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.

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.

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.

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.

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.

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.

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.

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.

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.

Assessment of Sodium Diamine Fluoride (SDF) with Light Curing Technique: A Pilot Study of Antimicrobial Effects.

Silver diamine fluoride (SDF) has been useful in clinical dentistry for the purpose of caries arrest and prevention. Although methods for the application of SDF are well-known among dental professionals, such as microbrush applications, few studies have explored the effect of light curing, which accelerates precipitation onto dentin, and whether this has any effect on the antimicrobial properties of SDF. To assess this technique, single (Streptococcus gordonii) and polymicrobial (mixed salivary) colonies were grown and plated using SDF applied to hydroxyapatite discs with and without treatment with curing light. Kirby-Bauer Zone of Inhibition assay results revealed no significant differences in the areas between the two treatment groups (SDF: 1.27 mm, SDF plus curing light: 1.25 mm), p = 0.887 in the single culture (S. gordonii) experiments. In addition, no significant differences were found between the two treatment groups (SDF: 1.26 mm, SDF plus curing light: 1.24 mm), p = 0.771 in the polymicrobial culture experiments. Although there may be specific properties associated with SDF induced following light curing, these differences do not appear to be associated with the antimicrobial properties affecting gram-positive or polymicrobial films.

Molecular Screening and Analysis Reveal Novel Oral Site-Specific Locations for the Cariogenic Pathogen Scardovia wiggsiae.

INTRODUCTION: Scardovia wiggsiae (SW) is a newly identified cariogenic pathogen associated with severe early childhood caries and oral disease. New studies have confirmed the presence of this organism among clinical samples from both pediatric and adult patients. However, the recent discovery of this organism has left researchers with only limited information available regarding the prevalence of this organism-and virtually no information regarding oral site-specific locations. Based upon this lack of information, the overall objective of this study was to perform an oral site-specific analysis of SW prevalence from clinical samples. METHODS: Using an approved human subjects protocol, samples (n = 60) from an existing saliva and site-specific biorepository were identified and screened for SW presence using quantitative polymerase chain reaction (qPCR). These data were summarized and subsequently analyzed for correlations with demographic (age, sex, race or ethnicity) or clinical (body mass index or BMI, primary/mixed/permanent dentition, orthodontic brackets) variables. RESULTS: These data revealed that average DNA concentrations from all sample sites (saliva, dorsum of tongue, gingival crevicular fluid (GCF), biofilm of upper buccal molar, and biofilm of lower lingual incisor) ranged between 13.74 and 14.69 µg/µL, with an overall average of 14.30 µg/µL ± 1.12 (standard error or SE). qPCR screening revealed a total of n = 34/60 or 56.7% of patient samples harboring SW. A total of n = 71/170 specific oral sites harbored this organism, with the majority of the SW-positive participant samples harboring SW at more than one oral site, n = 22/34 or 64.7%, including non-traditional sites such as GCF and the dorsum of the tongue. Weak correlations were found between specific SW outcomes in GCF and type of dentition (permanent; R = 0.2444), as well as SW outcomes in saliva with age (R = 0.228) and presence of orthodontic brackets (R = 0.2118). CONCLUSIONS: This study may be among the first to provide oral site-specific analysis to reveal the prevalence and location of Scardovia among clinical patient samples. Moreover, these data also provide some of the first evidence to suggest this organism may be present not only in traditional supragingival tooth-associated biofilm sites, but also in non-traditional oral sites including the dorsum of the tongue and the gingival crevice. Based upon these results, these data may represent a significant advance in our understanding of the potential sites and locations that harbor this organism and may help contribute to our understanding of the prevalence, distribution and potential for the development of oral disease among clinic patients.

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.

Differential MicroRNA Expression of miR-21 and miR-155 within Oral Cancer Extracellular Vesicles in Response to Melatonin.

OBJECTIVE: Extracellular vesicles derived from oral cancer cells, which include Exosomes and Oncosomes, are membranous vesicles secreted into the surrounding extracellular environment. These extracellular vesicles can regulate and modulate oral squamous cell carcinoma (OSCC) progression through the horizontal transfer of bioactive molecules including proteins, lipids and microRNA (miRNA). The primary objective of this study was to examine the potential to isolate and evaluate extracellular vesicles (including exosomes) from various oral cancer cell lines and to explore potential differences in miRNA content. METHODS: The OSCC cell lines SCC9, SCC25 and CAL27 were cultured in DMEM containing 10% exosome-free fetal bovine serum. Cell-culture conditioned media was collected for exosome and extracellular vesicle isolation after 72 hours. Isolation was completed using the Total Exosome Isolation reagent (Invitrogen) and extracellular vesicle RNA was purified using the Total Exosome RNA isolation kit (Invitrogen). Extracellular vesicle miRNA content was evaluated using primers specific for miR-16, -21, -133a and -155. RESULTS: Extracellular vesicles were successfully isolated from all three OSCC cell lines and total extracellular vesicle RNA was isolated. Molecular screening using primers specific for several miRNA revealed differential baseline expression among the different cell lines. The addition of melatonin significantly reduced the expression of miR-155 in all of the OSCC extracellular vesicles. However, miR-21 was significantly increased in each of the three OSCC isolates. No significant changes in miR-133a expression were observed under melatonin administration. CONCLUSIONS: Although many studies have documented changes in gene expression among various cancers under melatonin administration, few studies have evaluated these effects on microRNAs. These results may be among the first to evaluate the effects of melatonin on microRNA expression in oral cancers, which suggests the differential modulation of specific microRNAs, such as miR-21, miR-133a and miR-155, may be of significant importance when evaluating the mechanisms and pathways involved in melatonin-associated anti-tumor effects.

