The oral microbiome is increasingly recognized as a key factor in the development and progression of oral squamous cell carcinoma (OSCC). Dysbiosis has been associated with inflammation and tumorigenesis, highlighting the potential of microbial alterations and salivary biomarkers as tools for early, non-invasive diagnosis. This review examines recent advancements in understanding the oral microbiome's role in OSCC. A comprehensive synthesis of studies from 2016 to 2024 was conducted to identify emerging themes and significant findings in the field. Key topics included the interplay between microbiome-driven mechanisms and cancer development, with a focus on microbial communities and their metabolic byproducts. The findings emphasize the importance of specific microbial alterations in modulating immune responses and tumor microenvironments, as well as the promise of biomarkers such as interleukins and miRNA signatures in improving diagnostic accuracy. Recent research trends indicate growing interest in the therapeutic potential of targeting the oral microbiome in OSCC management. Despite significant advancements, gaps remain in the understanding of the precise mechanisms linking dysbiosis to cancer progression. This review underscores the need for continued research to develop personalized diagnostic and therapeutic strategies based on the oral microbiome, with the potential to transform OSCC management.

During development, tooth germs undergo various morphological changes resulting from interactions between the oral epithelium and ectomesenchyme. These processes are influenced by the extracellular matrix, the composition of which, along with cell adhesion and signaling, is regulated by metalloproteinases. Notably, these include matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs). Our analysis of previously published scRNAseq datasets highlight that these metalloproteinases show dynamic expression patterns during tooth development, with expression in a wide range of cell types, suggesting multiple roles in tooth morphogenesis. To investigate this, Marimastat, a broad-spectrum inhibitor of MMPs, ADAMs, and ADAMTSs, was applied to ex vivo cultures of mouse molar tooth germs. The treated samples exhibited significant changes in tooth germ size and morphology, including an overall reduction in size and an inversion of the typical bell shape. The cervical loop failed to extend, and the central area of the inner enamel epithelium protruded. Marimastat treatment also disrupted proliferation, cell polarization, and organization compared with control tooth germs. In addition, a decrease in laminin expression was observed, leading to a disruption in continuity of the basement membrane at the epithelial-mesenchymal junction. Elevated hypoxia-inducible factor 1-alpha gene (Hif-1α) expression correlated with a disruption to blood vessel development around the tooth germs. These results reveal the crucial role of metalloproteinases in tooth growth, shape, cervical loop elongation, and the regulation of blood vessel formation during prenatal tooth development.NEW & NOTEWORTHY Inhibition of metalloproteinases during tooth development had a wide-ranging impact on molar growth affecting proliferation, cell migration, and vascularization, highlighting the diverse role of these proteins in controlling development.

In crime investigations, the unambiguous identification of biological traces can be decisive for framing the events. In this study, we applied proteomics to analyze scant amounts of biological residues in the context of an alleged rape case, focusing on the detection of traces of vomit. We used high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and two distinct proteomic workflows to identify proteins and possible proteolytic peptides in biological residues from clothing, bedding, and car upholstery from the alleged crime scene. Specifically, a fragment of pillowcase contained a protein pattern indicative of human saliva and a complex panel of peptides resulting from extensive hydrolysis of salivary proteins. The presence of partly digested proteins from bovine meat, wheat, and eggs, along with salivary and gastric enzymes, demonstrated the presence of vomit on the alleged victim's trousers, also providing insights into the recently consumed meal. A drop of cow's milk on the seat of the suspect's car was likely irrelevant to the criminal act. Other fabric samples showed only common contaminants, excluding significant biological traces or food-derived proteins. These findings support the judicial decision regarding consent to sexual intercourse, for which DNA individualization lacks evidentiary power, and establish a reference for annotating saliva and vomit traces in forensic investigations.

Oral cancer progresses from asymptomatic to advanced stages, often involving cervical lymph node metastasis, resistance to chemotherapy, and an unfavorable prognosis. Clarifying its potential mechanisms is vital for developing effective theraputic strategies. Recent research suggests a substantial involvement of non-coding RNA (ncRNA) in the initiation and advancement of oral cancer. However, the underlying roles and functions of various ncRNA types in the growth of this malignant tumor remain unclear. Competing endogenous RNAs (ceRNAs) refer to transcripts that can mutually regulate each other at the post-transcriptional level by vying for shared miRNAs. Networks of ceRNAs establish connections between the functions of protein-coding mRNAs and non-coding RNAs, including microRNA, long non-coding RNA, pseudogenic RNA, and circular RNA, piwi-RNA, snoRNA. A growing body of research has indicated that imbalances in ceRNAs networks play a crucial role in various facets of oral cancer, including development, metastasis, migration, invasion, and inflammatory responses. Hence, delving into the regulatory pathways of ceRNAs in oral cancer holds the potential to advance our understanding of the pathological mechanisms, facilitate early diagnosis, and foster targeted drug development for this malignancy. The present review summarized the fundamental role of ceRNA network, discussed the limitations of current ceRNA applications, which have been improved through chemical modification and carrier delivery as new biomarkers for diagnosis and prognosis is expected to offer a groundbreaking therapeutic approach for individuals with oral cancer.

