Human oral mucosa cell atlas reveals a stromal-neutrophil axis regulating tissue immunity.

The oral mucosa remains an understudied barrier tissue. This is a site of rich exposure to antigens and commensals, and a tissue susceptible to one of the most prevalent human inflammatory diseases, periodontitis. To aid in understanding tissue-specific pathophysiology, we compile a single-cell transcriptome atlas of human oral mucosa in healthy individuals and patients with periodontitis. We uncover the complex cellular landscape of oral mucosal tissues and identify epithelial and stromal cell populations with inflammatory signatures that promote antimicrobial defenses and neutrophil recruitment. Our findings link exaggerated stromal cell responsiveness with enhanced neutrophil and leukocyte infiltration in periodontitis. Our work provides a resource characterizing the role of tissue stroma in regulating mucosal tissue homeostasis and disease pathogenesis.

Co-optation of Tandem DNA Repeats for the Maintenance of Mesenchymal Identity.

Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.

ILC1: guardians of the oral mucosa against enemy viruses.

Type 1 innate lymphoid cells (ILC1s) regulate inflammation in the tissues; however, their role in anti-viral immunity remains largely unknown. In this issue of Immunity, Shannon et al. report that ILC1s invoke an anti-viral effect by producing interferon (IFN)γ at homeostasis, thereby limiting viral replication in the oral mucosa.

The broadening scope of oral mucositis and oral ulcerative mucosal toxicities of anticancer therapies.

Oral mucositis (OM) is a common, highly symptomatic complication of cancer therapy that affects patients' function, quality of life, and ability to tolerate treatment. In certain patients with cancer, OM is associated with increased mortality. Research on the management of OM is ongoing. Oral mucosal toxicities are also reported in targeted and immune checkpoint inhibitor therapies. The objective of this article is to present current knowledge about the epidemiology, pathogenesis, assessment, risk prediction, and current and developing intervention strategies for OM and other ulcerative mucosal toxicities caused by both conventional and evolving forms of cancer therapy.

On-going Mechanical Damage from Mastication Drives Homeostatic Th17 Cell Responses at the Oral Barrier.

Immuno-surveillance networks operating at barrier sites are tuned by local tissue cues to ensure effective immunity. Site-specific commensal bacteria provide key signals ensuring host defense in the skin and gut. However, how the oral microbiome and tissue-specific signals balance immunity and regulation at the gingiva, a key oral barrier, remains minimally explored. In contrast to the skin and gut, we demonstrate that gingiva-resident T helper 17 (Th17) cells developed via a commensal colonization-independent mechanism. Accumulation of Th17 cells at the gingiva was driven in response to the physiological barrier damage that occurs during mastication. Physiological mechanical damage, via induction of interleukin 6 (IL-6) from epithelial cells, tailored effector T cell function, promoting increases in gingival Th17 cell numbers. These data highlight that diverse tissue-specific mechanisms govern education of Th17 cell responses and demonstrate that mechanical damage helps define the immune tone of this important oral barrier.

Individual-specific levels of CTG•CAG somatic instability are shared across multiple tissues in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a complex disease caused by a genetically unstable CTG repeat expansion in the 3'-untranslated region of the DMPK gene. Age-dependent, tissue-specific somatic instability has confounded genotype-phenotype associations, but growing evidence suggests that it also contributes directly toward disease progression. Using a well-characterized clinical cohort of DM1 patients from Costa Rica, we quantified somatic instability in blood, buccal cells, skin and skeletal muscle. Whilst skeletal muscle showed the largest expansions, modal allele lengths in skin were also very large and frequently exceeded 2000 CTG repeats. Similarly, the degree of somatic expansion in blood, muscle and skin were associated with each other. Notably, we found that the degree of somatic expansion in skin was highly predictive of that in skeletal muscle. More importantly, we established that individuals whose repeat expanded more rapidly than expected in one tissue (after correction for progenitor allele length and age) also expanded more rapidly than expected in other tissues. We also provide evidence suggesting that individuals in whom the repeat expanded more rapidly than expected in skeletal muscle have an earlier age at onset than expected (after correction for the progenitor allele length). Pyrosequencing analyses of the genomic DNA flanking the CTG repeat revealed that the degree of methylation in muscle was well predicted by the muscle modal allele length and age, but that neither methylation of the flanking DNA nor levels of DMPK sense and anti-sense transcripts could obviously explain individual- or tissue-specific patterns of somatic instability.