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.

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.

Loss of miRNAs during processing and storage of cow's (Bos taurus) milk.

MicroRNAs (miRs, miRNAs) play central roles in gene regulation. Previously, we reported that miRNAs from pasteurized, store-bought bovine milk have biological activity in humans. Here, we assessed the effects of milk processing, storage, somatic cell content, and handling by consumers on the degradation of miRNAs in milk; we also quantified miRNAs in dairy products. Pasteurization and homogenization caused a 63% loss of miR-200c, whereas a 67% loss observed for miR-29b was statistically significant only in skim milk. Effects of cold storage and somatic cell content were quantitatively minor (<2% loss). Heating in the microwave caused a 40% loss of miR-29b but no loss of miR-200c. The milk fat content had no effect on miRNA stability during storage and microwave heating. The concentrations of miRNAs in dairy products were considerably lower than in store-bought milk. We conclude that processing of milk by dairies and handling by consumers causes a significant loss of miRNAs.

Macrophage colony stimulating factor-induced macrophage differentiation influences myotube elongation.

BACKGROUND: Unaccustomed exercise, high-intensity dynamic sports activities, or the resumption of normal weight-bearing after a period of disuse can induce skeletal muscle injury, which activates an inflammatory response followed by muscle regeneration. Specific subsets of macrophages are involved in muscle regeneration. But the exact role of macrophage differentiation during muscle regeneration remains to be elucidated. OBJECTIVE: The objective of the study was to examine the effect of macrophage colony stimulating factor (M-CSF)-differentiated, lipopolysaccharides (LPS)-stimulated-macrophage-conditioned medium on muscle-cell proliferation, fusion, and elongation, which are key events during muscle regeneration and myogenesis. METHOD: Murine C2C12 myoblasts were cultured in conditioned medium obtained from PU5-1R macrophages that were (a) undifferentiated, unstimulated; (b) M-CSF-differentiated, unstimulated; (c) undifferentiated, LPS-stimulated; or (d) M-CSF-differentiated, LPS-stimulated. Myoblast proliferation ratio, nuclei number, and length were measured. RESULTS: C2C12 cells cultured in conditioned medium from M-CSF-differentiated, LPS-stimulated macrophages had significantly more nuclei and greater length than cells cultured in conditioned medium from undifferentiated, LPS-stimulated macrophages. Dilution and denaturization of the M-CSF-differentiated, LPS-stimulated-macrophage medium prevented a marked increase in C2C12 nuclei number and length. However, the C2C12 myoblast proliferation ratio was significantly greater in conditioned medium from undifferentiated, LPS-stimulated macrophages than in conditioned medium from M-CSF-differentiated, LPS-stimulated macrophages. CONCLUSIONS: M-CSF-differentiated, LPS-stimulated macrophages may influence myogenesis and the early and terminal stages of muscle regeneration. This knowledge may aid in developing therapies that will directly expedite muscle repair and lead to faster rehabilitation and reduced rehabilitation costs.

A molecular survey of S. mutans and P. gingivalis oral microbial burden in human saliva using relative endpoint polymerase chain reaction (RE-PCR) within the population of a Nevada dental school revealed disparities among minorities.

BACKGROUND: The University of Nevada, Las Vegas School of Dental Medicine recently opened an orthodontic treatment clinic to address the needs of the racially and ethnically diverse population of Southern Nevada, primarily focusing on the treatment and care of low-income and minority patients. Although orthodontic treatment and therapy has been shown to induce changes in the oral cavity, much of this evidence was collected from traditional White, teenage orthodontic clinic populations. The primary goal of this study was to describe the microbial burden of the cariogenic and periodontal pathogens, Streptococcus mutans and Porphyromonas gingivalis within the UNLV-SDM patient population. METHODS: Representative saliva samples were collected from healthy adult patients for DNA isolation. Relative endpoint polymerase chain reaction (RE-PCR) was performed to ascertain the presence and relative microbial burden of these oral pathogens. RESULTS: Nearly one quarter (13/56) or 23.3% of these patients had elevated levels of S. mutans, while (10/56) and 17.8% of these samples were found to have elevated levels of P. gingivalis, - with (90%) of P. gingivalis-positive samples from minority patients (X2=17.921, d.f. = 1; p<0.0001). CONCLUSIONS: These findings of elevated P. gingivalis levels, primarily among minority patients, may suggest underlying oral health practices contributing to adverse oral health conditions within this population. Oral health knowledge and practices among minority patients may be strongly influenced by other factors, including education and socioeconomic status, suggesting additional research may be needed to accurately determine the most appropriate standards for care and oral health education within this patient population.