The incidence of growth hormone deficiency (GHD) after subarachnoid hemorrhage (SAH) is significantly higher than that of other neuroendocrine disorders, but the mechanism is still elusive. We used mass spectrometry to identify differentially expressed proteins in cerebrospinal fluid samples from a well-characterized cohort of patients. A total of 683 proteins were identified, including 39 upregulated proteins in the GHD group. ADAM9 was most highly associated with GHD. In vivo, ADAM9 colocalized with M1 microglia markers, GH and cognitive ability of mice decreased significantly, and microglia secreted ADAM9 significantly. ADAM9 regulates pyroptosis of GHRH neurons by the Mad2L2-JNK-caspase-1 pathway. Sorafenib inhibits ADAM9 secretion by microglia and improves GH levels and the cognitive ability of mice. This study found that the crosstalk between GHRH neurons and neuroglial cells in the hypothalamic arcuate nucleus, i.e., microglia, is an essential factor in the formation of GHD in SAH. We propose that neutralization of ADAM9 production by microglia might be a potential therapy for GHD after SAH.

Salivaomics has emerged as a ground-breaking field in the detection and management of oral cancer (OC), offering a non-invasive, efficient, and patient-friendly alternative to traditional diagnostic methods. This innovative approach leverages the comprehensive molecular insights provided by genomics, transcriptomics, proteomics, metabolomics, and microbiomics. The potential of salivaomics lies in its ability to enable early detection, predict malignant transformation, and monitor treatment outcomes and disease recurrence. Advancing salivary diagnostics necessitates the standardization of saliva collection and processing protocols, identification and validation of robust biomarkers, and development of cutting-edge detection technologies. A single biomarker is unlikely to fulfill all diagnostic requirements; thus, research should focus on developing a panel of biomolecules to enhance diagnostic accuracy and management of OC. Salivaomics stands at the forefront of non-invasive diagnostic methods, with the promise to revolutionize early detection and management of OC. Future research directions should emphasize the integration of multi-omics data for superior biomarker discovery, the development of portable and cost-effective point-of-care devices, and the fostering of interdisciplinary collaborations to drive innovation. Overcoming these challenges will facilitate the translation of salivaomics into routine clinical practice, significantly improving early diagnosis, treatment, and prognosis of OC. This review provides a comprehensive overview of salivaomics, detailing the use of saliva as a diagnostic fluid. It covers saliva collection, preparation, transportation, storage methods, and various analytical techniques. Additionally, the review discusses the current challenges and future directions of this transformative technology, emphasizing its potential to enhance clinical outcomes in OC.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths. Saliva diagnosis is an essential approach for clinical applications owing to its noninvasive and material-rich features. The purpose of this study was to investigate differences in wheat germ agglutinin (WGA)-based recognition of salivary protein N-linked glycan profiles to distinguish non-small cell lung cancer (NSCLC) patients from controls. We used WGA-magnetic particle conjugates to isolate glycoproteins in the pooled saliva of healthy volunteers (HV, n = 35), patients with benign pulmonary disease (BPD, n = 35), lung adenocarcinoma (ADC, n = 35), and squamous cell carcinoma (SCC, n = 35), following to release the N-linked glycans from the isolated proteins with PNGase F, and further identified and annotated the released glycans by MALDI-TOF/TOF-MS, respectively. The results showed that 34, 35, 39, and 44 N-glycans recognized by WGA were identified and annotated from pooled saliva samples of HV, BPD, ADC, and SCC, respectively. Furthermore, the proportion of N-glycans recognized by WGA in BPD (81.2 %), ADC (90.1 %), and SCC (88.7 %), increased compared to HV (71.9 %). Two N-glycan peaks (m/z 2286.799, and 3399.211) specifically recognized by WGA were present only in NSCLC. These findings suggest that altered salivary glycopatterns such as sialic acids and GlcNAc containing N-glycans recognized by WGA might serve as potential personalized biomarkers for the diagnosis of NSCLC patients.

This study investigates the expressions of ADAM9, CDCP1 and t-PA in OSCC and their impacts on patient prognosis. Previous research has demonstrated the overexpression of ADAM9 and activation of plasminogen activator in OSCC, but CDCP1's role remains unexplored. While these biomolecules are known to contribute to lung cancer metastasis, their concurrent expressions in OSCC have not been thoroughly examined. Our aim is to assess the expressions of ADAM9, CDCP1, and t-PA in OSCC specimens, compare them with normal oral tissues, and explore their correlation with OSCC's clinicopathological features and patient survival outcomes.

Head and neck cancers (HNCs) are the seventh most common cancer worldwide, accounting for 4-5% of all malignancies. Salivary metabolites, which serve as key metabolic intermediates and cell-signalling molecules, are emerging as potential diagnostic biomarkers for HNC. While current research has largely concentrated on these metabolites as biomarkers, a critical gap remains in understanding their fluctuations before and after treatment, as well as their involvement in oral side effects. Recent studies emphasise the role of the oral microbiome and its metabolic activity in cancer progression and treatment efficacy by bacterial metabolites and virulence factors. Oral bacteria, such as P. gingivalis and F. nucleatum, contribute to a pro-inflammatory environment that promotes tumour growth. Additionally, F. nucleatum enhances its virulence through flagellar assembly and iron transport mechanisms, facilitating tumour invasion and survival. Moreover, alterations in the oral microbiome can influence chemotherapy efficacy and toxicity through the microbiota-host irinotecan axis, highlighting the complex interplay between microbial communities and therapeutic outcomes. Salivary metabolite profiles are influenced by factors such as gender, methods, and patient habits like smoking-a major risk factor for HNC. Radiotherapy (RT), a key treatment for HNC, often causes side effects such as xerostomia, oral mucositis, and swallowing difficulties which impact survivors' quality of life. Intensity-modulated radiotherapy (IMRT) aims to improve treatment outcomes and minimise side effects but can still lead to significant salivary gland dysfunction and associated complications. This review underscores the microbial and host interactions affecting salivary metabolites and their implications for cancer treatment and patient outcomes.

