Activation of human RNA lariat debranching enzyme Dbr1 by binding protein TTDN1 occurs though an intrinsically disordered C-terminal domain.

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2'-5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2'-5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease.

Hereditary cancer testing in a diverse sample across three breast imaging centers.

PURPOSE: Up to 10% of all breast cancers (BC) are attributed to inherited pathogenic variants (PV) in BC susceptibility genes; however, most carriers of PVs remain unidentified. Here, we sought to determine the yield of hereditary cancer gene PVs among diverse women attending breast imaging centers, who could benefit from enhanced surveillance and/or risk reduction interventions. METHODS: This cross-sectional retrospective cohort study included consecutive women, unselected for personal or family cancer history, who were offered genetic testing for hereditary cancer genes at the time of breast imaging at three centers (November 2020-March 2022). RESULTS: Among 1943 patients (median age: 66 years), self-reported race/ethnicity was White (34.5%), Hispanic (27.7%), African American (17.9%), Asian (4.5%), Ashkenazi Jewish (0.6%), Other (3.5%), and missing (13.0%). Thirty-nine patients (2%) were identified as carriers of a PV in an autosomal dominant clinically actionable hereditary breast and ovarian cancer (HBOC)-related or Lynch syndrome gene, most frequently, BRCA2 (6/39; 15.4%), PALB2 (8/39; 20.5%), CHEK2 (10/39; 25.6%), and PMS2 (5/39; 12.8%). Of the 34 PVs with known race/ethnicity, 47% were detected among non-White patients. Overall, 354/1,943 (18.2%) of patients met NCCN guidelines for HBOC gene testing and only 15/39 (38.5%) patients with an autosomal dominant clinically actionable PV met guidelines. CONCLUSION: This population health approach extended the reach of genetic cancer risk assessment in a diverse population and highlighted the limits of a guideline-based approach. This may help address inequity in access to risk-appropriate screening and cancer prevention.

Differential Expression of MicroRNA MiR-145 and MiR-155 Downstream Targets in Oral Cancers Exhibiting Limited Chemotherapy Resistance.

New evidence has suggested that non-coding microRNAs play a significant role in mediating and modulating chemotherapy resistance, particularly among oral cancers. One recent study found that the upregulation of miR-145 and the downregulation of miR-155 strongly correlated with a limited chemotherapy resistance to Cisplatin, 5-Fluorouracil, and Paclitaxel, although the mechanism(s) responsible for these observations remain unidentified. Using commercially available cell lines of oral squamous cell carcinoma, RNA was isolated, converted into cDNA, and subsequently screened for the expression of downstream targets of miR-145 and miR-155 using qPCR. These results demonstrated the upregulation of miR-21, miR-125, miR-133, miR-365, miR-720, and miR-1246, as well as the downregulation of miR-140, miR-152, miR-218, miR-221, and miR-224. This screening also confirmed the differential expression and regulation of mir-145 and miR-155 among the cell lines with limited chemotherapy resistance (SCC15). In addition, several downstream targets of these specific microRNAs were upregulated by all oral cancer cell lines, such as MBTD1 and FSCN1, or downregulated in all cell lines, such as CLCN3, FLI-1, MRTFB, DAB, SRGAP1, and ABHD17C. However, three miR-145 downstream targets were identified in the least chemotherapy-resistant cells, exhibiting the differential upregulation of KCNA4 and SRGAP2, as well as the downregulation of FAM135A, with this expression pattern not detected in any of the other oral cancer cell lines. These data strongly support that the differential regulation of these three downstream targets may be related to the chemosensitivity of this oral cancer cell line. The potential involvement of these targets must be further investigated to determine how and whether mechanisms of these cellular pathways may be involved in the observed lack of chemotherapy resistance. These data may be important to design targets or treatments to reduce chemotherapy resistance and improve patient treatment outcomes.

