Factor
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Chiara Baldini,
Laura Giusti,
Federica Ciregia,
Ylenia Da Valle,
Camillo Giacomelli, Elena Donadio,
Francesco Ferro,
Sara Galimberti,
Valentina Donati,
Laura Bazzichi,
Stefano Bombardieri,
Antonio Lucacchini
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ABSTRACT: In the last years human proteomic has represented a promising tool to promote the communication between basic and clinical science.
To explore the correspondence between salivary proteomic profile and clinical response, herein, we used a proteomic approach to analyse the whole saliva of a patient with primary Sjögren's Syndrome (pSS) and non-Hodgkin's-MALT type parotid lymphoma before, during and after a standard treatment with cyclophosphamide (CTX) and rituximab (RTX). To identify any discriminatory therapeutic salivary biomarker patient's whole saliva was collected at the baseline, after the fourth infusion of rituximab, and on remission and analysed combining two-dimensional electrophoresis (2DE) and MALDI-TOF/TOF mass spectrometry.
Proteomic results obtained from the comparison of salivary samples indicated several qualitative and quantitative modifications in the salivary expression of putative albumin, immunoglobulin J chain, Ig kappa chain C region, alpha-1-antitrypsin, haptoglobin and Ig alpha-1 chain C region.
This study suggests that clinical and functional changes of the salivary glands driven by autoimmune and lymphoproliferative processes might be reflected in patients' whole saliva proteins, shedding new light on the potential usefulness of salivary proteomic analysis in the identification of prognostic and therapeutic biomarkers for patients with pSS and non Hodgkin's lymphomas.
Journal of Translational Medicine 11/2011; 9:188. · 3.41 Impact Factor
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Laura Giusti,
Filomena Cetani,
Federica Ciregia,
Ylenia Da Valle, Elena Donadio,
Gino Giannaccini,
Chiara Banti,
Elena Pardi,
Federica Saponaro,
Fulvio Basolo,
Piero Berti,
Paolo Miccoli,
Aldo Pinchera,
Claudio Marcocci,
Antonio Lucacchini
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ABSTRACT: Parathyroid tumours are heterogeneous and in some cases the diagnosis may be difficult using histological features. In this study we used a two-dimensional electrophoresis (2D)/mass spectrometry (MS)-based approach to examine the global changes of parathyroid adenoma tissues protein profile compared to the parathyroid normal tissues. Validation of protein expression was performed by immunoblotting using specific antibodies. Ingenuity software was used to identify the biological processes to which these proteins belong and to construct a potential network. A total of 30 proteins were found to be differentially expressed, of which 22 resulted in being over-expressed. Proteins identified by 2D/MS/MS proteomics were classified into functional categories and a major change (≥ 2-fold) in terms of expression was found in proteins involved in response to biotic stimuli, cell organization and signal transduction. After Ingenuity analysis, 14-3-3 ζ/δ appears to be a key protein in the network of parathyroid adenoma, where it is linked to other proteins such as annexin A2, B box and SPRY domain-containing protein (BSPRY), p53 and epidermal growth factor receptor (EGFR). Our results suggest that the proteomic approach was able to differentiate the protein profiles of normal parathyroid and parathyroid adenoma and identify a panel of proteins which are differentially expressed. The functional role of these proteins in the network of intracellular pathways is discussed.
Molecular BioSystems 03/2011; 7(3):687-99. · 3.53 Impact Factor
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Elena Donadio,
Laura Giusti,
Filomena Cetani,
Ylenia Da Valle,
Federica Ciregia,
Gino Giannaccini,
Elena Pardi,
Federica Saponaro,
Liborio Torregrossa,
Fulvio Basolo,
Claudio Marcocci,
Antonio Lucacchini
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ABSTRACT: Proteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification.
The best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin.
In spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining.
Proteome Science 01/2011; 9(1):29. · 2.33 Impact Factor
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ABSTRACT: The goal of this study was to detect modification in the expression of plasma proteins and/or post-translational modifications of their structure in patients with end stage renal disease.