RFX transcription factor in the human-associated yeast Candida albicans regulates adhesion to oral epithelium.

Adhesion to mucosal surfaces is a critical step in many bacterial and fungal infections. Here, using a mouse model of oral infection by the human fungal pathobiont Candida albicans, we report the identification of a novel regulator of C. albicans adhesion to the oral mucosa. The regulator is a member of the regulatory factor X (RFX) family of transcription factors, which control cellular processes ranging from genome integrity in model yeasts to tissue differentiation in vertebrates. Mice infected with the C. albicans rfx1 deletion mutant displayed increased fungal burden in tongues compared to animals infected with the reference strain. High-resolution imaging revealed RFX1 transcripts being expressed by C. albicans cells during infection. Concomitant with the increase in fungal burden, the rfx1 mutant elicited an enhanced innate immune response. Transcriptome analyses uncovered HWP1, a gene encoding an adhesion protein that mediates covalent attachment to buccal cells, as a major RFX1-regulated locus. Consistent with this result, we establish that C. albicans adhesion to oral cells is modulated by RFX1 in an HWP1-dependent manner. Our findings expand the repertoire of biological processes controlled by the RFX family and illustrate a mechanism whereby C. albicans can adjust adhesion to the oral epithelium.

Growth modulatory effects of fenretinide encompass keratinocyte terminal differentiation: a favorable outcome for oral squamous cell carcinoma chemoprevention.

Oral squamous cell carcinoma (OSCC) is worldwide health problem associated with high morbidity and mortality. From both the patient and socioeconomic perspectives, prevention of progression of premalignant oral intraepithelial neoplasia (OIN) to OSCC is clearly the preferable outcome. Optimal OSCC chemopreventives possess a variety of attributes including high tolerability, bioavailability, efficacy and preservation of an intact surface epithelium. Terminal differentiation, which directs oral keratinocytes leave the proliferative pool to form protective cornified envelopes, preserves the protective epithelial barrier while concurrently eliminating growth-aberrant keratinocytes. This study employed human premalignant oral keratinocytes and an OSCC cell line to evaluate the differentiation-inducing capacity of the synthetic retinoid, fenretinide (4HPR). Full-thickness oral mucosal explants were evaluated for proof of concept differentiation studies. Results of this study characterize the ability of 4HPR to fulfill all requisite components for keratinocyte differentiation, i.e. nuclear import via binding to cellular RA binding protein-II (molecular modeling), binding to and subsequent activation of retinoic acid nuclear receptors (receptor activation assays), increased expression and translation of genes associated with keratinocyte differentiation [Reverse transcription polymerase chain reaction (RT-PCR), immunoblotting] upregulation of a transglutaminase enzyme essential for cornified envelope formation (transglutaminase 3, functional assay) and augmentation of terminal differentiation in human oral epithelial explants (image-analyses quantified corneocyte desquamation). These data build upon the chemoprevention repertoire of 4HPR that includes function as a small molecule kinase inhibitor and inhibition of essential mechanisms necessary for basement membrane invasion. An upcoming clinical trial, which will assess whether a 4HPR-releasing mucoadhesive patch induces histologic, clinical and molecular regression in OIN lesions, will provide essential clinical insights.

Langerhans cells in the skin and oral mucosa: Brothers in arms?

