Saliva Supernatant Research Articles

Oral Lactobacilli and salivary acidic proline-rich proteins (APRP-1/2) in dental caries.

A previous study shows that levels of acidic salivary proline-rich phosphoproteins-1/2 (APRP-1/2) increase with caries severity. The aim of this study was to examine whether this relationship also depends on the presence of H2O2-producing strains of Lactobacillus spp. Adults with severe caries (decayed, missing, and filled teeth (DMFT) > 13.9, n = 28) were compared with similarly aged adults who had minimal caries (DMFT < 5, n = 20). A total of 48 samples of whole unstimulated saliva were collected in the morning and centrifuged. Lactobacillus spp. were isolated from the sediment in Rogosa agar and peroxide (H2O2) production was determined by growing the isolates on TMB-Plus agar. Salivary APRP-1/2 content in the saliva supernatant was estimated using an enzyme-linked immunosorbent sandwich assay (ELISA). Lactobacilli were present in 67% of both caries groups but were H2O2 positive only in the minimal caries group. Irrespective of the presence of Lactobacilli, the total content of APRP-1/2 proteins was 34.5 ± 4.9 ng/ml in severe caries but just under half this in minimal caries. We conclude that Lactobacillus spp. was absent from about a third of the severe and minimal caries groups, and H2O2-producing strains were present only in the minimal caries group. The severe caries group possessed twice the content of salivary APRP 1/2 proteins as the minimal caries group. The implications of these findings for caries development are discussed.

CIP2A immunosensor comprised of vertically-aligned carbon nanotube interdigitated electrodes towards point-of-care oral cancer screening

Vertically aligned carbon nanotube array (VANTA) coatings have recently garnered significant attention due in part to their unique material properties including light absorption, chemical inertness, and electrical conductivity. Herein we report the first use of VANTAs grown via chemical vapor deposition in a 2D interdigitated electrode (IDE) footprint with a high height-to-width aspect ratio (3:1 or 75:25 µm). The VANTA-IDEs were functionalized with an antibody (Ab) specific to the human cancerous inhibitor PP2A (CIP2A)—an oncoprotein that is associated with a variety of malignancies such as oral, breast, and multiple myeloma cancers. The resultant label-free immunosensor was capable of detecting CIP2A across a wide linear sensing range (1−100 pg/mL) with a detection limit of 0.24 pg/mL within saliva supernatant—a range that is more sensitive than the corresponding CIP2A enzyme linked immunosorbent assay (ELISA). These results help pave the way for rapid cancer screening tests at the point-of-care (POC) such as for the early-stage diagnosis of oral cancer at a dentist's office.

Fluoride Levels in Unstimulated Whole Saliva following Clinical Application of Different 5% NaF Varnishes

The aim of this study was to evaluate the fluoride release from differently formulated 5% NaF varnishes into unstimulated whole saliva in vivo. The fluoride concentration in unstimulated whole saliva was determined after the application of 3 different 5% NaF varnishes (5% NaF, 5% NaF + tricalcium phosphate [TCP], and 5% NaF + amorphous calcium phosphate [ACP]) or a placebo. Fifteen subjects were recruited and enrolled following Institutional Review Board approval based upon the inclusion/exclusion criteria of this study. A cross-over study design was used for the application of either one of the 5% NaF varnishes or a placebo. Unstimulated whole saliva was collected at baseline and at 1, 4, 6, 26, and 50 h following application and analyzed for supernatant ionic fluoride and whole fluoride by microdiffusion. Linear mixed-effects models (5% significance level) were used to determine the effects of varnish and time on the salivary fluoride concentration. The highest amount of fluoride in saliva was found 1 h after application of the fluoride varnishes, with no significant differences among the treatment varnishes with respect to whole fluoride but with lower levels for 5% NaF + ACP in the saliva supernatant. Salivary fluoride levels at 4, 6, and 26 h decreased at each time point and were generally significantly higher for 5% NaF and 5% NaF + TCP. After 50 h, fluoride levels in saliva for all groups were at or below baseline levels. In conclusion, the formulation of other ingredients in fluoride varnishes can affect the fluoride concentration in saliva. The reasons for this phenomenon warrant further investigation since it might affect efficacy of the treatment. This trial is registered at ClinicalTrials.gov (NCT01629290).

