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The oral microbial flora may be significantly altered by orthodontic therapy and the use of fixed orthodontic brackets. Most orthodontic research has focused on cariogenic pathogens, while some evidence has demonstrated an increase in many known periodontal pathogens. However, little is known about the prevalence of the Gram-negative periodontal pathogen Selenomonas noxia (SN) among these patients. Using an existing saliva biorepository, n = 208 samples from adult and pediatric orthodontic and non-orthodontic patients were identified and screened for the presence of SN using qPCR and validated primers. In the pediatric study sample ( n = 89), 36% tested positive for the presence of SN, with orthodontic patients comprising more SN-positive samples (87.5%) than SN-negative samples (78.9%), p = 0.0271. In the adult study sample ( n = 119), SN was found in 28.6%, with orthodontic patients comprising 58.8% of positive samples and only 28.2% of negative samples ( p < 0.0001). These data demonstrated that both pediatric and adult orthodontic patients exhibited higher prevalence of SN compared with age-matched non-orthodontic controls. As this microorganism is associated not only with periodontal disease but also long-term health issues such as obesity, more research is needed regarding the factors that increase the prevalence of this microbe.

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.

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.

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.

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.

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.

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.

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 Fe 2+ 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., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

Coronavirus disease 2019 (COVID-19) is associated with a wide spectrum of disease presentation, ranging from asymptomatic infection to acute respiratory distress syndrome (ARDS). Paradoxically, a direct relationship has been suggested between COVID-19 disease severity and the levels of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, including virus-neutralizing titers. A serological analysis of 536 convalescent healthcare workers reveals that SARS-CoV-2-specific and virus-neutralizing antibody levels are elevated in individuals that experience severe disease. The severity-associated increase in SARS-CoV-2-specific antibody is dominated by immunoglobulin G (IgG), with an IgG subclass ratio skewed toward elevated receptor binding domain (RBD)- and S1-specific IgG3. In addition, individuals that experience severe disease show elevated SARS-CoV-2-specific antibody binding to the inflammatory receptor FcɣRIIIa. Based on these correlational studies, we propose that spike-specific IgG subclass utilization may contribute to COVID-19 disease severity through potent Fc-mediated effector functions. These results may have significant implications for SARS-CoV-2 vaccine design and convalescent plasma therapy., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

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.

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.

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.

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., Competing Interests: The authors declare no conflict of interest.

I'm a nursing auxiliary, soon to be degree student, and I have a different hair colour pretty much every week. Like Mary Walls Penney, the nursing home nurse who was confronted about her appearance (Nursing Standard online), I also have tattoos on show. However, I work in a day surgery unit and have never received negative comments about my appearance. Actually, patients often comment on my hair or tattoos; it's usually a nice ice breaker. What matters is being professional and caring.

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.

In the structure of the title complex, [Fe(C5H7O2)3] or Fe(acac)3, the asymmetric unit contains one mol-ecule in a general position. The coordination sphere of the Fe(III) atom is that of a slightly distorted octahedron. The crystal under investigation was a two-component pseudo-merohedral twin in the monoclinic system with a β angle close to 90°. Twin law [100/0-10/00-1] reduced the R1 residual [I > 2σ(I)] from 0.0769 to 0.0312, and the mass ratio of twin components refined to 0.8913 (5):0.1087 (5). In the crystal, mol-ecules are arranged in sheets normal to [001] via non-classical C-H⋯O hydrogen bonding. No other significant inter-molecular inter-actions are observed. The structure is a new polymorph of Fe(acac)3 and is isotypic with one polymorph of its gallium analog.

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.

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.

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.

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.