Chemotherapeutic Drug Resistance Associated with Differential miRNA Expression of miR-375 and miR-27 among Oral Cancer Cell Lines.

Recent advances have suggested that non-coding miRNAs (such as miR-21, miR-27, miR-145, miR-155, miR-365, miR-375 and miR-494) may be involved in multiple aspects of oral cancer chemotherapeutic responsiveness. This study evaluated whether these specific miRNAs are correlated with oral cancer responsiveness to chemotherapies, including Paclitaxel, Cisplatin and Fluorouracil (5FU). Commercially available and well-characterized oral squamous cell carcinoma cell lines (SCC4, SCC9, SCC15, SCC25 and CAL27) revealed differing resistance and chemosensitivity to these agents-with SCC9 and SCC25 demonstrating the most resistance to all chemotherapeutic agents. SCC9 and SCC25 were also the only cell lines that expressed miR-375, and were the only cell lines that did not express miR-27. In addition, the expression of miR-375 was associated with the upregulation of Rearranged L-myc fusion (RLF) and the downregulation of Centriolar protein B (POC1), whereas lack of miR-27 expression was associated with Nucleophosmin 1 (NPM1) expression. These data have revealed important regulatory pathways and mechanisms associated with oral cancer proliferation and resistance that must be explored in future studies of potential therapeutic interventions.

Positive predictive value of a single nucleotide polymorphism (SNP)-based NIPT for aneuploidy in twins: Experience from clinical practice.

OBJECTIVE: Twins account for approximately 1 in 30 live births in the United States. However, there are limited clinical experience studies published in noninvasive prenatal testing (NIPT) for detecting aneuploidies in twins. This study reports the performance of an SNP-based NIPT in the largest cohort with known outcomes for high-risk aneuploidy results. METHOD: This is a retrospective analysis of 18,984 results from commercial single-nucleotide polymorphism (SNP)-based NIPT tests performed in twins between October 2, 2017 and December 31, 2019. Follow-up for all 211 high-risk cases was solicited. RESULTS: Follow-up outcomes were obtained in 105 cases. Positive predictive values (PPVs) for high-risk results were 88.7% (63/71, 95% Confidence Interval [CI]: 79.0%-95.0%) for trisomy 21% and 72.7% (8/11, 95% CI: 39.0%-94.0%) for trisomy 18. The results were stratified into monozygotic (MZ) and dizygotic (DZ). The PPVs in MZ were 100% for both trisomy 21 (4/4, 95% CI: 40%-100%) and trisomy 18 (1/1, 95% CI: 2.5%-100%). No trisomy 13 cases were detected in the MZ group. The PPVs in DZ were 88.1% (59/67, 95% CI: 77.8%-94.7%), 70.0% (7/10, 95% CI: 34.8%-93.3%), and 66.7% (2/3, 95% CI: 9.4%-99.2%) for trisomy 21, trisomy 18, and trisomy 13, respectively. CONCLUSION: The performance of SNP-based NIPT in this large twin cohort was comparable to previously reported twin NIPT studies. SNP-based NIPT allows for zygosity-based PPV assessment.

Microbial Screening Reveals Oral Site-Specific Locations of the Periodontal Pathogen Selenomonas noxia.