The skin and the oral mucosa represent interfaces to the environment that are constantly exposed to pathogens and harmless foreign antigens such as commensal bacteria. Both barrier organs share the presence of Langerhans cells (LC), distinctive members of the heterogeneous family of antigen-presenting dendritic cells (DC) that have the unique ability to promote tolerogenic as well as inflammatory immune responses. While skin LC have been extensively studied in the past decades, less is known about the function of oral mucosal LC. Despite similar transcriptomic signatures, skin and oral mucosal LC differ greatly in their ontogeny and development. In this review article, we will summarize the current knowledge on LC subsets in the skin compared to the oral mucosa. We will discuss the similarities and differences in their development, homeostasis, and function in the two barrier tissues, including their interaction with the local microbiota. In addition, this review will update recent advances on the role of LC in inflammatory skin and oral mucosal diseases.

Activating the healing process: three-dimensional culture of stem cells in Matrigel for tissue repair.

BACKGROUND: To establish a strategy for stem cell-related tissue regeneration therapy, human gingival mesenchymal stem cells (hGMSCs) were loaded with three-dimensional (3D) bioengineered Matrigel matrix scaffolds in high-cell density microtissues to promote local tissue restoration. METHODS: The biological performance and stemness of hGMSCs under 3D culture conditions were investigated by viability and multidirectional differentiation analyses. A Sprague‒Dawley (SD) rat full-thickness buccal mucosa wound model was established, and hGMSCs/Matrigel were injected into the submucosa of the wound. Autologous stem cell proliferation and wound repair in local tissue were assessed by histomorphometry and immunohistochemical staining. RESULTS: Three-dimensional suspension culture can provide a more natural environment for extensions and contacts between hGMSCs, and the viability and adipogenic differentiation capacity of hGMSCs were significantly enhanced. An animal study showed that hGMSCs/Matrigel significantly accelerated soft tissue repair by promoting autologous stem cell proliferation and enhancing the generation of collagen fibers in local tissue. CONCLUSION: Three-dimensional cell culture with hydrogel scaffolds, such as Matrigel, can effectively improve the biological function and maintain the stemness of stem cells. The therapeutic efficacy of hGMSCs/Matrigel was confirmed, as these cells could effectively stimulate soft tissue repair to promote the healing process by activating the host microenvironment and autologous stem cells.

Candida albicans commensalism in the oral mucosa is favoured by limited virulence and metabolic adaptation.

As part of the human microbiota, the fungus Candida albicans colonizes the oral cavity and other mucosal surfaces of the human body. Commensalism is tightly controlled by complex interactions of the fungus and the host to preclude fungal elimination but also fungal overgrowth and invasion, which can result in disease. As such, defects in antifungal T cell immunity render individuals susceptible to oral thrush due to interrupted immunosurveillance of the oral mucosa. The factors that promote commensalism and ensure persistence of C. albicans in a fully immunocompetent host remain less clear. Using an experimental model of C. albicans oral colonization in mice we explored fungal determinants of commensalism in the oral cavity. Transcript profiling of the oral isolate 101 in the murine tongue tissue revealed a characteristic metabolic profile tailored to the nutrient poor conditions in the stratum corneum of the epithelium where the fungus resides. Metabolic adaptation of isolate 101 was also reflected in enhanced nutrient acquisition when grown on oral mucosa substrates. Persistent colonization of the oral mucosa by C. albicans also correlated inversely with the capacity of the fungus to induce epithelial cell damage and to elicit an inflammatory response. Here we show that these immune evasive properties of isolate 101 are explained by a strong attenuation of a number of virulence genes, including those linked to filamentation. De-repression of the hyphal program by deletion or conditional repression of NRG1 abolished the commensal behaviour of isolate 101, thereby establishing a central role of this factor in the commensal lifestyle of C. albicans in the oral niche of the host.