Saliva contains a diverse array of biomarkers indicative of various diseases. Saliva testing has been a major advancement towards non-invasive point-of-care diagnosis with clinical significance. However, there are challenges associated with salivary diagnosis from sample treatment and standardization. This review highlights the biomarkers in saliva and their role in identifying relevant diseases. It provides an overview and discussion about the current practice of saliva collection and processing, and advancements in saliva detection systems from in vitro methods to wearable oral devices. The review also addresses challenges in saliva diagnostics and proposes solutions, aiming to offer a comprehensive understanding and practical guidance for improving saliva-based detection in clinical diagnosis. Saliva diagnosis provides a rapid, effective, and safe alternative to traditional blood and urine tests for screening large populations and enhancing infectious disease diagnosis and surveillance. It meets the needs of various fields such as disease management, drug screening, and personalized healthcare with advances in saliva detection systems offering high sensitivity, fast response times, portability, and automation. Standardization of saliva collection, treatment, biomarker discovery, and detection between different laboratories needs to be implemented to obtain reliable salivary diagnosis in clinical practice.

Early childhood caries (ECC) is one of the most prevalent diseases in children worldwide. Early childhood caries is driven by a dysbiotic state of oral microorganisms, mainly caused by a sugar-rich diet. Additionally, poor oral hygiene or insufficient dental plaque removal leads to the rapid progression of ECC. Early childhood caries leads not only to dental destruction and pain in children but also affects the quality of life of the caregivers.Additionally, upon neutrophil activation at inflammatory locations, these proteases are externalized in an active state, aiding in the control of inflammatory and immunological responses. Any enzyme that catalyzes proteolysis reactions is known as a protease. Proteases are produced by human glands or derived from microbes in the oral cavity. Additionally, the oropharyngeal mucosae and crevicular fluids are sources of protease.

This study is aimed at the estimation and correlation of salivary protease enzymatic activity in the saliva of children with and without ECC.

A total of 50 children were included in the study, which was divided into two groups: group I (caries-active) and group II (caries-free)-each consisting of 25 children. Unstimulated saliva samples were collected and subjected to a spectrophotometer for analysis. Salivary protease levels were estimated and correlated between caries-active and caries-free children.

The correlation between caries score and salivary protease activity was statistically significant with a moderate correlation. The comparison of mean salivary protease activity between caries-active and caries-free groups was statistically significant. However, the comparison of salivary protease activity based on different age-groups was not statistically significant, whereas gender and caries scores in group A were statistically significant.

In conclusion, there is a substantial correlation between salivary protease enzyme levels and the severity of dental caries, and an increase in salivary protease enzyme levels is linked to a considerable rise in caries severity. As a result, prevention may be possible with early detection.

Thimmegowda U, Kuri PN, Dhamnekar P. Role of Salivary Protease Enzymatic Activity in Saliva of Children with and without Early Childhood Caries: A Randomized Clinical Trial. Int J Clin Pediatr Dent 2024;17(8):877-880.

Lung cancer (LC) is the malignant tumor with the highest mortality rate worldwide, and precise early diagnosis can improve patient prognosis. The purpose of this study was to investigate whether alterations in the glycopatterns recognized by the Hippeastrum hybrid lectin (HHL) in salivary proteins are associated with the development of LC.

First, we collected saliva samples from LC (15 lung adenocarcinoma (ADC); 15 squamous cell carcinoma (SCC); 15 small cell lung cancer (SCLC)) and 15 benign pulmonary disease (BPD) for high-throughput detection of abundance levels of HHL-recognized glycopatterns using protein microarrays, and then validated the pooled samples from each group with lectin blotting analysis. Finally, the N-glycan profiles of salivary glycoproteins isolated from the pooled samples using HHL-magnetic particle conjugates were characterized separately using MALDI-TOF/TOF-MS.

The results showed that the abundance level of glycopatterns recognized by HHL in salivary proteins was elevated in LC compared to BPD. The proportion of mannosylated N-glycans was notably higher in ADC (31.7%), SCC (39.0%), and SCLC (46.6%) compared to BPD (23.3%).

The altered salivary glycopatterns such as oligomannose, Manα1-3Man, or Manα1-6Man N-glycans recognized by HHL might serve as potential biomarkers for the diagnosis of LC patients.

This study provides crucial information for studying changes in salivary to differentiate between BPD and LC and facilitate the discovery of biomarkers for LC diagnosis based on precise alterations of mannosylated N-glycans in saliva.

Drug-induced liver injury (DILI) is one of the most common adverse events of medication use, and its incidence is increasing. However, early detection of DILI is a crucial challenge due to a lack of biomarkers and noninvasive tests.

To identify salivary metabolic biomarkers of DILI for the future development of noninvasive diagnostic tools.

