Massimo Cordaro

Top-down platform for deciphering the human salivary proteome.

Proteomic platforms can be classified in bottom-up strategies, which analyze the sample after proteolytic digestion, and top-down strategies, which analyze the intact naturally occurring proteome. Bottom-up platforms are high-throughput because they can investigate a large number of proteins, regardless of their dimension. Nonetheless, information on post-translational modifications (PTMs) can be lost, especially those regarding naturally occurring cleavages and alternative splicing. Top-down platforms cannot cover vast proteomes, however, they can disclose subtle structural variations occurring during protein maturation and allow label-free relative quantifications in an unlimited number of samples. A repertoire of 256 masses belonging to naturally occurring proteins and peptides consistently detected by RP-HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva is presented in this study. Of them, 233 have been identified, while 23 are still pending for the definitive characterization. The present review reports average and mono-isotopic masses of the peptides and proteins detected, RP-HPLC elution times, PTMs, origin and quali-quantitative variations observed in several physiological and pathological conditions. The information reported can be a reference for users of top-down RP-HPLC-ESI-MS proteomic platforms applied to the study of the human salivary proteome as well as of other human bodily fluids.

Influence of the shape of artificial canals on the fatigue resistance of NiTi rotary instruments

AIM To investigate the influence of the trajectory of NiTi rotary instruments on the outcome of cyclic fatigue tests. METHODOLOGY Ten ProFile and Mtwo instruments tip size 20, taper 0.06 and tip size 25, taper 0.06 were tested in two simulated root canals with an angle of curvature of 60 degrees and radius of curvature of 5 mm but with different shape. Geometrical analysis of the angle and radius of the curvature that each instrument followed inside the two different artificial canals was performed on digital images. The instruments were then rotated until fracture at a constant speed of 300 rpm to calculate the number of cycles to failure (NCF) and the length of the fractured fragment. Mean values were calculated and analysed using two different multivariate linear regression models and an independent sample t-test. RESULTS The shape of the artificial root canal used in cyclic fatigue studies influenced the trajectory of the instrument. This difference is reflected by the NCF measured for the same instrument in the different artificial root canals and by the impact of the type of canal on both the NCF (St.beta = 0.514) and fragment length (St.beta = -0.920). CONCLUSIONS Small variations in the geometrical parameters of the curvature of an instrument subjected to flexural fatigue could have a significant influence on the results of fatigue tests.

Characterization of salivary proteins of schizophrenic and bipolar disorder patients by top-down proteomics

UNLABELLED The analysis of whole saliva of 32 subjects with diagnosis of schizophrenia (SZ), 17 with diagnosis of bipolar disorder (BD), and 31 healthy subjects divided in non-smokers (HN; n=19) and smokers (HS; n=12) using an HPLC-ESI-MS top-down platform is reported in this study. Both SZ and BD revealed more than 10 fold mean increase of α-defensins 1-4, S100A12, cystatin A and S-derivatives of cystatin B levels with respect to the HN and HS control groups. No differences of protein levels were observed between SZ and BD groups and between HN and HS groups. Moreover, the correlation coefficients among the different proteins were significantly better in BD group than in SZ group. BIOLOGICAL SIGNIFICANCE This study on whole saliva confirms a schizophrenia-associated dysregulation of immune pathway of peripheral white blood cells and suggests that the dysregulation of BD group could involve the activation of more specific cell type than that of SZ group.

RP-HPLC-ESI-MS evidenced that salivary cystatin B is detectable in adult human whole saliva mostly as S-modified derivatives: S-Glutathionyl, S-cysteinyl and S-S 2-mer

An HPLC-ESI-MS analysis of adult human whole saliva evidenced three protein masses (M average 11,487±2, 11,301±2 and 22,362±3Da) eluting in the 32.5-35.0min range. Treatment in reducing conditions allowed establishing that they are S-derivatives of N-terminal acetylated cystatin B, namely its S-glutathionyl, S-cysteinyl and S-S dimer. The identification was confirmed by high resolution HPLC-ESI-MS-MS experiments on the intact naturally occurring proteins and their tryptic digests. S-unmodified cystatin B is rarely detectable in whole saliva of healthy adults (5 subjects out of 65) and its percentage does not overcome approximately 20% of total cystatin B (11±9%). In the majority of subjects (60 out of 65) the mean percentages of the S-modified derivatives were S-glutathionyl 53±13%, S-cysteinyl 15±5%, S-S 2-mer 32±13%. Variations of the percentages of these S-modified derivatives of cystatin B could be indicative of oral oxidative stress. As we are aware, this is the first time that S-glutathionylation and S-cysteinylation were described as extensive PTM of a salivary protein and the first time that these PTMs were detected in naturally occurring cystatin B.

