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Background: The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) represents a major threat to human health, which impairs the functionality of several organs. One of the hardest challenges in the fight against COVID-19 is the development of wide-scale, effective, and rapid laboratory tests to control disease severity, progression, and possible sudden worsening. Monitoring patients in real-time is indeed highly demanded in this pandemic era when physicians need reliable and quantitative tools to prioritize patients’ access to intensive care departments. In this regard, salivary biomarkers are extremely promising, as they allow for a fast and non-invasive specimens’ collection, which can be repeated multiple times. Methods: We compare salivary levels of immunoglobulin A subclasses (IgA1 and IgA2) and free-light chains (FLC k and λ) in a cohort of 29 SARS-CoV-2 patients and 21 healthy subjects. Results: We found that each biomarkers differs significantly between the two groups, with p-values ranging from 10-8 to 10-4. The performance ranking of these markers, shows that λFLC level (p=1.4e-8) is the best-suited candidate to discriminate the two groups, with an accuracy of 0.94 (0.87-1.00 95% CI), a precision of 0.91 (0.81-1.00 95% CI), a sensitivity of 1.00 (0.96-1.00 95% CI) and a specificity of 0.86 (0.70-1.00 95% CI). Conclusion: These results suggest λFLC as an ideal indicator of patient conditions. This is more strengthened in consideration that λFLC half-life (approximately 6 hours) is significantly shorter than the IgA one (21 days): thus λFLC appears displaying the potential to effectively monitor patients fluctuation in real-time.

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 +/- 3 Da) eluting in the 32.5-35.0 mwin 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. (C) 2011 Elsevier B.V. All rights reserved.

This study describes the identification and structural characterization of Sus scrofa statherin. HPLC–electrospray ionization mass spectrometry analysis on pig parotid secretory granule extracts evidenced a peptide with a molecular mass value of 5381.1 ± 0.6 Da and its truncated form, devoid of the C-terminal Ala residue, with a molecular mass value of 5310.1 ± 0.6 Da. The complete sequence of pig statherin gene was determined by sequencing the full-length cDNA obtained by rapid amplification of cDNA ends. The gene is 549 base pairs long and contains an open reading frame of 185 nucleotides, encoding a 42-amino acid secretory polypeptide with a signal peptide of 19 residues. This sequence presents some typical features of the four statherins characterized till now, showing the highest degree of amino acid identity with bovine (57%) and human statherin (39%). Pig statherin is mono-phoshorylated on Ser-3, while primate statherins already characterized are di-phosphorylated on Ser-2 and Ser-3. This difference, probably connected to the Asp-4 Glu substitution, suggests the involvement of the Golgi-casein kinase, which strictly recognizes the SX(E/pS) consensus sequence. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.

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 post-translational 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 proline-rich 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.

Basic proline-rich proteins (bPRPs) are a class of proteins widely present in saliva of humans and other mammals. They are synthesized as preproproteins and enzymatically cleaved into small peptides before secretion from the salivary glands. Recently, we characterized two proline-rich peptides (SP-A and SP-B) in parotid secretory granules of pig (Sus Scrofa) that are derived from three isoforms of a PRP proprotein (Swiss-Prot data bank: Q95JC9-1, Q95JC9-2 and Q95JC9-3). Together the coding regions for SP-A and SP-B, which are repeated many times, account for 52-70% of the coding regions of the PRP proproteins. This study was undertaken to identify peptides encoded by unassigned regions of the PRP proproteins. RP-HPLC-ESI-IT-MS analysis of enriched granule preparations from pig parotid glands by two different analytical strategies identified ten new proline-rich peptides derived from the three proproteins. Together with the coding regions for SP-A and SP-B already identified it was possible to assign 68-75% of the proproteins coding regions. The peptide sequences indicated a number of unusual proteolytic cleavage sites suggesting the presence of unknown proprotein convertases.

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.

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.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS) also in noise induced hearing loss (NIHL) and anti-oxidants and free-radicals scavengers have been shown to attenuate the damage. Coenzyme Q(10) (CoQ(10)) or ubiquinone has a bioenergetic role as a component of the mithocondrial respiratory chain, it inhibits mitochondrial lipid peroxidation, inducing ATP production and it is involved in ROS removal and prevention of oxidative stress-induced apoptosis. However the therapeutic application of CoQ(10) is limited by the lack of solubility and poor bio- availability, therefore it is a challenge to improve its water solubility in order to ameliorate the efficacy in tissues and fluids. This study was conducted in a model of acoustic trauma in the guinea pig where the effectiveness of CoQ(10) was compared with a soluble formulation of CoQ(10) (multicomposite CoQ(10) Terclatrate, Q-ter) given intraperitoneally 1 h before and once daily for 3 days after pure tone noise exposure (6 kHz for 1 h at 120 dB SPL). Functional and morphological studies were carried out by measuring auditory brainstem responses, scanning electron microscopy for hair cell loss count, active caspase 3 staining and terminal deoxynucleotidyl transferase-mediated dUTP labelling assay in order to identify initial signs of apoptosis. Treatments decreased active caspase 3 expression and the number of apoptotic cells, but animals injected with Q-ter showed a greater degree of activity in preventing apoptosis and thus in improving hearing. These data confirm that solubility of Coenzyme Q(10) improves the ability of CoQ(10) in preventing oxidative injuries that result from mitochondrial dysfunction.

This work reports the successful recombinant expression of human statherin in Escherichia coli , its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.