Lycopene and bone: an in vitro investigation and a pilot prospective clinical study.

BACKGROUND: There are several effective therapies for osteoporosis but these agents might cause serious adverse events. Lycopene intake could prevent bone loss, however studies on its effects on bone are scarce. Our aim was to investigate the effects of lycopene on osteoblast cells as well as bone mineral density and bone turnover markers in postmenopausal women. METHODS: We investigated the effect of lycopene on the Wnt/ß-catenin and ERK 1/2 pathways, RUNX2, alkaline phosphatase, RANKL and COL1A of Saos-2. We also carried out a pilot controlled clinical study to verify the feasibility of an approach for bone loss prevention through the intake of a lycopene-rich tomato sauce in 39 postmenopausal women. RESULTS: Lycopene 10 µM resulted in higher ß-catenin and phERK1/2 protein Vs the vehicle (p = 0.04 and p = 0.006). RUNX2 and COL1A mRNA was induced by both 5 and 10 µM doses (p = 0.03; p = 0.03 and p = 0.03; p = 0.05) while RANKL mRNA was reduced (p < 0.05). A significant bone density loss was not detected in women taking the tomato sauce while the control group had bone loss (p = 0.002). Tomato sauce intake resulted in a greater bone alkaline phosphatase reduction than the control (18% vs 8.5%, p = 0.03). CONCLUSIONS: Lycopene activates the WNT/ß-catenin and ERK1/2 pathways, upregulates RUNX2, alkaline phosphatase, COL1A and downregulates RANKL Saos-2. These processes contributed to prevent bone loss in postmenopausal women.

Hexagonal Mesoporous Silica as a Rapid, Efficient and Versatile Tool for MALDI-TOF MS Sample Preparation in Clinical Peptidomics Analysis: A Pilot Study.

Improvement in high-throughput MALDI-TOF MS analysis requires practical and efficient sample preparation protocols for high acquisition rates. The use of hexagonal mesoporous silica (HMS) sorbents in combination with MALDI-TOF MS was explored as a versatile tool for peptidomic profiling of clinical specimens difficult to process, but considered important sources of disease biomarkers: synovial fluid and sputum. A rapid and efficient procedure, based on dispersive solid-phase extraction of peptides using commercially available wormhole mesostructured HMS, was tested for: a) pre-concentration of standard peptides in serially diluted solution up to the sub-nanomolar range; b) peptidome profiling of sputum and synovial fluid. The use of HMS, as dispersed sponges, significantly amplified the peptidic repertoire of sputum and synovial fluid by excluding from the adsorptive process large size proteins, which mask and/or suppress peptidome signals. The protocol proposed, as dispersive solid phase extraction, ensures good analytical performances. Moreover, it is economical and rapid, as it avoids the use of less reproducible and prolonged sample preparation procedures, such as the use of ultrafiltration filter devices. These findings may contribute to defining a high-throughput screening MS-based platform for monitoring key peptidic features of difficult to analyse bodily fluids in a clinical setting.

Rapid Detection and Identification of Antimicrobial Peptide Fingerprints of Nasal Fluid by Mesoporous Silica Particles and MALDI-TOF/TOF Mass Spectrometry: From the Analytical Approach to the Diagnostic Applicability in Precision Medicine.

BACKGROUND: Antimicrobial peptides (AMP) play a pivotal role in innate host defense and in immune response. The delineation of new MS-based profiling tools, which are able to produce panels of AMP of the nasal fluid (NF), may be attractive for the discovery of new potential diagnostic markers of respiratory disorders. METHODS: Swabs collected NF from healthy patients and from patients with respiratory disorders. We used a fast procedure based on mesoporous silica particles (MPS) to enrich NF in its AMP component in combination with MALDI-TOF/TOF MS as a key tool for rapidly analyzing clinical samples. RESULTS: Reproducible MS peptide fingerprints were generated for each subject and several AMP were detected including (Human Neutrophil Peptides) HNPs, Statherin, Thymosin-ß4, Peptide P-D, II-2, ß-MSP, SLPI, Lysozyme-C, and their proteo-forms. In particular, Statherin, Thymosin-ß4, and Peptide P-D were accurately identified by direct MS/MS sequencing. Examples of applicability of this tool are shown. AMP fingerprints were obtained before and after a nasal polypectomy as well as before and post-treatment with azelastine/fluticasone in one case of allergic rhinitis. CONCLUSION: The potential of our platform to be implemented by new mesoporous materials for capturing a wider picture of AMP might offer an amazing opportunity for diagnostic clinical studies on individual and population scales.

An Analytical Method for Assessing Optimal Storage Conditions of Gingival Crevicular Fluid and Disclosing a Peptide Biomarker Signature of Gingivitis by MALDI-TOF MS.

