[Modern approaches to studying the molecular mechanisms of lung functioning in normal and pathological conditions]. / Sovremennye podkhody k izucheniyu molekulyarnykh mekhanizmov funktsionirovaniya legkikh v norme i pri patologii.

Problems with breathing and lung function are caused by the development of various lung diseases associated with lifestyle, harmful environmental factors and genetic predisposition. Knowledge of the molecular mechanisms of the development of the pathological process will allow on time identification of the disease or the development of targeted therapy. The article provides an overview of modern methods that make it possible to most accurately reproduce the structural, functional and mechanical properties of the lung (organ-on-a-chip), to perform non-invasive molecular studies of biomarkers of bronchopulmonary pathology using saliva diagnostics, as well as using DNA and RNA aptamers, verify tumor markers in biological samples of human tissue. Analysis of alterations in the pattern of protein glycosylation using glycodiagnostic methods makes it possible to detect lung cancer in the early stages.

Potential relevance of salivary legumain for the clinical diagnostic of hand, foot, and mouth disease.

The fight against hand, foot, and mouth disease (HFMD) remains an arduous challenge without existing point-of-care (POC) diagnostic platforms for accurate diagnosis and prompt case quarantine. Hence, the purpose of this salivary biomarker discovery study is to set the fundamentals for the realization of POC diagnostics for HFMD. Whole salivary proteome profiling was performed on the saliva obtained from children with HFMD and healthy children, using a reductive dimethylation chemical labeling method coupled with high-resolution mass spectrometry-based quantitative proteomics technology. We identified 19 upregulated (fold change = 1.5-5.8) and 51 downregulated proteins (fold change = 0.1-0.6) in the saliva samples of HFMD patients in comparison to that of healthy volunteers. Four upregulated protein candidates were selected for dot blot-based validation assay, based on novelty as biomarkers and exclusions in oral diseases and cancers. Salivary legumain was validated in the Singapore (n = 43 healthy, 28 HFMD cases) and Taiwan (n = 60 healthy, 47 HFMD cases) cohorts with an area under the receiver operating characteristic curve of 0.7583 and 0.8028, respectively. This study demonstrates the feasibility of a broad-spectrum HFMD POC diagnostic test based on legumain, a virus-specific host systemic signature, in saliva.

Saliva in the "Omics" era: A promising tool in paediatrics.

In vulnerable populations, such as infants and children, saliva makes the perfect diagnostic medium because of its noninvasive collection, easy handling and storage of samples. Its unique biomarker profiles help tremendously in the diagnosis of many diseases and conditions. In fact, saliva genomics, proteomics, transcriptomics, metabolomics and microbiome-based discoveries have led to complementary and powerful diagnostic information. In children and neonates, saliva is the preferred medium not only for diagnosis of caries and aggressive periodontitis but also for a number of systemic conditions, metabolic diseases, cognitive functions, stress assessment and evaluation of immunological and inflammatory responses to vaccination. In this review, we provide an overview of current and future applications of saliva diagnostics to various diseases and conditions and highlight studies in paediatrics across the "omic" spectrum. Emerging frontiers in salivary diagnostics research that may significantly advance the field are also highlighted.

Noninvasive saliva-based EGFR gene mutation detection in patients with lung cancer.

RATIONALE: Constitutive activation of the epidermal growth factor receptor (EGFR) is prevalent in epithelial cancers, particularly in non-small cell lung carcinoma (NSCLC). Mutations identified in EGFR predict the sensitivity to EGFR-targeted therapy. Detection of these mutations is mainly based on tissue biopsy, which is invasive, expensive, and time consuming. OBJECTIVES: Noninvasive, real-time, inexpensive detection and monitoring of EGFR mutations in patients with NSCLC is highly desirable. METHODS: We developed a novel core technology, electric field-induced release and measurement (EFIRM), which relies on a multiplexible electrochemical sensor that can detect EGFR mutations directly in bodily fluids. MEASUREMENTS AND MAIN RESULTS: We established EFIRM for the detection of the EGFR mutations in vitro and correlated the results with tumor size from xenografted mice. In clinical application, we demonstrated that EFIRM could detect EGFR mutations in the saliva and plasma of 22 patients with NSCLC. Finally, a blinded test was performed on saliva samples from 40 patients with NSCLC. The receiver operating characteristic analysis indicated that EFIRM detected the exon 19 deletion with an area under the curve of 0.94 and the L858R mutation with an area under the curve of 0.96. CONCLUSIONS: Our data indicate that EFIRM is effective, accurate, rapid, user-friendly, and cost effective for the detection of EGFR mutations in the saliva of patients with NSCLC. We termed this saliva-based EGFR mutation detection (SABER).

