Used Toothbrush as a Potential Source of Gene Expression Among Subjects With Systemic Disease and Adverse Habits.

Purpose To explore the viability of used toothbrush as a source of gene expression and comparison of the same among tobacco, alcohol, diabetic and hypertensive subjects. Materials and methods Fourteen subjects with a history of type 2 diabetes mellitus (T2DM) and hypertension were allocated to Group I, 14 subjects with tobacco and alcohol habits allocated to Group II and 14 healthy subjects allocated to Group III. Genetic materials retrieved from the used toothbrush of the study subjects were assessed for genetic expression of cyclin-dependent kinase 4 (CDK4), Bcl-2-like protein 4 (BAX), cyclin-dependent kinase inhibitor 2A (CDKN2A), B-cell lymphoma 2 (BCL2), G-protein beta-3 (GNB3), and subunit gene and transcription factor 7-like 2 (TCF7L2) by polymerase chain reaction (PCR). Results BAX gene showed reduced expression in tobacco and alcohol users (p=0.0497). BCL2, CDK4, and GNB3 showed no significant difference in expression in both the groups and with control. CDKN2A was expressed at a lower level in Group I and II participants. TCF7L2 showed higher expression in Group 2 participants (p=0.001). Conclusion The study concluded that used toothbrush is a reliable source for genetic expression. There was no difference in BCL2, CDK4, and GNB3 gene expression between subjects with systemic disease, adverse habits and healthy controls. There is a downregulation of BAX and upregulation of TCF7L2 gene in subjects with adverse habits.

Role of Toothbrushes as Gene Expression Profiling Tool for Oral Cancer Screening in Tobacco and Alcohol Users.

AIM: The use of toothbrushes was investigated as a potential RNA source and gene expression profiling tool for oral cancer screening in tobacco and alcohol users. METHODOLOGY: A total of 20 subjects were selected on the basis of inclusion and exclusion criteria. They were divided into two groups: group I-healthy controls (n = 6); group II-individuals who consume tobacco and alcohol (n = 14). After the volunteers brushed their teeth using a soft-bristle toothbrush with ~0.5 gm of toothpaste, the toothbrushes were collected, and the gene expression of BAX, BCL2, CDK4, CKDN2A, GNB3, and TCF7L2 was assessed. RESULTS: The gene expression of BAX decreased significantly in alcoholics and smokers (0.13867 ± 0.12014), while the gene expression of BCL2 increased in alcoholics and smokers (1.91001 ± 0.90425) in comparison with healthy controls (p = 0.0054 and p = 0.0055). Although there was increased expression of CDK4, CKDN2A, and TCF7L2 and decreased expression of GNB3 in smokers and alcoholics, the results were not significant. CONCLUSIONS: A toothbrush is a good source of RNA, and gene expression analysis can be performed using the genetic material retrieved from toothbrushes, which can aid in the early diagnosis of oral squamous cell carcinoma among tobacco and alcohol users. Further studies with a larger sample size and different durations of toothbrush use should be conducted to explore the role of toothbrushes as a noninvasive tool for disease diagnosis.

Evaluation and Comparison of Genomic DNA Extraction Methods and PCR Optimization on Archival Formalin-Fixed and Paraffin-Embedded Tissues of Oral Squamous Cell Carcinoma.

Recovery and amplification of nucleic acids from archived formalin-fixed tissue samples is the most developing field in retrospective genetic studies. We compared different deparaffinization methods and DNA isolation techniques, and intergroup comparisons were performed to evaluate the effectiveness of different storing methods for archival OSCC samples based on obtained mean DNA quantity, quality, and PCR amplification of the P53 gene. The study comprised 75 archival histologically diagnosed OSCC samples which were divided into Group I: Formalin-fixed paraffin-embedded tissue blocks and Group II: Long-term formalin-fixed tissue. A comparison of different deparaffinization methods showed that xylene deparaffinization is an efficient method to obtain suitable DNA. Comparing different DNA isolation techniques illustrated that the conventional phenol-chloroform method gives better integrity to DNA in contrast with the kit method. Comparison between FFPET and long-term FFT samples demonstrated that samples fixed in formalin overnight and embedded in wax yield better quality and quantity DNA in comparison with long-term samples fixed in formalin. To obtain suitable integrity of DNA, tissue samples should be stored by fixing in formalin overnight followed by preparation of paraffin tissue blocks, deparaffinization by xylene, and subjecting them to the conventional phenol-chloroform DNA isolation protocol.

