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The most heterogeneous family of human salivary proteins is represented by proline-rich proteins (PRPs) divided in acidic, basic, and basic glycosylated (aPRPs, bPRPs, gPRPs). They are encoded by six genes, clustered on chromosome 12p13.2: PRH1-2 encode aPRPs, PRB1-4 encode bPRPs and gPRPs. Each gene exists in different allelic forms: two for PRH2, three for PRH1, PRB2, and PRB4, four for PRB1, and PRB3. During granule maturation, PRP proproteins undergo proteolysis by the action of convertases and carboxypeptidases. Differently from bPRPs, proteolysis of aPRPs is not complete, and, besides fragments, entire protein species are also secreted. Maturation process generates ten aPRPs (PRP-1, PRP-2, PIF-s, Db-s, Pa, PRP-3, PRP-4, PIF-f, Db-f, P-C), and at least 18 bPRPs (II-2, P-E, IB-6, Ps-1, Ps-2, IB-1, P-J, IB-8a, P-F, P-H, P-D, II-1, protein glycosylated A, CD-IIg, and Gl1-4). In addition, single nucleotide and length polymorphisms, and differentially spliced transcripts originate several natural variants. Phosphorylation, N-pyroglutaminylation, dimerization, and N-/O-glycosylation also occur during maturation, enlarging the number of protein species, further increased by proteolytic events governed by carboxy- and endo-peptidases during and after secretion, and giving rise to a huge number of small peptides. The PRP functional role is still poorly understood. Significance: The high polymorphism of PRPs gives an important contribution to the high heterogeneity and inter-individual variability of the human salivary proteome. The products of six genes clustered on chromosome 12p13.2 comprise a mixture of entire, truncated, phosphorylated, glycosylated and dimerized protein/peptide species, sharing large part of their sequences, and possibly involved in different biological activities. Whatever the role of PRP species is, it should be crucial, given that PRPs are the most conserved oral salivary proteins among mammals.

This review is addressed to scientists working outside the field of proteomics and wishes to shed a light on the possibility offered by the latest proteomics strategies. Bottom-up and top-down platforms are critically examined outlining advantages and limitations of their application to qualitative and quantitative investigations. Discovery, directed and targeted proteomics as different options for the management of the MS instrument are defined emphasizing their integration in the experimental plan to accomplish meaningful results. The issue of data validation is analyzed and discussed. The most common qualitative proteomic platforms are described, with a particular emphasis on enrichment methods to elucidate PTMs codes (i.e. ubiquitin and histone codes). Label-free and labeled methods for relative and absolute quantification are critically compared. The possible contribution of proteomics platforms to the transition from structural proteomics to functional proteomics (study of the functional connections between different proteins) and to the challenging system biology (integrated study of all the functional cellular functions) is also briefly discussed.

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.

Histatin 1 (His-1) derivatives showing serial mass increases of 80.0 ( 0.1 Da were detected in human saliva by HPLC-ESI-MS. The same derivatives were also found in granules of submandibular glands and secretions of submandibular/sublingual glands, but not in granules and secretions of parotid glands. Only one phosphate group was present in His-1 and its derivatives, since treatment with alkaline phosphatase provided an 80.0 Da mass decrease. His-1 derivatives were almost completely transformed into His-1 by treatment with 1 M HCl at 100 °C, suggesting the presence of O-sulfotyrosine, which is more labile than phospho-Tyr to acidic hydrolysis. CE-MS analysis of pronase extensive digestion of derivatives confirmed the presence of sulfotyrosine. Derivatives were digested by trypsin, proteinase K, and protease V-8 and analyzed by different MS strategies. The results allowed locating sulfation on the last four tyrosines (Tyr 27, 30, 34, and 36). This study is the first report of the gland-specific sulfation of a salivary phosphopeptide in vivo.

Thirteen samples of human normal whole saliva were analyzed by RP-HPLC-ESI-MS and MALDI-TOF-MS to investigate the basic proline-rich protein complex. Between known basic-PRPs the P-B, P-C (or IB-8b), P-D (or IB-5), P-E (or IB-9), P-F (or IB-8), P-H (or IB-4), IB-6,II-2, IB-1, and IB-8a glucosylated were identified, whereas the II-1, IB-7, PA, and D1-A peptides were not detected. Some detected masses not attributable to known basic-PRPs were putatively ascribed to II-2 and IB-1 nonphosphorylated, II-2 and IB-1 missing the C-terminal arginine residue, and the 1-62 fragment of IB-6, named P-J peptide. A correlation matrix analysis revealed a cluster of correlation among all the basic PRF's (apart from the P-B peptide) which is in agreement with their common parotid origin.

Thermodynamics of the binding of Ni2+, Cu2+ and Zn2+ to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni2+ > Cu2+ > Zn2+, with association constant values of (2.3+/- 0.1) X 10(4), (4.9+/-0.2) x 10(3), and (1.5 +/- 0.1) x 10(3) m(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P-O peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni2+ and Cu2+ binding, with a shift of the pK(a), of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group Of D-Orn-7 from 9.7 to about 7. Even though on Zn2+ binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift Of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 Angstrom lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.