Saliva samples from 31 DILI patients and 35 healthy controls (HCs) were subjected to untargeted metabolomics using ultrahigh-pressure liquid chromatography coupled with tandem mass spectrometry. Subsequent analyses, including partial least squares-discriminant analysis modeling, t tests and weighted metabolite coexpression network analysis (WMCNA), were conducted to identify key differentially expressed metabolites (DEMs) and metabolite sets. Furthermore, we utilized least absolute shrinkage and selection operato and random fores analyses for biomarker prediction. The use of each metabolite and metabolite set to detect DILI was evaluated with area under the receiver operating characteristic curves.

We found 247 differentially expressed salivary metabolites between the DILI group and the HC group. Using WMCNA, we identified a set of 8 DEMs closely related to liver injury for further prediction testing. Interestingly, the distinct separation of DILI patients and HCs was achieved with five metabolites, namely, 12-hydroxydodecanoic acid, 3-hydroxydecanoic acid, tetradecanedioic acid, hypoxanthine, and inosine (area under the curve: 0.733-1).

Salivary metabolomics revealed previously unreported metabolic alterations and diagnostic biomarkers in the saliva of DILI patients. Our study may provide a potentially feasible and noninvasive diagnostic method for DILI, but further validation is needed.

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.

Saliva has gained increasing attention in the quest for disease biomarkers. Because it is a biological fluid that can be collected is an easy, painless, and safe way, it has been increasingly studied for the identification of oral cancer biomarkers. This is particularly important because oral cancer is often diagnosed at late stages with a poor prognosis.

The review addresses the evolution of the experimental approaches used in salivary proteomics studies of oral cancer over the years and outlines advantages and pitfalls related to each one. In addition, examines the current landscape of oral cancer biomarker discovery and translation focusing on salivary proteomic studies. This discussion is based on an extensive literature search (PubMed, Scopus and Google Scholar).

The introduction of mass spectrometry has revolutionized the study of salivary proteomics. In the future, the focus will be on refining existing methods and introducing powerful experimental techniques such as mass spectrometry with selected reaction monitoring, which, despite their effectiveness, are still underutilized due to their high cost. In addition, conducting studies with larger cohorts and establishing standardized protocols for salivary proteomics are key challenges that need to be addressed in the coming years.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

Different efforts have been made to find better and less invasive methods for the diagnosis and prediction of oral cancer, such as the study of saliva as a source of biomarkers. The aim of this study was to perform a scoping review about salivary molecules that have been assessed as possible biomarkers for the diagnosis of oral squamous cell carcinoma (OSCC). A search was conducted using EBSCO, PubMed (MEDLINE), Scopus, and Web of Science. The research question was as follows: which molecules present in saliva have utility to be used as biomarkers for the early detection of oral cancer? Sixty-two studies were included. Over 100 molecules were assessed. Most of the markers were oriented towards the early diagnosis of OSCC and were classified based on their ability for detecting OSCC and oral potentially malignant disorders (OPMDs), OSCC outcome prediction, and the prediction of the malignant transformation of OPMDs. TNF-α, IL-1β, IL-6 IL-8, LDH, and MMP-9 were the most studied, with almost all studies reporting high sensitivity and specificity values. TNF-α, IL-1β, IL-6 IL-8, LDH, and MMP-9 are the most promising salivary biomarkers. However, more studies with larger cohorts are needed before translating the use of these biomarkers to clinical settings.

Intestinal ischemia/reperfusion injury (IIRI) has the potential to be life threatening and is associated with significant morbidity and serious damage to distant sites in the body on account of disruption of the intestinal mucosal barrier. In the present study, we have explored this line of research by comparing and identifying peptides that originated from the intestinal segments of IIRI model rats by using liquid chromatography-mass spectrometry (LC-MS). We also analyzed the basic characteristics, cleavage patterns, and functional domains of differentially expressed peptides (DEPs) between the IIRI model rats and control (sham-operated) rats and identified bioactive peptides that are potentially associated with ischemia reperfusion injury. We also performed bioinformatics analyses in order to identify the biological roles of the DEPs based on their precursor proteins. Enrichment analysis demonstrated the role of several DEPs in impairment of the intestinal mucosal barrier caused by IIRI. Based on the results of comprehensive ingenuity pathway analysis, we identified the DEPs that were significantly correlated with IIRI. We identified a candidate precursor protein (Actg2) and seven of its peptides, and we found that Actg2-6 had a more significant difference in its expression, a longer half-life, and better lipophilicity, hydrophobicity, and stability than the other candidate Actg2 peptides examined. Furthermore, we observed that Actg2-6 might play critical roles in the protection of the intestinal mucosal barrier during IIRI. In summary, our study provides a better understanding of the peptidomics profile of IIRI, and the results indicate that Actg2-6 could be a useful target in the treatment of IIRI.

Oral squamous cell carcinoma (OSCC) is one of the most frequent types of head and neck cancer. Despite the genetic and environmental risk factors, OSCC is also associated with microbial infections and/or dysbiosis. The secreted saliva serves as the chemical barrier of the oral cavity and, since OSCC can alter the protein composition of saliva, our aim was to analyze the effect of OSCC on the salivary chemical barrier proteins. Publicly available datasets regarding the analysis of salivary proteins from patients with OSCC and controls were collected and examined in order to identify differentially expressed chemical barrier proteins. Network analysis and gene ontology (GO) classification of the differentially expressed chemical barrier proteins were performed as well. One hundred and twenty-seven proteins showing different expression pattern between the OSCC and control groups were found. Protein-protein interaction networks of up- and down-regulated proteins were constructed and analyzed. The main hub proteins (IL-6, IL-1B, IL-8, TNF, APOA1, APOA2, APOB, APOC3, APOE, and HP) were identified and the enriched GO terms were examined. Our study highlighted the importance of the chemical barrier of saliva in the development of OSCC.