INTRODUCTION: Selenomonas noxia (SN) is an important periodontal pathogen, associated with gingivitis and periodontitis. Many studies have found associations between SN and indicators of poor health outcomes, such as smoking, low socioeconomic status and obesity. However, less is known about the prevalence of this organism and more specifically about other oral site-specific locations that may harbor this organism. METHODS: Using an existing patient repository (n = 47) of DNA isolated from saliva and other oral sites (n = 235), including the dorsum of the tongue, lower lingual incisor, upper buccal molar and gingival crevicular fluid (GCF), molecular screening for SN was performed. Screening results were analyzed for associations between demographic variables (age, sex, race/ethnicity) and clinical information (body mass index or BMI, presence of orthodontic brackets, primary/mixed/permanent dentition). RESULTS: qPCR screening revealed a total of n = 62/235 sites or 26.3% harboring SN with saliva and GCF (either alone or in combination with one or more sites) most often observed (Saliva, n = 23/27 or 85.18%, GCF, n = 14/27 or 51%). Analysis of site-specific data revealed most positive results were found among saliva and GCF alone or in combination, with fewer positive results observed among the tongue (33.3%), lower lingual incisor (29.6%), and upper buccal molar (25.9%). No significant associations were found between demographic or clinical variables and presence of SN at any site. CONCLUSIONS: These results may be among the first to describe site-specific locations of S. noxia among various additional oral biofilm sites. These data may represent a significant advancement in our understanding of the sites and locations that harbor this organism, which may be important for our understanding of the prevalence and distribution of these organisms among patients of different ages undergoing different types of oral treatments, such as orthodontic treatment or therapy.

miR-365 (microRNA): Potential Biomarker in Oral Squamous Cell Carcinoma Exosomes and Extracellular Vesicles.

INTRODUCTION: miR-365 is a non-coding microRNA that regulates transcription and has been demonstrated to promote oncogenesis and metastasis in some cancers, while suppressing these effects in others. Many microRNAs are produced and then exported extracellularly in exosomes, which are small extracellular vesicles ranging from 30 to 100 nm that are found in eukaryotic fluids and facilitate many cellular functions. Exosomes and extracellular vesicles are produced by many cell types, including oral cancer cells-although no study to date has evaluated miR-365 and oral cancer exosomes or extracellular vesicles. Based on this information, our research question was to evaluate whether oral cancers produce exosomes or extracellular vesicles containing miR-365. MATERIALS AND METHODS: Two commercially available oral cancer cell lines (SCC25 and CAL27) and a normal oral keratinocyte (OKF4) were grown in serum-free media, supplemented with exosome-depleted fetal bovine serum. Extracellular vesicles and exosomes were then isolated using the Invitrogen total exosome RNA and protein isolation kit for processing using the hsa-miR-365a-5p microRNA qPCR assay kit. RESULTS: RNA was successfully isolated from the exosome-depleted supernatant from each cell line-SCC9, SCC15, SCC25, and CAL27 (oral squamous cell carcinomas) and OKF4 (oral epithelial cell line). Relative concentrations of RNA were similar among each cell line, which were not significantly different, p = 0.233. RNA quality was established by A260:A280 absorbance using a NanoDrop, revealing purity ranging 1.73-1.86. Expression of miR-16 was used to confirm the presence of microRNA from the extracted exosomes and extracellular vesicles. The presence of miR-365 was then confirmed and normalized to miR-16 expression, which demonstrated an increased level of miR-365 in both CAL27 and SCC25. In addition, the normalized relative quantity (RQ) for miR-365 exhibited greater variation among SCC25 (1.382-4.363) than CAL27 cells (1.248-1.536). CONCLUSIONS: These results confirm that miR-365 is not only expressed in oral cancer cell lines, but also is subsequently exported into exosomes and extracellular vesicles derived from these cultures. These data may help to contextualize the potential for this microRNA to contribute to the phenotypes and behaviors of oral cancers that express this microRNA. Future research will begin to investigate these potential mechanisms and pathways and to determine if miR-365 may be useful as an oral cancer biomarker for salivary or liquid biopsies.

Robotic transanal minimally invasive rectal mucosa harvest.