Clinical and Radiological Outcomes for Guided Implant Placement in Sites Preserved with Bioactive Glass Bone Graft after Tooth Extraction: A Controlled Clinical Trial.

The goal of the study was to evaluate marginal bone loss (MBL) after 1-year implant placement using a guided implant surgical (GIS) protocol in grafted sockets compared to non-grafted sites. We followed a parallel study design with patients divided into two groups: grafted group (Test group, n = 10) and non-grafted group (Control, n = 10). A bioactive glass bone graft was used for grafting. A single edentulous site with a minimum bone height ≥11 mm and bone width ≥6 mm confirmed by cone-beam computerized tomography (CBCT) was chosen for implant placement. Tapered hybrid implants that were sandblasted and acid-etched (HSA) were placed using the GIS protocol and immediately loaded with a provisional prosthesis. MBL and implant survival rates (ISR) were assessed based on standardized radiographs and clinical exams. Patients were followed up for 1-year post-loading. MBL after one year, in the control group, was −0.31 ± 0.11 mm (mesial) and −0.28 ± 0.09 mm (distal); and in the test group was −0.35 ± 0.11 mm (mesial) and −0.33 ± 0.13 mm (distal), with no statistical significance (p > 0.05). ISR was 100% in both groups after one year. ISR was similar between groups and the marginal bone changes were comparable one year after functional loading, without statistical significance, suggesting that bioactive glass permitted adequate bone formation. The GIS protocol avoided raising flaps and provided a better position to place implants, preserving the marginal bone around implants.

Impact of Red Complex Bacteria and TNF-α Levels on the Diabetic and Renal Status of Chronic Kidney Disease Patients in the Presence and Absence of Periodontitis.

Scientific evidence shows a positive association in the etiopathogenesis of periodontitis and chronic kidney disease (CKD). Various confounding factors, such as obesity, diabetes, and inflammation, also play a significant role in the progression of CKD, which remains unexplored. We hypothesise the role of red complex bacteria with various confounding factors associated with chronic kidney disease. The study comprised a total of 120 participants categorised into 4 groups: the control group (C), periodontitis subjects without CKD (P), periodontally healthy chronic kidney disease subjects (CKD), and subjects having both periodontitis and CKD (P + CKD), with 30 subjects in each group. Demographic variables, and periodontal, renal, and diabetic parameters were recorded. Tumour necrosis factor (TNF)-α levels and those of red complex bacteria such as Prophyromonas gingivalis (P.g), Treponema denticola (T.d), and Tonerella forsythia (T.f) were assessed, and the obtained results were statistically analysed. Among the various demographic variables, age showed a level of significance. Mean PI, GI, CAL, and PPD (the proportion of sites with PPD ≥ 5 mm and CAL ≥ 3 mm) were elevated in the P + CKD group. Diabetic parameters such as fasting blood sugar (FBS) and HbA1c levels were also greater in the P + CKD group. Renal parameters such as eGFR and serum creatinine levels were greater in CKD patients. The estimation of red complex periodontal pathogens such as Pg, Td and Tf levels were significantly greater in the P and P + CKD groups. Pearson correlation analysis revealed significant correlation of red complex bacteria with all variables. Greater levels of P.g, T.d and T.f were found in the P groups, thus indicating their important role in the initiation and progression of inflammation of periodontitis and CKD, with diabetes as one of the confounding factors. The study also confirmed a log-linear relationship between TNF-α levels and red complex bacteria, thereby demonstrating the role of inflammatory biomarkers in periodontal disease progression that could contribute to the development of systemic inflammation such as CKD.

In-vitro analysis on the potential use of dental pulp mesenchymal stem cells on arecoline-induced oral epithelial cells.