Serum samples from 19 adult patients treated by maintenance hemodialysis (MHD) were analyzed in comparison to sera from six healthy controls using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Spots of interest were identified by mass spectrometry analysis. In addition, the 2DE maps were incubated with a human anti-albumin polyclonal antibody.
SDS-PAGE gels, 2DE maps and matrix-assisted laser desorption/ionization time of flight analysis indicated over-expression of low-molecular weight proteins (LMWP) in sera from patients. Unexpectedly, another 15 spots with estimated M(r) of 12.5-29 kDa from the 2DE maps of six patients were identified as fragments of albumin. 2D immunoblotting of sera from 12 other patients detected numerous albumin fragments.
These results indicate that in addition to increased expression of LMWP, a relevant amount of albumin fragments are detectable in the serum of patients undergoing MHD. Uremia appears to facilitate the fragmentation of albumin and/or the retention of albumin fragments in blood.
Clinical Chemistry and Laboratory Medicine 09/2009; 47(11):1373-9. · 2.15 Impact Factor
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ABSTRACT: An expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process.
FEMS Microbiology Letters 05/2007; 269(1):77-84. · 2.04 Impact Factor
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ABSTRACT: Cancer and treatment by chemotherapy often produce abnormalities in endogenous cytokine, chemokine, and inflammatory mediator production. Sorbent-based adsorption therapies have been used to remove cytokines in diverse human diseases.
The aim of this study was to evaluate the effects of chemotherapy on serum proteomic pattern and cytokine concentration, and to evaluate the ex vivo feasibility of using sorbents to remove cytokines, chemokines and other proteins in adult cancer patients undergoing chemotherapy with fluorouracil or carboplatin-taxane combinations.
Serum samples of three female adult patients (one affected by rectal cancer and two by ovarian cancer) were examined before and on the fourth day of the first cycle of chemotherapy with fluorouracil (rectal cancer patient) or carboplatin-taxane combination (ovarian cancer patients). The analysis was performed, by means of luminex technology and with a proteomic approach, on native serum samples, and on the same sera after 2 hours of in vitro incubation with a synthetic based styrenic divinylbenzene resin.
Chemotherapy determined variable effects on serum concentration of cytokines, while the incubation in vitro of patients serum samples with the resin induced a significant decrease (>80%) in serum concentration of different chemokines, cytokines, growth factors and proteins. The proteomic approach, using SDS PAGE and 2-DE highlighted differences in protein expression between sera from healthy controls and cancer patients. Proteomic analysis demonstrated also variations in the expression of proteins, particularly those with low-molecular weight, due to chemotherapy. Finally, the incubation in vitro of serum samples with sorbents induced a general reduction of protein expression. Within the cancer patients maps, 10 spots were chosen for identification with MALDI-TOF analysis.
The incubation in vitro with sorbents normalized the over-expression of different proteins and cytokines induced by chemotherapy, suggesting further evaluation as a possible adjuvant treatment.
Biomedecine [?] Pharmacotherapy 10/2006; 60(8):463-7. · 2.00 Impact Factor
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ABSTRACT: The recent availability of the honey-bee Apis mellifera genome and trascriptome of both the female castes, has stimulated new efforts in investigating the protein composition of royal jelly (RJ), its role in caste differentiation and its quality and typicality by a proteomic approach. This study is aimed both to separate and identify proteins of royal jelly and to detect some of them in honey-bee pollen-bread by using two-dimensional gel electrophoresis, mass spectrometry and by de novo sequencing. All the identified proteins belonged to the Apis mellifera genome. Apalbumin 1 was also confirmed to be present in honey-bee pollen-bread where the presence of apalbumin 2 was also found. In addition several fragments of apalbumin 1 and apalbumin 3 were also found in RJ. These could be the result of protease activity other than that of serine-protease. This study is a contribution to the description of royal jelly proteome.