Assessment of saliva, hair and toenails as biomarkers of low level exposure to manganese from drinking water in children

We evaluated hair, toenails, and saliva (whole and supernatant) as biomarkers of exposure to manganese (Mn) in 274 school age children (6–13 years) consuming well water in southeastern New Brunswick, Canada. Mn concentrations in tap water ranged from <0.03 to 1046μgL−1 (geometric mean 5.96μgL−1). The geometric mean of Mn intake resulting from the consumption of water was 0.25 (0–34.95) μg kg−1day−1. Both Mn concentration in water and Mn intake were significantly correlated with Mn in hair (r=0.60 and r=0.53, respectively), Mn in toenail (r=0.29 and r=0.37 respectively) and to a lesser extent with Mn in saliva supernatant (r=0.14 and r=0.18, respectively). Mn in whole saliva did not correlate with Mn in water or Mn intake. Both Mn in hair and Mn in toenail allowed to discriminate the most exposed group from the least exposed group, based on Mn in water and Mn intake from water. In this group of children with low level Mn exposure, Mn concentrations in hair, and toenails reflected reasonably well Mn exposure from drinking water, whereas Mn content in saliva correlated less strongly.

Salivary extracellular RNA biomarkers for insulin resistance detection in hispanics

Insulin resistance (IR) detection is challenging and no test is currently used in clinical practice. We developed salivary biomarkers that could be used for IR detection. We collected saliva from 186 healthy and 276 pre-diabetic participants, divided them into high and low IR groups based on a HOMA cutoff of 2.5. We profiled extracellular transcriptome by microarray in saliva supernatant from 23 high IR and 15 low IR participants, and pre-validated the top ten extracellular mRNA (exRNA) markers in a new cohort of 40 high and 40 low IR participants. A prediction panel was then built and validated in an independent cohort of 149 high and 195 low IR participants. Transcriptomic analyses identified 42 exRNA candidates differentially present in saliva of high and low IR participants. From the top ten candidates, six were individually validated (PRKCB, S100A12, IL1R2, CAMP, VPS4B, CAP1) (p<0.01) and yielded AUC values ranging from 0.66 to 0.76. Body mass index (BMI) was significant higher in high compared to low IR group with AUC of 0.66, and showed no correlation with any of candidate biomarkers. The combination of four exRNA markers (IL1R2, VPS4B, CAP1, LUZP6) with BMI achieved excellent results in the prediction panel building dataset (AUC=0.79, sensitivity=79%, specificity=64%). The prediction model was validated in an independent cohort (AUC=0.82, sensitivity=63%, specificity=92%). A panel of four salivary exRNA biomarkers (IL1R2, VPS4B, CAP1, LUZP6) and BMI was validated that can distinguish high and low IR participants, overall and in subgroups of healthy and pre-diabetic participants.

The Potential Use of Salivary miRNAs as Promising Biomarkers for Detection of Cancer: A Meta-Analysis

BackgroundAccumulating evidence has demonstrated that microRNAs (miRNAs) could serve as promising molecular biomarkers for cancer detection. This study aims to systematically assess the diagnostic performance of salivary miRNAs in detection of cancer through a comprehensive meta-analysis.MethodsEligible studies were identified using PubMed and other computerized databases up to October 31, 2015, supplemented by a manual search of references from retrieved articles. The pooled sensitivity, specificity, and other measurements of accuracy of salivary miRNAs in the diagnosis of cancer were analyzed using the bivariate binomial mixed model.ResultsSeventeen studies from 8 articles with 694 subjects were included in this meta-analysis. All studies have a relatively high score of quality assessment. The overall sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) of salivary miRNAs in detection of cancer were 0.77 (95% confidence intervals [CI]: 0.69–0.84), 0.77 (95%CI: 0.65–0.88), 3.37 (95%CI: 2.26–5.02), 0.29 (95%CI: 0.23–0.38), and 11.41 (95%CI: 7.35–17.73), respectively. The AUC was 0.84 (95%CI: 0.80–0.87). Moreover, both whole saliva and saliva supernatant could be used as sources of clinical specimens for miRNAs detection.ConclusionsOur meta-analysis demonstrated that salivary miRNAs may serve as potential noninvasive biomarkers for cancer detection. The findings need to be confirmed with further research before it can be applied in the clinic.

