Discovery and Validation of Salivary Extracellular RNA Biomarkers for Noninvasive Detection of Gastric Cancer.

BACKGROUND: Biomarkers are needed for noninvasive early detection of gastric cancer (GC). We investigated salivary extracellular RNA (exRNA) biomarkers as potential clinical evaluation tools for GC. METHODS: Unstimulated whole saliva samples were prospectively collected from 294 individuals (163 GC and 131 non-GC patients) who underwent endoscopic evaluation at the Samsung Medical Center in Korea. Salivary transcriptomes of 63 GC and 31 non-GC patients were profiled, and mRNA biomarker candidates were verified with reverse transcription quantitative real-time PCR (RT-qPCR). In parallel, microRNA (miRNA) biomarkers were profiled and verified with saliva samples from 10 GC and 10 non-GC patients. Candidate biomarkers were validated with RT-qPCR in an independent cohort of 100/100 saliva samples from GC and non-GC patients. Validated individual markers were configured into a best performance panel. RESULTS: We identified 30 mRNA and 15 miRNA candidates whose expression pattern associated with the presence of GC. Among them, 12 mRNA and 6 miRNA candidates were verified with the discovery cohort by RT-qPCR and further validated with the independent cohort (n = 200). The configured biomarker panel consisted of 3 mRNAs (SPINK7, PPL, and SEMA4B) and 2 miRNAs (MIR140-5p and MIR301a), which were all significantly down-regulated in the GC group, and yielded an area under the ROC curve (AUC) of 0.81 (95% CI, 0.72-0.89). When combined with demographic factors, the AUC of the biomarker panel reached 0.87 (95% CI, 0.80-0.93). CONCLUSIONS: 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.

Coupling high-throughput genetics with phylogenetic information reveals an epistatic interaction on the influenza A virus M segment.

BACKGROUND: Epistasis is one of the central themes in viral evolution due to its importance in drug resistance, immune escape, and interspecies transmission. However, there is a lack of experimental approach to systematically probe for epistatic residues. RESULTS: By utilizing the information from natural occurring sequences and high-throughput genetics, this study established a novel strategy to identify epistatic residues. The rationale is that a substitution that is deleterious in one strain may be prevalent in nature due to the presence of a naturally occurring compensatory substitution. Here, high-throughput genetics was applied to influenza A virus M segment to systematically identify deleterious substitutions. Comparison with natural sequence variation showed that a deleterious substitution M1 Q214H was prevalent in circulating strains. A coevolution analysis was then performed and indicated that M1 residues 121, 207, 209, and 214 naturally coevolved as a group. Subsequently, we experimentally validated that M1 A209T was a compensatory substitution for M1 Q214H. CONCLUSIONS: This work provided a proof-of-concept to identify epistatic residues by coupling high-throughput genetics with phylogenetic information. In particular, we were able to identify an epistatic interaction between M1 substitutions A209T and Q214H. This analytic strategy can potentially be adapted to study any protein of interest, provided that the information on natural sequence variants is available.

Salivary biomarkers: toward future clinical and diagnostic utilities.

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.

Catalytic activities of NifEN: implications for nitrogenase evolution and mechanism.

NifEN is a key player in the biosynthesis of nitrogenase MoFe protein. It not only shares a considerable degree of sequence homology with the MoFe protein, but also contains clusters that are homologous to those found in the MoFe protein. Here we present an investigation of the catalytic activities of NifEN. Our data show that NifEN is catalytically competent in acetylene (C(2)H(2)) and azide (N(3)(-)) reduction, yet unable to reduce dinitrogen (N(2)) or evolve hydrogen (H(2)). Upon turnover, C(2)H(2) gives rise to an additional S = 1/2 signal, whereas N(3)(-) perturbs the signal originating from the NifEN-associated FeMoco homolog. Combined biochemical and spectroscopic studies reveal that N(3)(-) can act as either an inhibitor or an activator for the binding and/or reduction of C(2)H(2), while carbon monoxide (CO) is a potent inhibitor for the binding and/or reduction of both N(3)(-) and C(2)H(2). Taken together, our results suggest that NifEN is a catalytic homolog of MoFe protein; however, it is only a "skeleton" version of the MoFe protein, as its associated clusters are simpler in structure and less versatile in function, which, in turn, may account for its narrower range of substrates and lower activities of substrate reduction. The resemblance of NifEN to MoFe protein in catalysis points to a plausible, sequential appearance of the two proteins in nitrogenase evolution. More importantly, the discrepancy between the two systems may provide useful insights into nitrogenase mechanism and allow reconstruction of a fully functional nitrogenase from the "skeleton" enzyme, NifEN.

