Scientific frontiers: emerging technologies for salivary diagnostics.

Saliva, a biofluid historically well-studied biochemically and physiologically, has entered the post-genomic 'omics' era, where its proteomic, genomic, and microbiome constituents have been comprehensively deciphered. The translational path of these salivary constituents has begun toward a variety of personalized individual medical applications, including early detection of cancer. Salivary diagnostics is a late-comer, but it is catching up where dedicated resources, like the Salivaomics Knowledge Base (SKB), now have taken center stage in the dissemination of the diagnostic potentials of salivary biomarkers and other translational and clinical utilities.

Saliva, diagnostics, and dentistry.

Saliva, a scientific and clinical entity familiar to every oral health researcher and dental practitioner, has emerged as a translational and clinical commodity that has reached national visibility at the National Institutes of Health and the President's Office of Science and Technology. "Detecting dozens of diseases in a sample of saliva" was issued by President Obama as one of the 14 Grand Challenges for biomedical research in the 21(st) Century (National Economic Council, 2010). In addition, NIH's 2011 Government Performance Report Act (GPRA) listed 10 initiatives in the high-risk long-term category (Collins, 2011). The mandate is to determine the efficacy of using salivary diagnostics to monitor health and diagnose at least one systemic disease by 2013. The stage is set for the scientific community to capture these national and global opportunities to advance and substantiate the scientific foundation of salivary diagnostics to meet these goals. A specific calling is to the oral, dental, and craniofacial health community. Three areas will be highlighted in this paper: the concept of high-impact diagnostics, the role of dentists in diagnostics, and, finally, an infrastructure currently being developed in the United Kingdom--The UK Biobank--which will have an impact on the translational and clinical utilizations of saliva.

Role of Saliva and Salivary Diagnostics in the Advancement of Oral Health.

The objective of this article was to provide an account of some of the developments related to saliva over the first 100 years of the Journal of Dental Research and to outline some of the many biomarkers identified in saliva in the last few years. The first section covers findings in salivary physiology, biochemistry, calcium phosphate chemistry related to saliva, microbiology, and the role of saliva in maintaining oral health. The second section highlights salivary diagnostics, salivaomics, and saliva exosomics in the context of the emerging theme of personalized and precision medicine.

Liquid Biopsy in Head and Neck Cancer: Promises and Challenges.

Head and neck cancer is the sixth most common cancer worldwide. It remains one of the leading causes of death, and its early detection is crucial. Liquid biopsy has emerged as a promising tool for detecting and monitoring the disease status of patients with early and advanced cancers. Circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomal miRNAs have received enormous attention because of their apparent clinical implications. Analyses of these circulating biomarkers have paved the way for novel therapeutic approaches and precision medicine. A growing number of reports have implicated the use of circulating biomarkers for detection, treatment planning, response monitoring, and prognosis assessment. Although these new biomarkers can provide a wide range of possible clinical applications, no validated circulating biomarkers have yet been integrated into clinical practice for head and neck cancer. In this review, we summarize the current knowledge of circulating biomarkers in this field, focusing on their feasibility, limitations, and key areas of clinical applications. We also highlight recent advances in salivary diagnostics and their potential application in head and neck cancer.

Human tRNA-Derived Small RNAs Modulate Host-Oral Microbial Interactions.

Coevolution of the human host and its associated microbiota has led to sophisticated interactions to maintain a delicate homeostasis. Emerging evidence suggests that in addition to small molecules, peptides, and proteins, small regulatory noncoding RNAs (sRNAs) might play an important role in cross-domain interactions. In this study, we revealed the presence of diverse host transfer RNA-derived small RNAs (tsRNAs) among human salivary sRNAs. We selected 2 tsRNAs (tsRNA-000794 and tsRNA-020498) for further study based on their high sequence similarity to specific tRNAs from a group of Gram-negative oral bacteria, including Fusobacterium nucleatum, a key oral commensal and opportunistic pathogen. We showed that the presence of F. nucleatum triggers exosome-mediated release of tsRNA-000794 and tsRNA-020498 by human normal oral keratinocyte cells. Furthermore, both tsRNA candidates exerted a growth inhibition effect on F. nucleatum, likely through interference with bacterial protein biosynthesis, but did not affect the growth of Streptococcus mitis, a health-associated oral Gram-positive bacterium whose genome does not carry sequences bearing high similarity to either tsRNA. Our data provide the first line of evidence for the modulatory role of host-derived tsRNAs in the microbial-host interaction.

