Elisabetta Pisano | Università degli Studi di Cagliari (original) (raw)

Papers by Elisabetta Pisano

Molecular &amp Cellular Proteomics

People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health p... more People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Since saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10-17 yrs and 18-50 yrs, was analyzed by a top-down proteomic approach, based on the HPLC-ESI-MS analysis of the intact proteins/peptides, and the qualitative and quantitative profiles were compared with sex and age-matched control groups. The results showed the following interesting features: i) differently from controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase wit...

This study assesses human papillomavirus (HPV) detection and genotyping in self-sampled genital s... more This study assesses human papillomavirus (HPV) detection and genotyping in self-sampled genital smears applied to an indicating FTA elute cartridge (FTA cartridge). The study group consisted of 96 women, divided into two sample sets. All samples were analyzed by the HPV SPF 10 -Line Blot 25. Set 1 consisted of 45 women attending the gynecologist; all obtained a self-sampled cervicovaginal smear, which was applied to an FTA cartridge. HPV results were compared to a cervical smear (liquid based) taken by a trained physician. Set 2 consisted of 51 women who obtained a self-sampled cervicovaginal smear at home, which was applied to an FTA cartridge and to a liquid-based medium. DNA was obtained from the FTA cartridges by simple elution as well as extraction. Of all self-obtained samples of set 1, 62.2% tested HPV positive. The overall agreement between self-and physician-obtained samples was 93.3%, in favor of the self-obtained samples. In sample set 2, 25.5% tested HPV positive. The overall agreement for high-risk HPV presence between the FTA cartridge and liquid-based medium and between DNA elution and extraction was 100%. This study shows that HPV detection and genotyping in self-obtained cervicovaginal samples applied to an FTA cartridge is highly reliable. It shows a high level of overall agreement with HPV detection and genotyping in physician-obtained cervical smears and liquid-based self-samples. DNA can be obtained by simple elution and is therefore easy, cheap, and fast. Furthermore, the FTA cartridge is a convenient medium for collection and safe transport at ambient temperatures. Therefore, this method may contribute to a new way of cervical cancer screening.

European journal of oral sciences, 2005

The acidic-soluble protein content of human gingival crevicular fluid was analyzed by reverse-pha... more The acidic-soluble protein content of human gingival crevicular fluid was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), and the eluent deriving from the chromatography separation was directly introduced into an ion-trap mass spectrometer through electrospray ionization (ESI-IT MS). By this technique the molecular weight of peptides/proteins was determined with a precision of approximately 1/10,000 amu. On the basis of the chromatographic behavior and the knowledge of the molecular mass value, some peptides and proteins soluble in acidic solution were unambiguously recognized. Besides high quantities of human serum albumin, alpha-defensins 1-4 and minor amounts of cystatin A, statherin, basic PB salivary peptide and other unidentified components were detected. The presence of alpha-defensins in gingival crevicular fluid is in agreement with their relevant contribution to protein composition deriving from granulocyte secretions. Other peptides and protein...

Journal of Proteome Research, 2015

An important contribution to the variability of any proteome is given by the time dimension that ... more An important contribution to the variability of any proteome is given by the time dimension that should be carefully considered to define physiological modifications. To this purpose, whole saliva proteome was investigated in a wide age range. Whole saliva was collected from 17 preterm newborns with a postconceptional age at birth of 178-217 days. In these subjects sample collection was performed serially starting immediately after birth and within about 1 year follow-up, gathering a total of 111 specimens. Furthermore, whole saliva was collected from 182 subjects aged between 0 and 17 years and from 23 adults aged between 27 and 57 years. The naturally occurring intact salivary proteome of the 316 samples was analyzed by low- and high-resolution HPLC-ESI-MS platforms. Proteins peculiar of the adults appeared in saliva with different time courses during human development. Acidic proline-rich proteins encoded by PRH2 locus and glycosylated basic proline-rich proteins encoded by PRB3 locus appeared following 180 days of postconceptional age, followed at 7 months (±2 weeks) by histatin 1, statherin, and P-B peptide. The other histatins and acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (±3 months), and basic proline-rich proteins appeared at 4 years (±1 year) of age. All of the proteinases involved in the maturation of salivary proteins were more active in preterm than in at-term newborns, on the basis of the truncated forms detected. The activity of the Fam20C kinase, involved in the phosphorylation of various proteins, started around 180 days of postconceptional age, slowly increased reaching values comparable to adults at about 2 years (±6 months) of age. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns.