The impact of stannous, fluoride ions and its combination on enamel pellicle proteome and dental erosion prevention.

ObjectivesTo compare the effects of stannous (Sn) and fluoride (F) ions and their combination on acquired enamel pellicle (AEP) protein composition (proteome experiment), and protection against dental erosion (functional experiment).MethodsIn the proteome experiment, bovine enamel specimens were incubated in whole saliva supernatant for 24h for AEP formation. They were randomly assigned to 4 groups (n=10), according to the rinse treatment: Sn (800ppm/6.7mM, SnCl2), F (225ppm/13mM, NaF), Sn and F combination (Sn+F) and deionized water (DIW, negative control). The specimens were immersed 3× in the test rinses for 2min, 2h apart. Pellicles were collected, digested, and analyzed for protein content using liquid chromatography electrospray ionization tandem mass spectrometry. In the functional experiment, bovine enamel specimens (n=10) were similarly treated for pellicle formation. Then, they were subjected to a five-day erosion cycling model, consisting of 5min erosive challenges (15.6 mM citric acid, pH 2.6, 6×/d) and 2min treatment with the rinses containing Sn, F or Sn+F (3×/d). Between the treatments, all specimens were incubated in whole saliva supernatant. Surface loss was determined by profilometry.ResultsOur proteome approach on bovine enamel identified 72 proteins that were common to all groups. AEP of enamel treated with Sn+F demonstrated higher abundance for most of the identified proteins than the other groups. The functional experiment showed reduction of enamel surface loss for Sn+F (89%), Sn (67%) and F (42%) compared to DIW (all significantly different, p<0.05).ConclusionThis study highlighted that anti-erosion rinses (e.g. Sn+F) can modify quantitatively and qualitatively the AEP formed on bovine enamel. Moreover, our study demonstrated a combinatory effect that amplified the anti-erosive protection on tooth surface.

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Salivary MicroRNAs as Promising Biomarkers for Detection of Esophageal Cancer

Background and PurposeTissue microRNAs (miRNAs) can detect cancers and predict prognosis. Several recent studies reported that tissue, plasma, and saliva miRNAs share similar expression profiles. In this study, we investigated the discriminatory power of salivary miRNAs (including whole saliva and saliva supernatant) for detection of esophageal cancer.Materials and MethodsBy Agilent microarray, six deregulated miRNAs from whole saliva samples from seven patients with esophageal cancer and three healthy controls were selected. The six selected miRNAs were subjected to validation of their expression levels by RT-qPCR using both whole saliva and saliva supernatant samples from an independent set of 39 patients with esophageal cancer and 19 healthy controls.ResultsSix miRNAs (miR-10b*, miR-144, miR-21, miR-451, miR-486-5p, and miR-634) were identified as targets by Agilent microarray. After validation by RT-qPCR, miR-10b*, miR-144, and miR-451 in whole saliva and miR-10b*, miR-144, miR-21, and miR-451 in saliva supernatant were significantly upregulated in patients, with sensitivities of 89.7, 92.3, 84.6, 79.5, 43.6, 89.7, and 51.3% and specificities of 57.9, 47.4, 57.9%, 57.9, 89.5, 47.4, and 84.2%, respectively.ConclusionsWe found distinctive miRNAs for esophageal cancer in both whole saliva and saliva supernatant. These miRNAs possess discriminatory power for detection of esophageal cancer. Because saliva collection is noninvasive and convenient, salivary miRNAs show great promise as biomarkers for detection of esophageal cancer in areas at high risk.