Biomarker detection has attracted increasing interest in recent years due to the minimally or non-invasive sampling process. Single entity analysis of biomarkers is expected to provide real-time and accurate biological information for early disease diagnosis and prognosis, which is critical to the effective disease treatment and is also important in personalized medicine. As an innovative single entity analysis method, nanopore sensing is a pioneering single-molecule detection technique that is widely used in analytical bioanalytical fields. In this review, we overview the recent progress of nanopore biomarker detection as new approaches to disease diagnosis. In highlighted studies, nanopore was focusing on detecting biomarkers of different categories of communicable and noncommunicable diseases, such as pandemic Covid-19, AIDS, cancers, neurologic diseases, etc. Various sensitive and selective nanopore detecting strategies for different types of biomarkers are summarized. In addition, the challenges, opportunities, and direction for future development of nanopore-based biomarker sensors are also discussed.

Matrix metalloproteinase-12 (MMP12), or macrophage metalloelastase, plays important roles in extracellular matrix (ECM) component degradation. Recent reports show MMP12 has been implicated in the pathogenesis of periodontal diseases. To date, this review represents the latest comprehensive overview of MMP12 in various oral diseases, such as periodontitis, temporomandibular joint dysfunction (TMD), orthodontic tooth movement (OTM), and oral squamous cell carcinoma (OSCC). Furthermore, the current knowledge regarding the distribution of MMP12 in different tissues is also illustrated in this review. Studies have implicated the association of MMP12 expression with the pathogenesis of several representative oral diseases, including periodontitis, TMD, OSCC, OTM, and bone remodelling. Although there may be a potential role of MMP12 in oral diseases, the exact pathophysiological role of MMP12 remains to be elucidated. Understanding the cellular and molecular biology of MMP12 is essential, as MMP12 could be a potential target for developing therapeutic strategies targeting inflammatory and immunologically related oral diseases.

Saliva is a complex biological fluid with a variety of biomolecules, such as DNA, RNA, proteins, metabolites and microbiota, which can be used for the screening and diagnosis of many diseases. In addition, saliva has the characteristics of simple collection, non-invasive and convenient storage, which gives it the potential to replace blood as a new main body of fluid biopsy, and it is an excellent biological diagnostic fluid. This review integrates recent studies and summarizes the research contents of salivaomics and the research progress of saliva in early diagnosis of oral and systemic diseases. This review aims to explore the value and prospect of saliva diagnosis in clinical application.

Inherited copy number variations (CNVs) can provide valuable information for cancer susceptibility and prognosis. However, their association with oropharynx squamous cell carcinoma (OPSCC) is still poorly studied. Using microarrays analysis, we identified three inherited CNVs associated with OPSCC risk, of which one was validated in 152 OPSCC patients and 155 controls and related to pseudogene-microRNA-mRNA interaction. Individuals with three or more copies of ADAM3A and ADAM5 pseudogenes (8p11.22 chromosome region) were under 6.49-fold increased risk of OPSCC. ADAM5 shared a highly homologous sequence with the ADAM9 3'-UTR, predicted to be a binding site for miR-122b-5p. Individuals carrying more than three copies of ADAM3A and ADAM5 presented higher ADAM9 expression levels. Moreover, patients with total deletion or one copy of pseudogenes and with higher expression of miR-122b-5p presented worse prognoses. Our data suggest, for the first time, that ADAM3A and ADAM5 pseudogene-inherited CNV could modulate OPSCC occurrence and prognosis, possibly through the interaction of ADAM5 pseudogene transcript, miR-122b-5p, and ADAM9.

Oral squamous cell carcinoma (OSCC) is an aggressive malignancy with increased mortality, in which the early diagnosis is the most important step in increasing patients' survival rate. Extensive research has evaluated the role of saliva as a source of diagnostic biomarkers, among which matrix metalloproteinases (MMPs) have shown a valuable potential for detecting even early stages of OSCC. The aim of this review was to present recent clinical data regarding the significance of salivary MMPs in the detection of early malignant transformation of the oral mucosa. A narrative review was conducted on articles published in PubMed, Cochrane Library, Web of Science, EBSCO and SciELO databases, using specific terms. Our search revealed that MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-12 and MMP-13 had significantly higher levels in saliva from patients with OSCC compared to controls. However, the strength of evidence is limited, as most information regarding their use as adjuvant diagnostic tools for OSCC comes from studies with a low number of participants, variable methodologies for saliva sampling and diagnostic assays, and insufficient adjustment for all covariates. MMP-1, MMP-3 and MMP-9 were considered the most promising candidates for salivary diagnosis of OSCC, but larger studies are needed in order to validate their clinical application.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Mtb can overcome macrophage intracellular killing and lead to persistent infections. The proteases of Mtb are critical virulence factors that participate in immune responses. We determined that Rv3090 is a cell wall-associated protease and a potential pathogenic factor. To characterize the role of Rv3090 in Mtb, recombinant Msg_Rv3090 and Msg_pAIN strains were constructed to infect macrophages and mice. Lactate dehydrogenase assays and flow cytometry results showed that Rv3090 induces late macrophage apoptosis. In vivo infection experiments indicated that Rv3090 could induce hepatocyte and lung cell apoptosis and cause pathological damage to the spleen, livers and lungs. Msg_Rv3090 specifically stimulated the secretion of inflammatory cytokines including TNF-α, IL-6 and IL-1β. Overexpression of Rv3090 significantly promoted the survival of Msg in livers and lungs. Thus, Rv3090 protease triggered late cell apoptosis and contributed to the pathogenicity and dissemination of Mtb.