Identification of discrete chromosomal deletion by binary recursive partitioning of microarray differential expression data.

DNA copy number abnormalities (CNA) are characteristic of tumours, and are also found in association with congenital anomalies and mental retardation. The ultimate impact of copy number abnormalities is manifested by the altered expression of the encoded genes. We previously developed a statistical method for the detection of simple chromosomal amplification using microarray expression data. In this study, we significantly advanced those analytical techniques to allow detection of localised chromosomal deletions based on differential gene expression data. Using three cell lines with known chromosomal deletions as model system, mRNA expression in those cells was compared with that observed in diploid cell lines of matched tissue origin. Results show that genes from deleted chromosomal regions are substantially over-represented (p<0.000001 by chi2) among genes identified as underexpressed in deletion cell lines relative to normal matching cells. Using a likelihood based statistical model, we were able to identify the breakpoint of the chromosomal deletion and match with the karyotype data in each cell line. In one such cell line, our analyses refined a previously identified 10p chromosomal deletion region. The deletion region was mapped to between 10p14 and 10p12, which was further confirmed by subtelomeric fluorescence in situ hybridisation. These data show that microarray differential expression data can be used to detect and map the boundaries of submicroscopic chromosomal deletions.

DNA hybridization arrays for gene expression analysis of human oral cancer.

DNA hybridization arrays permit global gene expression profiling to be done in a single experiment. The evolution and challenges of DNA hybridization arrays are reflected in the variety of experimental platforms, probe composition, hybridization/signal detection methods, and bioinformatic interpretation. In tumor biology, DNA hybridization arrays are being used for gene/gene pathway discovery, diagnosis, and therapeutic design. Similar applications are advancing our understanding of oral cancer cell biology.

RNA profiling of cell-free saliva using microarray technology.

Saliva, like other bodily fluids, has been used to monitor human health and disease. This study tests the hypothesis that informative human mRNA exists in cell-free saliva. If present, salivary mRNA may provide potential biomarkers to identify populations and patients at high risk for oral and systemic diseases. Unstimulated saliva was collected from ten normal subjects. RNA was isolated from the cell-free saliva supernatant and linearly amplified. High-density oligonucleotide microarrays were used to profile salivary mRNA. The results demonstrated that there are thousands of human mRNAs in cell-free saliva. Quantitative PCR (Q-PCR) analysis confirmed the present of mRNA identified by our microarray study. A reference database was generated based on the mRNA profiles in normal saliva. Our finding proposes a novel clinical approach to salivary diagnostics, Salivary Transcriptome Diagnostics (STD), for potential applications in disease diagnostics as well as normal health surveillance.

Emerging horizons of salivary diagnostics for periodontal disease.

The field of salivary diagnostics to allow risk determination for periodontal diseases is advancing. New technologies in proteomics, genomics and nanotechnologies have continued the discovery of discriminatory periodontal disease biomarkers. This review briefly overviews biomarker studies that have been completed in saliva for the detection of periodontal disease since 2010. Disease specific biomarkers could be used in risk determination, treatment planning and disease progression. Currently, diagnostic tests are commercially available, and the development of point-of-care tests is expanding. Even though challenges remain, salivary diagnostics for periodontal disease is promising and could facilitate the diagnostics and treatment in a clinical practice by dental practitioners.

Salivary diagnostics.

Saliva is a noninvasive and accessible biofluid that permits early detection of oral and systemic diseases. Recent scientific and technologic advances have uncovered specific salivary biomarkers for a number of clinical conditions, including cancers, autoimmune diseases, and cardiovascular disorders. The availability of highly sensitive and high-throughput assays such as microarray, mass spectrometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and nano-scale sensors that can measure proteins and nucleic acids are poising saliva as an emerging biofluid for translational and clinical applications. This paper will discuss development of salivary biomarkers for the detection of oral and systemic diseases and the translational application of these markers for clinical applications.

Double edge: CDK2AP1 in cell-cycle regulation and epigenetic regulation.