INTRODUCTION: Buccal mucosal grafts (BMG) are traditionally used in urethral reconstruction. There may be insufficient BMG for applications requiring large grafts, such as urethral stricture after gender-affirming phalloplasty. Rectal mucosa in lieu of BMG avoids oral impairment, while potentially affording less postoperative pain and larger graft dimensions. Transanal minimally invasive surgery (TAMIS) using laparoscopic instruments has been described. Due to technical challenges of harvesting a sizable graft within the rectal lumen, we adopted a new robotic approach. We demonstrate the feasibility and safety of a novel technique of Robotic TAMIS (R-TAMIS) in the harvest of rectal mucosa for the purpose of onlay graft urethroplasty. METHODS: Six patients (ages 28-60) presenting with urethral stricture and one vaginal stricture underwent robotic rectal mucosal harvest. The procedure, which was first studied on an inanimate bovine colon model, was performed under general anesthesia in lithotomy position using the GelPOINTTM Path Transanal Access. Mucosa was harvested robotically after submucosal hydrodissection. Graft size harvested correlated with surface area needed for urethral or vaginal reconstruction. Following specimen retrieval, flexible sigmoidoscopy confirmed hemostasis. The graft was placed as an onlay for urethroplasty. RESULTS: There were no intraoperative or postoperative complications. Mean graft size was 11.4 × 3.0 cm. All reconstructions had excellent graft take. All patients recovered without morbidity or mortality. They reported minimal postoperative pain and all regained bowel function on postoperative day one. Patients with prior BMG harvests subjectively self-reported less postoperative pain and greater quality of life. There have been no long-term complications at a median follow-up of 17 months. CONCLUSIONS: To our knowledge, this is the first use of R-TAMIS for rectal mucosa harvest. Our preliminary series indicates this approach is feasible and safe, constituting a promising minimally invasive technique for urethral reconstruction. Prospective studies evaluating graft outcomes and donor site morbidity with more long-term follow-up are needed.

The periodontal war: microbes and immunity.

Maintenance of periodontal health or transition to a periodontal lesion reflects the continuous and ongoing battle between the vast microbial ecology in the oral cavity and the array of resident and emigrating inflammatory/immune cells in the periodontium. This war clearly signifies many 'battlefronts' representing the interface of the mucosal-surface cells with the dynamic biofilms composed of commensal and potential pathogenic species, as well as more recent knowledge demonstrating active invasion of cells and tissues of the periodontium leading to skirmishes in connective tissue, the locality of bone and even in the local vasculature. Research in the discipline has uncovered a concerted effort of the microbiome, using an array of survival strategies, to interact with other bacteria and host cells. These strategies aid in colonization by 'ambushing, infiltrating and outflanking' host cells and molecules, responding to local environmental changes (including booby traps for host biomolecules), communicating within and between genera and species that provide MASINT (Measurement and Signature Intelligence) to enhance sustained survival, sabotage the host inflammatory and immune responses and by potentially adopting a 'Fabian strategy' with a war of attrition and resulting disease manifestations. Additionally, much has been learned regarding the ever-increasing complexity of the host-response armamentarium at both cellular and molecular levels that is addressed in this review. Knowledge regarding how these systems fully interact requires both new laboratory and clinical tools, as well as sophisticated modeling of the networks that help maintain homeostasis and are dysregulated in disease. Finally, the triggers resulting in a 'coup de main' by the microbiome (exacerbation of disease) and the characteristics of susceptible hosts that can result in 'pyrrhic victories' with collateral damage to host tissues, the hallmark of periodontitis, remains unclear. While much has been learned, substantial gaps in our understanding of the 'parameters of this war' remain elusive toward fulfilling the Sun Tzu adage: 'If you know the enemy and know yourself, you need not fear the result of a hundred battles.'

Gene regulation by dietary microRNAs.