Oral cancer is the 16th most common cancer worldwide. It commonly arises from painless white or red plaques within the oral cavity. Clinical outcome is highly related to the stage when diagnosed. However, early diagnosis is complex owing to the impracticality of biopsying every potentially premalignant intraoral lesion. Therefore, there is a need to develop a non-invasive cost-effective diagnostic technique to differentiate non-malignant and early-stage malignant lesions. Optical spectroscopy may provide an appropriate solution to facilitate early detection of these lesions. It has many advantages over traditional approaches including cost, speed, objectivity, sensitivity, painlessness, and ease-of use in clinical setting for real-time diagnosis. This review consists of a comprehensive overview of optical spectroscopy for oral cancer diagnosis, epidemiology, and recent improvements in this field for diagnostic purposes. It summarizes major developments in label-free optical spectroscopy, including Raman, fluorescence, and diffuse reflectance spectroscopy during recent years. Among the wide range of optical techniques available, we chose these three for this review because they have the ability to provide biochemical information and show great potential for real-time deep-tissue point-based in vivo analysis. This review also highlights the importance of saliva-based potential biomarkers for non-invasive early-stage diagnosis. It concludes with the discussion on the scope of development and future demands from a clinical point of view.

The senescence-associated secretory phenotype (SASP), which accumulates over the course of normal aging and in age-related diseases, is a crucial driver of chronic inflammation and aging phenotypes. It is also responsible for the pathogenesis of multiple oral diseases. However, the pathogenic mechanism underlying SASP has not yet been fully elucidated. Here, relevant articles on SASP published over the last five years (2017-2022) were retrieved and used for bibliometric analysis, for the first time, to examine SASP composition. More than half of the relevant articles focus on various cytokines (27.5%), growth factors (20.9%), and proteases (20.9%). In addition, lipid metabolites (13.1%) and extracellular vesicles (6.5%) have received increasing attention over the past five years, and have been recognized as novel SASP categories. Based on this, we summarize the evidences demonstrating that SASP plays a pleiotropic role in oral immunity and propose a four-step hypothetical framework for the progression of SASP-related oral pathology-1) oral SASP development, 2) SASP-related oral pathological alterations, 3) pathological changes leading to oral immune homeostasis disruption, and 4) SASP-mediated immune dysregulation escalating oral disease. By targeting specific SASP factors, potential therapies can be developed to treat oral and age-related diseases.

Biopsy is the gold standard for oral squamous cell carcinoma (OSCC) diagnosis. Salivary biomarkers provide promising complementary alternative diagnostic adjunct for its simple non- invasive collection and technique and to screen large population.

To summarize and compare the existing evidence on diagnostic accuracy of salivary biomarkers with their estimation method in detecting early oral squamous cell carcinoma.

The review protocol is registered under PROSPERO(CRD42021225704). PubMed, Google Scholar, EBSCOhost were searched from 2000 to 2020 to identify the screening potential of eight salivary biomarkers: mRNA, miRNA, DUSP100, s100P, IL-8, IL-1B, TNF-a and MMP-9. True-positive, false-positive, true-negative, false-negative, sensitivity, specificity values were extracted or calculated if not present for each study. Quality of selected studies was evaluated based on QUADAS 2 tool. Meta-analysis was performed using a bivariate model parameter for the sensitivity and specificity and summary points, summary receiver operating curve (SROC), confidence region, and prediction region were calculated.

Eighteen studies were included for qualitative synthesis and out of that 13 for meta-analysis. Sensitivity and specificity were calculated with AUC. For mRNA it was 91% and 90% with 0.96 AUC, miRNA had 91% and 91% with 0.95 AUC for PCR. IL-1B had 46% and 60% with 0.61 AUC, S100p had 45% and 90% with 0.57 AUC for ELISA. IL-8 had 54% and 74% for ELISA and 89% and 90% for PCR with 0.79 AUC and DUSP1 had 32% and 87% for ELISA and 76% and 83% for PCR with 0.83 AUC respectively.

Early detection of OSCC was best achieved by screening for salivary mRNA and miRNA estimated by PCR. Further investigation is required into salivary RNA as novel biomarkers and these salivary biomarkers may be potentially used for non-invasive diagnosis of early OSCC.

Due to poor targeting ability of anti-tumor drugs and self-adaptation of tumors, the chemotherapy of ovarian cancer is still poorly effective. In recent years, the treatment of tumor with nano-targeted agents has become a potential research focus. In this study, a new type of short cell-penetrating peptide RPV-modified paclitaxel plus schisandrin B liposomes were constructed to disrupt VM channels, angiogenesis, proliferation and migration for the treatment of ovarian cancer.

