Proteomic Analysis of Human Saliva via Solid-Phase Microextraction Coupled with Liquid Chromatography-Mass Spectrometry.

Proteomics of human saliva samples was achieved for the first time via biocompatible solid-phase microextraction (bio-SPME) devices. Upon introduction of a porogen to a conventional C18 coating, porous C18/polyacrylonitrile (PAN) SPME blades were able to extract peptides up to 3.0 kDa and more peptides than commercial SPME blades. Following Trypsin digestion, salivary proteomic analysis was achieved via SPME-LC-MS/MS. Seven endogenous proteins were consistently identified in all saliva samples via bio-SPME. Taking advantage of this strategy, untargeted peptidomics was applied for the comparison of saliva samples between healthy and SARS-CoV-2 positive individuals. The results showed clear peptidomic differences between the viral and healthy saliva samples. This proof-of-concept study demonstrates the potential of bio-SPME-LC-MS/MS for peptidomics and proteomics in biomedical applications.

Progression from healthy periodontium to gingivitis and periodontitis: Insights from bioinformatics-driven proteomics - A systematic review with meta-analysis.

AIM: The current study aimed to: (1) systematically review the published literature regarding the proteomics analyses of saliva and gingival crevicular fluid (GCF) in healthy humans and gingivitis and/or periodontitis patients; and (2) to identify the differentially expressed proteins (DEPs) based on the systematic review, and comprehensively conduct meta-analyses and bioinformatics analyses. METHODS: An online search of Web of Science, Scopus, and PubMed was performed without any restriction on the year and language of publication. After the identification of the DEPs reported by the included human primary studies, gene ontology (GO), the Kyoto encyclopedia of genes and genomes pathway (KEGG), protein-protein interaction (PPI), and meta-analyses were conducted. The risk of bias among the included studies was evaluated using the modified Newcastle-Ottawa quality assessment scale. RESULTS: The review identified significant differences in protein expression between healthy individuals and those with gingivitis and periodontitis. In GCF, 247 proteins were upregulated and 128 downregulated in periodontal diseases. Saliva analysis revealed 79 upregulated and 70 downregulated proteins. There were distinct protein profiles between gingivitis and periodontitis, with 159 and 31 unique upregulated proteins in GCF, respectively. Meta-analyses confirmed significant upregulation of various proteins in periodontitis, including ALB and MMP9, while CSTB and GSTP1 were downregulated. AMY1A and SERPINA1 were upregulated in periodontitis saliva. HBD was upregulated in gingivitis GCF, while DEFA3 was downregulated. PPI analysis revealed complex networks of interactions among DEPs. GO and KEGG pathway analyses provided insights into biological processes and pathways associated with periodontal diseases. CONCLUSION: The ongoing MS-based proteomics studies emphasize the need for a highly sensitive and specific diagnostic tool for periodontal diseases. Clinician acceptance of the eventual diagnostic method relies on its ability to provide superior or complementary information to current clinical assessment procedures. Future research should prioritize the multiplex measurement of multiple biomarkers simultaneously to enhance diagnostic accuracy and large study cohorts are necessary to ensure the validity and reliability of research findings.

Identification of SARS-CoV-2 biomarkers in saliva by transcriptomic and proteomics analysis.

The detection of SARS-CoV-2 biomarkers by real time PCR (rRT-PCR) has shown that the sensitivity of the test is negatively affected by low viral loads and the severity of the disease. This limitation can be overcome by the use of more sensitive approaches such as mass spectrometry (MS), which has not been explored for the detection of SARS-CoV-2 proteins in saliva. Thus, this study aimed at assessing the translational applicability of mass spectrometry-based proteomics approaches to identify viral proteins in saliva from people diagnosed with COVID-19 within fourteen days after the initial diagnosis, and to compare its performance with rRT-PCR. After ethics approval, saliva samples were self-collected by 42 COVID-19 positive and 16 healthy individuals. Samples from people positive for COVID-19 were collected on average on the sixth day (± 4 days) after initial diagnosis. Viable viral particles in saliva were heat-inactivated followed by the extraction of total proteins and viral RNA. Proteins were digested and then subjected to tandem MS analysis (LC-QTOF-MS/MS) using a data-dependent MS/MS acquisition qualitative shotgun proteomics approach. The acquired spectra were queried against a combined SARS-CoV-2 and human database. The qualitative detection of SARS-CoV-2 specific RNA was done by rRT-PCR. SARS-CoV-2 proteins were identified in all COVID-19 samples (100%), while viral RNA was detected in only 24 out of 42 COVID-19 samples (57.1%). Seven out of 18 SARS-CoV-2 proteins were identified in saliva from COVID-19 positive individuals, from which the most frequent were replicase polyproteins 1ab (100%) and 1a (91.3%), and nucleocapsid (45.2%). Neither viral proteins nor RNA were detected in healthy individuals. Our mass spectrometry approach appears to be more sensitive than rRT-PCR for the detection of SARS-CoV-2 biomarkers in saliva collected from COVID-19 positive individuals up to 14 days after the initial diagnostic test. Based on the novel data presented here, our MS technology can be used as an effective diagnostic test of COVID-19 for initial diagnosis or follow-up of symptomatic cases, especially in patients with reduced viral load.