MicroRNAs (miRNAs) silence genes through destabilizing mRNA or preventing translation of mRNA, thereby playing an essential role in gene silencing. Traditionally, miRNAs have been considered endogenous regulators of genes, i.e., miRNAs synthesized by an organism regulate the genes in that organism. Recently, that dogma has been challenged in studies suggesting that food-borne miRNAs are bioavailable and affect gene expression in mice and humans. While the evidence in support of this theory may be considered weak for miRNAs that originate in plants, there is compelling evidence to suggest that humans use bovine miRNAs in cow's milk and avian miRNAs in chicken eggs for gene regulation. Importantly, evidence also suggests that mice fed a miRNA-depleted diet cannot compensate for dietary depletion by increased endogenous synthesis. Bioinformatics predictions implicate bovine miRNAs in the regulation of genes that play roles in human health and development. Current challenges in this area of research include that some miRNAs are unable to establish a cause-and-effect between miRNA depletion and disease in miRNA knockout mice, and sequence similarities and identities for bovine and human miRNAs render it difficult to distinguish between exogenous and endogenous miRNAs. Based on what is currently known about dietary miRNAs, the body of evidence appears to be sufficient to consider milk miRNA bioactive compounds in foods, and to increase research activities in this field.

Development of a polymerase chain reaction assay for the rapid detection of the oral pathogenic bacterium, Selenomonas noxia.

BACKGROUND: In recent studies, periodontal health has been linked to being overweight and/or obese. Among common oral bacteria, Selenomonas noxia has been implicated in converting periodontal health to disease, and Selenomonas species have also been found in gastric ulcers. The objective of this study was to develop and validate a quantitative polymerase chain reaction (qPCR) assay for the specific and rapid detection of S. noxia. METHODS: Two oligonucleotide primer pairs and one probe were designed and tested to determine optimal amplification signal with three strains of S. noxia. The PCR assay was tested against fourteen non-target organisms, including closely related oral Selenomonads, one phylogenetically closely related bacterium, and two commonly isolated oral bacteria. RESULTS: One of the primer sets was more sensitive at detecting the target organism and was selected for optimization and validation experiments. The designed primers and probe amplified the target organism with 100% specificity. PCR inhibition was observed with an internal positive control, and inhibition was resolved by diluting the DNA extract. CONCLUSIONS: The qPCR assay designed in this study can be used to specifically detect S. noxia in the clinical setting and in future research involving the enhanced detection of S. noxia. The assay can also be used in epidemiological studies for understanding the role of S. noxia in disease processes including, but not limited to, oral health and obesity of infectious origin.

Screening for Selenomonas noxia in a Pediatric and Adolescent Patient Population Reveals Differential Oral Prevalence across Age Groups.

Selenomonas noxia, a gram-negative anaerobe usually present in periodontitis, may be linked to overweight and obese adults. Recent advancements include a valid qPCR screening, enabling an effective prevalence study among pediatric patients aged 7 to 17 years. The aim of this study was to complete a retrospective screening of saliva samples from an existing biorepository using a validated qPCR screening protocol. The pediatric study sample (n = 87) comprised nearly equal numbers of males and females, mostly minority patients (67%), with an average age of 13.2 years. Screening for Selenomonas noxia revealed 34.4% (n = 30/87) positive samples, evenly distributed between males and females (p = 0.5478). However, an age-dependent association was observed with higher percentages of positive samples observed with higher ages (13.3% among 7 to 10 years; 34.6% among 11 to 13 years; 54.8% among 14-17 years), which was statistically significant (p = 0.0001). Although these findings revealed no noteworthy distinctions between males or females and minorities and non-minorities, the notable contrast between younger (7 to 10 years) and older (11 to 17 years) participants, possibly influenced by factors such as hormones and behavioral traits, will require further investigation of this patient population.

Higher Prevalence of the Periodontal Pathogen Selenomonas noxia among Pediatric and Adult Patients May Be Associated with Overweight and Obesity.