PROTEOMICS 03/2005; 5(3):769-76. · 4.51 Impact Factor
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ABSTRACT: The aim of this study was to evaluate the relationship between serum levels of beta-trace protein (BTP), a low molecular weight (MW) protein, and glomerular filtration rate (GFR). GFR and serum levels of BTP, and for comparison creatinine (Creat), cystatin C (Cys) and beta 2-microglobulin (beta 2M), were measured in 60 patients, with renal function ranging from normality to advanced renal failure. Serum levels of BTP progressively increased with the reduction of GFR. A good correlation was found between GFR and serum levels of BTP (r=0.918), Creat (r=0.932), Cys (r=0.937), and beta 2M (r=0.924). Furthermore, no statistically significant difference was found between BTP and Creat, Cys, beta 2M, as indicators of a moderate GFR impairment. These preliminary data indicate that BTP might be suitable as an indicator of GFR.
Journal of Pharmaceutical and Biomedical Analysis 09/2003; 32(4-5):1099-104. · 2.97 Impact Factor
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ABSTRACT: RIASSUNTO
L’allevamento in pieno campo di Osmia cornuta è ancora necessario a causa della mancanza
di informazioni sui meccanismi molecolari che regolano la nutrizione di tali apoidei.
Lo scopo di questo studio è stato quello di dare un contributo nella descrizione della variazione
del pattern proteasico così da dirottare, in parte, le strategie di allevamento in pieno
campo verso quelle di laboratorio.
Da celle pedotrofiche prelevate da un nido di Osmia sono stati campionati gli stadi larvali
I, III, V e V maturo di O. cornuta. Le proteine delle larve sono state estratte in presenza
di un tampone nativo, così da preservare l’eventuale presenza dell’attività enzimatica delle
proteasi; l’omogenato così ottenuto è stato centrifugato e il surnatante analizzato mediante
elettroforesi per attività. Il gel è stato preparato facendo copolimerizzare l’acrilammide in
presenza di gelatina, e dopo la corsa il gel è stato incubato per 1 ora a pH 8 a 37°C. Infine il
gel è stato colorato con Blue di Coomassie.
L’analisi degli zimogrammi rileva che il pattern proteasico varia durante l’ontogenesi larvale, in
particolare nel I stadio larvale sono risultate ben visibili 3 bande di attività proteasica, nel III stadio
tali bande sono diventate 4, nel V stadio larvale sono risultate visibili ancora 4 bande di attività,
di cui 3 in comune con il terzo stadio, infine nel V stadio larvale maturo è visibile una sola
banda di attività.
Questo approccio molecolare può dare un contributo allo studio dei meccanismi di digestione
di questo apoideo solitario nell’ottica della messa a punto di una dieta artificiale.
SUMMARY
Field rearing of Osmia cornuta is still necessary due to lack of knowledge in nutrition
molecular mechanisms. The aim of this investigation was to give a contribute in describing
the changes in the expression of the proteinase pattern in order to direct the osmia bee rearing
strategy from nest-trapping (NT) and releasing-and-rearing (RR), towards more controlled conditions such as the laboratory (captivity), whilst optimising their use for crop pollination
in both open field and confined environments.
I, III, V and mature V instar larvae of Osmia cornuta were sampled from a pedotrophic
nest. Proteins were extracted in a native buffer, in order to preserve the proteolytic activity;
homogenate was centrifuged and the surnatant was analysed by zymography. Acrylamide gel
has been precasted with gelatine. After electrophoresis, gel was incubated for at 37°C 1 hour
in a pH 8 buffer. After incubation gel was stained with Blue Coomassie.
Zymograms show that proteinase pattern change during larval ontogenesis. In particular,
in the I instar 3 activity bands are well detectable, in the III and V instar 4 bands are
detectable, of these 3 are in common, in the V mature instar only one band is detectable.
This molecular approach could help in investigating the digestion mechanisms of this
solitary bee in order to gain more information to project an artificial diet.