Objectives: This study was designed to investigate and characterize the ability of platelet-activating factor (PAF) to induce the expression of platelet-activating factor acetylhydrolase (PAF-AH)., Methods: Ribonuclease protection assays and quantitative real-time PCR were used to investigate the ability of lipopolysaccharide (LPS) and PAF to regulate PAF-AH mRNA expression in human monocyte-macrophage 6 (MM6) cells. Pharmacological inhibitors of mitogen activated protein kinases (MAPK) and PAF receptor antagonists were used to investigate the mechanism of regulation of PAF-AH., Results: PAF-AH mRNA levels were increased upon exposure to LPS or PAF in a dose-dependent manner. LPS elicited a more potent and rapid increase in PAF-AH expression than the PAF-stimulated response. However, when administered concomitantly, PAF augmented the LPS-stimulated response. LPS-stimulated PAF-AH expression was susceptible to partial inhibition by a p38 MAPK inhibitor and PAF receptor antagonists. PAF-induced up-regulation of PAF-AH levels was solely mediated via the PAF receptor and was p38 MAPK-independent., Conclusion: The proinflammatory mediators, LPS and PAF, increased levels of PAF-AH mRNA via distinct signaling pathways.

Background: The objective of this study was to measure the validity and reliability of a multifactorial Risk Factor Model developed for use in predicting future caries risk in Nevada adolescents in a public health setting., Methods: This study examined retrospective data from an oral health surveillance initiative that screened over 51,000 students 13-18 years of age, attending public/private schools in Nevada across six academic years (2002/2003-2007/2008). The Risk Factor Model included ten demographic variables: exposure to fluoridation in the municipal water supply, environmental smoke exposure, race, age, locale (metropolitan vs. rural), tobacco use, Body Mass Index, insurance status, sex, and sealant application. Multiple regression was used in a previous study to establish which significantly contributed to caries risk. Follow-up logistic regression ascertained the weight of contribution and odds ratios of the ten variables. Researchers in this study computed sensitivity, specificity, positive predictive value (PVP), negative predictive value (PVN), and prevalence across all six years of screening to assess the validity of the Risk Factor Model., Results: Subjects' overall mean caries prevalence across all six years was 66%. Average sensitivity across all six years was 79%; average specificity was 81%; average PVP was 89% and average PVN was 67%., Conclusions: Overall, the Risk Factor Model provided a relatively constant, valid measure of caries that could be used in conjunction with a comprehensive risk assessment in population-based screenings by school nurses/nurse practitioners, health educators, and physicians to guide them in assessing potential future caries risk for use in prevention and referral practices.

