Salivary biomarkers of oxidative stress: A critical review

Human saliva is an increasingly attractive medium for biomarker discovery due to its amenability to noninvasive and repeated sampling, ease of collection and processing, and suitability for single analyte or metabolomic measurements. Salivary biomarkers of oxidative stress reflect local and systemic pathologies and may inform on the diagnosis, prognosis, and therapeutic responsiveness of numerous human diseases. However, for many of the disorders investigated, data reporting on alterations in salivary redox homeostasis are often highly conflicted across studies. We surveyed the available biomedical literature on this topic and noted significant discrepancies in the study designs, target populations, and operating procedures which likely contribute to the discordant data sets reported. Based on these observations, guidelines are provided to minimize interlaboratory variability in redox biomarker discovery based on human saliva.     

Salivary and urinary metabolome analysis for pre-puberty-related biomarkers identification in porcine

Estrus synchronization is important for optimal management of gilt reproduction in pig farms. Hormonal treatments, such as synthetic progestogens, are used on a routine basis, but there is a growing demand for non-hormonal alternative breeding tools. Before puberty, gilts exhibit a ‘waiting period,’ related to the ovarian development and gonadotrophin secretions, during which external stimulations, such as boar exposure, could induce and synchronize first ovulation. Practical non-invasive tools for identification of this period in farms are lacking. During this period, urinary oestrone levels are high, but urine sampling is difficult in group-housed females. The aim of this work was to search for specific biomarkers of the ‘waiting period’ in saliva and urine. In total, nine 144- to 147-day-old Large White gilts were subjected to trans-abdominal ultrasonography three times a week for 5 weeks until puberty detection (week –5 to week –1 before puberty). Urine and saliva samples were collected for oestrone assay to detect the ‘waiting period’ and for metabolome analysis using ¹H-nuclear magnetic resonance spectroscopy to detect potential biomarkers of the ‘waiting period.’ Gilts were slaughtered 7 days after puberty detection for puberty confirmation. Results were consistent with ultrasonography data for six gilts. Urine and saliva samples from these six gilts were analyzed. Urinary estrone concentration significantly increased 2 weeks before puberty detection. Metabolome analysis of urine samples allowed the identification of 78 spectral bins, among them, 42 low-molecular-weight metabolites were identified. Metabolome analysis of salivary samples allowed the identification of 59 spectral bins, among them, 23 low-molecular-weight metabolites were detected and 17 were identified. No potential biomarker was identified in urinary samples. In saliva, butyrate and 2HOvalerate, 5.79 ppm (putatively uridine), formate, malonate and propionate could be biomarker candidates to ascertain the pre-puberty period in gilt reproduction. These results confirm that non-invasive salivary samples could allow the identification of the physiological status of the gilts and presumably the optimal time for application of the boar effect. This could contribute to synchronize puberty onset and hence to develop non-hormonal breeding tools.     

Genomic medicine: bringing biomarkers to clinical medicine

An important by-product of sequencing the human genome has been the development of a novel 'toolbox' for biomarker discovery and development. Genomic medicine is an emerging discipline in the genome sciences that integrates these tools to interrogate genomic variation in well-defined populations in order to develop predictors of disease susceptibility, progression and drug response. Several important classes of biomarkers result from these analyses which, when translated to clinical medicine and drug development, will have an important impact on human health and disease. This review highlights both the opportunities and challenges in bringing biomarkers into clinical medicine.     

Personalized medicine using DNA biomarkers: a review

Biomarkers are of increasing importance for personalized medicine, with applications including diagnosis, prognosis, and selection of targeted therapies. Their use is extremely diverse, ranging from pharmacodynamics to treatment monitoring. Following a concise review of terminology, we provide examples and current applications of three broad categories of biomarkers-DNA biomarkers, DNA tumor biomarkers, and other general biomarkers. We outline clinical trial phases for identifying and validating diagnostic and prognostic biomarkers. Predictive biomarkers, more generally termed companion diagnostic tests predict treatment response in terms of efficacy and/or safety. We consider suitability of clinical trial designs for predictive biomarkers, including a detailed discussion of validation study designs, with emphasis on interpretation of study results. We specifically discuss the interpretability of treatment effects if a large set of DNA biomarker profiles is available and the number of therapies is identical to the number of different profiles.     