Fibrotic Matrix Induces Mesenchymal Transformation of Epithelial Cells in Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is a potentially malignant disorder of the oral mucosa; however, whether and how the fibrotic matrix of OSF is involved in the malignant transformation of epithelial cells remains unknown. Herein, oral mucosa tissue from patients with OSF, OSF rat models, and their controls were used to observe the extracellular matrix changes and epithelial-mesenchymal transformation (EMT) in fibrotic lesions. Compared with controls, oral mucous tissues from patients with OSF showed an increased number of myofibroblasts, a decreased number of blood vessels, and increased type I and type III collagen levels. In addition, the oral mucous tissues from humans and OSF rats showed increased stiffness, accompanied by increased EMT activities of epithelial cells. The EMT activities of stiff construct-cultured epithelial cells were increased significantly by exogenous piezo-type mechanosensitive ion channel component 1 (Piezo1) activation, and decreased by yes-associated protein (YAP) inhibition. During ex vivo implantation, oral mucosal epithelial cells of the stiff group showed increased EMT activities and increased levels of Piezo1 and YAP compared with those in the sham and soft groups. These results indicate that increased stiffness of the fibrotic matrix in OSF led to increased proliferation and EMT of mucosal epithelial cells, in which the Piezo1-YAP signal transduction is important.

Affirming Long-Term Outcomes After Contemporary Urethroplasty: The Adverse Impact of Increasing Stricture Length, Lichen Sclerosus, Radiation, and Infectious Strictures.

PURPOSE: There is a paucity of reported long-term outcomes after contemporary urethroplasty. Our objective is to determine the long-term success of modern urethroplasty and identify factors associated with stricture recurrence in this context. MATERIALS AND METHODS: Patients undergoing urethroplasty from July 2003 to May 2013 with at least 100 months of follow-up were identified. Long-term outcomes including stricture recurrence and patient satisfaction were evaluated by review of regional/provincial electronic records and telephone interview. Urethroplasty failure was defined as a recurrent stricture (<16F) confirmed on cystoscopy. Cox regression was used to evaluate variables associated with long-term stricture recurrence. RESULTS: A total of 733 patients were identified with ≥ 100 months follow-up. Median patient age was 45 years, stricture length was 4.7 cm, and 85.8% failed prior endoscopic treatment. At a median follow-up of 12.3 years, 89 recurrences were observed. Cumulative incidence of stricture recurrence was 6%, 10%, and 12% after 1, 5, and 10 years, respectively. From a patient-reported perspective, 89% of patients reported being satisfied with the outcome of surgery. On multivariable analyses, increasing stricture length (HR 1.1, 95% CI 1.05-1.15; P < .001) and stricture etiology (P < .001), in particular lichen sclerosus (HR 4.46, 95% CI 2.25-9.53), radiation (HR 4.25, 95% CI 1.65-10.9), and infectious strictures (HR 5.27, 95% CI 2.03-13.7), were independently associated with stricture recurrence. CONCLUSIONS: This study affirms the widely held belief that modern urethroplasty provides high long-term patency and patient-reported satisfaction. Patients with longer strictures as well as those with lichen sclerosus, radiation, and infectious etiologies have a higher hazard of stricture recurrence in the long term.

Multi-Institutional Outcomes of Dorsal Onlay Buccal Mucosal Graft Urethroplasty in Patients With Postprostatectomy, Postradiation Anastomotic Stenosis.

PURPOSE: The treatment of urethral stenosis after a combination of prostatectomy and radiation therapy for prostate cancer is understudied. We evaluate the clinical and patient-related outcomes after dorsal onlay buccal mucosal graft urethroplasty (D-BMGU) in men who underwent prostatectomy and radiation therapy. MATERIALS AND METHODS: A multi-institutional, retrospective review of men with vesicourethral anastomotic stenosis or bulbomembranous urethral stricture disease after radical prostatectomy and radiation therapy from 8 institutions between 2013 to 2021 was performed. The primary outcomes were stenosis recurrence and development of de novo stress urinary incontinence. Secondary outcomes were surgical complications, changes in voiding, and patient-reported satisfaction. RESULTS: Forty-five men were treated with D-BMGU for stenosis following prostatectomy and radiation. There was a total of 7 recurrences. Median follow-up in patients without recurrence was 21 months (IQR 12-24). There were no incidents of de novo incontinence, 28 patients were incontinent pre- and postoperatively, and of the 6 patients managed with suprapubic catheter preoperatively, 4 were continent after repair. Following repair, men had significant improvement in postvoid residual, uroflow, International Prostate Symptom Score, and International Prostate Symptom Score quality-of-life domain. Overall satisfaction was +2 or better in 86.6% of men on the Global Response Assessment. CONCLUSIONS: D-BMGU is a safe, feasible, and effective technique in patients with urethral stenosis after a combination of prostatectomy and radiation therapy. Although our findings suggest this technique may result in lower rates of de novo urinary incontinence compared to conventional urethral transection and excision techniques, head-to-head comparisons are needed.

