iTRAQ-based quantitative analysis of age-specific variations in salivary proteome of caries-susceptible individuals.

BACKGROUND: Human saliva is a protein-rich, easily accessible source of potential biomarkers for the diagnosis of oral and systemic diseases. However, little is known about the changes in salivary proteome associated with aging of patients with dental caries. Here, we applied isobaric tags for relative and absolute quantitation (iTRAQ) in combination with multiple reaction monitoring mass spectrometry (MRM-MS) to characterize the salivary proteome profiles of subjects of different ages, presenting with and without caries, with the aim of identifying age-related biomarkers for dental caries. METHODS: Unstimulated whole saliva samples were collected from 40 caries-free and caries-susceptible young adults and elderly individuals. Salivary proteins were extracted, reduced, alkylated, digested with trypsin and then analyzed using iTRAQ-coupled LC-MS/MS, followed by GO annotation, biological pathway analysis, hierarchical clustering analysis, and protein-protein interaction analysis. Candidate verification was then conducted using MRM-MS. RESULTS: Among 658 salivary proteins identified using tandem mass spectrometry, 435 proteins exhibited altered expression patterns in different age groups with and without caries. Of these proteins, 96 displayed age-specific changes among caries-susceptible adults and elderly individuals, and were mainly associated with salivary secretion pathway, while 110 age-specific proteins were identified among healthy individuals. It was found that the age factor caused significant variations and played an important role in both healthy and cariogenic salivary proteomes. Subsequently, a total of 136 target proteins with complex protein-protein interactions, including 14 age-specific proteins associated with caries, were further successfully validated using MRM analysis. Moreover, non-age-specific proteins (histatin-1 and BPI fold-containing family B member 1) were verified to be important candidate biomarkers for common dental caries. CONCLUSIONS: Our proteomic analysis performed using the discovery-through-verification pipeline revealed distinct variations caused by age factor in both healthy and cariogenic salivary proteomes, highlighting the significance of age in the great potential of saliva for caries diagnosis and biomarker discovery.

Promoting effect of a calcium-responsive self-assembly ß-sheet peptide on collagen intrafibrillar mineralization.

Recently, a de novo synthetic calcium-responsive self-assembly ß-sheet peptide ID8 (Ile-Asp-Ile-Asp-Ile-Asp-Ile-Asp) has been developed to serve as the template inducing hydroxyapatite nucleation. The aim of this study was to evaluate the effect of ID8 on intrafibrillar mineralization of collagen making full use of its self-assembly ability. The mineralization experiments were carried out in vitro on both bare Type I collagen and fully demineralized dentin samples. The calcium-responsive self-assembly of ID8 was revealed by circular dichroism spectrum, 8-anilino-1-naphthalenesulfonic acid ammonium salt hydrate assay, attenuated total reflection Fourier transform infrared spectrum (ATR-FTIR) and transmission electron microscope (TEM). Polyacrylic acid (450 kDa) with a concentration of 100 µg ml-1 was selected as the nucleation inhibitor based on the determination of turbidimetry and TEM with selected area electron diffraction (TEM-SAED). The results showed that collagen intrafibrillar mineralization was significantly promoted with the pretreatment of self-assembly ID8 detected by TEM-SAED, SEM, X-ray diffraction and ATR-FTIR. The pretreatment of collagen utilizing self-assembly ID8 not only enhanced intermolecular hydrogen bonding but also contributed to calcium retention inside collagen and significantly increased the hydrophilicity of collagen. These results indicated that peptides with self-assembly properties like ID8 are expected to be potential tools for biomimetic mineralization of collagen.

Rational Design of ß-sheet Peptides with Self-Assembly into Nanofibres on Remineralisation of Initial Caries Lesions.

OBJECTIVE: To explore the self-assembly and gelation properties of synthetic peptides, and their efficacy on hydroxyapatite (HAP) nucleation and in situ remineralisation of initial caries lesions. METHODS: Mass spectrometry and reversed-phase high performance liquid chromatography (RPHPLC) were used to confirm the successful synthesis of peptides. Their self-assembly properties and conformation stability were evaluated using circular dichroism (CD) spectroscopy and Fourier-transform infrared spectroscopy (FTIR). Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used to evaluate their cytotoxicity. The efficacy of the peptides on HAP nucleation and in situ remineralisation of initial caries lesions was explored using FTIR, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction and transverse microradiography analysis. RESULTS: Two kinds of self-assembly ß-sheet peptides named ID4 and ID8, respectively, were successfully synthesised with purities greater than 95%. Both were stable under neutral physiological conditions and had low cytotoxicity. ID4 and ID8 showed calcium responsive self-assembly properties and could self-assemble into nanofibres. Compared with ID4, ID8 resulted in the rapid formation of hydrogel with a lower concentration of calcium, and self-assembled ID8 hydrogel induced the formation of flower-like HAP and significantly promoted the remineralisation of initial enamel caries. CONCLUSION: ID8 could serve as the template to induce HAP nucleation and promote biomimetic remineralisation of initial caries lesions. These results underpin future research on peptide design, and ID8 may be a promising bioactive component for anti-caries applications.

Clog-free cell filtration using resettable cell traps.

The separation of cells by filtration through microstructured constrictions is limited by clogging and adsorption, which reduce selectivity and prevent the extraction of separated cells. To address this key challenge, we developed a mechanism for simply and reliably adjusting the cross-section of a microfluidic channel to selectively capture cells based on a combination of size and deformability. After a brief holding period, trapped cells can then be released back into flow, and if necessary, extracted for subsequent analysis. Periodically clearing filter constrictions of separated cells greatly improves selectivity and throughput, and minimizes adsorption of cells to the filter microstructure. This mechanism is capable of discriminating cell-sized polystyrene microspheres with <1 µm resolution. Rare cancer cells doped into leukocytes can be enriched ~1800× with ~90% yield despite a significant overlap in size between these cell types. An important characteristic of this process is that contaminant leukocytes are captured by non-specific adsorption and not mechanical constraint, enabling repeated filtration to improve performance. The throughput of this mechanism is 900,000 cells per hour for 32 multiplexed microchannels, or ~1,200,000 cells cm⁻² h⁻¹ on a per area basis, which exceeds existing micropore filtration mechanisms by a factor of 20.