S100 proteins: Diagnostic and prognostic biomarkers in laboratory medicine

S100 proteins are members of the superfamily of Ca²⁺-binding proteins characterized by the specific Ca²⁺-binding motif, the EF-hand. Proteins of this superfamily are of clinical use as important diagnostic and prognostic biomarkers in adult and pediatric Laboratory Medicine. For example, measurements of troponin are nowadays the ‘gold standard’ in the diagnosis of patients with acute coronary syndrome. Parvalbumins were identified as major fish allergens and blocking antibodies, induced by immunization with a hypoallergenic parvalbumin mutant, were shown to reduce allergic symptoms. Mutations in calmodulin are linked to inherited ventricular tachycardia, and cardiac arrhythmias. S100 proteins, the largest sub-group within the EF-hand protein family, are closely associated with cardiovascular diseases, various types of cancer, inflammation and autoimmune pathologies and brain diseases. The intention of this review is to focus on the clinical use of S100 proteins as biomarkers and potential drug targets helping to improve the diagnosis of these human diseases in children and adults leading to more selective therapeutic interventions.     

Mining the fecal proteome: from biomarkers to personalised medicine

Introduction: Fecal proteomics has gained increased prominence in recent years. It can provide insights into the diagnosis and surveillance of many bowel diseases by both identifying potential biomarkers in stool samples and helping identify disease-related pathways. Fecal proteomics has already shown its potential for the discovery and validation of biomarkers for colorectal cancer screening, and the analysis of fecal microbiota by MALDI-MS for the diagnosis of a range of bowel diseases is gaining clinical acceptance.

Areas covered: Based on a comprehensive analysis of the current literature, we introduce the range of sensitive and specific proteomics methods which comprise the current ‘Proteomics Toolbox’, explain how the integration of fecal proteomics with data processing/bioinformatics has been used for the identification of potential biomarkers for both CRC and other gut-related pathologies and analysis of the fecal microbiome, outline some of the current fecal assays in current clinical practice and introduce the concept of personalised medicine which these technologies will help inform.

Expert commentary: Integration of fecal proteomics with other proteomics and genomics strategies as well as bioinformatics is paving the way towards personalised medicine, which will bring with it improved global healthcare.     

Schizophrenia proteomics: biomarkers on the path to laboratory medicine?

Over two million Americans are afflicted with schizophrenia, a debilitating mental health disorder with a unique symptomatic and epidemiological profile. Genomics studies have hinted towards candidate schizophrenia susceptibility chromosomal loci and genes. Modern proteomic tools, particularly mass spectrometry and expression scanning, aim to identify both pathogenic-revealing and diagnostically significant biomarkers. Only a few studies on basic proteomics have been conducted for psychiatric disorders relative to the plethora of cancer specific experiments. One such proteomic utility enables the discovery of proteins and biological marker fingerprinting profiling techniques (SELDI-TOF-MS), and then subjects them to tandem mass spectrometric fragmentation and de novo protein sequencing (MALDI-TOF/TOF-MS) for the accurate identification and characterization of the proteins. Such utilities can explain the pathogenesis of neuro-psychiatric disease, provide more objective testing methods, and further demonstrate a biological basis to mental illness. Although clinical proteomics in schizophrenia have yet to reveal a biomarker with diagnostic specificity, methods that better characterize the disorder using endophenotypes can advance findings. Schizophrenia biomarkers could potentially revolutionize its psychopharmacology, changing it into a more hypothesis and genomic/proteomic-driven science.     