Muscle and bone form a functional unit. While muscle size is a useful surrogate of mechanical load on bone, the independent contributions to bone strength of muscle force, muscle size, gravitational load (body weight), and physical activity have not been assessed. Three hundred twenty-one healthy participants (32% black, 47% male), aged 5-35 years were assessed. Peak dorsiflexion muscle torque (ft-lbs) of the ankle was assessed using isometric dynamometry. Tibia peripheral quantitative computed tomography measures included polar section modulus (Zp; mm(3)), periosteal and endosteal circumference (mm), cortical area (mm(2)), and volumetric bone mineral density (vBMD; mg/cm(3)) at the 38% site, and muscle cross-sectional area (CSA; mm(2)), at the 66% site. Physical activity (average hours per week) was assessed by questionnaire. Log linear regression was used to assess determinants of muscle specific force (MSF; torque relative to muscle CSA) and Zp adjusted for age and tibia length. MSF was greater in blacks than whites (p<0.05) and lower in females than males (p<0.001). Zp was greater in blacks than whites (p=0.002) in Tanner stages 1-4, but the difference was attenuated in Tanner 5 (interaction, p=0.02); R(2)=0.87. Muscle CSA, muscle torque, body weight, and physical activity were added to the model and each load covariate was independently and significantly (all, p<0.02) associated with Zp (R(2)=0.92), periosteal circumference, and cortical area. Inclusion of these measures attenuated but did not eliminate the significant race differences. Only muscle CSA was positively associated with endosteal circumference, while none of the load covariates were associated with vBMD. In conclusion, bone geometry is associated with several factors that define the mechanical load on bone, independent of age, tibia length, maturation, race, and sex. Race differences in Zp were not explained by these measures of mechanical load. Given that inclusion of muscle torque, body weight, and physical activity resulted in a nominal increase in the R(2), muscle size is an adequate surrogate for the mechanical load on bone in healthy participants., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background & Aims: Pediatric Crohn's disease (CD) is associated with growth, lean mass (LM), and fat mass (FM) deficits. This study assessed and identified determinants of changes in height and body composition in children with CD following., Methods: Whole-body LM and FM were assessed using dual-energy x-ray absorptiometry in 78 CD subjects at diagnosis, 6, 12, and a median of 43 months (range, 24-63) later. Race- and sex-specific Z scores for lean mass (LM-ht-Z) and fat mass (FM-ht-Z) relative to height were derived using reference data in >900 controls. Serum cytokines and growth factors were measured, and quasi-least squares regression was used to identify determinants of changes in height and body composition., Results: LM-ht-Z and FM-ht-Z (both P<.005) improved significantly after diagnosis; however, female patients had persistent LM deficits vs controls (-0.50+/-1.02, P<.05). Serum interleukin-6, tumor necrosis factor-alpha, and lipopolysaccharide binding protein decreased significantly (all P<.001). Greater increases in LM-ht-Z were associated with infliximab therapy (P<.05), increases in albumin (P<.001) and decreases in erythrocyte sedimentation rate (P<.05), interleukin-6 (P<.005), and lipopolysaccharide binding protein (P<.05). Greater increases in FM-ht-Z were associated with glucocorticoid, methotrexate, and infliximab therapy, and increases in albumin (P<.05) and growth hormone binding protein (P<.05). Overall, height-Z did not improve; however, greater increases in insulin-like growth factor-1 (P<.05) and decreases in tumor necrosis factor-alpha (P<.05), interleukin-6 (P<.05), and lipopolysaccharide binding protein (P<.05) levels were associated with increases in height-Z., Conclusions: Immune-mediated mechanisms contribute to growth and body composition deficits in CD. Therapies should target these deficits., (Copyright (c) 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Platelet-activating factor (PAF) acetylhydrolase plays a crucial role inactivating the potent inflammatory mediator, PAF. PAF is implicated in the initiation and propagation of acute lung injury. Although PAF acetylhydrolase is a constitutively active plasma protein, increased PAF production during inflammatory events may necessitate an increase in PAF acetylhydrolase activity in the local environment. A series of experiments were conducted to determine whether the systemic administration of LPS to Sprague-Dawley rats resulted in enhanced expression of PAF acetylhydrolase in lung tissue. Ribonuclease protection assays revealed a dramatic increase in PAF acetylhydrolase mRNA, which peaked at 24 h following in vivo LPS administration. The increase in PAF acetylhydrolase mRNA was dose dependent and was detected when as little as 10 microg/kg of LPS was administered. Western blot analyses of lung tissue homogenates confirmed an increased production of PAF acetylhydrolase protein in response to LPS. In addition, Western blot analyses revealed the rat PAF acetylhydrolase protein exhibited heterogeneous molecular weights with predominant species migrating at 63 and 67 kDa. Some of the molecular weight heterogeneity likely resulted from extensive glycosylation of the secreted protein. Immunohistochemical analyses of lung tissue sections and colocalization experiments revealed a heterogenous population of cells that express the plasma-type PAF acetylhydrolase. Lung interstitial macrophages were PAF acetylhydrolase positive, but surprisingly, alveolar macrophages did not increase expression of PAF acetylhydrolase in response to systemic LPS administration. In addition, rat granulocytes consisting primarily of neutrophils were strongly positive for PAF acetylhydrolase in the LPS-exposed lung tissue. The absence of immunoreactive PAF acetylhydrolase in alveolar macrophages obtained from bronchial alveolar lavage confirmed that systemic LPS administration resulted in enhanced PAF acetylhydrolase expression only in a subset of lung macrophages.