BMC infectious diseases, 2006

The aim of our study is to describe a fast molecular method, able to distinguish and quantize the... more The aim of our study is to describe a fast molecular method, able to distinguish and quantize the two different genotypes (652 and JP2) of an important periodontal pathogen: Actinobacillus actinomycetemcomitans. The two genotypes show differences in the expression of an important pathogenic factor: the leukotoxin (ltx). In order to evidence this, we performed a real time PCR procedure on the ltx operon, able to recognize Aa clinical isolates with different leukotoxic potentials. The specificity of the method was confirmed in subgingival plaque and saliva specimens collected from eighty-one Italian (Sardinian) subjects with a mean age of 43.9, fifty five (68 %) of whom had various clinical forms of periodontal disease. This procedure showed a good sensitivity and a high linear dynamic range of quantization (10(7)-10(2) cells/ml) for all genotypes and a good correlation factor (R2 = 0.97-0.98). Compared with traditional cultural methods, this real time PCR procedure is more sensitive;...

The Open Virology Journal, 2010

Objective: HIV-positive women are known to be at high-risk of human papillomavirus (HPV) infectio... more Objective: HIV-positive women are known to be at high-risk of human papillomavirus (HPV) infection and its associated cervical pathology. Here, we describe the prevalence and distribution of HPV genotypes among HIV-positive and -negative women in South Africa, with and without cervical intraepithelial neoplasia (CIN).

Molecular & Cellular Proteomics, 2010

The acidic soluble fraction of whole saliva of type 1 diabetic children was analyzed by reversed ... more The acidic soluble fraction of whole saliva of type 1 diabetic children was analyzed by reversed phase (RP)(1)-HPLC-ESI-MS and compared with that of sex- and age-matched control subjects. Salivary acidic proline-rich phosphoproteins (aPRP), histatins, α-defensins, salivary cystatins, statherin, proline-rich peptide P-B (P-B), beta-thymosins, S100A8 and S100A9*(S100A9* corresponds to S100A9 vairant lacking the first four amino acids), as well some naturally occurring peptides derived from salivary acidic proline-rich phosphoproteins, histatins, statherin, and P-B peptide, were detected and quantified on the basis of the extracted ion current peak area. The level of phosphorylation of salivary acidic proline-rich phosphoproteins, histatin-1 (Hst-1), statherin and S100A9* and the percentage of truncated forms of salivary acidic proline-rich phosphoproteins was also determined in the two groups. The study revealed that statherin, proline-rich peptide P-B, P-C peptide, and histatins, were significantly less concentrated in saliva of diabetic subjects than in controls, while concentration of α-defensins 1, 2 and 4 and S100A9* was higher. The low concentration of P-C peptide was paralleled by high levels of some of its fragments. On the whole, the study highlighted the severe impairment of the repertoire of peptides involved in the safeguard of the oral cavity in children who have diabetes, as well as an higher concentration of the proinflammatory mediator S100A9* with respect to healthy children.

Journal of Separation Science, 2012

This study describes the characterization of the glycan moieties and the peptide backbone of six ... more This study describes the characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1 + , a basic proline-rich protein present in human saliva. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1 + and the variant IB-8a CON1 − , lacking of the glycosylation site, have been also detected in human saliva.

Journal of Separation Science, 2009

HPLC-ESI-MS analysis of oral human fluids reveals that gingival crevicular fluid is the main sour... more HPLC-ESI-MS analysis of oral human fluids reveals that gingival crevicular fluid is the main source of oral thymosins b 4 and b 10 Thymosin b 4 (Tb 4 ), its sulfoxide, and thymosin b 10 (Tb 10 ) were detected in human saliva and identified by different strategies based on RP HPLC coupled to electrospray multidimensional IT MS. Tb 4 was almost always detected in whole saliva, its sulfoxide sporadically, Tb 10 rarely. Tb 4 was undetectable in parotid saliva and less concentrated in submandibular/sublingual saliva than in whole saliva. Analysis of gingival crevicular fluid revealed high relative amounts of Tb 4 , Tb 4 sulfoxide, and Tb 10 in all the samples. Tb 4 mean concentration was 200 times higher in crevicular fluid (20 lmol/L, N = 9) than in whole saliva (0.1 lmol/L, N = 9). Crevicular fluid concentration of Tb 4 (ca. 5% represented by its sulfoxide) and b 10 significantly correlated (r = 0.856; N = 9), and their ratio was about 5. A significant correlation was also observed between Tb 4 concentrations in whole saliva and gingival crevicular fluid (r = 0.738; N = 9). Immunohistochemical analysis of the major salivary glands showed that immunoreactivity for Tb 4 is restricted to ductal cells, with minor degree of focal positivity in some acinar cells. On the whole, results indicate that gingival sulcus is a main, although not the sole, source for oral Tb 4 and Tb 10 .