To assess the protective role of the secretome of dental pulp mesenchymal stem cells on arecoline-induced epithelial-mesenchymal transition and senescence on epithelial cells of the oral mucosa. Effect of varying concentrations of arecoline extract and dental pulp mesenchymal stem cell condition media (DPSC-CM) were noted on oral mucosal epithelial cells. MTT assay, Annexin V-FITC/PI assay, and the quantitative gene expressions of BCL2, PUMA, BAD, BAX, CASP3, CASP9, CASP12, TGFB1, CST3, COL1A2, COL3A1, TIMP1, TIMP2, CDH1, and CDH2 were assessed. Oral mucosal epithelial cells exposed only to the arecoline were the control. 50% and 100% DPSC-CM decreased apoptosis-related gene expression in the cells exposed with 25 µM arecoline compared to the control. 50% DPSC-CM attenuated the expression of all fibrotic genes and EMT-related genes. 20% and 100% DPSC-CM showed differential effects on fibrotic and EMT-related genes. DPSC-CM inhibited apoptosis, and attenuated expression of fibrotic and EMT-related genes on arecoline treated human oral epithelial cells.

The SARS-CoV-2 Virus could Downregulate Dual Specificity Phosphatase-1 Expression in Oral Cancer: A Plausible Hypothesis that could Explain Oral Cancer Progression in the COVID-19 Pandemic and Post-pandemic Situation.

This study explores the downregulation of Dual Specificity Phosphatase-1 (DUSP-1) expression in oral cancer progression during the pandemic and post-pandemic situations. Keywords: COVID-19, Dual specificity phosphatase-1, Oral cancer, SARS-CoV-2.

Melatonin levels in periodontitis vs. the healthy state: A systematic review and meta-analysis.

The aim was to provide a comprehensive qualitative and quantitative assessment of any potential differences in melatonin levels in periodontitis vs. the healthy state. The keyword combination "melatonin" AND "periodontitis" was searched in Web of Science, PubMed, and Scopus. Qualitative analysis and quantitative analysis were performed on articles satisfying the inclusion criteria. Only 14 studies were included in the systematic review, out of which only 10 had quantitative data compatible with a meta-analysis. Ten studies demonstrated low melatonin in periodontitis, three studies demonstrated an initial reduction in melatonin levels followed by elevation with worsening of periodontitis, and one study showed an elevation in melatonin levels in the transition from a healthy state to periodontitis. Grading of recommendations assessment, development, and evaluation revealed that all the included studies had low to very low overall evidence. The meta-analysis revealed a significant reduction (p < .0001) in salivary melatonin levels in chronic periodontitis (3.26 ± 3.44 pg/ml) compared with healthy controls (5.27 ± 5.39 pg/ml), with a mean difference of 2.65 ± 7.84 and a confidence interval of 1.94-3.36. The significantly lower salivary melatonin levels in periodontitis must be inferred with caution given the low quality of the included studies.

Anticancer Effects of Carica papaya L. and Benzyl Isothiocyanate on an Oral Squamous Cell Carcinoma Cell Line: An In Vitro Study.

AIM: The study aimed to assess the anticancer effects of leaves of the male and female plant and seeds Carica papaya L. extract and the active compound benzyl isothiocyanate on oral squamous cell carcinoma (OSCC) cell line. MATERIALS AND METHODS: Extracts of CO2 strain C. papaya L. seeds were prepared using water, ethanol, and ethanol:water by maceration, and benzyl isothiocyanate was quantified. Alkaloid fractions of leaves of male and female plants of C. papaya L. were prepared and quantified. The anticancer effects of the test substances on the SCC-25 cell line were assessed by MTT, apoptosis assay, cell cycle analysis, and determination of mitochondrial membrane potential. RESULTS: The ethanol:water extract of C. papaya L. (seeds) demonstrated the highest quantity of benzyl isothiocyanate. Male plant leaves demonstrated greater alkaloid content. The leaves of the male plant exhibited apoptosis induction and S-phase arrest, whereas the leaves of the female plant and seeds of C. papaya L. demonstrated G2M-phase arrest and apoptosis induction. CONCLUSION: C. papaya L. and benzyl isothiocyanate demonstrated anticancer effects. There was a difference in the anticancer effects of leaves of male and female plants of C. papaya L. CLINICAL SIGNIFICANCE: The anticancer effects of papaya leaves and seeds could be further explored to develop an adjunct therapy for oral cancer to improve prognosis and reduce recurrence rates.