Glucocorticoid (GC) effects on skeletal development have not been established. The objective of this pQCT study was to assess volumetric BMD (vBMD) and cortical dimensions in childhood steroid-sensitive nephrotic syndrome (SSNS), a disorder with minimal independent deleterious skeletal effects. Tibia pQCT was used to assess trabecular and cortical vBMD, cortical dimensions, and muscle area in 55 SSNS (age, 5-19 yr) and >650 control participants. Race-, sex-, and age-, or tibia length-specific Z-scores were generated for pQCT outcomes. Bone biomarkers included bone-specific alkaline phosphatase and urinary deoxypyridinoline. SSNS participants had lower height Z-scores (p < 0.0001) compared with controls. In SSNS, Z-scores for cortical area were greater (+0.37; 95% CI = 0.09, 0.66; p = 0.01), for cortical vBMD were greater (+1.17; 95% CI = 0.89, 1.45; p < 0.0001), and for trabecular vBMD were lower (-0.60; 95% CI, = -0.89, -0.31; p < 0.0001) compared with controls. Muscle area (+0.34; 95% CI = 0.08, 0.61; p = 0.01) and fat area (+0.56; 95% CI = 0.27, 0.84; p < 0.001) Z-scores were greater in SSNS, and adjustment for muscle area eliminated the greater cortical area in SSNS. Bone formation and resorption biomarkers were significantly and inversely associated with cortical vBMD in SSNS and controls and were significantly lower in the 34 SSNS participants taking GCs at the time of the study compared with controls. In conclusion, GCs in SSNS were associated with significantly greater cortical vBMD and cortical area and lower trabecular vBMD, with evidence of low bone turnover. Lower bone biomarkers were associated with greater cortical vBMD. Studies are needed to determine the fracture implications of these varied effects.

Background & Aims: The impact of childhood Crohn's disease (CD) on volumetric bone mineral density (vBMD), bone structure, and muscle mass have not been established. The objective of this longitudinal study was to assess musculoskeletal outcomes in an incident cohort of children with CD using peripheral quantitative computed tomography (pQCT)., Methods: Tibia pQCT was performed in 78 CD subjects (ages, 5-18 years) at diagnosis and in 67 over the subsequent year. pQCT outcomes were converted to sex- and race-specific z scores based on reference data in over 650 controls. Multivariable linear regression models identified factors associated with changes in bone outcomes., Results: At diagnosis, CD subjects had significant deficits in trabecular vBMD (z score, -1.32+/-1.32; P< .001), cortical section modulus (a measure of bone geometry and strength) (z score, -0.44+/-1.11; P< .01), and muscle (z score, -0.96+/-1.02; P< .001) compared with controls. Over the first 6 months, trabecular vBMD and muscle z scores improved significantly (both, P< .001); however, section modulus worsened (P= .0001), and all 3 parameters remained low after 1 year. Increases in muscle z scores were associated with less severe declines in cortical section modulus z scores. Improvements in trabecular vBMD z scores were greater in prepubertal subjects. Glucocorticoids were associated with increases in cortical vBMD., Conclusions: Substantial deficits in trabecular vBMD, cortical bone geometry, and muscle were observed at CD diagnosis. Trabecular vBMD improved incompletely; however, cortical deficits progressed despite improvements in muscle. Glucocorticoids were not associated with bone loss. Therapies to improve bone accrual in childhood CD are needed.

Background: Research programs within medical and dental schools are important vehicles for biomedical and clinical discovery, serving as effective teaching and learning tools by providing situations in which predoctoral students develop problem-solving and critical-thinking skills. Although research programs at many medical and dental schools are well-established, they may not be well integrated into the predoctoral curriculum to effectively support the learning objectives for their students., Methods: A series of structured seminars, incorporating faculty research, was designed for first-year dental students at the University of Nevada, Las Vegas, School of Dental Medicine to reinforce and support the concepts and skills taught in concurrent courses. A structured research enrichment period was also created to facilitate student engagement in active research using faculty and student curricular release time. Course evaluations and surveys were administered to gauge student perceptions of the curricular integration of research, the impact of these seminars on recruitment to the research program, and overall levels of student satisfaction with research enrichment., Results: The analysis of course surveys revealed that students perceived the research-containing seminars effectively illustrated concepts, were logically sequenced, and were well-integrated into their curriculum. In addition, analysis of surveys revealed that the Integration Seminar courses motivated students to engage in research enrichment. Finally, this analysis provided evidence that students were very satisfied with their overall learning experience during research enrichment., Conclusion: Curricular integration is one method of improving the teaching and learning of complicated and inter-related concepts, providing an opportunity to incorporate research training and objectives into traditionally separate didactic courses. Despite the benefits of curricular integration, finding the most appropriate points of integration, obtaining release time for curricular development and for research engagement, and funding predoctoral student research remain issues to be addressed in ways that reflect the character of the faculty and the goals of each institution.

