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.

Assembly of nitrogenase MoFe protein is arguably one of the most complex processes in the field of bioinorganic chemistry, requiring, at least, the participation of nifS, nifU, nifB, nifE, nifN, nifV, nifQ, nifZ, nifH, nifD, and nifK gene products. Previous genetic studies have identified factors involved in MoFe protein assembly; however, the exact functions of these factors and the precise sequence of events during the process have remained unclear until the recent characterization of a number of assembly-related intermediates that provided significant insights into this biosynthetic "black box". This review summarizes the recent advances in elucidation of the mechanism of FeMoco biosynthesis in four aspects: (1) the ex situ assembly of FeMoco on NifEN, (2) the incorporation of FeMoco into MoFe protein, (3) the in situ assembly of P-cluster on MoFe protein, and (4) the stepwise assembly of MoFe protein.

The P-cluster of nitrogenase is one of the most complex biological metallocenters known to date. Despite the recent advances in the chemical synthesis of P-cluster topologs, the biosynthetic mechanism of P-cluster has not been well defined. Here, we present a combined biochemical, electron paramagnetic resonance, and Xray absorption spectroscopy/extended X-ray absorption fine-structure investigation of the maturation process of P-clusters in ⌬nifH molybdenum-iron (MoFe) protein. Our data indicate that the previously identified, [Fe 4S4]-like cluster pairs in ⌬nifH MoFe protein are indeed the precursors to P-clusters, which can be reductively coupled into the mature [Fe8S7] structures in the presence of Fe protein, MgATP, and dithionite. Moreover, our observation of a biphasic maturation pattern of P-clusters in ⌬nifH MoFe protein provides dynamic proof for the previously hypothesized, stepwise assembly mechanism of the two P-clusters in the ␣2␤2-tetrameric MoFe protein, i.e., one P-cluster is formed in one ␣␤ dimer before the other in the second ␣␤ dimer.assembly ͉ biosynthesis B iological nitrogen fixation is a remarkable chemical feat accomplished by a select group of microorganisms. These microorganisms have a complex metalloenzyme, nitrogenase, which is capable of reducing atmospheric dinitrogen (N 2 ) to bioavailable ammonia (NH 3 ) under ambient conditions. The most extensively studied member of this enzyme family is the molybdenum (Mo)-nitrogenase of Azotobacter vinelandii, which consists of two redox-active proteins (1). One, designated iron (Fe) protein (encoded by nifH), is a 60-kDa ␣ 2 homodimer containing one [Fe 4 S 4 ] cluster at the subunit interface and one MgATP binding site in each subunit. The other, termed molybdenum-iron (MoFe) protein (encoded by nifD and nifK), is a 230-kDa ␣ 2 ␤ 2 heterotetramer containing one P-cluster ([Fe 8 S 7 ]) at each ␣/␤-subunit interface and one iron-molybdenum cofactor (FeMoco) ([MoFe 7 S 9 X-homocitrate], where X ϭ C, N, or O) within each ␣ subunit (2). It is believed that, concomitant with ATP hydrolysis, Fe protein undergoes repeated association/ dissociation processes with MoFe protein, donating electrons from its [Fe 4 S 4 ] cluster, through the P-cluster, to the FeMoco of the MoFe protein, where substrate reduction eventually takes place.The structure of P-cluster can be viewed as a symmetric double cubane in which two [Fe 4 S 4 ] cubanes share a central 6 -sulfur (S) atom. Such a geometry suggests that the P-cluster is likely assembled by the fusion of two [Fe 4 S 4 ]-like subclusters (3). This reaction mechanism is well established in synthetic inorganic chemistry and successfully realized by the recent synthesis of P-cluster topologs (4-6). Biological evidence in this regard was supplied by a FeMoco-deficient form of MoFe protein (designated ⌬nifH MoFe protein), which was isolated from a nifHdeletion strain of A. vinelandii (7). Extended X-ray absorption fine structure (EXAFS) (8) and magnetic circular dichroism (MCD) (9) ]-like clusters are likely th...

Mo-nitrogenase catalyzes the reduction of dinitrogen to ammonia at the cofactor (i.e., FeMoco) site of its MoFe protein component. Biosynthesis of FeMoco involves NifEN, a scaffold protein that hosts the maturation of a precursor to a mature FeMoco before it is delivered to the target location in the MoFe protein. Previously, we have shown that the NifEN-bound precursor could be converted, in vitro, to a fully complemented “FeMoco” in the presence of 2 mM dithionite. However, such a conversion was incomplete and Mo was only loosely associated to the NifEN-bound “FeMoco”. Here we report the optimized maturation of NifEN-associated precursor in 20 mM dithionite. Activity analyses show that, upon the optimal conversion of precursor to “FeMoco”, NifEN is capable of activating a FeMoco-deficient form of MoFe protein to the same extent as the isolated FeMoco. Further, EPR and XAS/EXAFS analyses reveal the presence of a tightly organized Mo site in NifEN-bound “FeMoco”, which allows the observation of a FeMoco-like, S = 3/2 EPR signal and the modeling of a NifEN-bound “FeMoco” that adopts a very similar conformation to that of the MoFe protein-associated FeMoco. The sensitivity of FeMoco maturation to dithionite concentration suggests an essential role of redox chemistry in this process, and the optimal potential of dithionite solution could serve as a guideline for future identification of in vivo electron donors for FeMoco maturation.

NifEN is a key player in the biosynthesis

MicroRNAs (miRNAs) in human saliva have recently become an emerging field in saliva research for diagnostics applications and its potential role in biological implications. miRNAs are short noncoding RNA molecules that play important roles in regulating a variety of cellular processes. Dysregulation of miRNAs are known to be associated with many diseases. miRNAs were found present in the saliva of OSCC patients and could serve as potential biomarkers for oral cancer detection. Understanding the biological function of miRNAs in association with diseases is important towards utilizing miRNAs as diagnostic markers. There are currently a variety of profiling methods available for detecting miRNA expression levels. In this chapter, we overview the Applied Biosystem Stem-loop RT based Taqman MicroRNA Assay for salivary miRNA profiling. Using this highly sensitive and specific assay, miRNAs in saliva are profiled with only a few nanograms of starting RNA. This method is also applicable for studying biomarkers in other body fluids or clinical samples that contain small amounts of RNA.

We have discovered and validated a panel of salivary exRNA biomarkers with credible clinical performance for the detection of GC. Our study demonstrates the potential utility of salivary exRNA biomarkers in screening and risk assessment for GC.

Novel biomarkers and non-invasive diagnostic methods are urgently needed for the screening of gastric cancer to reduce its high mortality. We employed quantitative proteomics approach to develop discriminatory biomarker signatures from human saliva for the detection of gastric cancer. Salivary proteins were analyzed and compared between gastric cancer patients and matched control subjects by using tandem mass tags (TMT) technology. More than 500 proteins were identified with quantification, and 48 of them showed significant difference expression (p < 0.05) between normal controls and gastric cancer patients, including 7 up-regulated proteins and 41 down-regulated proteins. Five proteins were selected for initial verification by ELISA and three were successfully verified, namely cystatin B (CSTB), triosephosphate isomerase (TPI1), and deleted in malignant brain tumors 1 protein (DMBT1). All three proteins could differentiate gastric cancer patients from normal control subjects, dramatically (p < 0.05). The combination of these three biomarkers could reach 85% sensitivity and 80% specificity for the detection of gastric cancer with accuracy of 0.93. This study provides the proof of concept of salivary biomarkers for the non-invasive detection of gastric cancer. It is highly encouraging to turn these biomarkers into an applicable clinical test after large scale validation.