Allicin May Promote Reversal of T-Cell Dysfunction in Periodontitis via the PD-1 Pathway.

We evaluated the role of allicin in periodontitis using an in silico and in vitro design. An in silico docking analysis was performed to assess the plausible interactions between allicin and PD-L1. The cytokine profile of gingival crevicular fluid (GCF) samples obtained from periodontitis patients was estimated by cytometric bead array. CD3+ lymphocytes isolated from the peripheral blood were sorted and characterized using immunomagnetic techniques. Cultured and expanded lymphocytes were treated with the GCF samples to induce T-cell exhaustion. Optimum concentrations of allicin were added to exhausted lymphocytes to compare the expression of TIM-3 and LAG-3 gene expression at baseline and post-treatment. Allicin was found to bind to the PD-L1 molecule as revealed by the in-silico experiment, which is possibly an inhibitory interaction although not proven. GCF from periodontitis patients had significantly higher concentrations of TNF-α, CCL2, IL-6, IFN-γ, and CXCL8 than controls. GCF treatment of CD3+ lymphocytes from the periodontitis patients significantly increased expression of T-cell exhaustion markers TIM-3 and LAG-3. Allicin administration with GCF treatment resulted in significant lowering of the expression of exhaustion markers. Allicin may exert an immunostimulatory role and reverse immune-destructive mechanisms such as T-cell exhaustion.

Tinospora cordifolia (Thunb.) Miers (Giloy) inhibits oral cancer cells in a dose-dependent manner by inducing apoptosis and attenuating epithelial-mesenchymal transition.

BACKGROUND: Tinospora cordifolia (Thunb.) Miers (Giloy) has been applied successfully as an anti-inflammatory, anti-diabetic, and even as an anti-cancer agent. Yet, to date, the application of Giloy has not been explored concerning oral cancer. OBJECTIVES: To assess the effect of T cordifolia (Thunb.) Miers (Giloy) extract (TcE) on an oral cancer cell line. METHODS: AW13516 (oral cancer cell line) cells were treated with the prepared aqueous extract of TcE for 24 h at various concentrations ranging between 5 µg/ml and 100 µg/ml and compared with control (cells without treatment). Thee effect of the extracts on apoptosis was assessed by through Annexin V flow cytometry assay and Luminometry based assessment of Caspase 8, 9 and caspase 3/7 activity. RNA was isolated from treated cells and gene expression of selected metastatic genes (MMP1, MMP10, and CXCL8); epithelial-mesenchymal stem cell genes (TWIST1, SNAIL, ZEB1, Oct4) and stemness related genses (Nanog, Sox2) were analyzed by using a quantitative real-time PCR system. The experiments were performed in triplicates. RESULTS: Aqueous extract of TcE was found to induce apoptosis inducer in AW13516 cells in a concentration-dependent manner and was potent even at a low concentration of 5 µg/ml. The apoptosis induction was confirmed with the caspase activity assay. Treatment of the cells with the extract for 24 h exhibited a significant decrease in the expression of EMT genes in a dose-dependent manner without an effect on the metastatic genes. CONCLUSION: Aqueous extract of TcE induces apoptosis-mediated cell death in the oral cancer cell line AW13516 while attenuating its potential for epithelial mesenchymal transition.

Taurodontism in maxillary and mandibular molars using cone beam computed tomography in a dental center in Saudi Arabia.