Salivary protein homology between humans and dogs: Mass spectrometry-based proteomics analysis.

OBJECTIVE: This benchmark study aimed to investigate sex-related differences based on the identification and characterization of the salivary proteome of healthy male and female dogs using mass spectrometry (MS) technique and a homology-driven approach to analyze salivary proteins in both human and dog species utilizing protein sequence alignment technique. METHODS: Unstimulated whole saliva was collected from 10 healthy Beagles. After processing the samples and determining the total protein content, in-solution protein digestion was performed involving denaturation, reduction of disulfide bonds, alkylation, and removal of interfering compounds. Samples were analyzed using LC-ESI-MS/MS. RESULTS: LC-ESI-MS/MS analysis identified 327 and 341 unique proteins in male and female dog saliva, respectively, of which 318 (97.25 %) in male dogs and 326 (95.60 %) in female dogs were characterized. Abundant shared proteins included albumin, BPI fold-containing family A member 2, and VWFD domain-containing protein. A notable uncharacterized protein, VWFD domain-containing protein, was among the most abundant in both sexes. Comparative analysis of 69 abundant shared proteins indicated an upregulation of CES5A, EFHD, GC, IGHM, LOC100653049, KRT10, LCP1, PGD, TPI1 in male dogs, while LOC100855593 was upregulated in female dogs. In total, 84 % (n = 229/274) and 86 % (n = 235/275) salivary proteins identified in male and female dogs, respectively, were homologous to human proteins, with an overall homology of 86 % (n = 364/423), including 15 with 100 % homology. CONCLUSION: The study revealed clear differences in the salivary proteomics profile of healthy male and female dogs. However, most of the salivary proteins in both male and female dogs showed homology with human salivary proteins. CLINICAL RELEVANCE: The identification of unique salivary proteome profiles in male and female dogs, coupled with substantial homology to human proteins, provides promising biomarkers for health assessment, highlighting its clinical significance for diagnostics and therapeutic exploration not only in veterinary and human dentistry, but across mammalian species.

Metabolic reprogramming by Acly inhibition using SB-204990 alters glucoregulation and modulates molecular mechanisms associated with aging.

ATP-citrate lyase is a central integrator of cellular metabolism in the interface of protein, carbohydrate, and lipid metabolism. The physiological consequences as well as the molecular mechanisms orchestrating the response to long-term pharmacologically induced Acly inhibition are unknown. We report here that the Acly inhibitor SB-204990 improves metabolic health and physical strength in wild-type mice when fed with a high-fat diet, while in mice fed with healthy diet results in metabolic imbalance and moderated insulin resistance. By applying a multiomic approach using untargeted metabolomics, transcriptomics, and proteomics, we determined that, in vivo, SB-204990 plays a role in the regulation of molecular mechanisms associated with aging, such as energy metabolism, mitochondrial function, mTOR signaling, and folate cycle, while global alterations on histone acetylation are absent. Our findings indicate a mechanism for regulating molecular pathways of aging that prevents the development of metabolic abnormalities associated with unhealthy dieting. This strategy might be explored for devising therapeutic approaches to prevent metabolic diseases.

Validation of a cariogenic biofilm model by evaluating the effect of fluoride on enamel demineralization.