New evidence has suggested that oral and gut microflora may have significant impacts on the predisposition, development, and stability of obesity in adults over time-although less is known about this phenomenon in children. Compared with healthy-weight controls, overweight and obese adult patients are now known to harbor specific pathogens, such as Selenomonas noxia (S. noxia), that are capable of digesting normally non-digestible cellulose and fibers that significantly increase caloric extraction from normal dietary intake. To evaluate this phenomenon, clinical saliva samples (N = 122) from subjects with a normal BMI (18-25) and a BMI over 25 (overweight, obese) from an existing biorepository were screened using qPCR. The prevalence of S. noxia in samples from normal-BMI participants were lower (21.4%) than in overweight-BMI (25-29; 46.1%) and obese-BMI (30 and above; 36.8%) samples-a strong, positive correlation that was not significantly affected by age or race and ethnicity. These data strongly suggest that S. noxia may be intricately associated with overweight and obesity among patients, and more research will be needed to determine the positive and negative feedback mechanisms that may be responsible for these observations as well as the interventions needed to remove or reduce the potential effects of this oral pathogen.

Downstream Target Analysis for miR-365 among Oral Squamous Cell Carcinomas Reveals Differential Associations with Chemoresistance.

Expression of microRNAs, such as miR-365, is known to be dysregulated in many tumors, including oral cancers, although little is known about their role or functions. The objective of this project is to evaluate the downstream targets of miR-365 to determine any potential pathways or effects. Downstream targets for miR-365 (miRdatabase target scores > 90) were used for qPCR screening of oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, CAL27). Each oral cancer cell line expressed miR-365 downstream targets molybdenum cofactor synthesis-2 (MOCS2), erythropoietin receptor (EPOR), IQ motif containing-K (IQCK), carboxypeptidase A3 (CPA3), solute carrier family 24 member-3 (SLC24A3), and coiled-coil domain containing 47 (CCDC47)-although the expression levels varied somewhat. However, differential results were observed with ubiquitin protein ligase E3 component n-recognin-3 (UBR3), nudix hydrolase-12 (NUDT12), zinc finger CCHC-type containing-14 (ZCCHC14), and homeobox and leucine zipper encoding (HOMEZ). These data suggest that many of the miR-365 targets are expressed in the oral cancers screened, with the differential expression of UBR3, ZCCHC14, HOMEZ, and NUDT12, which may be correlated with chemoresistance among two specific oral cancer cell lines (SCC25, SCC9). These results suggest this differential expression may signal potential targets for patient treatment with tumors exhibiting miR-365 and chemotherapeutic resistance.

Screening for High-Risk Human Papillomavirus Reveals HPV52 and HPV58 among Pediatric and Adult Patient Saliva Samples.

Previous research has demonstrated that the human papillomavirus (HPV) can infect a wide range of human tissues, including those within the oral cavity. High-risk oral HPV strains have been associated with the development and progression of oral cancers, including oral squamous cell carcinomas. Although many studies have examined the prevalence of the high-risk strains HPV16 and HPV18, far fewer have assessed the prevalence of other high-risk HPV strains. An approved study protocol was used to identify HPV52 and HPV58 among clinical samples (n = 87) from a saliva biorepository. Quantitative polymerase chain reaction (qPCR) and validated primers for HPV52 and HPV58 were used to facilitate this screening. This screening demonstrated that a total of n = 4/45 or 8.9% of adult saliva samples harbored high-risk HPV52, and n = 2/45 or 4.4% tested positive for high-risk HPV58. In addition, a total of n = 6/42 or 14.3% of the pediatric saliva samples tested positive for high-risk HPV, including n = 5/42 or 11.9% with HPV52 and n = 3/42 or 7.1% for HPV58. These data demonstrate the presence of the high-risk oncogenic HPV52 and HPV58 strains among both adult and pediatric clinical patient samples. More detailed longitudinal research must be conducted to determine whether this prevalence may be increasing or decreasing over time. In addition, these data strongly support public health prevention efforts, such as knowledge and awareness of the nine-valent HPV vaccine covering additional high-risk strains, including HPV52 and HPV58.

Administration of Epidermal Growth Factor (EGF) and Basic Fibroblast Growth Factor (bFGF) to Induce Neural Differentiation of Dental Pulp Stem Cells (DPSC) Isolates.