Stepwise formation of P-cluster in nitrogenase 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 X-ray absorption spectroscopy/extended X-ray absorption fine-structure investigation of the maturation process of P-clusters in DeltanifH molybdenum-iron (MoFe) protein. Our data indicate that the previously identified, [Fe(4)S(4)]-like cluster pairs in DeltanifH MoFe protein are indeed the precursors to P-clusters, which can be reductively coupled into the mature [Fe(8)S(7)] structures in the presence of Fe protein, MgATP, and dithionite. Moreover, our observation of a biphasic maturation pattern of P-clusters in DeltanifH MoFe protein provides dynamic proof for the previously hypothesized, stepwise assembly mechanism of the two P-clusters in the alpha(2)beta(2)-tetrameric MoFe protein, i.e., one P-cluster is formed in one alphabeta dimer before the other in the second alphabeta dimer.

Polymerase translocation with respect to single-stranded nucleic acid: looping or wrapping of primer around a poly(A) polymerase.

Vaccinia virus protein VP55 translocates continuously with respect to single-stranded nucleic acid while extending its 3'end. Here, all key sites of polymerase-primer interaction were identified, demonstrating the wrapping or looping of polyadenylation primer around the polymerase during translocation. Side-chain substitutions at one of the sites indicated its requirement for tail extension beyond approximately 12 nucleotides in length, and conformational changes observed upon oligonucleotide binding suggested allosteric connectivity during translocation. Conformational changes in VP39 upon VP55 binding suggested that, within the VP55-VP39 complex, VP39's mRNA 5' cap binding site closes. The crystallographic structure showed a PAPase catalytic center without side-chain substitutions, possessing two metal ions and with all known reactive and catalytic groups represented, fitting a classical two-metal ion mechanism for phosphoryl transfer.

Insertion of heterometals into the NifEN-associated iron-molybdenum cofactor precursor.

The cofactors of Mo-, V-, Fe-dependent nitrogenases are believed to be highly homologous in structure despite the different types of heterometals (Mo, V, and Fe) they contain. Previously, a precursor form of the FeMo cofactor (FeMoco) was captured on NifEN, a scaffold protein for FeMoco biosynthesis. This all-Fe precursor closely resembles the Fe/S core structure of the FeMoco and, therefore, could reasonably serve as a precursor for all nitrogenase cofactors. Here, we report the heterologous incorporation of V and Fe into the NifEN-associated FeMoco precursor. EPR and activity analyses indicate that V and Fe can be inserted at much reduced efficiencies compared with Mo, and incorporation of both V and Fe is enhanced in the presence of homocitrate. Further, native polyacrylamide gel electrophoresis experiments suggest that NifEN undergoes a significant conformational rearrangement upon metal insertion, which allows the subsequent NifEN-MoFe protein interactions and the transfer of the cofactor between the two proteins. The combined outcome of these in vitro studies leads to the proposal of a selective mechanism that is utilized in vivo to maintain the specificity of heterometals in nitrogenase cofactors, which is likely accomplished through the redox regulation of metal mobilization by different Fe proteins (encoded by nifH, vnfH, and anfH, respectively), as well as the differential interactions between these Fe proteins and their respective scaffold proteins (NifEN and VnfEN) in the Mo-, V-, and Fe-dependent nitrogenase systems.

Optimization of FeMoco maturation on NifEN.

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 with the NifEN-bound "FeMoco". Here we report the optimized maturation of the 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. Furthermore, 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 conformation very similar 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.

Nucleic Acid Isolation and Quality Control.