Measurement of the trajectory of different NiTi rotary instruments in an artificial canal specifically designed for cyclic fatigue tests

OBJECTIVE The objective of this study was to investigate the trajectory of NiTi rotary instruments of the same size but different design in an artificial root canal manufactured for cyclic fatigue test. STUDY DESIGN Eight groups of instruments with tip size 25, 0.06 taper with different design were tested in a simulated root canal with an angle of curvature of 60 degrees and radius of curvature of 5 mm milled in a stainless steel block with a tapered shape corresponding to the dimensions of the instruments tested. Geometric analysis of the trajectory that each instrument followed inside the artificial canal was performed on digital images, determining 3 parameters: angle and radius of the curvature and the position of the center of the curvature. Mean values were then calculated for each group. Data were analyzed using 1-way ANOVA and Holm t test to determine any statistical difference (P < .05). RESULTS All the instruments tested followed the curvature established with little variations and ANOVA test did not show significant difference among the different groups for all the parameters analyzed (P > .05). CONCLUSIONS The results of the present study emphasized the importance of using an artificial canal specifically constructed on the dimensions of the instrument tested to reduce this approximation in cyclic fatigue tests. The artificial canal manufactured for the present study seems to guarantee that different NiTi rotary instruments may follow a precise and repeteable trajectory in terms of radius and angle of curvature.

Chrono-Proteomics of Human Saliva: Variations of the Salivary Proteome during Human Development

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.

Top-down HPLC-ESI-MS detection of S-glutathionylated and S-cysteinylated derivatives of cystatin B and its 1-53 and 54-98 fragments in whole saliva of human preterm newborns.

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.

Effects of Dental Methacrylates on Oxygen Consumption and Redox Status of Human Pulp Cells

Several studies have already demonstrated that the incomplete polymerization of resin-based dental materials causes the release of monomers which might affect cell metabolism. The aim of this study was to investigate the effects of triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, urethane dimethacrylate, and 2-hydroxyethyl methacrylate on (1) cellular energy metabolism, evaluating oxygen consumption rate, glucose consumption, glucose 6-phosphate dehydrogenase activity, and lactate production, and (2) cellular redox status, through the evaluation of glutathione concentration and of the activities of enzymes regulating glutathione metabolism. Methods. Human pulp cells were used and oxygen consumption was measured by means of a Clark electrode. Moreover, reactive oxygen species production was quantified. Enzymatic activity and glucose and lactate concentrations were determined through a specific kit. Results. Triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, and 2-hydroxyethyl methacrylate induced a decrease in oxygen consumption rate, an enhancement of glucose consumption, and lactate production, whilst glucose 6-phosphate dehydrogenase and glutathione reductase activity were not significantly modified. Moreover, the monomers induced an increase of reactive oxygen species production with a consequent increase of superoxide dismutase and catalase enzymatic activities. A depletion of both reduced and total glutathione was also observed. Conclusion. The obtained results indicate that dental monomers might alter energy metabolism and glutathione redox balance in human pulp cells.

Surgery-first orthognathic approach vs traditional orthognathic approach: Oral health-related quality of life assessed with 2 questionnaires

Introduction The purposes of the study were to investigate and evaluate the differences detected by the patients between the traditional orthognathic approach and the surgery‐first one in terms of level of satisfaction and quality of life. Methods A total of 30 patients who underwent orthognathic surgery for correction of malocclusions were selected and included in this study. Fifteen patients were treated with the conventional orthognathic surgery approach, and 15 patients with the surgery‐first approach. Variables were assessed through the Orthognathic Quality of Life Questionnaire and the Oral Health Impact Profile questionnaire and analyzed with 2‐way repeated‐measures analysis of variance. Results The results showed significant differences in terms of the Orthognathic Quality of Life Questionnaire (P <0.001) and the Oral Health Impact Profile (P <0.001) scores within groups between the first and last administrations of both questionnaires. Differences in the control group between first and second administrations were also significant. Questionnaire scores showed an immediate increase of quality of life after surgery in the surgery‐first group and an initial worsening during orthodontic treatment in the traditional approach group followed by postoperative improvement. Conclusions This study showed that the worsening of the facial profile during the traditional orthognathic surgery approach decompensation phase has a negative impact on the perception of patients’ quality of life. Surgeons should consider the possibility of a surgery‐first approach to prevent this occurrence. HighlightsBenefits of traditional orthognathic surgery and surgery‐first approach were studied.Self‐esteem, body image, level of satisfaction, and quality of life were compared.The results showed significant benefits with the surgery‐first approach.

N- and O-linked glycosylation site profiling of the human basic salivary proline-rich protein 3M.

In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline-rich protein 3M, encoded by PRB3-M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed-phase high-performance liquid chromatography with high-resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N-deglycosylation with Peptide-N-Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N- and O-glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O-linked to Threonine 50, and 33 different glycans N-linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.