The diagnostic potential of salivary protease activities in periodontal health and disease

Periodontal disease is characterised by proteolytic processes involving enzymes that are released by host immune cells and periodontal bacteria. These enzymes, when detectable in whole saliva, may serve as valuable diagnostic markers for disease states and progression. Because the substrate specificities of salivary proteases in periodontal health and disease are poorly characterised, we probed these activities using several relevant substrates: (i) gelatin and collagen type IV; (ii) the Arg/Lys-rich human salivary substrate histatin-5; and (iii) a histatin-derived synthetic analog benzyloxycarbonyl-Arg-Gly-Tyr-Arg-methyl cumaryl amide (Z-RGYR-MCA). Substrate degradation was assessed in gel (zymography) and in solution. Whole saliva supernatant enzyme activities directed at gelatin, quantified from the 42 kDa, 92 kDa and 130 kDa bands in the zymograms, were 1.3, 1.4 and 2.0-fold higher, respectively, in the periodontal patient group (P<0.01), consistent with enhanced activities observed towards collagen type IV. On the other hand, histatin 5 degraded equally fast in healthy and periodontal patients' whole saliva supernatant samples (P>0.10). Likewise, the hydrolysis rates of the Z-RGYR-MCA substrate were the same in the healthy and periodontal patient groups (P>0.10). In conclusion, gelatinolytic/collagenolytic activities but not trypsin-like activities in human saliva differentiate health from periodontal disease and may thus provide an adjuvant to diagnosis for monitoring disease activity.

Saliva Supernatant miR-21: a Novel Potential Biomarker for Esophageal Cancer Detection

To identify whether saliva supernatant miR-21 can serve as a novel potential biomarker in patients with esophageal cancer (EC). 32 patients with EC and 16 healthy controls were recruited in this study. Total RNA was extracted from saliva supernatant samples for measurement of miR-21 levels using RT-qPCR and relationships between miR-21 levels and clinical characteristics of EC patients were analyzed. miR-21 was significantly higher in the EC than control groups. The sensitivity and specificity were 84.4% and 62.5% respectively. Supernatant miR-21 levels showed no significant correlation with cancer stage, differentiation and nodal metastasis. Saliva supernatant miR-21 may be a novel biomarker for EC.

Direct Saliva Transcriptome Analysis

Current standard operating procedures for salivary transcriptomic analysis require low temperatures and lengthy mRNA isolation, which substantially hamper its use in the clinic. We developed a streamlined, ambient-temperature processing, stabilization, and storage protocol for clinical analysis of salivary RNA. The direct saliva transcriptome analysis (DSTA) used cell-free saliva supernatant instead of isolated mRNA for saliva transcriptomic detection, and all procedures, including processing, stabilization, and storage of saliva samples, were performed at ambient temperature without a stabilizing reagent. We evaluated this streamlined protocol by comparing the mRNA expression levels of 3 saliva internal reference genes [glyceraldehyde-3-phosphate dehydrogenase (GAPDH); actin, beta (ACTB); and ribosomal protein S9 (RPS9)] to levels measured with standard procedures, and detecting the variation of their expression levels under long-term ambient temperature storage. The clinical utility of DSTA was assessed by use of 7 oral cancer salivary mRNA biomarkers in a clinical study. Each saliva internal reference gene mRNA showed similar expression levels when assayed by the DSTA or standard procedures, and remained stable under ambient temperature storage for at least 10 weeks without significant degradation (P = 0.918, 0.288, and 0.242 for GAPDH, ACTB, and RPS9, respectively). Compared with standard procedures, the performance characteristics of oral cancer salivary transcriptomic markers were retained as assayed by DSTA after 10 weeks of storage at ambient temperature. These results indicate that the DSTA is a suitable alternative method for saliva transcriptomic analysis and is feasible for use in clinical cancer research applications. The streamlined DSTA protocol can impact the saliva-handling method and improve the standard operating procedures for clinical saliva transcriptomic diagnostics.