Salivary miRNAs as auxiliary liquid biopsy biomarkers for diagnosis in patients with oropharyngeal squamous cell carcinoma: a systematic review and meta-analysis.

Objective: The healthcare system needs a novel approach to improve and diagnose early oropharyngeal squamous cell carcinoma against its low survival rate. We conduct a systematic review and a comprehensive meta-analysis for the diagnostic role of blood and salivary microRNAs (miRNAs). Methods: An unbiased and thorough literature search in PubMed yielded appropriate data from qualified articles regarding different miRNA biomarkers, method of extraction, research location, and year of publication. Stata was used to calculate the sensitivity, specificity, diagnostic odds ratio, and summary receiver operating characteristic curve. Results: We included 9 studies with 399 qualified oropharyngeal squamous cell carcinoma patients, which yielded a high diagnostic accuracy of blood miRNAs in combination with salivary miRNAs with a sensitivity of 0.70 (p < 0.001), specificity of 0.75 (p = 0.26), diagnostic odds ratio of 7, and an area under the curve of 0.78. Conclusion: Combined blood- and saliva-derived miRNAs demonstrated a high diagnostic accuracy in detecting oropharyngeal squamous cell carcinoma. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42024509424.

Saliva diagnostics: Salivaomics, saliva exosomics, and saliva liquid biopsy.

BACKGROUND: Each day, humans produce approximately 0.5 through 1.5 liters of saliva, a biofluid that is rich in biological omic constituents. Our lack of understanding how omic biomarkers migrate from diseased tissue to the saliva has impeded the clinical translation of saliva testing. Although such biomarkers must be conveyed via the vascular and lymphatic systems to the salivary glands, the molecular mechanisms that underlie this transport remain unclear. Although COVID-19 highlighted the need for rapid and reliable testing for infectious diseases, it represents only one of the many health conditions that potentially can be diagnosed using a saliva sample. TYPES OF STUDIES REVIEWED: The authors discuss salivaomics, saliva exosomics, and the mechanisms on which saliva diagnostics are based and introduce a novel electrochemical sensing technology that may be exploited for saliva liquid biopsy. RESULTS: The utility of saliva for screening for lung cancer is under investigation. Saliva testing may be used to stratify patients, monitor treatment response, and detect disease recurrence. The authors also highlight the landscapes of saliva-based SARS-CoV-2 testing and ultrashort cell-free DNA and outline how these fields are likely to evolve in the near future. PRACTICAL IMPLICATIONS: Breakthroughs in the study of saliva research, therefore, will facilitate clinical deployment of saliva-based testing.

Development of a Biosensor Based on Angiotensin-Converting Enzyme II for Severe Acute Respiratory Syndrome Coronavirus 2 Detection in Human Saliva.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus responsible for COVID-19. Infection in humans requires angiotensin-converting enzyme II (hACE2) as the point of entry for SARS-CoV-2. PCR testing is generally definitive but expensive, although it is highly sensitive and accurate. Biosensor-based monitoring could be a low-cost, accurate, and non-invasive approach to improve testing capacity. We develop a capacitive hACE2 biosensor for intact SARS-CoV-2 detection in saliva. Laser-induced graphene (LIG) electrodes were modified with platinum nanoparticles. The quality control of LIG electrodes was performed using cyclic voltammetry. Truncated hACE2 was used as a biorecognition element and attached to the electrode surface by streptavidin-biotin coupling. Biolayer interferometry was used for qualitative interaction screening of hACE2 with UV-attenuated virions. Electrochemical impedance spectroscopy (EIS) was used for signal transduction. Truncated hACE2 binds wild-type SARS-CoV-2 and its variants with greater avidity than human coronavirus (common cold virus). The limit of detection (LoD) is estimated to be 2,960 copies/ml. The detection process usually takes less than 30 min. The strength of these features makes the hACE2 biosensor a potentially low-cost approach for screening SARS-CoV-2 in non-clinical settings with high demand for rapid testing (for example, schools and airports).

