SUMMARY The pursuit of timely, cost-effective, accurate, and noninvasive diagnostic methodologies is an endeavor of urgency among clinicians and scientists alike. Detecting pathologies at their earliest stages can significantly affect patient discomfort, prognosis, therapeutic intervention, survival rates, and recurrence. Diagnosis and monitoring often require painful invasive procedures such as biopsies and repeated blood draws, adding undue stress to an already unpleasant experience. The discovery of saliva-based microbial, immunologic, and molecular biomarkers offers unique opportunities to bypass these measures by utilizing oral fluids to evaluate the condition of both healthy and diseased individuals. Here we discuss saliva and its significance as a source of indicators for local, systemic, and infectious disorders. We highlight contemporary innovations and explore recent discoveries that deem saliva a mediator of the body's physiological condition. Additionally, we examine the current state of salivary diagnostics and its associated technologies, future aspirations, and potential as the preferred route of disease detection, monitoring, and prognosis.

Background: Salivary biomarkers for systemic diseases have been undermined due to lack of mechanistic and biological rationale. Results: Suppression of exosome biogenesis leads to ablation of salivary biomarkers. Conclusion: Tumor-derived exosomes provide a mechanism for discriminatory biomarkers in saliva. Significance: Tumor-derived exosomes provide the scientific rationale that connects pancreatic tumors and the oral cavity leading to salivary biomarkers.

Vascular endothelial growth factor A (VEGFA) and the type III receptor tyrosine kinase receptors (RTKs) are both required for the differentiation of endothelial cells (vasculogenesis) and for the sprouting of new capillaries (angiogenesis). We have isolated a duplicated zebrafish VegfA locus, termed VegfAb, and a duplicate RTK locus with homology to KDR/FLK1 (named Kdrb). Morpholinodisrupted VegfAb embryos develop a normal circulatory system until approximately 2 to 3 days after fertilization (dpf), when defects in angiogenesis permit blood to extravasate into many tissues. Unlike the VegfAa 121 and VegfAa 165 isoforms, the VegfAb isoforms VegfAb 171 IntroductionDifferentiation of endothelial cells to form the vascular network of arteries and veins requires a critical mitogenic signal, vascular endothelial growth factor A (VEGFA), which is transduced to the nucleus through a number of functionally redundant receptors. These receptors include the type III receptor tyrosine kinases VEGFR1/FLT1, VEGFR2/FLK1 (human KDR), and type I nonkinase transmembrane receptors neuropilin 1 (NRP1) and NRP2, 1 which are critical mediators of both vasculogenesis (de novo formation of blood vessels) and angiogenesis (sprouting of vessels from preexisting vessels). 2 Mice hemizygous at their single VegfA locus show defective vasculogenesis and die between 11 and 12 days after conception (dpc). [3][4][5] Embryos homozygous for a targeted disruption of Flk1 (Vegfr2) die between 8.5 and 9.5 dpc from defects in both hematopoiesis and vasculogenesis, but disruption of Flt1 (Vegfr1) only leads to perturbed vascular patterning in full-term embryos. 6,7 The tight regulation of VEGFA levels and the requirement for its receptors in hematopoiesis and vasculogenesis point to its critical role in the formation of blood vessels during development.Differential splicing of the single VEGFA locus in humans gives rise to multiple protein isoforms, ranging in size from 121 to 206 amino acids (aa's) (VEGFA 121 , VEGFA 145 , VEGFA 165 , VEGFA 189 , and VEGF 206 ). 8,9 The VEGF isoforms differ mainly by the presence or absence of 2 heparin-binding domains encoded within exon 6 and 7 sequences. [10][11][12] Expression of a single VEGFA isoform rescues VegfA mutant mice through birth, although the vascular networks differ with expression of different isoforms. [13][14][15][16] VEGFA splicing is regulated during development and in disease, resulting in tissue-and stage-specific isoform ratios, which allow for distinct, context-dependent VEGFA signaling. 17 Zebrafish have proven useful for dissecting vascular developmental pathways. 18 A single zebrafish vegfA gene has been previously described, encoding the 121-and 165-aa isoforms. 19,20 Although VegfA 121 is the predominant isoform in early embryos and VegfA 165 in adults, other alternate transcripts exist in specific tissues. 21 The initial establishment of axial vasculature patterning does not require VegfA, but the sprouting of intersegmental vessels does. 22 Together, these results show that VegfA...