This chapter describes the most practical manual and automated methods of isolating genomic DNA and RNA from different sources. It also summarizes currently popular methods of quality control (QC) for genomic DNA, RNA, and next generation sequencing (NGS) libraries.

Effects of Mutations on Replicative Fitness and Major Histocompatibility Complex Class I Binding Affinity Are Among the Determinants Underlying Cytotoxic-T-Lymphocyte Escape of HIV-1 Gag Epitopes.

Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8+ cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity.IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape.

Quantifying perinatal transmission of Hepatitis B viral quasispecies by tag linkage deep sequencing.

Despite full immunoprophylaxis, mother-to-child transmission (MTCT) of Hepatitis B Virus still occurs in approximately 2-5% of HBsAg positive mothers. Little is known about the bottleneck of HBV transmission and the evolution of viral quasispecies in the context of MTCT. Here we adopted a newly developed tag linkage deep sequencing method and analyzed the quasispecies of four MTCT pairs that broke through immunoprophylaxis. By assigning unique tags to individual viral sequences, we accurately reconstructed HBV haplotypes in a region of 836 bp, which contains the major immune epitopes and drug resistance mutations. The detection limit of minor viral haplotypes reached 0.1% for individual patient sample. Dominance of "a determinant" polymorphisms were observed in two children, which pre-existed as minor quasispecies in maternal samples. In all four pairs of MTCT samples, we consistently observed a significant overlap of viral haplotypes shared between mother and child. We also demonstrate that the data can be potentially useful to estimate the bottleneck effect during HBV MTCT, which provides information to optimize treatment for reducing the frequency of MTCT.

Differential Proteomic Analysis of Human Saliva using Tandem Mass Tags Quantification for Gastric Cancer Detection.

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.

Correction: Human Salivary Micro-RNA in Patients with Parotid Salivary Gland Neoplasms.

[This corrects the article DOI: 10.1371/journal.pone.0142264.].

Human Salivary Micro-RNA in Patients with Parotid Salivary Gland Neoplasms.

BACKGROUND: Currently, clinical examination, ultrasound scanning (with or without fine needle aspiration cytology), preoperative CT-scan and MRI are available for the differential diagnosis of parotid gland swelling. A preliminary non-invasive salivary diagnostic tool may be helpful in the clinical decision making process. Altered salivary micro-RNA (miRNA) expression levels have been observed in saliva from patients with various cancers. Therefore, we investigated miRNA expression levels in saliva samples from patients with a parotid gland neoplasm using Human miRNA cards in comparison to controls. RESULTS: In the discovery phase, eight miRNAs were identified having different expression levels in patients compared to controls. In the validation phase, the differences in miRNA expression levels between patients and controls were confirmed for seven out of eight discovered miRNAs (p < 0.001). A combination of two miRNAs yielded a receiver-operator-characteristics curve with an AUC of 0.94 (95% CI: 0.87-1.00; sensitivity 91%; specificity 86%). Validation of discovered miRNAs in segregated collected parotid saliva revealed that expression of these miRNAs differ between whole saliva and parotid saliva. CONCLUSIONS: A two miRNA combination can predict the presence of a parotid gland neoplasm. Furthermore, this study suggested that the identified, patient-specific, salivary miRNAs were not derived from the parotid gland itself.

Salivary microRNAs and oral cancer detection.

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.

Discovery and prevalidation of salivary extracellular microRNA biomarkers panel for the noninvasive detection of benign and malignant parotid gland tumors.