Salivary β-galactosidase activity affects physiological oral malodour

ObjectivesPrevious reports have associated salivary β-galactosidase activity with non-periodontopathic oral malodour. In this study, we investigated the localization of β-galactosidase and elucidated the relationship between its enzymatic activity and physiological oral malodour. Study designFifty-six patients complaining of halitosis were separated into two groups: periodontally healthy and periodontitis. Saliva samples from the subjects were separated by centrifugation, and the level of β-galactosidase activity was measured in the supernatant, pellet lysate, and whole saliva using the chromogenic substrate o-nitrophenyl-β-d-galactopyranoside. The correlation of salivary β-galactosidase activity with breath odour and associated parameters was examined. ResultsSimilar levels of β-galactosidase activity were detected in the pellet lysate and whole saliva, but not in the saliva supernatant. Positive correlations were observed between the β-galactosidase activity in whole saliva and oral malodour levels in the periodontally healthy group, but not in the periodontitis group. In addition, the plaque index and tongue coating score were positively correlated with β-galactosidase activity in the periodontally healthy group. Overall, stimulated salivary flow and salivary pH were negatively correlated with enzyme activity. The amounts of total bacteria, Fusobacterium nucleatum, and Streptococcus salivarius were positively associated with β-galactosidase activity in the periodontally healthy group. Furthermore, the amounts of total bacteria and S. salivarius were positively associated with the amount of volatile sulphur compounds. ConclusionsOur results indicate that β-galactosidase is located on the cell surface of oral bacteria derived from dental plaque and tongue coating, and it plays an important role in producing the malodour underlying physiological oral malodour.

Mass Spectrometric Identification of Key Proteolytic Cleavage Sites in Statherin Affecting Mineral Homeostasis and Bacterial Binding Domains

Human salivary statherin inhibits both primary and secondary calcium phosphate precipitation and, upon binding to hydroxyapatite, associates with a variety of oral bacteria. These functions, crucial in the maintenance of tooth enamel integrity, are located in defined regions within the statherin molecule. Proteases associated with saliva, however, cleave statherin effectively, and it is of importance to determine how statherin functional domains are affected by these events. Statherin was isolated from human parotid secretion by zinc precipitation and purified by reversed-phase high performance liquid chromatography (RP-HPLC). To characterize the proteolytic process provoked by oral proteases, statherin was incubated with whole saliva and fragmentation was monitored by RP-HPLC. The early formed peptides were structurally characterized by reversed phase liquid chromatography electrospray-ionization tandem mass spectrometry. Statherin was degraded 3.6× faster in whole saliva than in whole saliva supernatant. The main and primary cleavage sites were located in the N-terminal half of statherin, specifically after Arg(9), Arg(10), and Arg(13); after Phe(14) and Tyr(18); and after Gly(12), Gly(15), Gly(17) and Gly(19) while the C-terminal half of statherin remained intact. Whole saliva protease activities separated the charged N-terminus from the hydrophobic C-terminus, negatively impacting on full length statherin functions comprising enamel lubrication and inhibition of primary calcium phosphate precipitation. Cryptic epitopes for bacterial binding residing in the C-terminal domain were likewise affected. The full characterization of the statherin peptides generated facilitates the elucidation of their novel functional roles in the oral and gastro-intestinal environment.

Abstract 2726: Salivary biomarkers for breast cancer detection

Abstract Objectives: The high burden of breast cancer in women worldwide underscores the unmet potential of biomarker for early detection. New strategies and biomarkers for early detection of breast cancer are sorely needed. In this study, we have conducted a prospective sample collection and retrospective blinded validation (PRoBE design) to evaluate the performance and translational utilities of salivary biomarkers for the non-invasive detection of breast cancer. Methods: The Affymetrix HG U133 Plus 2.0 Array and 2D-DIGE were used to profile transcriptomes and proteomes in saliva supernatant from patients with invasive ductal carinoma and matched controls respectively. The discovered transcriptomic and proteomic candidate biomarkers were subjected to a clinical validation using an independent sample set of 31 breast cancer (invasive ductal carcinoma, stages I and II) and 62 healthy controls. Transcriptomic biomarkers were validated by qPCR and proteomic biomarkers were validated by quantitative immuno-blot. Results: Significant variations of salivary transcriptomic and proteomic profiles were observed between breast cancer patients and healthy controls. Eleven mRNA biomarker candidates and two protein biomarker candidates were selected for independent validation. Eight mRNA biomarkers and one protein biomarker have been validated for breast cancer detection, yielding ROC-plot AUC values between 0.665 and 0.959. Six biomarkers have known associations with breast cancer. Conclusion: Salivary biomarkers possess discriminatory power for the detection of breast cancer, with high specificity and sensitivity. This report provides proof of concept of salivary transcriptomic and proteomic biomarkers for the non-invasive detection of breast cancer. The salivary biomarkers' discriminatory power paves the way for a pivotal clinical validation. Supported by NIH grant U01DE016275, U01DE017790 and R21CA126733 (DTW). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2726.