Staphylococcus aureus infections are known triggers for skin inflammation and can modulate immune responses. The present studies used model systems consisting of platelet-activating factor receptor-positive and -negative (PAF-R-positive and -negative) cells and PAF-R-deficient mice to demonstrate that staphylococcal lipoteichoic acid (LTA), a constituent of Gram-positive bacteria cell walls, acts as a PAF-R agonist. We show that LTA stimulates an immediate intracellular Ca2+ flux only in PAF-R-positive cells. Intradermal injections of LTA and the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine (CPAF) induced cutaneous inflammation in wild-type but not PAF-R-deficient mice. Systemic exposure to LTA or CPAF inhibited delayed-type hypersensitivity (DTH) reactions to the chemical dinitrofluorobenzene only in PAF-R-expressing mice. The inhibition of DTH reactions was abrogated by the addition of neutralizing antibodies to IL-10. Finally, we measured levels of LTA that were adequate to stimulate PAF-R in vitro on the skin of subjects with infected atopic dermatitis. Based on these studies, we propose that LTA exerts immunomodulatory effects via the PAF-R through production of the Th2 cytokine IL-10. These findings show a novel mechanism by which staphylococcal infections can inhibit Th1 reactions and thus worsen Th2 skin diseases, such as atopic dermatitis.

The eukaryotic trifunctional enzyme, C(1)-tetrahydrofolate (THF) synthase, interconverts folic acid derivatives between various oxidation states and is critical for normal cellular function, growth, and differentiation. Using a rat C(1)-THF synthase cDNA and synthetic oligonucleotides, the rat C(1)-THF synthase gene was isolated and characterized. The gene consists of 28 exons and spans 67.5 kbp. Primer extension, RNase protection, and rapid amplification of cDNA ends (RACE) experiments indicate the presence of multiple transcription start points (tsp) within a 250-bp window located between 50 and 300 bp upstream from the start codon. The 5' flanking region is devoid of a TATA consensus sequence motif, but putative regulatory elements, including NF-kappabeta, HNF-4alpha1, RARalpha1, C/EBP, and PPAR are present in the promoter region. The 5' flanking region also contains two sets of tetranucleotide repeats and two short interspersed nuclear elements (SINES). The initial 2500 bp of 5' flanking sequences of the rat and mouse cytoplasmic C(1)-THF synthase genes share 70% identity. However, comparison with the human gene from the Human Genome Data Bank revealed no significant homology in the 5' flanking region. The gene structure characterization led to the identification of a pseudogene that is 94% identical to the C(1)-THF synthase gene and probably diverged 10-12 million years ago. In addition, the gene expression patterns of C(1)-THF synthase were investigated during liver regeneration and liver and kidney organogenesis, two highly regulated events. In both processes, C(1)-THF synthase expression correlated with increased nucleotide metabolism. This pattern suggests that the gene is regulated in response to changes in the demand for folate-dependent one-carbon units.