The aging populations in many countries have developed many chronic illnesses and diseases, including chronic neurologic conditions such as Parkinson's and Azheimer's diseases. Many new lines of research and treatment are focusing on the potential for neurologic regeneration using mesenchymal stem cells (MSCs) in the rapidly growing field of regenerative medicine. This may include dental pulp stem cells (DPSCs), which have recently been demonstrated to produce neuronal precursors. Based upon this evidence, the primary aim of this study was to determine if the growth factors used in MSC-based studies are sufficient to induce neuronal differentiation among DPSCs. Using an existing biorepository, n = 16 DPSC isolates were thawed and cultured for this study, which revealed several subpopulations of rapid-, intermediate-, and slowly dividing DPSCs. Administration of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were sufficient to induce differential changes in growth and viability mainly among some of the rapidly growing DPSCs (n = 4). These phenotypic changes included expression of neural differentiation markers including Sox1, Pax6 and NF-M, which were observed only among those DPSC isolates not expressing early odontoblast-specific biomarkers such as ALP and DSPP. Future studies will be needed to confirm if these methods are sufficient to induce consistent and reliable induction of DPSCs towards neuronal specific differentiation.

Differential Expression of MicroRNA (MiR-27, MiR-145) among Dental Pulp Stem Cells (DPSCs) Following Neurogenic Differentiation Stimuli.

This study sought to evaluate the expression of previously identified microRNAs known to regulate neuronal differentiation in mesenchymal stem cells (MSCs), including miR-27, miR-125, miR-128, miR-135, miR-140, miR-145, miR-218 and miR-410, among dental pulp stem cells (DPSCs) under conditions demonstrated to induce neuronal differentiation. Using an approved protocol, n = 12 DPSCs were identified from an existing biorepository and treated with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), which were previously demonstrated to induce neural differentiation markers including Sox1, Pax6 and NFM among these DPSCs. This study revealed that some microRNAs involved in the neuronal differentiation of MSCs were also differentially expressed among the DPSCs, including miR-27 and miR-145. In addition, this study also revealed that administration of bFGF and EGF was sufficient to modulate miR-27 and miR-145 expression in all of the stimulus-responsive DPSCs but not among all of the non-responsive DPSCs-suggesting that further investigation of the downstream targets of these microRNAs may be needed to fully evaluate and understand these observations.

The RNA cargo in small extracellular vesicles from chicken eggs is bioactive in C57BL/6 J mice and human peripheral blood mononuclear cells ex vivo.

Small extracellular vesicles (sEVs) and their RNA cargo in milk are bioavailable in humans, pigs, and mice, and their dietary depletion and supplementation elicits phenotypes. Little is known about the content and biological activity of sEVs in foods of animal origin other than milk. Here we tested the hypothesis that sEVs in chicken eggs (Gallus gallus) facilitate the transfer of RNA cargo from an avian species to humans and mice, and their dietary depletion elicits phenotypes. sEVs were purified from raw egg yolk by ultracentrifugation and authenticated by transmission electron microscopy, nano-tracking device, and immunoblots. The miRNA profile was assessed by RNA-sequencing. Bioavailability of these miRNAs in humans was assessed by egg feeding study in adults, and by culturing human peripheral blood mononuclear cells (PBMCs) with fluorophore-labeled egg sEVs ex vivo. To further assess bioavailability, fluorophore-labeled miRNAs, encapsulated in egg sEVs, were administered to C57BL/6 J mice by oral gavage. Phenotypes of sEV RNA cargo depletion were assessed by feeding egg sEV and RNA-defined diets to mice and using spatial learning and memory in the Barnes and water mazes as experimental readouts. Egg yolk contained 6.30 × 1010 ± 6.06 × 109 sEVs/mL, which harbored eighty-three distinct miRNAs. Human PBMCs internalized sEVs and their RNA cargo. Egg sEVs, loaded with fluorophore-labeled RNA and administered orally to mice, accumulated primarily in brain, intestine and lungs. Spatial learning and memory (SLM) was compromised in mice fed on egg sEV- and RNA-depleted diet compared to controls. Egg consumption elicited an increase of miRNAs in human plasma. We conclude that egg sEVs and their RNA cargo probably are bioavailable. The human study is registered as a clinical trial and accessible at https://www.isrctn.com/ISRCTN77867213.