In this study, clone assay, TUNEL, Transwell, wound-healing, CAM and mimics assay were used to detect the effects of RPV-modified liposomes on ovarian cancer SK-OV-3 cells before and after treatment. HE-staining, immunofluorescence and ELISA were used to further detect the expression of tumor-related proteins.

RPV-modified paclitaxel plus schisandrin B liposomes can inhibit angiogenesis, VM channel formation, invasion and proliferation of ovarian SK-OV-3 cells. In vitro and in vivo studies showed that tumor-related protein expression was down-regulated. Modification of RPV can prolong the retention time of liposome in vivo and accumulate in the tumor site, increasing the anti-tumor efficacy.

The RPV-modified paclitaxel plus schisandrin B liposomes have good anti-tumor effect, thus may provide a new avenue for the treatment of ovarian cancer.

Oral squamous cell carcinoma (OSCC) usually originates from oral potentially malignant disorders (OPMD), such as oral leukoplakia (OLK) and oral lichen planus (OLP). Identifying biomarkers for the early diagnosis and evaluation of malignant transformation in OPMD could improve the survival rate of OSCC patients.

The present study aimed to screen for potential salivary biomarkers for evaluating the malignant transformation of OPMD.

Salivary proteases from OLK and OSCC patients or healthy donors and proteases in cultural medium from DOK and Cal-27 cells were detected with a human protease array kit. The concentrations of the salivary Kallikrein 5 (KLK5) and urokinase-type plasminogen activator (uPA) proteases were measured by ELISA. Receiver operating characteristics (ROC) to determine the potential value of these proteases in clinical diagnosis were calculated using SPSS software. Immunohistochemistry was used to detect the KLK5 and uPA expression in the oral organizations.

The salivary protease spectrum was different among patients with OLK and OSCC and healthy donors. KLK5 and uPA levels in saliva tended to increase as the disease progressed (healthy < OPMD [OLK and OLP] < OSCC). ROC curves showed the optimum diagnostic cutoffs for KLK5 as a biomarker for OLK, OLP, and OSCC were 5.97, 6.03, and 9.45 pg/mL, respectively, while the cutoffs for uPA were 17.19, 17.26, and 20.96 pg/mL. Their combined analysis showed a higher sensitivity for the differential diagnosis of disease. Furthermore, higher levels of KLK5 and uPA were observed in OSCC tissues than in OLK and OLP.

Salivary KLK5 and uPA are potential biomarkers for evaluating OLK and OLP malignant transformation and early diagnosis of OSCC.

The oral cavity hosts over 700 different microbial species that produce a rich reservoir of bioactive metabolites critical to oral health maintenance. Over the last two decades, new insights into the oral microbiome and its importance in health and disease have emerged mainly due to the discovery of new oral microbial species using next-generation sequencing (NGS). This advancement has revolutionized the documentation of unique microbial profiles associated with different niches and health/disease states within the oral cavity and the relation of the oral bacteria to systemic diseases. However, less work has been done to identify and characterize the unique oral microbial metabolites that play critical roles in maintaining equilibrium between the various oral microbial species and their human hosts. This article discusses the most significant microbial metabolites produced by these diverse communities of oral bacteria that can either foster health or contribute to disease. Finally, we shed light on how advances in genomics and genome mining can provide a high throughput platform for discovering novel bioactive metabolites derived from the human oral microbiome to tackle emerging human infections and systemic diseases.

Melatonin's role in circadian rhythm is well documented, as are its' anti-oxidant, oncostatic and anti-inflammatory properties. Poor sleep quality has been associated as a potential risk factor for several malignancies, including head and neck cancers. The purpose of this study is to determine salivary melatonin (MLT) levels in oral squamous cell carcinoma (OSCC) patients, compare the salivary MLT levels with those in healthy individuals and compare the salivary and serum levels in OSCC patients. Furthermore, the aim is to investigate the potential relationship between sleep quality and salivary MLT levels in OSCC patients. Unstimulated (UWS) and stimulated (SWS) whole saliva was sampled from patients with T1N0M0 and T2N0M0 OSCC (N = 34) and 33 sex and age matched healthy subjects. Serum samples were taken from 11 OSCC patients. Sleep quality was measured using Pittsburgh Sleep Quality Index (PSQI) questionnaire. Melatonin levels in UWS and SWS were significantly higher in the OSCC group. Sleep quality was significantly lower in patients with OSCC (P = 0.0001). ROC analysis was found to be significant (P < 0.001) in evaluating MLT concentration limit in diagnosing OSCC. The expected relationship between sleep quality and salivary MLT levels in OSCC patients was not observed. Our results suggest salivary MLT as a potential biomarker that might facilitate non-invasive detection of early stage OSCC.

Matrix metalloproteinase-1 (MMP-1) is associated with many types of cancers, including oral, colorectal, and brain cancers. This paper describes the fabrication of an MMP-1 immunosensor based on a gold nanoparticle/polyethyleneimine/reduced graphene oxide (AuNP/PEI/rGO)-modified disposable screen-printed electrode (SPE). A microwave-assisted single-step method was employed for the simultaneous reduction of gold and graphene oxide in a PEI environment to avoid AuNP agglomeration. The crystal structure, chemical composition, optical properties, and interior morphology of the materials were probed by X-ray diffraction, Raman spectroscopy, UV-visible spectrometry, and transmission electron microscopy techniques. To assemble a label-free MMP-1 immunosensor layer-by-layer, 3-mercaptopropionic acid was utilized due to its strong sulfur-gold bonding ability, and its tail end was attached to a carboxyl group, allowing the MMP-1 antibody (anti-MMP-1) to be subsequently cross-linked using the traditional N-(3-dimethylaminopropyl) and N' ethylcarbodiimide hydrochloride method. Differential pulse voltammetry analysis showed a linear relationship with MMP-1 concentration in the range of 1-50 ng ml-1 with an R2 value of ∼0.996 (n = 5, RSD < 5%). This immunosensor was successfully applied for MMP-1 detection in urine, saliva, bovine serum, and cell culture media (HSC-3 & C6) of oral and brain cancers showing results comparable to those of the credible ELISA method.