Directed Transport of CRP Across In Vitro Models of the Blood-Saliva Barrier Strengthens the Feasibility of Salivary CRP as Biomarker for Neonatal Sepsis.

C-reactive protein (CRP) is a commonly used serum biomarker for detecting sepsis in neonates. After the onset of sepsis, serial measurements are necessary to monitor disease progression; therefore, a non-invasive detection method is beneficial for neonatal well-being. While some studies have shown a correlation between serum and salivary CRP levels in septic neonates, the causal link behind this correlation remains unclear. To investigate this relationship, CRP was examined in serum and saliva samples from 18 septic neonates and compared with saliva samples from 22 healthy neonates. While the measured blood and saliva concentrations of the septic neonates varied individually, a correlation of CRP levels between serum and saliva samples was observed over time. To clarify the presence of active transport of CRP across the blood-salivary barrier (BSB), transport studies were performed with CRP using in vitro models of oral mucosa and submandibular salivary gland epithelium. The results showed enhanced transport toward saliva in both models, supporting the clinical relevance for salivary CRP as a biomarker. Furthermore, CRP regulated the expression of the receptor for advanced glycation end products (RAGE) and the addition of soluble RAGE during the transport studies indicated a RAGE-dependent transport process for CRP from blood to saliva.

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice.

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients' general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk's potential and suitability for inclusion in larger prospective implementation studies in dental care settings.

Microbial Analysis of Saliva to Identify Oral Diseases Using a Point-of-Care Compatible qPCR Assay.

Oral health is maintained by a healthy microbiome, which can be monitored by state-of-the art diagnostics. Therefore, this study evaluated the presence and quantity of ten oral disease-associated taxa (P. gingivalis, T. forsythia, T. denticola, F. nucleatum, C. rectus, P. intermedia, A. actinomycetemcomitans, S. mutans, S. sobrinus, oral associated Lactobacilli) in saliva and their clinical status association in 214 individuals. Upon clinical examination, study subjects were grouped into healthy, caries and periodontitis and their saliva was collected. A highly specific point-of-care compatible dual color qPCR assay was developed and used to study the above-mentioned bacteria of interest in the collected saliva. Assay performance was compared to a commercially available microbial reference test. Eight out of ten taxa that were investigated during this study were strong discriminators between the periodontitis and healthy groups: C. rectus, T. forsythia, P. gingivalis, S. mutans, F. nucleatum, T. denticola, P. intermedia and oral Lactobacilli (p < 0.05). Significant differentiation between the periodontitis and caries group microbiome was only shown for S. mutans (p < 0.05). A clear distinction between oral health and disease was enabled by the analysis of quantitative qPCR data of target taxa levels in saliva.

Tracking the functional meaning of the human oral-microbiome protein-protein interactions.

The interactome - the network of protein-protein interactions (PPIs) within a cell or organism - is technically difficult to assess. Bioinformatic tools can, not only, identify potential PPIs that can be later experimentally validated, but also be used to assign functional meaning to PPIs. Saliva's potential as a non-invasive diagnostic fluid is currently being explored by several research groups. But, in order to fully attain its potential, it is necessary to achieve the full characterization of the mechanisms that take place within this ecosystem. The onset of omics technologies, and specifically of proteomics, delivered a huge set of data that is largely underexplored. Quantitative information relative to proteins within a given context (for example a given disease) can be used by computational algorithms to generate information regarding PPIs. These PPIs can be further analyzed concerning their functional meaning and used to identify potential biomarkers, therapeutic targets, defense and pathogenicity mechanisms. We describe a computational pipeline that can be used to identify and analyze PPIs between human and microbial proteins. The pipeline was tested within the scenario of human PPIs of systemic (Zika Virus infection) and of oral conditions (Periodontal disease) and also in the context of microbial interactions (Candida-Streptococcus) and showed to successfully predict functionally relevant PPIs. The pipeline can be applied to different scientific areas, such as pharmacological research, since a functional meaningful PPI network can provide insights on potential drug targets, and even new uses for existing drugs on the market.