PURPOSE: This study was conducted to explore the differences in salivary microRNA (miRNA) profiles between patients with malignant or benign parotid gland tumors as a potential preoperative diagnostic tool of tumors in the salivary glands. EXPERIMENTAL DESIGN: Whole saliva samples from patients with malignant (n = 38) or benign (n = 29) parotid gland tumors were obtained from the Salivary Gland Tumor Biorepository (SGTB). After total RNA isolation, human miRNA cards were used for miRNA profiling. The differential miRNA expression was analyzed using two-sided Wilcoxon test. Quantitative real-time PCR (qRT-PCR) was used to validate selected miRNAs in an independent sample set. Receiver-operating characteristics curve and probability of malignancy was exploited to evaluate the diagnostic power of the validated miRNAs. RESULTS: With miRNA profiling, 57 of 750 investigated miRNAs were differently expressed, of which 54 showed higher miRNA expression in samples from patients with malignant tumors than those from patients with benign tumors. Validating the expression in an independent sample set of 9 miRNAs revealed indeed higher expression of miRNAs in malignant samples compared with benign samples. The expression of 6 validated miRNAs was statistically significantly different between the two groups (P < 0.05). A four miRNA combination was able to discriminate between saliva samples from patients with malignant tumors from those of patients with benign parotid gland tumors (sensitivity 69%, specificity 95%). CONCLUSIONS: Salivary miRNA profiles differ in saliva from patients with malignant from saliva from patients with a benign parotid gland tumor. These preliminary results are promising to develop a noninvasive diagnostic tool for diagnosing tumors in the salivary glands.

Formation of a homocitrate-free iron-molybdenum cluster on NifEN: implications for the role of homocitrate in nitrogenase assembly.

Molybdenum (Mo)-dependent nitrogenase is a complex metalloprotein that catalyzes the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) at the molybdenum-iron cofactor (FeMoco) site of its molybdenum-iron (MoFe) protein component. Here we report the formation of a homocitrate-free, iron-molybdenum ("FeMo") cluster on the biosynthetic scaffold of FeMoco, NifEN. Such a NifEN-associated "FeMo" cluster exhibits EPR features similar to those of the NifEN-associated, fully-complemented "FeMoco", which originate from the presence of Mo in both cluster species; however, "FeMo" cluster and "FeMoco" display different temperature-dependent changes in the line shape and the signal intensity of their respective EPR features, which reflect the impact of homocitrate on the redox properties of these clusters. XAS/EXAFS analysis reveals that the Mo centers in both "FeMo" cluster and "FeMoco" are present in a similar coordination environment, although Mo in "FeMo" cluster is more loosely coordinated as compared to that in "FeMoco" with respect to the Mo-O distances in the cluster, likely due to the absence of homocitrate that normally serves as an additional ligand for the Mo in the cluster. Subsequent biochemical investigation of the "FeMo" cluster not only facilitates the determination of the sequence of events in the mobilization of Mo and homocitrate during FeMoco maturation, but also permits the examination of the role of homocitrate in the transfer of FeMoco between NifEN and MoFe protein. Combined outcome of these studies establishes a platform for future structural analysis of the interactions between NifEN and MoFe protein, which will provide useful insights into the mechanism of cluster transfer between the two proteins.

Assembly of nitrogenase MoFe protein.

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.

Saltatory forward movement of a poly(A) polymerase during poly(A) tail addition.

Vaccinia poly(A) polymerase (VP55) interacts with > or = 33-nucleotide (nt) primers via uridylates at two sites (-27/-26 and -10). It adds approximately 30-nt poly(A) tails with a rapid, processive burst in which the first few nt are added without substantial primer movement, and addition of the remaining adenylates is dependent upon a six-uridylate tract at the extreme 3' end of the primer and accompanied by polymerase translocation. Interaction of VP55 with 2-aminopurine (2-AP)-containing primers was associated with a 3-fold enhancement in 2-AP fluorescence. In stopped-flow experiments, fluorescence intensity changed with time during the polyadenylation burst in a manner dependent upon the position of 2-AP, indicating a non-uniform isomerization of the polymerase-primer complex with time consistent with a discontinuous (saltatory) translocation mechanism. Three distinct translocatory phases could be discerned: a -10(U)-binding site forward movement, a -27/-26(UU)-binding site jump to -10, then a -27/-26(UU)-binding site movement further downstream. Poly(A) tail elongation showed no apparent pauses during these isomerizations. Fluorescence changes during polyadenylation of 2-AP-containing primers with short preformed oligo(A) tails reinforced the above observations. Primers composed entirely of oligo(U) (apart from the 2-AP sensor), in which the polymerase modules might be most able to "slide" uniformly, also showed the characteristic saltatory pattern of translocation. These data indicate, for the first time, a discontinuous mode of translocation for a non-templated polymerase.