Oral squamous cell carcinoma is a global threat and accounts for approximately 90% of malignant oral lesions. The emergence of oral carcinoma is linked to precancerous lesions, which act as precursors of the disease. Matrix metalloproteinases appear to play a significant role in the pathogenesis of both precancerous conditions and oral malignancies due to their participation in remodeling of the extracellular matrix.

This is an analytical study conducted at Dow University of Health Sciences, Pakistan. Unstimulated saliva samples were collected from healthy, oral submucous fibrosis and oral squamous cell carcinoma patients. The level of MMP-12 was estimated using enzyme-linked immunosorbent assay. One-way Analysis of variance was run to determine if MMP-12 levels differ between the three groups, which was preceded by post hoc Tuckey test. MMP-12 cut off values were determined using Receiver operating characteristic curve.

A significant difference in salivary MMP-12 expression was observed in OSF and OSCC (p < 0.001). The expression of salivary MMP-12 was higher in OSF and OSCC patients as compared to the healthy group (p < 0.001). The mean MMP-12 expression in OSCC appeared higher than in OSF cases (p < 0.05). MMP-12 value of [Formula: see text] 4.05 ng/ml and [Formula: see text] 4.20 ng/ml is predictive of OSF and OSCC respectively, with 100% sensitivity and specificity (p < 0.001).

Increased expression of MMP-12 appears as the healthy patient advances to OSF and OSCC. The study results also demonstrate higher MMP-12 expression in OSCC patients as compared to OSF. Therefore, the estimation of salivary MMP-12 serves as a valuable non-invasive early diagnostic tool in diagnosing oral submucous fibrosis and oral squamous cell carcinoma.

Tobacco exposure (through smoking or chewing) is one of the predominant risk factors associated with the development of oral squamous cell carcinoma (OSCC). Despite the growing number of patients diagnosed with OSCC, there are few circulating biomarkers for identifying individuals at a higher risk of developing the disease. Successful identification of candidate molecular markers for risk assessment could aid in the early detection of oral lesions and potentially be used for community screening of high-risk populations.

Identification of differentially expressed proteins in the serum of oral cancer patients which can serve as biomarkers for the diagnosis of the onset of oral cancer among tobacco users.

We employed a tandem mass tag (TMT)-based quantitative proteomics approach to study alterations in the serum proteomes of OSCC patients based on their tobacco exposure habits (chewing and smoking) compared to healthy individuals with no history of using any form of tobacco or any symptoms of the disease.

Mass spectrometry-based analysis resulted in the identification of distinct signatures in the serum of OSCC patients who either chewed or smoked tobacco. Pathway analysis revealed opposing effects of dysregulated proteins enriched in the complement-coagulation signaling cascades with a high expression of the Serpin family of proteins observed in OSCC patients who chewed tobacco compared to healthy individuals whereas these proteins showed decreased levels in OSCC patients who smoked. ELISA-based validation further confirmed our findings revealing higher expression of SERPINA6 and SERPINF1 across serum of OSCC patients who chewed tobacco compared to healthy individuals.

This study serves as a benchmark for the identification of serum-based protein markers that may aid in the identification of high-risk patients who either chew tobacco or smoke tobacco.

Protease activity has been associated with pathological processes that can lead to cancer development and progression. However, understanding the pathological unbalance in proteolysis is challenging because changes can occur simultaneously at protease, their inhibitor, and substrate levels. Here, we present a pipeline that combines peptidomics, proteomics, and peptidase predictions for studying proteolytic events in the saliva of 79 patients and their association with oral squamous cell carcinoma (OSCC) prognosis. Our findings revealed differences in the saliva peptidome of patients with (pN+) or without (pN0) lymph-node metastasis and delivered a panel of ten endogenous peptides correlated with poor prognostic factors plus five molecules able to classify pN0 and pN+ patients (area under the receiver operating characteristic curve > 0.85). In addition, endopeptidases and exopeptidases putatively implicated in the processing of differential peptides were investigated using cancer tissue gene expression data from public repositories, reinforcing their association with poorer survival rates and prognosis in oral cancer. The dynamics of the OSCC-related proteolysis were further explored via the proteomic profiling of saliva. This revealed that peptidase/endopeptidase inhibitors exhibited reduced levels in the saliva of pN+ patients, as confirmed by selected reaction monitoring-mass spectrometry, while minor changes were detected in the level of saliva proteases. Taken together, our results indicated that proteolytic activity is accentuated in the saliva of patients with OSCC and lymph-node metastasis and, at least in part, is modulated by reduced levels of salivary peptidase inhibitors. Therefore, this integrated pipeline provided better comprehension and discovery of molecular features with implications in the oral cancer metastasis prognosis.