BACKGROUND: Taurodontism is a dental anomaly characterized by altered crown root ratio that is often diagnosed by radiographic evaluation. A three-dimensional cone beam computed tomography (CBCT) can aid in the diagnosis and treatment of taurodontic teeth. Only one study has reported the prevalence of taurodontism in a Saudi population. OBJECTIVE: Determine prevalence and other characteristics of taurodontism in permanent maxillary and mandibular molars, by CBCT in a dental center in Saudi Arabia. DESIGN: Cross-sectional. SETTING: College of dentistry. SUBJECTS AND METHODS: The first and second molars, maxillary and mandibular, from study participants of Saudi origin from Jazan region of Saudi Arabia were evaluated for taurodontism based on the criteria of Shifman and Chanannel. The degree of taurodontism was determined by the taurodontism index. Results were tabulated for analysis and the chi-square test was applied for the differences between age groups, genders, and maxillary and mandibular teeth. MAIN OUTCOME MEASURES: Characteristics of taurodontism. SAMPLE SIZE: 1839 teeth in 300 individuals. RESULTS: Taurodontism was seen in 24 (8%) of the study participants and in 71 teeth (3.9%). Taurodontism was significantly more prevalent in individuals between 21 and 40 years of age. Hypotaurodontism (67.6%, n=48) was most prevalent, followed by mesotaurodontism (23.9%, n=17) and hypertaurodontism (8.5%, n=6). The maxillary molars were more commonly involved than mandibular, but the results were not statistically significant. No significant differences in gender were observed. CONCLUSIONS: Dental clinicians should be familiar with the condition due to the clinical implications in oral surgery, endodontics and prosthodontics. The possibilities of a complex root canal system, an additional canal, difficulties in canal negotiation, instrumentation and subsequent obturation can be challenging for the clinician. LIMITATIONS: Conducted in a single dental center hence future studies with larger sample sizes in different regions of Saudi Arabia should be conducted to more accurately measure the prevalence. CONFLICTS OF INTEREST: None.

miRNA-146a and miRNA-126 as Potential Biomarkers in Patients with Coronary Artery Disease and Generalized Periodontitis.

The present study aims to compare the levels of micro-RNA-146a and micro-RNA-126 in oral subgingival plaque and coronary plaque from artery walls in patients with coronary artery disease who suffer from generalized periodontitis. A total of 75 participants were selected and grouped into three categories of 25 patients each: GP+CAD, GP, and HP groups. GP+CAD consisted of patients diagnosed with generalized periodontitis (GP) and coronary artery disease (CAD). The GP+CAD group was further divided into two groups-GP+CADa: where subgingival plaque samples were collected; GP+CADb group: where coronary plaque samples were collected while the patient underwent a coronary artery bypass grafting surgery. The GP group consisted of 25 patients diagnosed with only generalized periodontitis. The HP group consisted of 25 systemically and periodontally healthy controls. miRNA-146a and miRNA126 levels were assessed in subgingival plaque (SP) samples from all groups. Results revealed that miRNA-146a was expressed at higher levels and miRNA-126 was downregulated in the GP+CAD group. microRNAs in subgingival plaque samples showed a significant correlation with the coronary plaque samples in the GP+CAD group. miRNA-146a and miRNA-126 were present in coronary artery disease patients with periodontitis. These micro-RNAs may serve as risk biomarkers for coronary artery disease and generalized periodontitis.

Reply to the letter regarding "Chronic mechanical irritation and oral squamous cell carcinoma: A systematic review and meta-analysis".

Dear Editor, We thank Dr. Jian Xie for the valuable inputs on our paper titled 'Chronic mechanical irritation and oral squamous cell carcinoma: A systematic review and meta-analysis [1].' The first concern of Dr. Xie was that we had included two studies that were based on the same population. We re-examined these papers, one was published in 2010 [2] and the other in 2017 [3] by the same group of authors. Given the significant time difference between the two papers, we did not want to presume they were from the same sample population. There is no clear evidence that they are from the same sample population. Read more in PDF.

Chronic mechanical irritation and oral squamous cell carcinoma: A systematic review and meta-analysis.

The objective of the present article was to qualitatively and quantitatively review the association between chronic mechanical irritation and oral squamous cell carcinoma (OSCC). PubMed, SCOPUS, and Web of Science databases were searched using the keyword combinations "chronic trauma and oral squamous cell carcinoma; chronic irritation and oral squamous cell carcinoma; chronic irritation and oral cancer; and chronic trauma and oral cancer." Duplicates and irrelevant articles were excluded after the title and abstract screening. The full texts of the remaining articles were assessed using selection criteria. A total of 375 (PubMed-126; SCOPUS-152; WOS-97) articles were screened, and 343 duplicates and irrelevant articles were excluded from the study. Only 9 of the remaining 32 articles met the selection criteria and were included in the qualitative analysis. Buccal mucosa and tongue, being highly prone to chronic irritation through the dental prosthesis, were the common sites for OSCC. Edentulous subjects with ill-fitting dentures were at a high risk of developing chronic irritation associated-OSCC. According to the Joanna Briggs Institute of risk assessment, eight of the nine included studies had a low risk of bias. The quantitative analysis showed a significant association (p < 0.00001) between the chronic oral mucosal irritation and OSCC with an overall risk ratio of 2.56 at a confidence interval of 1.96-3.35. Chronic oral mucosa irritation has a significant association with OSCC, and the nature of association could be that of a potential co-factor (dependent risk factor) rather than an independent risk factor.