In an effort to understand the morphogenetic forces that shape the bones of the skull, we inactivated Msx1 and Msx2 conditionally in neural crest. We show that Wnt1-Cre inactivation of up to three Msx1/2 alleles results in a progressively larger defect in the neural crest-derived frontal bone. Unexpectedly, in embryos lacking all four Msx1/2 alleles, the large defect is filled in with mispatterned bone consisting of ectopic islands of bone between the reduced frontal bones, just anterior to the parietal bones. The bone is derived from neural crest, not mesoderm, and, from DiI cell marking experiments, originates in a normally non-osteogenic layer of cells through which the rudiment elongates apically. Associated with the heterotopic osteogeneis is an upregulation of Bmp signaling in this cell layer. Prevention of this upregulation by implantation of noggin-soaked beads in head explants also prevented heterotopic bone formation. These results suggest that Msx genes have a dual role in calvarial development: They are required for the differentiation and proliferation of osteogenic cells within rudiments, and they are also required to suppress an osteogenic program in a cell layer within which the rudiments grow. We suggest that the inactivation of this repressive activity may be one cause of Wormian bones, ectopic bones that are a feature of a variety of pathological conditions in which calvarial bone development is compromised.

The discovery of disease-specific biomarkers in oral fluids has revealed a new dimension in molecular diagnostics. Recent studies have reported the mechanistic involvement of tumor cells derived mediators, such as exosomes, in the development of saliva-based mRNA biomarkers. To further our understanding of the origins of disease-induced salivary biomarkers, we here evaluated the hypothesis that tumor-shed secretory lipidic vesicles called exosome-like microvesicles (ELMs) that serve as protective carriers of tissue-specific information, mRNAs, and proteins, throughout the vasculature and bodily fluids. RNA content was analyzed in cell free-saliva and ELM-enriched fractions of saliva. Our data confirmed that the majority of extracellular RNAs (exRNAs) in saliva were encapsulated within ELMs. Nude mice implanted with human lung cancer H460 cells expressing hCD63-GFP were used to follow the circulation of tumor cell specific protein and mRNA in the form of ELMs in vivo. We were able to identify human GAPDH mRNA in ELMs of blood and saliva of tumor bearing mice using nested RT-qPCR. ELMs positive for hCD63-GFP were detected in the saliva and blood of tumor bearing mice as well as using electric field-induced release and measurement (EFIRM). Altogether, our results demonstrate that ELMs carry tumor cell–specific mRNA and protein from blood to saliva in a xenografted mouse model of human lung cancer. These results therefore strengthen the link between distal tumor progression and the biomarker discovery of saliva through the ELMs.

Imagine a time where your health status could be available to you without the pain, discomfort and inconvenience of a physical examination. Distant vision of an inconceivable future or impending reality with potentially immeasurable impact? Recent advancements in the field of molecular diagnostics indicate this is not only possible, but closer than we think. Novel discoveries and substantial advancements have revealed that saliva may contain real-time information describing our overall physiological condition. Researchers are now reporting that, like blood and tissue biopsies, oral fluids could be a source of biochemical data capable of detecting certain diseases. What is even more intriguing is that this phenomenon not only applies to local disorders like oral cancer and Sjögren's syndrome, but distant pathologies like autoimmune, cardiovascular and metabolic diseases as well as viral/bacterial infections and even some cancers. These revelations have provided a foundation for the burgeoning field of salivary diagnostics and hence spurred the onset of investigations poised at deciphering the salivary milieu. This paper overviews salivary diagnostics from biomarker development to the multitude of techniques utilized in identifying saliva-based molecular indicators of disease. In doing so, we present oral fluids as an easily accessible noninvasive alternative to traditional diagnostic avenues and not just an essential component of the digestive process. Determining saliva as a credible means of evaluating health status represents a considerable leap forward in health care, one that could lead to enormous translational advantages and significant clinical opportunities.

Cutaneous wounds in adult mammals typically heal by scarring. However, large full-thickness wounds undergo wound-induced hair follicle neogenesis (WIHN), a form of regeneration. Here, we show that WIHN requires transient expression of epidermal Msx2 in two phases: the wound margin early and the wound center late. Msx2 expression is present in the migrating epithelium during early wound healing and then presents in the epithelium and mesenchyme later in the wound center. WIHN is abrogated in germline and epithelial-specific Msx2 mutant mice. Unlike the full-length Msx2 promoter, a minimal Msx2 promoter fails activation in the wound center, suggesting complex regulation of Msx2 expression. The Msx2 promoter binding sites include Tcf/Lef, Jun/Creb, Pax3, and three SMAD sites. However, basal epithelial-induced BMP suppression by noggin overexpression did not affect WIHN. We propose that Msx2 signaling is required for the epidermis to acquire spatiotemporal competence during WIHN. Topologically, hair regeneration dominates in the wound center, coinciding with late Msx2 expression. Together, these results suggest that intrinsic Msx2 expression supports epithelial competency during hair follicle neogenesis. This work provides insight into endogenous mechanisms modulating competency of adult epidermal progenitors for mammalian ectodermal appendage neogenesis, and offers the target Msx2 for future regeneration-promoting therapies.

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