The production of platelet-activating factor (PAF) and PAF-like phospholipids that also bind the PAF receptor are implicated in numerous pathological situations including bacterial endotoxemia and injury-induced oxidative damage. PAF and PAF-like phospholipids are hydrolyzed and inactivated by the enzyme PAF acetylhydrolase. In the intact rat, infusion of lipopolysaccharide (LPS) into a mesenteric vein served as an acute, liver-focused model of endotoxemia. We determined that the liver responds to LPS exposure with the production of plasma-type PAF acetylhydrolase mRNA and protein expression specifically in the resident macrophages of the liver. Liver macrophages, defined immunohistochemically using antibodies against ED1, present in livers from saline-treated animals contained no detectable PAF acetylhydrolase. Twenty-four hours following in vivo LPS administration, immunohistochemistry detected a slight increase in the number of ED1 staining cells and the ED1-positive cells now contained an abundance of PAF acetylhydrolase. The systemic administration of LPS resulted in increased expression of PAF acetylhydrolase in several tissues. Of the tissues examined, the greatest increase in PAF acetylhydrolase expression was observed in lung followed by increases in spleen, liver, kidney, and thymus. Additionally, the expression of PAF acetylhydrolase mRNA increased in circulating leukocytes and in peritoneal macrophages in response to systemic exposure to LPS. We examined the regulation of PAF acetylhydrolase expression and demonstrated the administration of the PAF receptor antagonists, BN 50739 and WEB 2170, inhibited by 50% the increase in PAF acetylhydrolase expression in response to LPS. The up-regulation of the plasma-type PAF acetylhydrolase expression constitutes an important mechanism for elevating the local and systemic ability to inactivate PAF and oxidized phospholipids in order to minimize PAF-mediated pathophysiology consequent from exposure to endotoxin. The abundance of PAF acetylhydrolase production in the liver lobule likely limits endotoxin-mediated tissue damage due to PAF synthesis.

Objective: To determine the cause of an outbreak of Pseudomonas aeruginosa cerebral ventriculitis among eight patients at a community hospital neurosurgical intensive care unit. All had percutaneous external ventricular catheters (EVCs) to monitor cerebrospinal fluid (CSF) pressure., Methods: Cohort study of all patients who had EVCs placed during the epidemic period (August 8-October 22, 1997). A case-patient was any patient with P aeruginosa ventriculitis during the epidemic period. Pulsed-field gel electrophoresis (PFGE) was performed on all isolates., Results: P aeruginosa was significantly more likely to be isolated from CSF per EVC placed in the epidemic than pre-epidemic (January 1-August 7, 1997) periods (8/61 [13%] vs 2/131 [1.5%], P=.002). During the epidemic period, ventriculitis was significantly more likely after EVC placement in the operating room than in other units (8/24 vs 0/22, P=.004). EVC placement technique differed for EVCs placed in the operating room (little hair was removed, preventing application of an occlusive dressing) versus other hospital units (more hair was removed, and an occlusive dressing was applied). Among patients who had operating room EVC placement, contact with one healthcare worker was statistically significant (7/13 vs 0/8, P=.02). Hand cultures of this worker were negative. All isolates had closely related PFGE patterns., Conclusions: These data suggest that a single healthcare worker may have contaminated EVC insertion sites, resulting in an outbreak of P aeruginosa ventriculitis. Affected patients were unlikely to have had an occlusive dressing at the EVC insertion site. Application of a sterile occlusive dressing may decrease the risk of ventriculitis in patients with EVCs.

Patient injuries are thought to have a substantial financial impact on the health care system, but recent studies have been limited to estimating the costs of adverse drug events in teaching hospitals. This analysis estimated the costs of all types of patient injuries from a representative sample of hospitals in Utah and Colorado. We detected 459 adverse events (of which 265 were preventable) by reviewing the medical records of 14,732 randomly selected 1992 discharges from 28 hospitals. The total costs (all results are discounted 1996 dollars) were $661,889,000 for adverse events, and $308,382,000 for preventable adverse events. Health care costs totaled $348,081,000 for all adverse events and $159,245,000 for the preventable adverse events. Fifty-seven percent of the adverse event health care costs, and 46% of the preventable adverse event costs were attributed to outpatient medical care. Surgical complications, adverse drug events, and delayed or incorrect diagnoses and therapies were the most expensive types of adverse events. The costs of adverse events were similar to the national costs of caring for people with HIV/AIDS, and totaled 4.8% of per capita health care expenditures in these states.