Salivary Transcriptomic Biomarkers for Detection of Resectable Pancreatic Cancer

Lack of detection technology for early pancreatic cancer invariably leads to a typical clinical presentation of incurable disease at initial diagnosis. New strategies and biomarkers for early detection are sorely needed. In this study, we have conducted a prospective sample collection and retrospective blinded validation to evaluate the performance and translational utilities of salivary transcriptomic biomarkers for the noninvasive detection of resectable pancreatic cancer. The Affymetrix HG U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA) was used to profile transcriptomes and discover altered gene expression in saliva supernatant. Biomarkers discovered from the microarray study were subjected to clinical validation using an independent sample set of 30 pancreatic cancer patients, 30 chronic pancreatitis patients, and 30 healthy controls. Twelve messenger RNA biomarkers were discovered and validated. The logistic regression model with the combination of 4 messenger RNA biomarkers (KRAS, MBD3L2, ACRV1, and DPM1) could differentiate pancreatic cancer patients from noncancer subjects (chronic pancreatitis and healthy control), yielding a receiver operating characteristic plot, area under the curve value of 0.971 with 90.0% sensitivity and 95.0% specificity. The salivary biomarkers possess discriminatory power for the detection of resectable pancreatic cancer, with high specificity and sensitivity. This report provides the proof of concept of salivary biomarkers for the noninvasive detection of a systemic cancer and paves the way for prediction model validation study followed by pivotal clinical validation.

Activity‐based mass spectrometric characterization of proteases and inhibitors in human saliva

Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50-75 kDa region. Individual bands were excised, in-gel trypsinized and subjected to LC/ESI-MS/MS. The data obtained were searched against human, oral microbial and protease databases. A total of 13 proteases were identified all of which were of mammalian origin. Proteases detected in multiple fractions with cleavage specificities toward arginine and lysine residues, were lactotransferrin, kallikrein-1, and human airway trypsin-like protease. Unexpectedly, ten protease inhibitors were co-identified suggesting they were associated with the proteases in the same fractions. The inhibitors found most frequently were alpha-2-macroglobulin-like protein 1, alpha-1-antitrypsin, and leukocyte elastase inhibitor. Regulation of oral fluid proteolysis is highly important given that an inbalance in such activities has been correlated to a variety of pathological conditions including oral cancer.

Salivary biomarkers for pancreatic cancer detection

4630 Pancreatic cancer is the 4th leading cause of cancer death. Lack of early detection technology for pancreatic cancer invariably leads to a typical clinical presentation of incurable disease at initial diagnosis. We evaluated the performance and translational utilities of the salivary transcriptomic and microbial biomarkers for pancreatic cancer detection. Biomarker discovery strategies were used to profile transcriptome in saliva supernatant and microflora in saliva pellet. The Affymetrix Human Genome U133+2.0 array was used to discover altered gene expression in saliva supernatant. The Human Oral Microbe Identification Microarray (HOMIM) was used to investigate microflora shift in saliva pellet. Biomarkers selected from both studies were subjected to an independent clinical validation using a cohort of 30 pancreatic cancer, 30 chronic pancreatitis and 30 healthy matched-control saliva samples. Two panels of salivary biomarkers, including eleven mRNA biomarkers and two microbial biomarkers were discovered and validated for pancreatic cancer detection. The logistic regression model with the combination of three mRNA biomarkers (ACRV1, DMXL2 and DPM1) yielded a ROC-plot AUC value of 0.974 (95% CI, 0.896 to 0.997; P &lt; 0.0001) with 93.3% sensitivity and 90% specificity in distinguishing pancreatic cancer patients from healthy subjects. The logistic regression model with the combination of two bacterial biomarkers (Neisseria elongata and Streptococcus mitis) yielded a ROC-plot AUC value of 0.895 (95% CI, 0.784 to 0.961; P &lt; 0.0001) with 96.4% sensitivity and 82.1% specificity in distinguishing pancreatic cancer patients from healthy subjects. More importantly, the logistic regression model with the combination of four biomarkers (mRNA biomarkers, ACRV1, DMXL2 and DPM1; bacterial biomarker, S. mitis) could differentiate pancreatic cancer patients from all non-cancer subjects (chronic pancreatitis and healthy control), yielding a ROC-plot AUC value of 0.949 (95% CI, 0.877 to 0.985; P &lt; 0.0001) with 92.9% sensitivity and 85.5% specificity. This is the first report demonstrating the value of multiplex salivary biomarkers for the non-invasive detection of a high impact systemic cancer. No significant financial relationships to disclose.