PURPOSE: Gingival crevicular fluid (GCF) is an important diagnostic source of biomarkers for both periodontitis and gingivitis. However, GCF peptide signature may change depending on factors such as handling and storage. Here we propose a standardized methodology for GCF analysis by MALDI-TOF/TOF-MS in order to distinguish a characteristic peptide signature of gingivitis. EXPERIMENTAL DESIGN: The best storage/handling conditions which may ensure the stability of the endogenous peptidome in GCF is determined and then MALDI-TOF MS comparative analysis is performed. Reproducible GCF MALDI-TOF signatures between two groups of gingivitis (n = 10) and healthy (n = 10) subjects are compared. RESULTS: A pattern of five peptides resulted differentially expressed between gingivitis and healthy groups. Interestingly, among these biomarkers the C-terminal fragment of alpha-1-antitrypsin (AAT) namely C-36 peptide and two different PTMs of the full-length S100A9 protein are found. CONCLUSIONS AND CLINICAL RELEVANCE: The method described provides a rapid comparative analysis of GCF signatures between periodontally healthy and gingivitis subjects. A pattern based on the expression of endogenous peptides and their PTMs is identified in GCF as putative biomarkers of gingivitis. These findings improve the knowledge of the inflammatory, immune, and structural substrates which might have a key role in the pathogenesis of gingivitis.

A rapid differential display analysis of nasal swab fingerprints to distinguish allergic from non-allergic rhinitis subjects by mesoporous silica particles and MALDI-TOF mass spectrometry.

Discriminating different rhinitis cases can sometimes be difficult as the diagnostic criteria used to identify the various subgroups are not always unambiguous. The nasal fluid (NF) highly reflects the pathophysiology of these inflammatory diseases. However, its collection, as nasal lavage fluid, may cause discomfort. Due to the non-invasiveness and rapidity of collection, nasal swab might represent an alternative to overcome these problems and also an ideal source of biomarkers. In this study, we demonstrate that the combined use of mesoporous silica (MPS) with MALDI-TOF MS allows the rapid detection of differential nasal peptide profiles from nasal swabs of healthy (H), allergic rhinitis (AR) and non-allergic rhinitis (NAR) subjects. NF peptides from nasal swabs were captured by the mean of MPS then profiled by MALDI-TOF MS. As a proof-of-principle, we also explored the ability of our platform to discriminate between nasal swabs of patients with AR and NAR, and between these groups and H controls. Four peaks resulted differentially expressed between NAR and AR, two peaks discriminated AR from H while one peak segregated NAR from H group. Therefore, peptides selected and enriched by our platform could form a part of a diagnostic ''rhinomic'' profile of the allergic and non-allergic patients.

Influence of storage conditions on MALDI-TOF MS profiling of gingival crevicular fluid: Implications on the role of S100A8 and S100A9 for clinical and proteomic based diagnostic investigations.

Gingival crevicular fluid (GCF) may be a source of diagnostic biomarkers of periodontitis/gingivitis. However, peptide fingerprints may change, depending on GCF collection, handling and storage. We evaluated how storage conditions affect the quality and the reproducibility of MALDI-TOF profiles of this fluid. GCF was collected on paper strips from four subjects with healthy gingiva. Our findings demonstrated that sample storage conditions significantly affect GCF peptide pattern over time. Specifically, the storage of GCF immediately extracted from paper strips generates less variations in molecular profiles compared to the extraction performed after the storage. Significant spectral changes were detected for GCF samples stored at -20°C directly on the paper strips and extracted after three months, in comparison to the freshly extracted control. Noteworthy, a significant decrease in the peak area of HNP-3, S100A8, full-length S100A9 and its truncated form were detected after 3 months at -80°C. The alterations found in the "stored GCF" profile not only may affect the pattern-based biomarker discovery but also make its use not adequate for in vitro diagnostic test targeting S100A8, S100A9 proposed as potential diagnostic biomarkers for periodontal disease. In summary, this study shows that the best preserved signatures were obtained for the GCF samples eluted in trifluoroacetic acid and then immediately stored at -80°C for 1 month. The wealth of information gained from our data on protein/patterns stability after storage might be helpful in defining new protocols which enable optimal preservation of GCF specimen.

Assessment of pre-analytical and analytical variables affecting peptidome profiling of gingival crevicular fluid by MALDI-TOF mass spectrometry.

BACKGROUND: Gingival crevicular fluid (GCF) may be a source of new biomarkers of periodontitis/gingivitis. However, the minute volumes of GCF harvested in healthy sites are a serious drawback. We evaluated how pre-analytical and analytical variables concerning GCF collection and processing, could significantly influence quality and reproducibility of MALDI-TOF profiles. METHODS: GCF was collected from healthy sites. The use of paper strips vs paper points was compared. Peptides and proteins were extracted by centrifugal elution in different solutions, at different accelerations, with and without protease inhibitor cocktail (PIC). Finally, we evaluated how matrix composition and matrix/sample volume ratio affect the reproducibility of MALDI-TOF profiles. RESULTS: Trifluoroacetic acid elution generated richer gingival fingerprints compared to acetic acid, independently of the collection device. Centrifugation speed and PIC supplementation did not change GCF profiles. A fine modulation of matrix composition and matrix/sample volume ratio resulted in a satisfactory reproducibility (CV less than 10% for peak area and signal-to-noise ratio). CONCLUSION: An optimized procedure, enabling generation of reproducible MALDI-TOF profiles from limited volume of GCF, is proposed. These fingerprints may serve as reference for future studies oriented to the maintenance and preservation of good gingival status and to discovery biomarkers of periodontitis/gingivitis.