Potential role of salivary lactic acid bacteria in pathogenesis of oral lichen planus.

BACKGROUND: Emerging evidence emphasized the role of oral microbiome in oral lichen planus (OLP). To date, no dominant pathogenic bacteria have been identified consistently. It is noteworthy that a decreased abundance of Streptococcus, a member of lactic acid bacteria (LAB) in OLP patients has been commonly reported, indicating its possible effect on OLP. This study aims to investigate the composition of LAB genera in OLP patients by high-throughput sequencing, and to explore the possible relationship between them. METHODS: We collected saliva samples from patients with OLP (n = 21) and healthy controls (n = 22) and performed 16 S rRNA gene high-throughput sequencing. In addition, the abundance of LAB genera was comprehensively analyzed and compared between OLP and HC group. To verify the expression of Lactococcus lactis, real time PCR was conducted in buccal mucosa swab from another 14 patients with OLP and 10 HC. Furthermore, the correlation was conducted between clinical severity of OLP and LAB. RESULTS: OLP and HC groups showed similar community richness and diversity. The members of LAB, Lactococcus and Lactococcus lactis significantly decreased in saliva of OLP cases and negatively associated with OLP severity. In addition, Lactococcus and Lactococcus lactis showed negative relationship with Fusobacterium and Aggregatibacter, which were considered as potential pathogens of OLP. Similarly, compared with healthy controls, the amount of Lactococcus lactis in mucosa lesion of OLP patients was significantly decreased. CONCLUSIONS: A lower amount of Lactococcus at genus level, Lactococcus lactis at species level was observed in OLP cases and associated with disease severity. Further studies to verify the relationship between LAB and OLP, as well as to explore the precise mechanism is needed.

Primary Tumour Detection in Carcinoma of Unknown Primary with Transoral Robotic Surgery (TORS) Tongue Base Mucosectomy: A Meta-analysis.

BACKGROUND: Head and neck carcinoma of unknown primary (CUP) represents a challenging diagnostic process when standard work-up fails to identify the primary tumour site. The aim of this systematic review and meta-analysis was to evaluate the diagnostic utility and complication profile of transoral robotic surgery (TORS) tongue base mucosectomy (TBM) in the management of CUP. PATIENTS AND METHODS: An electronic database search was performed in the EMBASE, MEDLINE, PubMed and Cochrane databases. A meta-analysis of proportions was performed to obtain an estimate of the overall proportion for the detection and complication rates. RESULTS: Nine studies representing 235 patients with CUP who had TORS TBM were included in the final analysis. The overall pooled tumour detection rate was 66.2% [95% confidence interval (CI) 56.1-75.8]. The incidence of tumour detection in human papilloma virus (HPV)-positive cases (81.5%, 95% CI 60.8-96.4) was significantly higher than HPV-negative cases (2.3%, 95% CI 0.00-45.7). Weighted overall complication rate was 11.4% (95% CI 7.2-16.2). The majority were grade I or II (80%) according to the Clavien-Dindo classification. CONCLUSIONS: This meta-analysis suggests TORS to be safe and effective in localising the primary tumour site in patients with CUP. While the current data supports the use of TORS in patients who are HPV positive, larger numbers of HPV-negative cases are required to determine the true diagnostic effect with TORS before any valid conclusions can be inferred in this particular subgroup. Further research should focus on high quality prospective trials with stringent methodological work-up to minimise heterogeneity and allow for more accurate statistical analysis.