In vitro biofilm models have been extensively used, but only few of the models available to date had been validated in terms of the dose-response effect of anti-caries and/or antimicrobial substances. Additionally, none of the validated models allow the use of microliter volumes of the treatment solutions, needed mainly to test (screen) novel but expensive substances under development. This study aimed at modifying an in vitro cariogenic Streptococcus mutans biofilm model and validating it by assessing the dose-response effect of fluoride on enamel demineralization. S. mutans cariogenic biofilms were developed on saliva-coated enamel slabs previously bonded to acrylic holders fixed to a lid of a culture plate. Biofilms were incubated 8 h/day in culture medium supplemented with 1% sucrose and then overnight in culture medium with glucose 0.1 mM. Biofilms were also treated 2×/day with 2.0 mL of solutions containing 0, 125, 275 and 1250 µg F/mL (n = 10/group). The replaced culture medium was used to: determine the biofilm acidogenicity; estimate the demineralization of enamel; and monitor the fluoride concentration. At 144 h, biofilms were collected for fluoride concentration analyses, and the fluoride uptake by enamel was determined in each slab. The model showed a dose-response effect of fluoride (R2 = 0.96, p < 0.001) between enamel demineralization and the fluoride concentration of the treatments. Water-soluble and bound biofilm fluoride concentrations (p < 0.007), as well as the firmly-bound fluoride concentration found in enamel (p < 0.0001), increased in a dose-dependent manner. Our model constitutes a validated approach that would allow the assessment of the anticaries potential of novel biotechnological strategies, as in the case of expensive salivary peptides, because it would allow to test the treatment solutions using smaller volumes.

The incidence of COVID-19 among dentists practicing in the community in Canada: A prospective cohort study over a 6-month period.

BACKGROUND: Oral health care settings carry a potentially high risk of causing cross-infection between dentists and patients and among dental staff members due to close contact and use of aerosol-generating procedures. The authors aimed to estimate COVID-19 incidence rates among Canadian dentists over a 6-month period. METHODS: The authors conducted a prospective cohort study of 644 licensed dentists across Canada from July 29, 2020, through February 12, 2021. An online questionnaire, adapted from the World Health Organization's Unity Studies protocols for assessment of COVID-19 risk among health care workers, was used to collect data on self-reported severe acute respiratory syndrome coronavirus 2 infections every 4 weeks. A bayesian Poisson model was used to estimate the incidence rate and corresponding 95% credible intervals (CIs). RESULTS: Median age of participants was 47 years; most participants were women (56.4%) and general practitioners (90.8%). Median follow-up time was 188 days. Six participants reported COVID-19 infections during the study period, giving an incidence rate of 5.10 per 100,000 person-days (95% CI, 1.86 to 9.91 per 100,000 person-days). The incidence proportion was estimated to be 1,084 per 100,000 dentists (95% CI, 438 to 2,011 per 100,000 dentists) and 1,864 per 100,000 people (95% CI, 1,859 to 1,868 per 100,000 people) in the Canadian population during the same period. CONCLUSIONS: The low infection rate observed among Canadian dentists from July 29, 2020, through February 12, 2021, should be reassuring to the dental and general community. PRACTICAL IMPLICATIONS: Although the infection rates were low among Canadian dentists, it is important to continue to collect disease surveillance data.

Detection of periodontal disease activity based on histatin degradation in individuals with cerebral palsy.

Objectives: This proof-of-concept study aimed at evaluating the proteolytic profile of histatin 1 and 5 in saliva of adolescents with spastic cerebral palsy (CP) with gingivitis. Methods: This cross-sectional study included 24 individuals allocated into three groups: G1 (CP with gingivitis; n = 8), G2 (without CP and without gingivitis; n = 8), and G3 (without CP and with gingivitis; n = 8). The gingival index (GI) and simplified oral hygiene index (OHI-S) were evaluated. Whole saliva was collected and used to assess the rate and mode of histatin 1 and 5 at different times. The degradation products were visualized after cationic PAGE and the protein band densities (BDs) were compared with a protein standard. Fragmentation products were collected from the gel, pooled by group and characterized by mass spectrometry. BDs and gingival health parameters were analyzed by One-Way ANOVA or Kruskal Wallis tests, whereas poisson multilevel regression was used to the factors that influenced histatin degradation (α = 5%). Results: Groups G1 and G3 differed significantly on OHI-S, visible biofilm, oral calculus and GI (p < 0.001). Poisson Regression showed that: 1) CP and gingivitis influenced the degradation of histatin 1 and 5 (p < 0.05); 2) The degradation of histatin 5 was influenced by age and male sex (p < 0.05); and 3) GI influenced significantly the degradation of histatin 1 (p < 0.001). Unique histatin degradation peptides were identified in individuals with gingivitis. Conslusions: These data demonstrated that both the kinetics and pattern of histatins degradation differ according to the gingival health or disease conditions.

pH-Sensitive Chitosan Nanoparticles for Salivary Protein Delivery.