Hepatocytes and Kupffer cells in primary culture both secrete plasma-type platelet-activating factor-acetylhydrolase (pPAF-AH) into serum-free culture medium. The rate of secretion of pPAF-AH by Kupffer cells was 20 to 25 times higher than from hepatocytes, and Kupffer cells expressed a higher level of pPAF-AH mRNA than did hepatocytes. Purified liver cell-secreted pPAF-AH exhibited a major protein band of 65-67 kDa on SDS-PAGE; this was the band predominantly labeled when the enzyme catalytic center was reacted with [3H]diisopropylfluorophosphate ([3H]DFP). Rat bile collected from cannulated bile ducts contained significant PAF-AH activity, and bile samples possessed a prominent band at 30-32 kDa, which was the exclusive target for [3H]DFP. Experiments using tunicamycin, an inhibitor of N-linked glycosylation, and endoglycosidase H suggested that pPAF-AH secreted constitutively by cultured hepatocytes and Kupffer cells is glycosylated. The present study supports the notion that hepatic secretion of pPAF-AH into the blood contributes to the regulation of PAF and oxidized phospholipid levels in the circulation, whereas secretion of PAF-AH into the bile may allow hepatic control of these phospholipid signaling molecules in the gastrointestinal tract.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that causes hypotension, increases vascular permeability, and has been implicated in anaphylaxis, septic shock and several other inflammatory responses. PAF is hydrolyzed and inactivated by the enzyme PAF-acetylhydrolase. In the intact rat, a mesenteric vein infusion of lipopolysaccharide (LPS) served as an acute, liver-focused model of endotoxemia. Plasma PAF-acetylhydrolase activity increased 2-fold by 24 h following LPS administration. Ribonuclease protection experiments demonstrated very low levels of plasma-type PAF-acetylhydrolase mRNA transcripts in the livers of saline-infused rats; however, 24 h following LPS exposure, a 20-fold induction of PAF-acetylhydrolase mRNA was detected. In cells isolated from endotoxin-exposed rat livers, Northern blot analyses demonstrated that Kupffer cells but not hepatocytes or endothelial cells were responsible for the increased PAF-acetylhydrolase mRNA levels. In Kupffer cells, plasma-type PAF-acetylhydrolase mRNA was induced by 12 h, peaked at 24 h, and remained substantially elevated at 48 h. Induction of neutropenia prior to LPS administration had no effect on the increase in PAF-acetylhydrolase mRNA seen at 24 h. Although freshly isolated Kupffer cells contain barely detectable levels of plasma-type PAF-acetylhydrolase mRNA, when Kupffer cells were established in culture, PAF-acetylhydrolase expression became constitutively activated concomitant with cell adherence to the culture plates. Alterations in plasma-type PAF-acetylhydrolase expression may constitute an important mechanism for elevating plasma PAF-acetylhydrolase levels and an important component in minimizing PAF-mediated pathophysiology in livers exposed to endotoxemia.

A simple, semiautomated, nonhazardous procedure for the production of a magnetizable solid-phase support (MSPS) has been developed based on the extrusion of molten agarose-iron oxide mixtures, which enables manufacture of a range of differently sized spherical agarose-iron oxide beads. This system has enabled scale-up of an original manufacture procedure and reproducible preparation of kg quantities of MSPS suitable for biomolecular purifications. An improved protocol for the isolation of plasmid DNA directly from cell lysates using this MSPS, derivatized with diethylaminoethyl (DEAE) groups, is reported. This involves a modified alkaline lysis, followed by adsorption to and elution from the support, yielding plasmid DNA of a purity comparable with, or better than, other methods of plasmid isolation. Using the same procedure, plasmid DNA can be isolated from bacterial cell culture volumes of 1.5 mL and 100 mL with equal efficiency and purity.

Activation of endothelin (ET) receptors in the liver causes vasoconstriction, glucose production, and lipid and peptide mediator synthesis. In the intact rat, a bolus infusion of endotoxin into a mesenteric vein served as an acute exposure model of endotoxemia. In response to this challenge, a ninefold increase in hepatic ET-1 mRNA occurred within 3 h. The plasma level of immunoreactive ET-1 (irET-1) increased correspondingly by 8.5-fold within 6 h. ET-1 mRNA levels in liver endothelial cells (EC) isolated from livers of endotoxin-treated rats at various times after endotoxin challenge showed a more gradual increase. Northern blot analyses of the major liver cell types demonstrated that ET-1 mRNA was most abundant in the EC. The present results document a significant increase in the circulating level of irET-1 during episodes of endotoxemia. The increased hepatic ET-1 production in response to endotoxin infusion suggests that ET-1 produced in the liver could make a significant contribution to the plasma irET-1 and may be an important component in the hepatic responses to systemic trauma.