Causal attribution and psychobiological response to competition in young men

A contribution to a special issue on Hormones and Human Competition.Psychoneuroendocrine effects of competition have been widely accepted as a clear example of the relationship between androgens and aggressive/dominant behavior in humans. However, results about the effects of competitive outcomes are quite heterogeneous, suggesting that personal and contextual factors play a moderating role in this relationship. To further explore these dimensions, we aimed to examine (i) the effect of competition and its outcome on the psychobiological response to a laboratory competition in young men, and (ii) the moderating role of some cognitive dimensions such as causal attributions. To do so, we compared the responses of 56 healthy young men faced with two competitive tasks with different instructions. Twenty-eight men carried out a task whose instructions led subjects to think the outcome was due to their personal performance (“merit” task), whereas 28 other men faced a task whose outcome was attributable to luck (“chance” task). In both cases, outcome was manipulated by the experimenter. Salivary steroid hormones (testosterone and cortisol), cardiovascular variables (heart rate and blood pressure), and emotional state (mood and anxiety) were measured at different moments before, during and after both tasks. Our results did not support the “winner-loser effect” because no significant differences were found in the responses of winners and losers. However, significantly higher values on the testosterone and cardiovascular variables, along with slight decreases in positive mood, were associated with the merit-based competition, but not the chance-based condition. In addition, an exploratory factorial analysis grouped the response components into two patterns traditionally related to more active or more passive behaviors. Thus, our results suggest that the perception of contributing to the outcome is relevant in the psychobiological response to competition in men. Overall, our results reveal the importance of the appraisal of control and causal attribution in understanding human competitive interactions.     

Salivary and serum procalcitonin and C-reactive protein as biomarkers of periodontitis in United States veterans with osteoarthritis or rheumatoid arthritis

Serum procalcitonin (ProCT) is elevated in response to bacterial infections, whereas high sensitivity C-reactive protein (hsCRP) is a nonspecific inflammatory marker that is increased by excess adipose tissue. We examined the efficacy of ProCT and hsCRP as biomarkers of periodontitis in the saliva and serum of patients with arthritis, which is characterized by variable levels of systemic inflammation that potentially can confound the interpretation of inflammatory biomarkers. Blood and unstimulated whole saliva were collected from 33 patients with rheumatoid arthritis (RA) and 50 with osteoarthritis (OA). Periodontal status was assessed by full mouth examination and patients were categorized as having no/mild, moderate or severe periodontitis by standard parameters. Salivary and serum ProCT and hsCRP concentrations were compared. BMI, diabetes, anti-inflammatory medications and smoking status were ascertained from the patient records. Differences between OA and RA in proportionate numbers of patients were compared for race, gender, diabetes, adiposity and smoking status. Serum ProCT was significantly higher in arthritis patients with moderate to severe and severe periodontitis compared with no/mild periodontitis patients. There were no significant differences in salivary ProCT or salivary or serum hsCRP in RA patients related to periodontitis category. Most of the OA and RA patients were middle aged or older, 28.9% were diabetic, 78.3% were overweight or obese, and slightly more than half were either current or past smokers. The OA and RA groups differed by race, but not gender; blacks and males were predominant in both groups. The OA and RA groups did not differ in terms of controlled or uncontrolled diabetes, smoking status or BMI. The RA patients had been prescribed more anti-inflammatory medication than the OA patients. Our results demonstrate that circulating ProCT is a more discriminative biomarker for periodontitis than serum hsCRP in patients with underlying arthritis. Any elevation in salivary and serum hsCRP due to periodontitis apparently was overshadowed by differences among these patients in factors that influence CRP, such as the extent of inflammation between RA and OA, the extent of adipose tissue, the use of anti- inflammatory medications and smoking status. Although our study showed no differences in salivary ProCT related to severity of periodontitis, this biomarker also may be useful with further refinement.     

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 peptidomics

In recent years, there has been an increased interest in the study of saliva. This bodily fluid contains a vast number of protein species, the salivary peptidome, of low molecular weight, comprising approximately 40–50% of the total secreted proteins, in addition to peptides generated by proteolysis of proteins of different sources. Owing to the presence of other components, in particular mucins and enzymes, some distinctive requirements and precautions related to sample collection, time of analysis, sample preservation and treatment are necessary for the successful analysis of salivary peptides. More than 2000 peptides compose the salivary peptidome, from which only 400–600 are directly derived from salivary glands, suggesting an important qualitative peptide contribution of other sources, namely of epithelial cells. Proteolysis events are the main supply for the peptidome and considerable efforts have been made to identify the resulting fragments, the cleavage sites and the involved proteases. The salivary proteins more prone to proteolysis are proline-rich proteins (PRPs; acidic PRPs and basic PRPs), statherin, histatins and P-B peptide. Gln–Gly cleavages are largely associated with PRP classes, while Tyr–Gly cleavages are related to histatin 1 and to the P-B peptide. The interest in saliva has been growing for clinical purposes, as it is an alternative sample to other traditional bodily fluids, such as blood or urine, since it involves an easy and noninvasive collection. In fact, apart from its usefulness as a source of information for the prognosis, diagnosis and treatment of oral diseases, such as Sjögren’s syndrome, gum disease, tooth decay or oral cancer, saliva might also be seen as a potential tool to the diagnosis of systemic diseases. Owing to the enormous amount of previously discovered salivary peptide species, in this article, we attempt to harmonize the nomenclature, following International Union of Pure and Applied Chemistry recommendations.     