Administration of Clinical COVID-19 Mouthwashing Protocol and Potential Modulation of Pediatric Oral Bacterial Prevalence of Selenomonas noxia: A Pilot Study.

Dental office protocols to combat the SARS-CoV-2 (COVID-19) pandemic include mouth washing for an extended 60 s, thereby reducing detectable oral virus. However, it is unclear whether this protocol has any effects on the newly identified periodontal pathogen and obesity-related bacterium often found among pediatric patients, Selenomonas noxia. To determine if the mouthwash protocol has any measurable effect on S. noxia amongst pediatric patients, clinical pediatric saliva samples were obtained from pediatric patients during routine visits for clinical care and treatment. Using an approved protocol, two saliva samples were collected on the same visit before and after chlorhexidine mouthwash (Sample A, Sample B). The third sample (Sample C) was taken at the recall appointment-usually between two and eight weeks later. A total of n = 97 pre-mouthwash samples, and an equal number of matching post-mouthwash samples (n = 97) were collected, with a small number of matching recall samples (n = 36) that were subsequently collected and identified. The demographic composition of the study sample was analyzed using Chi square statistics. Sample DNA from the matching pre-, post-, and recall collections (Sample A, Sample B, and Sample C) was isolated and screened using qPCR and validated primers, which revealed that 11.1% (n = 4/36) from Sample A tested positive for S. noxia with 0% (n = 0/36) of Sample B testing positive and 13.9% (n = 5/36) of the recall (Sample C) testing positive. In addition, comparative analysis of the qPCR cycle threshold data revealed relatively lower expression (quantity) of S. noxia DNA among the recall samples, as determined by two-tailed t-tests (p=0.004). These data and results provide new evidence for the oral prevalence of S. noxia among pediatric patients, while also demonstrating that the COVID-19 protocol of mouth washing prior to clinical treatment for periods extending up to 60 s may be sufficient to reduce the levels of detectable S. noxia-at least temporarily. More research will be needed to determine whether these effects may be limited to the short- or may exhibit more lasting effects in the long-term.

Assessment of SARS-CoV-2 (COVID-19) Clinical Mouthwash Protocol and Prevalence of the Oral Pathogen Scardovia wiggsiae: A Pilot Study of Antibacterial Effects.

One protocol in healthcare facilities and dental offices due to the COVID-19 pandemic for reducing the amount of detectable oral SARS-CoV-2 has been gargling with mouthwash for 60 s. This protocol lasts longer than the daily routine for most patients and may have unexpected benefits in reducing oral microbes as a result. This project evaluated the prevalence of the newly identified oral pathogen Scardovia wiggsiae before and after this procedure to determine any measurable effects. Using an approved protocol, n = 36 pre-mouthwash patient samples, n = 36 matched post-mouthwash samples, and n = 36 matched recall samples were identified (total sample number n = 108). DNA was isolated from each sample (pre-, post-mouthwash, and recall). Screening using qPCR and validated primers revealed n = 10/36 or 27.8% tested positive for Scardovia among the pre-mouthwash (Sample A) isolates with n = 3/36 or 8.3% testing positive among the post-mouthwash (Sample B) isolates. Screening of the recall (Sample C) samples has revealed n = 10/36, or 27.8% once again tested positive for Scardovia, demonstrating that this pathogen was found among a significant proportion of pediatric patient samples. Moreover, the COVID-19-related procedure of requiring sustained mouth washing prior to clinical treatment appears to reduce the levels of detectable Scardovia, at least initially. However, this study found no long-term effects using this isolated protocol.