Necrotizing periodontal diseases in human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy: A review.

Human immunodeficiency virus-infected patients have depleted CD4 lymphocyte counts and are susceptible to a plethora of infections of bacterial, viral, and fungal etiology. In addition to a wide range of systemic manifestations, human immunodeficiency virus-infected patients also display several characteristic oral manifestations. Studies have shown a correlation between some of the oral manifestations and CD4 lymphocyte counts which in turn is an independent prognostic indicator. To tackle the human immunodeficiency virus numerous drugs have been discovered and implemented. To overcome any potential resistance, human immunodeficiency virus patients are prescribed highly active antiretroviral therapy, wherein a combination of antiretroviral regimens are used. Studies have shown that in addition to controlling the viral activity, the treatment regimen, has a significant effect on the oral manifestations of the human immunodeficiency virus-infected patients. The present paper highlights the effects of highly active antiretroviral therapy on periodontal diseases in human immunodeficiency virus-infected individuals.

Reviewing the oral pigmented lesions of human immunodeficiency virus with emphasis on the effect of highly active antiretroviral therapy.

Acquired immunodeficiency syndrome a disease with high mortality rates is caused by the well-known human immunodeficiency virus. The disease is characterized by several opportunistic infections owing to the decreased CD4 lymphocyte counts. Oral manifestations of human immunodeficiency virus are vital as they are one of the early manifestations of the disease. Also, they serve as prognostic markers as they correlate with the CD4 lymphocyte counts of the affected individuals. Human immunodeficiency virus is not only common in the adult population but also can affect pediatric patients through vertical transmission. The initial therapeutic strategy for the management of the virus was only the prevention of opportunistic infections. Later in the mid-1990s, antiretroviral therapy was introduced but there was no significant improvement in prognosis. After the advent of combination therapy or the use of three antiretroviral drugs also known as highly active antiretroviral therapy, there has been a marked reduction in human immunodeficiency virus-associated mortality rates. The highly active antiretroviral therapy has several effects on the oral manifestations of the human immunodeficiency virus. The present paper aims to review the oral pigmented lesions associated with human immunodeficiency virus with an emphasis on the effect of highly active antiretroviral therapy.

EMMPRIN/BASIGIN as a biological modulator of oral cancer and COVID-19 interaction: Novel propositions.

Extracellular matrix metalloproteinase inducer (EMMPRIN), which is also called BASIGIN/CD147, is a cell surface glycoprotein that belongs to the immunoglobulin superfamily and plays a significant role in intercellular recognition in immunology, cellular differentiation and development. Apart from ACE-2, recently EMMPRIN, has been regarded as a target for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attachment and entry into the host cell. Since one of the routes of entry for the virus is the oral cavity, it becomes imperative to percept oral comorbidities such oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs) in terms of EMMPRIN as a target for SARS-CoV-2. In the present paper, it is proposed that OSCC, by the virtue of upregulation of EMMPRIN expression, increases the susceptibility to coronavirus disease (COVID-19). In turn, COVID-19 in OSCC patients causes exhaustion of EMMPRIN receptor due to binding with 'S' receptor leading to a downregulation of related carcinogenesis events. We proposed that in the ACE-2 depleted situation in OSCC, EMMPRIN receptor might get high jacked by the COVID-19 virus for the entry into the host cells. Apart from the anti-monoclonal antibody, it is recommended to explore the use of grape seed and skin containing mouthwash as an adjunct, which could also have anti EMMPRIN effects in patients with OSCC and OPMDs.