Salivary proteins such as histatins (HTNs) have demonstrated critical biological functions directly related to tooth homeostasis and prevention of dental caries. However, HTNs are susceptible to the high proteolytic activities in the oral environment. Therefore, pH-sensitive chitosan nanoparticles (CNs) have been proposed as potential carriers to protect proteins from enzymatic degradation at physiological salivary pH. Four different types of chitosan polymers were investigated and the optimal formulation had good batch to batch reproducibility, with an average hydrodynamic diameter of 144 ± 6 nm, a polydispersity index of 0.15 ± 0.04, and a zeta potential of 18 ± 4 mV at a final pH of 6.3. HTN3 encapsulation and release profiles were characterized by cationic polyacrylamide gel electrophoresis. The CNs successfully encapsulated HTN3 and selectively swelled at acidic pH to facilitate HTN3 release. Protection of HTN3 against enzymatic degradation was investigated in diluted whole saliva. HTN3 encapsulated in the CNs had a prolonged survival time compared to the free HTN3. CNs with and without HTN3 also successfully reduced biofilm weight and bacterial viability. The results of this study have demonstrated the suitability of CNs as potential protein carriers for oral applications, especially for complications occurring at acidic conditions.

Protecting the Aging Genome.

Mounting evidence suggests that DNA damage plays a central role in aging. Multiple tiers of defense have evolved to reduce the accumulation of DNA damage, including reducing damaging molecules, repairing DNA damage, and inducing senescence or apoptosis in response to persistent DNA damage. Mutations in or failure of these pathways can lead to accelerated or premature aging and age-related decline in vital organs, supporting the hypothesis that maintaining a pristine genome is paramount for human health. Understanding how we cope with DNA damage could inform on the aging process and further on how deficient DNA maintenance manifests in age-related phenotypes. This knowledge may lead to the development of novel interventions promoting healthspan.

Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients.

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the antibody response in saliva and its relationship to systemic antibody levels. Here, we profiled by enzyme-linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor-binding domain (RBD) in serum and saliva of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3-115 days post-symptom onset (PSO), compared to negative controls. Anti-SARS-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16-30 days PSO. Longitudinal analysis revealed that anti-SARS-CoV-2 IgA and IgM antibodies rapidly decayed, while IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that serum and saliva IgG antibodies to SARS-CoV-2 are maintained in the majority of COVID-19 patients for at least 3 months PSO. IgG responses in saliva may serve as a surrogate measure of systemic immunity to SARS-CoV-2 based on their correlation with serum IgG responses.

Current cariology education in dental schools in Spanish-speaking Latin American countries.

This study sought to provide an overview of current cariology education in Spanish-speaking Latin American dental schools. Data collection was via an eighteen-item survey with questions about curriculum, methods of diagnosis and treatment, and instructors' perceptions about cariology teaching. The response rate was 62.1 percent (n=54), and distribution of participating schools by country was as follows: Bolivia (four), Chile (four), Colombia (twenty-four), Costa Rica (one), Cuba (one), Dominican Republic (two), El Salvador (two), Mexico (six), Panama (two), Peru (four), Puerto Rico (one), Uruguay (two), and Venezuela (one). Forty percent of the responding schools considered cariology the key axis of a course, with a cariology department in 16.7 percent. All schools reported teaching cariology, but with varying hours and at varying times in the curriculum, and 77.8 percent reported having preclinical practices. The majority reported teaching most main teaching topics, except for behavioral sciences, microbiology, saliva and systemic diseases, caries-risk factors, root caries, erosion, and early caries management strategies. The most frequently taught caries detection methods were visual-tactile (96.3 percent), radiographic (92.6 percent), and the International Caries Detection and Assessment System (ICDAS) (61.1 percent). Respondents said their schools' clinics make an operative treatment decision when radiolucency is in the inner half of enamel (42.3 percent) for radiographic criteria and when the lesion is visually non-cavitated (5.8 percent). All respondents reported that their schools teach preventive strategies, but only 43.4 percent said they tie it to risk assessment and 40.7 percent said they implement nonsurgical management regularly.