Journal of Separation Science, 2006

HPLC-MS characterization of cyclo-statherin Q-37, a specific cyclization product of human salivar... more HPLC-MS characterization of cyclo-statherin Q-37, a specific cyclization product of human salivary statherin generated by transglutaminase 2

Journal of Proteome Research, 2009

Physiological variability of the naturally occurring, human salivary secretory peptidome was stud... more Physiological variability of the naturally occurring, human salivary secretory peptidome was studied as a function of age. The qualitative and quantitative changes occurring in the secretion of proteins/ peptides specific to the oral cavity (i.e., basic salivary proline-rich proteins, salivary acidic proline-rich phosphoproteins, statherin, proline-rich peptide P-B, salivary cystatins, and histatins) were investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry in 67 subjects aged between 3 and 44 years. Subjects were divided into five age groups: group A, 8 donors, 3-5 years; group B, 11 donors, 6-9 years; group C, 20 donors, 10-12 years; group D, 15 donors, 13-17 years; group E, 13 donors, 24-44 years. Basic salivary proline-rich proteins, almost undetectable in the 3-5 and 6-9 years groups, reached salivary levels comparable to that of adults (24-44 years) around puberty. Levels of peptide P-D, basic peptide P-F, peptide P-H, peptide P-J (a new basic salivary proline-rich protein characterized in this study), and basic proline-rich peptide IB-1 were significantly higher in the 10-12-year-old group than in the 3-5-year-old group, whereas the increase of proline-rich peptide II-2 was significant only after the age of 12 years. The concentration of salivary acidic proline-rich phosphoproteins, histatin-3 1/24, histatin-3 1/25, and monophosphorylated and diphosphorylated cystatin S showed a minimum in the 6-9-year-old group. Finally, the histatin-1 concentration was significantly higher in the youngest subjects (3-5 years) than in the other groups.

Journal of Proteome Research, 2013

Analysis by a HPLC-ESI-MS top-down proteomic platform of specimens of human preterm newborn whole... more Analysis by a HPLC-ESI-MS top-down proteomic platform of specimens of human preterm newborn whole saliva evidenced high relative amounts of cystatin B and its S-glutathionylated,S-cysteinylated, and S-S 2-mer (on Cys(3)) derivatives, decreasing as a function of postconceptional age (PCA). The percentage of S-unmodified cystatin B was higher than the S-modified isoforms in the early PCA period, differently from adults where cystatin B was detectable only as S-modified derivatives. The percentage of S-modified derivatives increased as a function of PCA, reaching at the normal term of delivery values similar to those determined in at-term newborns, babies, and adults. Moreover, in the early PCA period, high relative amounts of the 1-53 and 54-98 cystatin B fragments were detected, decreasing as a function of PCA and disappearing at the normal term of delivery. In agreement with intact cystatin B, fragment 1-53 was detectable as S-unmodified and S-modified derivatives, and their percentages changed accordingly with the percentages of intact proteins, suggesting that the fragmentation process could be subsequent to and independent from the S-modification of the protein. This study highlights specific enzymatic activity in the oral cavity of preterm newborns not present in at-term newborns and adults, which can be a clue to specialized pathways occurring during fetal oral development.

Biochemical and Biophysical Research Communications, 2010

a b s t r a c t RP-HPLC-ESI-MS profile of saliva samples from human preterm newborn showed a prot... more a b s t r a c t RP-HPLC-ESI-MS profile of saliva samples from human preterm newborn showed a protein peak in the elution range 26.6-27.6 min. Deconvolution of ESI-MS spectra revealed the presence of two proteins with average molecular mass (Mav) values of 17,239 ± 3 Da and 18,065 ± 3 Da in 9 samples, with Mav value of 17,239 ± 3 Da in 4 samples and Mav value of 18,065 ± 3 Da in 2 samples. MALDI-TOF-MS analysis of tryptic digest allowed identifying the proteins as two isoforms of small proline-rich protein 3 and cDNA amplification of RNA extracts from oral mucosa, parotid and submandibular gland samples, obtained at fetal autopsy, provided two nucleotide sequences in agreement with those reported in the literature. The two proteins differ for an octapeptide repeat (GCTKVPEP) and the substitution Leu ? Val, at position 148 and 140 of the mature form of the 18,065 and 17,239 Da protein, respectively. During maturation the two proteins undergo two post-translational modifications, corresponding to N-terminal acetylation and removal of the initiator methionine. cDNA amplification did not allow to clarify if the proteins found in saliva originated from cellular shedding of the epithelium and/or secretion.

Molecular & Cellular Proteomics, 2008

To elucidate the localization of post-translational modifications of different classes of human s... more To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a posttranslational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small prolinerich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands. Molecular & Cellular Proteomics 7:911-926, 2008.