Salivary peptidomics

In recent years, there has been an increased interest in the study of saliva. This bodily fluid contains a vast number of protein species, the salivary peptidome, of low molecular weight, comprising approximately 40-50% of the total secreted proteins, in addition to peptides generated by proteolysis of proteins of different sources. Owing to the presence of other components, in particular mucins and enzymes, some distinctive requirements and precautions related to sample collection, time of analysis, sample preservation and treatment are necessary for the successful analysis of salivary peptides. More than 2000 peptides compose the salivary peptidome, from which only 400-600 are directly derived from salivary glands, suggesting an important qualitative peptide contribution of other sources, namely of epithelial cells. Proteolysis events are the main supply for the peptidome and considerable efforts have been made to identify the resulting fragments, the cleavage sites and the involved proteases. The salivary proteins more prone to proteolysis are proline-rich proteins (PRPs; acidic PRPs and basic PRPs), statherin, histatins and P-B peptide. Gln-Gly cleavages are largely associated with PRP classes, while Tyr-Gly cleavages are related to histatin 1 and to the P-B peptide. The interest in saliva has been growing for clinical purposes, as it is an alternative sample to other traditional bodily fluids, such as blood or urine, since it involves an easy and noninvasive collection. In fact, apart from its usefulness as a source of information for the prognosis, diagnosis and treatment of oral diseases, such as Sj?gren's syndrome, gum disease, tooth decay or oral cancer, saliva might also be seen as a potential tool to the diagnosis of systemic diseases. Owing to the enormous amount of previously discovered salivary peptide species, in this article, we attempt to harmonize the nomenclature, following International Union of Pure and Applied Chemistry recommendations.     

Salivary peroxidases

Summary Peroxidases are known to be involved in the intracellular metabolism of H₂O₂ coupled with various physiological functions. Apart from the thyroid gland, the enzyme has been isolated from various extrathyroidal sources of which salivary gland is one of the richest sources of the enzyme. The enzyme from bovine and goat submaxillary gland has been extensively studied in terms of their molecular, spectral, kinetic, catalytic and immunological properties and compared with the lactoperoxidase which is similar to the salivary peroxidase. The modulation of the salivary peroxidase by various factors and the probable mechanism of the modulation has been described. The enzyme has also been compared with the thyroid peroxidase as regards their physicochemical properties as well as on the immunological and functional aspects. The similarities and dissimilarities have been incorporated. The possible function of the enzyme in iodine metabolism and in bactericidal action has been discussed.[/p]     

cfRNAs as biomarkers in oncology – still experimental or applied tool for personalized medicine already?

Currently, the challenges of contemporary oncology are focused mainly on the development of personalized medicine and precise treatment, which could be achieved through the use of molecular biomarkers. One of the biological molecules with great potential are circulating free RNAs (cfRNAs) which are present in various types of body fluids, such as blood, serum, plasma, and saliva. Also, different types of cfRNA particles can be distinguished depending on their length and function: microRNA (miRNA), PIWI-interacting RNA (piRNA), tRNA-derived RNA fragments (tRFs), circular RNA (circRNA), long non-coding RNA (lncRNA), and messenger RNA (mRNA). Moreover, cfRNAs occur in various forms: as a free molecule alone, in membrane vesicles, such as exosomes, or in complexes with proteins and lipids. One of the modern approaches for monitoring patient's condition is a "liquid biopsy" that provides a non-invasive and easily available source of circulating RNAs. Both the presence of specific cfRNA types as well as their concentration are dependent on many factors including cancer type or even reaction to treatment. Despite the possibility of using circulating free RNAs as biomarkers, there is still a lack of validated diagnostic panels, defined protocols for sampling, storing as well as detection methods.In this work we examine different types of cfRNAs, evaluate them as possible biomarkers, and analyze methods of their detection. We believe that further research on cfRNA and defining diagnostic panels could lead to better and faster cancer identification and improve treatment monitoring.