A developmental validation of the Quick TargSeq 1.0 integrated system for automated DNA genotyping in forensic science for reference samples.

Analysis of short tandem repeats (STRs) is a global standard method for human identification. Insertion/Deletion polymorphisms (DIPs) can be used for biogeographical ancestry inference. Current DNA typing involves a trained forensic worker operating several specialized instruments in a controlled laboratory environment, which takes 6-8 h. We developed the Quick TargSeq 1.0 integrated system (hereinafter abbreviated to Quick TargSeq) for automated generation of STR and DIP profiles from buccal swab samples and blood stains. The system fully integrates the processes of DNA extraction, polymerase chain reaction (PCR) amplification, and electrophoresis separation using microfluidic biochip technology. Internal validation studies were performed using RTyper 21 or DIP 38 chip cartridges with single-source reference samples according to the Scientific Working Group for DNA Analysis Methods guidelines. These results indicated that the Quick TargSeq system can process reference samples and generate STR or DIP profiles in approximately 2 h, and the profiles were concordant with those determined using traditional STR or DIP analysis methods. Thus, reproducible and concordant DNA profiles were obtained from reference samples. Throughout the study, no lane-to-lane or run-to-run contamination was observed. The Quick TargSeq system produced full profiles from buccal swabs with at least eight swipes, dried blood spot cards with two 2-mm disks, or 10 ng of purified DNA. Potential PCR inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect the amplification reactions of the instrument. The overall success rate and concordance rate of 153 samples were 94.12% and 93.44%, respectively, which is comparable to other commercially available rapid DNA instruments. A blind test initiated by a DNA expert group showed that the system can correctly produce DNA profiles with 97.29% genotype concordance with standard bench-processing methods, and the profiles can be uploaded into the national DNA database. These results demonstrated that the Quick TargSeq system can rapidly generate reliable DNA profiles in an automated manner and has the potential for use in the field and forensic laboratories.

Oral epithelial dysplasia with lymphocytic immune response: clinicopathological characterisation of 44 cases.

AIMS: Oral epithelial dysplasia (OED) often exhibits a lymphocytic/lichenoid immune response (LIR), imparting histological resemblance to lichenoid mucositis and rendering diagnosis challenging. The clinical appearances of OED and lichenoid inflammatory processes are generally divergent, presenting as well-demarcated hyperkeratotic plaques and diffuse white and/or red mucosal change with variably prominent Wickham striae, respectively. To date, clinicopathological characterisation of OED with LIR, including clinical/gross appearance, has not been depicted. METHODS AND RESULTS: Cases of solitary OED with LIR for which a clinical photograph was available were identified in the authors' institutional files. Clinical and histological features were documented. In 44 identified cases, dysplasia was mild (19 of 44, 43.2%), moderate (19 of 44, 43.2%) and severe (six of 44, 13.6%). Clinically/grossly, all 44 cases (100.0%), presented as well-demarcated hyperkeratotic plaques lacking diffuse white-and-red mucosal change or Wickham striae. Histologically, OED with LIR exhibited numerous 'lichenoid' features beyond the lymphocytic band in the superficial lamina propria, including: leucocyte transmigration (38 of 44, 86.4%), spongiosis (37 of 44, 84.1%), Civatte/colloid bodies (36 of 44, 81.8%), basal cell degeneration (29 of 45, 65.9%), sawtooth rete ridges (11 of 44, 25.0%) and subepithelial clefting (7 of 44, 15.9%). CONCLUSIONS: Virtually any lichenoid histological feature may be seen in OED with LIR, representing a significant diagnostic pitfall. The typical clinical appearance of OED with LIR is of a well-demarcated hyperkeratotic plaque, characteristic of keratinising dysplasia and devoid of lichenoid features. This suggests that pathologist access to clinical photographs during diagnostic interpretation of biopsied white lesions, which represents opportunity to perform gross examination of the disease process, may reduce interobserver variability and improve diagnostic accuracy in this challenging differential diagnosis.