Saliva biomarkers in neurological disorders: A "spitting image" of brain health?

In this issue of the Biomedical Journal, we learn how biomarkers in saliva may be able to provide insight into the health of the brain and the central nervous system. We also discover how computational modeling can help to identify potential epitopes for vaccine development against Chlamydia, the world's most common sexually transmitted infection.

Immunodetection of salivary biomarkers by an optical microfluidic biosensor with polyethylenimine-modified polythiophene-C70 organic photodetectors.

This work reports a novel optical microfluidic biosensor with highly sensitive organic photodetectors (OPDs) for absorbance-based detection of salivary protein biomarkers at the point of care. The compact and miniaturized biosensor has comprised OPDs made of polythiophene-C70 bulk heterojunction for the photoactive layer; whilst a calcium-free cathode interfacial layer, made of linear polyethylenimine, was incorporated to the photodetectors to enhance the low cost. The OPDs realized onto a glass chip were aligned to antibody-functionalized chambers of a poly(methyl methacrylate) microfluidic chip, in where immunogold-silver assays were conducted. The biosensor has detected IL-8, IL-1ß and MMP-8 protein in spiked saliva with high detection specificity and short analysis time exhibiting detection limits between 80pgmL-1 and 120pgmL-1. The result for IL-8 was below the clinical established cut-off of 600pgmL-1, which revealed the potential of the biosensor to early detection of oral cancer. The detection limit was also comparable to other previously reported immunosensors performed with bulky instrumentation or using inorganic photodetectors. The optical detection sensitivity of the polythiophene-C70 OPD was enhanced by optimizing the thickness of the photoactive layer and anode interfacial layer prior to the saliva immunoassays. Further, the biosensor was tested with unspiked human saliva samples, and the results of measuring IL-8 and IL-1ß were in statistical agreement with those provided by two commercial assays of ELISA. The optical microfluidic biosensor reported hereby offers an attractive and cost-effective tool to diagnostics or screening purposes at the point of care.

A Pilot Study into the Association between Oral Health Status and Human Papillomavirus-16 Infection.

BACKGROUND: Over the next 20 years, oropharyngeal cancers (OPC) will represent the majority of head and neck cancers (HNCs) in the United States. It is estimated that human papillomavirus (HPV) may account for as much as 70% to 80% of OPCs in North America and in certain parts of Europe. It is hence crucial to understand the disease risk factors and natural history of oral HPV infections. We hypothesized that poor oral health (by measures such as poor oral hygiene and periodontal disease) leads to a higher degree of oral HPV-16 infections within a patient cohort from a dental school clinic. This study aims to test this hypothesis and gauge possible disease associations before larger scale studies. SUBJECTS AND METHODS: 223 participants were recruited in this study from the University of Queensland Dental School clinic. Clinical oral health parameters (such as oral hygiene measures and periodontal disease measurements) have been examined and determined by dental professionals. We have collected oral rinse samples from these volunteers. RESULTS: 10 (4.5%) out of 223 participants were found to have HPV-16 DNA in their oral rinse samples using NB2 endpoint PCR and Sanger sequencing. Within the HPV-16 DNA positive subjects, 7 (70%) and 3 (30%) were associated with poor oral hygiene and periodontal disease, respectively. CONCLUSION: Our results show a trend towards a positive correlation between oral HPV-16 infection and poor clinical oral health status.

Evaluation of a novel saliva-based epidermal growth factor receptor mutation detection for lung cancer: A pilot study.