Acute endotoxic shock is accompanied by an increase in the production of nitric oxide (NO) by several different hepatic cell types. Platelet-activating factor (PAF) is a potent proinflammatory mediator with many pathophysiological actions and, in fact, elevated plasma and tissue levels of PAF are observed in animal models of endotoxic shock. The current study demonstrates that PAF induced nitrite formation, the end product of nitric oxide synthesis, by Kupffer cells in a dose- and time-dependent manner. Moreover, PAF was seen to initiate NO synthase gene expression and protein synthesis. PAF augmented lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase messenger RNA (mRNA), protein, nitrite and cyclic guanosine monophosphate (cGMP) levels in Kupffer cells. Treatment of Kupffer cells with actinomycin D or cycloheximide inhibited PAF- and LPS-stimulated nitrite and nitric oxide synthase protein formation confirming that de novo synthesis of the enzyme occurred. In Kupffer cells, the presence of an arginine analog, NG-methyl-L-arginine, attenuated nitrite formation induced by PAF and LPS alone or in combination. L-arginine is the principal substrate for nitric oxide synthase. PAF and LPS individually and in combination induced a time-dependent uptake of L-[3H]-arginine into the Kupffer cell, and this response was sensitive to cycloheximide. The current study indicates that exogenous PAF contributes to the induction of nitric oxide synthase by LPS in cultured rat Kupffer cells.

In the present study, the interaction of the phospholipid mediator platelet-activating factor (PAF) with rat hepatocytes in primary culture was examined. Following exposure to hepatocytes, exogenous [3H]alkyl-PAF was metabolized rapidly to [3H]lyso-PAF, the content of which was raised in the outer leaflet of the plasma membrane within the initial 5 min of incubation. Thereafter [3H]lyso-PAF was translocated into cells with concomitant reacylation to [3H]alkyl-acyl-glycerophosphocholine. A portion of untransformed [3H]PAF accumulated in the outer leaflet, and only a small amount of the [3H]PAF was translocated into the inner leaflet of the plasma membrane. Detectable levels of [3H]lyso-PAF were found in the medium of hepatocyte cultures at all times of incubation. These findings suggest that at least a portion of the cellular PAF-acetylhydrolase (PAF-AH) activity is located in the outer leaflet of the plasma membrane and can be secreted into the medium. Indeed, rat hepatocytes in culture released PAF-AH into the medium in a time-dependent fashion, Incubation of hepatocytes with exogenous PAF increased secretion of PAF-AH, whereas lyso-PAF and the nonhydrolyzable analog methylcarbamyl-PAF significantly reduced secretion. The structurally related PAF receptor antagonist CV 3988 markedly inhibited the activity of PAF-AH and also diminished its release by hepatocytes. In contrast, BN 50739 amd WEB 2170, thienotriazolodiazepine PAF receptor antagonists, did not affect the PAF-AH activity, but increased its secretion by the cells. A full-length 3.8-kb mRNA encoding the cell surface PAF receptor was absent in hepatocytes as indicated by Northern blot analysis using the rat PAF receptor cDNA, whereas PAF receptor mRNA was readily detected in Kupffer cells. Upon incubation with hepatocytes, PAF induced tyrosine phosphorylation of proteins with molecular masses of 120-130 and 160-180 kDa and dephosphorylation of 80-90-kDa proteins; these responses were not inhibited by WEB 2170 and BN 50739. The protein tyrosine kinase inhibitor genistein abolished the release of free arachidonic acid, suggesting a crucial role for tyrosine phosphorylation in PAF-induced phospholipase A2 activation in rat hepatocytes. Taken together, our data indicate that the interaction of PAF with rat hepatocytes is dependent upon its metabolism, involves protein tyrosine phosphorylation/dephosphorylation and arachidonic acid release, and does not involve the heteromeric G-protein-coupled PAF receptor which has been characterized in Kupffer cells. This metabolically regulated mechanism for PAF action on hepatocytes may be of potential biological importance in the liver under normal and pathological conditions.