Development and validation of betel-year: a clinical tool for quantifying exposure to betel quid chewing, utilizing nuclear morphometric analysis and validated by cytomorphological analysis.

BACKGROUND: Betel quid chewing, prevalent in Southeast Asia and South Asia, involves components such as betel leaf, areca nut, slaked lime, and sometimes tobacco. This study aims to assess buccal mucosa changes in betel quid chewers, develop a clinical tool for assessing exposure, and investigate its usability in predicting dysplasia. METHODS: After obtaining ethical approval and informed consent, patients were recruited from the Out-Patient Department of Government Medical College, Omandurar, Government Estate, India. A target sample size of 200 was calculated. The data included the history of betel quid chewing, buccal mucosa cells obtained by oral cytology, and the severity of dysplasia of the slides assessed by pathologists. We utilized principal component analysis (PCA) and confirmatory factor analysis (CFA) to validate a new outcome variable reflecting nuclear morphometric parameters (NMPs). Multiplicative regression models were developed for betel years based on betel exposure and additives. Spearman correlation and Kruskal-Wallis test was used to check the association between betel years and dysplasia. RESULTS: Significant differences in NMPs were observed among different betel chewing groups. We derived multiplicative regression models for betel years. In the logarithmic transformation approach, betel year = 0.05×betel-exposure×0.09×slaked-lime use×0.11×tobacco-use. In the original variable approach, betel year = 5.05×betel-exposure^0.00048×slaked-lime-use^0.18133×tobacco-use^1.47513. Spearman correlation and Kruskal-Wallis tests confirmed associations with dysplasia. CONCLUSION: Betel year is a pioneering tool for assessing lifetime betel quid exposure, similar to pack years for smoking. It could aid in risk stratification, targeted interventions and shaping public health policies. Despite limitations, betel year holds promise for revolutionizing oral health risk assessment, and future research can expand its scope globally, considering diverse betel quid compositions.

Prenatal exposure to environmental air pollution and psychosocial stress jointly contribute to the epigenetic regulation of the serotonin transporter gene in newborns.

Antenatal exposures to maternal stress and to particulate matter with an aerodynamic diameter of less than 2.5 µm (PM2.5) have been independently associated with developmental outcomes in early infancy and beyond. Knowledge about their joint impact, biological mechanisms of their effects and timing-effects, is still limited. Both PM2.5 and maternal stress exposure during pregnancy might result in altered patterns of DNA methylation in specific stress-related genes, such as the serotonin transporter gene (SLC6A4 DNAm), that might, in turn, influence infant development across several domains, including bio-behavioral, cognitive and socio-emotional domains. Here, we investigated the independent and interactive influence of variations in antenatal exposures to maternal pandemic-related stress (PRS) and PM2.5 on SLC6A4 DNAm levels in newborns. Mother-infant dyads (N = 307) were enrolled at delivery during the COVID-19 pandemic. Infants' methylation status was assessed in 13 CpG sites within the SLC6A4 gene's region (chr17:28562750-28562958) in buccal cells at birth and women retrospectively report on PRS. PM2.5 exposure throughout the entire gestation and at each gestational trimester was estimated using a spatiotemporal model based on residential address. Among several potentially confounding socio-demographic and health-related factors, infant's sex was significantly associated with infants' SLC6A4 DNAm levels, thus hierarchical regression models were adjusted for infant's sex. Higher levels of SLC6A4 DNAm at 6 CpG sites were found in newborns born to mothers reporting higher levels of antenatal PRS and greater PM2.5 exposure across gestation, while adjusting for infant's sex. These effects were especially evident when exposure to elevated PM2.5 occurred during the second trimester of pregnancy. Several important brain processes (e.g., synaptogenesis and myelination) occur during mid-pregnancy, potentially making the second trimester a sensitive time window for the effects of stress-related exposures. Understanding the interplay between environmental and individual-level stressors has important implications for the improvement of mother-infant health during and after the pandemic.