BACKGROUND: This article describes a pilot study evaluating a novel liquid biopsy system for non-small cell lung cancer (NSCLC) patients. The electric field-induced release and measurement (EFIRM) method utilizes an electrochemical biosensor for detecting oncogenic mutations in biofluids. METHODS: Saliva and plasma of 17 patients were collected from three cancer centers prior to and after surgical resection. The EFIRM method was then applied to the collected samples to assay for exon 19 deletion and p.L858 mutations. EFIRM results were compared with cobas results of exon 19 deletion and p.L858 mutation detection in cancer tissues. RESULTS: The EFIRM method was found to detect exon 19 deletion with an area under the curve (AUC) of 1.0 in both saliva and plasma samples in lung cancer patients. For L858R mutation detection, the AUC of saliva was 1.0, while the AUC of plasma was 0.98. Strong correlations were also found between presurgery and post-surgery samples for both saliva (0.86 for exon 19 and 0.98 for L858R) and plasma (0.73 for exon 19 and 0.94 for L858R). CONCLUSION: Our study demonstrates the feasibility of utilizing EFIRM to rapidly, non-invasively, and conveniently detect epidermal growth factor receptor mutations in the saliva of patients with NSCLC, with results corresponding perfectly with the results of cobas tissue genotyping.

The Proteomics of Saliva in Sjögren's Syndrome.

One of the main characteristics of primary Sjögren's syndrome (pSS) is chronic dysfunction and destruction of the salivary and lacrimal glands; their secretory biofluids should reflect the glandular biological status. Saliva is a heterogeneous biofluid comprised of biomolecules and omics constituents that are altered in response to various diseases. Scientific effort has evaluated saliva proteome to diagnose, monitor, and prognosticate pSS. This article reviews the recent advances in salivary proteomics in the context of pSS, highlighting the most significant and promising findings. Determining saliva as a credible means of early disease detection could lead to translational advantages and significant clinical opportunities for pSS.

Sensing of Salivary Glucose Using Nano-Structured Biosensors.

The anxiety and pain associated with frequent finger pricking has always been troublesome for diabetics measuring blood glucose (BG) in their daily lives. For this reason, a reliable glucose monitoring system that allows noninvasive measurements is highly desirable. Our main objective is to develop a biosensor that can detect low-level glucose in saliva (physiological range 0.5-20 mg/dL). Salivary glucose (SG) sensors were built using a layer-by-layer self-assembly of single-walled carbon nanotubes, chitosan, gold nanoparticles, and glucose oxidase onto a screen-printed platinum electrode. An electrochemical method was utilized for the quantitative detection of glucose in both buffer solution and saliva samples. A standard spectrophotometric technique was used as a reference method to validate the glucose content of each sample. The disposable glucose sensors have a detection limit of 0.41 mg/dL, a sensitivity of 0.24 µA·s·dL·mg(-1), a linear range of 0.5-20 mg/dL in buffer solution, and a response time of 30 s. A study of 10 healthy subjects was conducted, and SG levels between 1.1 to 10.1 mg/dL were successfully detected. The results revealed that the noninvasive SG monitoring could be an alternative for diabetes self-management at home. This paper is not intended to replace regular BG tests, but to study SG itself as an indicator for the quality of diabetes care. It can potentially help patients control and monitor their health conditions, enabling them to comply with prescribed treatments for diabetes.

Current trends in the etiology and diagnosis of HPV-related head and neck cancers.

Human papilloma virus (HPV) infection is a major risk factor for a distinct subset of head and neck squamous cell carcinoma (HNSCC). The current review summarizes the epidemiology of HNSCC and the disease burden, the infectious cycle of HPV, the roles of viral oncoproteins, E6 and E7, and the downstream cellular events that lead to malignant transformation. Current techniques for the clinical diagnosis of HPV-associated HNSCC will also be discussed, that is, the detection of HPV DNA, RNA, and the HPV surrogate marker, p16 in tumor tissues, as well as HPV-specific antibodies in serum. Such methods do not allow for the early detection of HPV-associated HNSCC and most cases are at an advanced stage upon diagnosis. Novel noninvasive approaches using oral fluid, a clinically relevant biological fluid, allow for the detection of HPV and cellular alterations in infected cells, which may aid in the early detection and HPV-typing of HNSCC tumors. Noninvasive diagnostic methods will enable early detection and intervention, leading to a significant reduction in mortality and morbidity associated with HNSCC.