The systematic search by tandem mass spectrometry of human saliva from four different subjects, of 136 possible fragments originated from histatin 3, allowed the detection of 24 different peptides. They include, with the exception of histatin 4, all the known histatin 3 fragments, namely histatins 5-12 and the peptides corresponding to 15-24, 26-32, 29-32 residues, and 13 new fragments corresponding to 1-11, 1-12, 1-13, 5-13, 6-11, 6-13, 7-11, 7-12, 7-13, 14-24, 14-25, 15-25, and 28-32 residues of histatin 3. On the contrary, none of 119 possible fragments of histatin 1, including histatin 2, was detected. The results suggest that the genesis of histatin 3-related peptides, being under the principal action of trypsin-like activities, is probably not a random process but rather follows a sequential fragmentation pathway. Lack of detection of C-terminal fragments, with the exception of 26-32, 28-32, and 29-32 fragments, suggested that arginine 25 should be the first cleavage site, generating histatin 6 and 26-32 fragments. The genesis of 28-32 and 29-32 fragments and histatin 5 should implicate a subsequent exo-protease action. Similarly, lack of detection of fragments having Lys-5 and Arg-6 at the N terminus and Arg-25 at the C terminus strongly suggested that sequences KRKF (11-14 residues) and AKR (4-6 residues) should be the second and the third cleavage sites, respectively. Lys-17 and Arg-22 are not cleaved at all.

Statisticians often focus on fuzzy numbers with triangular or trapezoidal membership functions because they are very easy to apply. Although they offer a good approximation of a fuzzy variable, several doubts arise about the appropriateness of these kind of shapes. As known, fuzzy sets are useful for interval data when the “degree of truth” of the values varies within this range. In particular, they are desirable for translating human language into numbers. In this paper, we propose an alternative membership function that appears more appropriate to deal with linguistic variables. We refer to this function as “bell-shaped fuzzy number associated with the normal curve”. In particular, we highlight the specific properties of the proposed fuzzy number and illustrate the utility of linking this function with the normal distribution.

Bayesian networks are multivariate statistical models satisfying sets of conditional independence statements. The association structure can be learnt from data by a sequence of independence and conditional independence tests using the PC algorithm. The learning process is based on assumption of independent and identically distributed observations. This assumption is almost never valid for sample surveys data since most of the commonly used survey designs employ stratification and/or cluster sampling and/or unequal selection probabilities. Here, a PC algorithm correction is proposed for taking into account the sampling design complexity.

A parametric functional analysis of variance (FANOVA) is proposed to measure the functional varia- bility explained by other variables. This technique presents the advantage of using all the information in the curves, instead of some specific values on them. In particular, we refer to cases in which the functional observations belong to a parametric family of functions, whose functional form is known in advance. In this framework, the approximation of the function underlying the data is not required as in the classical functional approach. Therefore, we avoid the impact that smoothing techniques might have on FANOVA. The proposed method is applied to a real data set concerning lichen biodiversity in Liguria region, in northwestern Italy.

An important feature of statistical matching is the estimation of the underlying joint distribution of variables separately available from independent sample surveys. Unless special assumptions are made, the absence of joint information on the variables of interest leads to uncertainty about the data generating model. The aim of this paper is to analyze the uncertainty in statistical matching for complex survey data.

In the present work we propose to analyze, through Multiple Factorial Analysis (MFA), the relational structure embedded in collaboration networks emerging in Technological Districts (TD) and observed across time occasions. In our opinion, MFA allows to deal with complex relational structures that lead to highly heterogeneous networks. We show, through a case study, how the solutions provided by the MFA can be interpreted in a natural and suitable way in the relational setting. They can furnish valuable information about the strength and typology of the collaboration structure and its evolution. As case study we focus on the analysis of a TD located in South Italy

The present paper aims at describing the scientific collaboration patterns of the Italian academic statisticians by exploiting the information retrieved through different bibliographic archives (ISI-WoS, Current Index to Statistics), and the bibliographic information of the database of nationally funded research projects. A data merging procedure is adopted to obtain a unified dataset containing both topinternational as well as nationally oriented production. The unified dataset will be used as basis for network analysis.

This paper proposes a spatial functional formulation of the normal mixed effect model for the statistical classification of spatially dependent Gaussian curves. Fixed effect parameters are represented in terms of a functional mul- tiple regression model whose regression operators can change in space. Local spatial homogeneity of these operators is measured in terms of their Hilbert- Schmidt distances, leading to the classification of fixed effect curves in dif- ferent groups. A real data example in the financial context is analyzed as an illustration.

In this work we propose a nonparametric test for the identification of nonlinear dependence in time series. The approach is based on a combination of a test statistic based on an entropy dependence metric together with a suitable extension of surrogate data methods, a class of Monte Carlo tests introduced in the field of nonlinear dynamics. We focus on the null hypothesis of linear Gaussian processes and we derive the asymptotic theory for the test statistics. Since the asymptotic approximations depend on unknown quantities and require long series to be feasible we advocate the use of surrogate methods. We prove the asymptotic validity of the inference derived from the test and show the finite sample performance through a small simulation study.

Fuzzy regression models often consider triangular or trapezoidal membership functions. Of course this kind of fuzzy numbers has the advantage that they are much easy to use; nevertheless several doubts arise about the appropriateness of the triangular shape. As known, the fuzzy sets are very useful when there are imprecise data and in particular when, within an interval, the degree of truth of the values is not always the same. Fuzzy numbers are also useful to translate terms of human language into numbers. Triangular fuzzy numbers offer a good solution for dealing with this kind of data. However in this paper it is proposed an alternative membership function which appears to be much more appropriate; it has been called ”fuzzy number associated with the normal curve”. In this framework it will be justified the introduction of this membership function as a valid substitute for triangular fuzzy numbers.