Kinetics of histatin proteolysis in whole saliva and the effect on bioactive domains with metal‐binding, antifungal, and wound‐healing properties

The present study was undertaken to investigate the rate and mode of degradation of individual histatin proteins in whole saliva to establish the impact on its functional domains. Pure synthetic histatins 1, 3, and 5 were incubated with whole saliva supernatant as the enzyme source, and peptides in the resultant digests were separated by reverse-phase-HPLC and structurally characterized by electrospray ionization-tandem mass spectrometry. The overall V(max)/K(m) ratios, a measure of proteolytic efficiency, were on the order of histatin-5 > histatin-3 > histatin-1. Mathematical models predict that histatins 1, 3, and 5 levels in whole saliva stabilize at 5.1, 1.9, and 1.2 microM, representing 59, 27, and 11% of glandular histatins 1, 3, and 5 levels, respectively. Monitoring of the appearance and disappearance of histatin fragments yielded the identification of the first targeted enzymatic cleavage sites as K(13) and K(17) in histatin 1, R(22), Y(24), and R(25) in histatin 3, and Y(10), K(11), R(12), K(13), H(15), E(16), K(17), and H(18) in histatin 5. The data indicate that metal-binding, antifungal, and wound-healing domains are largely unaffected by the primary cleavage events in whole saliva, suggesting a sustained functional activity of these proteins in the proteolytic environment of the oral cavity.

A universal pre-analytic solution for concurrent stabilization of salivary proteins, RNA and DNA at ambient temperature

Objective Saliva is a biofluid that can be obtained from individuals without supervision by health care providers. To maximize this clinical advantage, it is highly desirable to have a global salivary analyte stabilizer for proteins, RNA and DNA at ambient temperature. Design Whole saliva, saliva supernatant and saliva filtrate (5.0 μm) were treated with RPS at room temperature (RT) for up to 6 days and then subjected to SDS-PAGE. Immunoblotting of β-actin and cystatin C were used to evaluate protein stability. For salivary DNA/RNA, whole saliva was incubated with RPS at RT for up to 10 weeks. After extracting total DNA/RNA in samples at week 0, 2, 6 and 10, DNA stability was assayed by chromosome 18 DNA qPCR and RNA stability by β-actin mRNA RT-qPCR. Results β-actin completely degraded in all types of saliva samples after 6-day incubation at RT. However, 24.0%, 91.4% and 89.3% of β-actin remained intact with RPS for whole saliva, saliva supernatant and filtrate, respectively. Similarly, 70.3% of cystatin C in supernatant remained intact in the presence of RPS. For salivary DNA/RNA, the cycle threshold (Ct) values showed no significant change for chromosome 18 DNA and β-actin mRNA in RPS-incubated saliva during the 10-week time course while significant increase in Ct values were observed in controls without RPS for both β-actin mRNA and DNA. Conclusions RPS provided effective concurrent stabilization to salivary DNA/RNA in whole saliva for up to 10 weeks and proteins in saliva filtrate for 6 days at RT. We also achieved separation of saliva supernatant from cellular elements by a simple filtration step (bypassing the need for centrifugation).