Cancer research has been devoted toward an understanding of the molecular regulation and functional significance of cell-cycle regulators in the pathogenesis and development of cancers. Cyclin-dependent Kinase 2-associated Protein 1 (CDK2AP1) is one such cell-cycle regulator, originally identified as a growth suppressor and a prognostic marker for human oral/head and neck cancers. Functional importance and the molecular mechanism of CDK2AP1-mediated cell-cycle regulation have been documented over the years. Recent progress has shown that CDK2AP1 is a competency factor in embryonic stem cell differentiation. Deletion of CDK2AP1 leads to early embryonic lethality, potentially through altered differentiation capability of embryonic stem cells. More intriguingly, CDK2AP1 exerts its effect on stem cell maintenance/differentiation through epigenetic regulation. Cancer cells and stem cells share common cellular characteristics, most prominently in maintaining high proliferative potential through an unconventional cell-cycle regulatory mechanism. Cross-talk between cellular processes and molecular signaling pathways is frequent in any biological system. Currently, it remains largely elusive how cell-cycle regulation is mechanistically linked to epigenetic control. Understanding the molecular mechanism underlying CDK2AP1-mediated cell-cycle regulation and epigenetic control will set an example for establishing a novel and effective molecular link between these two important regulatory mechanisms.

[Not Available].

Human saliva can be separated by centrifugation into cell pellet and cell-free supernatant, which are called cellular phase and liquid phase in this study. While it is well documented that the cellular phase of saliva contains hundreds of oral bacteria species, little is known whether the liquid phase of saliva contains any information related to oral microbiota. In this study, we analyzed the bacterial nucleic acid contents of the liquid phase of saliva. Using primers universal to most eubacterial 16S rDNA, we detected large amounts of bacterial 16S rRNA and rDNA in the cell-free phase of saliva. Random sequencing analysis of forty PCR amplicons from the cell-free phase of saliva led to 15 operational taxonomic unit (OTU) groups. Furthermore, using denaturing gradient gel electrophoresis (DGGE), we compared 16S rRNA/rDNA profiles derived from liquid phases and cellular phases of saliva samples, and found positive correlations (Pearson Correlation=0.822, P<0.001) between these sample groups. These findings indicate that the liquid phase of saliva contains numerous bacterial 16S rRNA/rDNA molecules that have correlations with bacteria existing in the cellular phase.

Expression of cell-cycle regulator CDK2-associating protein 1 (p12CDK2AP1) in transgenic mice induces testicular and ovarian atrophy in vivo.

The novel cell-cycle regulator p12(CDK2AP1) (p12) gene encodes a cyclin-dependent kinase 2 (CDK2) partner that participates in cell-cycle regulation, apoptosis, and proliferation. CDK2 has been implicated in maintenance of gonadal homeostasis, as knockout mice display reproductive abnormalities. To investigate the role of p12 in homeostasis of gonadal tissues in vivo, we generated a transgenic mouse model driven by the human keratin 14 promoter, reported to target transgene expression to gonadal tissues and also stratified epithelia. Overexpression of the transgene was associated with a gonadal atrophy phenotype in mice of both sexes, yet fertility was not impaired. Histological evaluation of testes showed seminiferous tubule degeneration and decreased tubule diameter. Female transgenic mice had small ovaries, with a higher number of atretic follicles/mm(2) as compared to control nontransgenic mice. Also observed was increased germ cell apoptosis in both sexes (TUNEL). These results suggest that overexpression of p12 leads to testicular and ovarian abnormalities, a phenotype closely related to that of cdk2-/- mice. In combination, these observations suggest that the p12/CDK2 signaling pathways are carefully orchestrated to maintain proper gonadal tissue homeostasis. We suggest that the mechanisms of this regulation may be through p12-mediated altered expression of gonadal-specific genes and apoptotic pathways.

Quantitative analyses of Streptococcus mutans biofilms with quartz crystal microbalance, microjet impingement and confocal microscopy.

Microbial biofilm formation can be influenced by many physiological and genetic factors. The conventional microtiter plate assay provides useful but limited information about biofilm formation. With the fast expansion of the biofilm research field, there are urgent needs for more informative techniques to quantify the major parameters of a biofilm, such as adhesive strength and total biomass. It would be even more ideal if these measurements could be conducted in a real-time, non-invasive manner. In this study, we used quartz crystal microbalance (QCM) and microjet impingement (MJI) to measure total biomass and adhesive strength, respectively, of S. mutans biofilms formed under different sucrose concentrations. In conjunction with confocal laser scanning microscopy (CLSM) and the COMSTAT software, we show that sucrose concentration affects the biofilm strength, total biomass, and architecture in both qualitative and quantitative manners. Our data correlate well with previous observations about the effect of sucrose on the adherence of S. mutans to the tooth surface, and demonstrate that QCM is a useful tool for studying the kinetics of biofilm formation in real time and that MJI is a sensitive, easy-to-use device to measure the adhesive strength of a biofilm.