Fluorescent Plaque Research Articles

Fluorescence-based Evaluation of the Efficacy of Augmented Reality-assisted Toothbrush on Oral Hygiene Practices Among 6–8 Years Old Children

Aim: Effective toothbrushing is the most simple way to maintain a child’s oral hygiene for a cavity-free mouth. Yet, most children find it an uninteresting task and they fail to understand its scientific basis. Augmented reality (A-R)-assisted toothbrushes for pediatric population are commercially available to make toothbrushing interactive, interesting, and educative for the child, aided by gamification providing an interactive videogame platform between the real and the virtual world. The aim of the study was to evaluate the efficacy of A-R-assisted brushing experience of children with respect to fluorescence imaging data and plaque Streptococcus mutans count at clinical and microbiological levels, respectively. Materials and Methods: A total of 32 children in the age group of 6-8 years were included and allocated in two study groups (A-R-assisted toothbrushing and manual toothbrushing). Fluorescence-based lesion area scoring was done using FC, Duerr Dental, Vistaproof, Germany. Microbiological analysis of plaque Streptococcus mutans was done to evaluate change in bacterial count. The parameters were recorded again after two weeks. Pre- and post-intervention scores were subjected to statistical analysis. Results: Significantly higher reduction of fluorescent scores and mean bacterial count were found in A-R-assisted tooth-brushing group when compared to that of manual toothbrushing. Conclusion: Based on the findings of the study, A-R-assisted toothbrushing proves to be an effective tool for motivating and maintaining oral hygiene among children and efforts should be made in incorporating this practice in daily domestic healthcare.

Effects of pH alteration on respiratory syncytial virus in human airway epithelial cells.

Respiratory syncytial virus (RSV) is a leading cause of respiratory distress and hospitalisation in the paediatric population. Low airway surface pH impairs antimicrobial host defence and worsens airway inflammation. Inhaled Optate safely raises airway surface pH in humans and raises intracellular pH in primary human airway epithelial cells (HAECs) in vitro. We aimed to determine whether raising intracellular pH with Optate would decrease infection and replication of RSV in primary HAECs. We cultured HAECs from healthy subjects in both air-liquid interface and submerged conditions. We infected HAECs with green fluorescent protein-labelled RSV (GFP-RSV; multiplicity of infection=1) and treated them with Optate or PBS control. We collected supernatant after a 4-h incubation and then every 24 h. We used fluorescence intensity, fluorescent particle counts, plaque assays, Western blots and ELISA to quantitate infection. In submerged culture, fluorescence intensity decreased in Optate-treated cells (48 h p=0.0174, 72 h p≤0.001). Similarly, Optate treatment resulted in decreased fluorescent particle count (48 h p=0.0178, 72 h p=0.0019) and plaque-forming units (48 h p=0.0011, 72 h p=0.0148) from cell culture supernatant. In differentiated HAECs cultured at ALI, Optate treatment decreased fluorescence intensity (p≤0.01), GFP via Western blot and ELISA (p<0.0001), and RSV-fusion protein via ELISA (p=0.001). Additionally, RSV infection decreased as Optate concentration increased in a dose-dependent manner (p<0.001). Optate inhibits RSV infection in primary HAECs in a dose-dependent manner. These findings suggest that Optate may have potential as an inhaled therapeutic for patients with RSV.

Effectiveness of an Oral Health Education Program Using a Smart Toothbrush with Quantitative Light-Induced Fluorescence Technology in Children

This randomized controlled trial aimed to educate patients and manage oral hygiene remotely via a smart toothbrush (ST) by using mobile applications and to improve oral hygiene and habits by evaluating the plaque state via images from a Q-ray cam, which is a quantitative light-induced fluorescence (QLF) digital imaging device. In total, 68 children (aged 6-12 years) were randomly divided into three groups. Group I was assigned an electro-toothbrush (ET), group II was assigned a smart toothbrush (ST), and group III used a manual toothbrush (MT). Each group used an assigned toothbrush and was checked after 1 and 3 months. Oral hygiene status was evaluated using the patient hygiene performance (PHP) index and fluorescent plaque index (FPI), which are presented in the QLF program. In addition, questionnaires on oral health behaviors and attitudes were also evaluated. There was a 0.24 decrease in the PHP index and a 1.40 decrease in the simple hygiene score three months later compared with the baseline in the ST group, with no significant difference between the three methods (p = 0.518, p = 0.626). Many group II participants said that ST helped with oral hygiene, and they were willing to use it continuously. In addition, all participants' brushing times and attitudes toward oral hygiene improved after oral hygiene education using a Q-ray cam. Therefore, the use of STs provided good dental health education and a plaque-decreasing effect to children aged 6-12 years old. Furthermore, the QLF device can be used as a useful tool for maintaining good oral hygiene in children.

Evaluation of Periodontal Risk Factors with Quantitative Light-Induced Fluorescence Based Fluorescent Plaque Index, in Comparison to Radiographic and Oral Health Habit Scoring: A Retrospective Case Study.

The aim of this study was to evaluate periodontal risk factors with oral health habits and fluorescent plaque index (FPI) using quantitative light-induced fluorescence (QLF) images, and to evaluate their effect on the degree of radiographic bone loss (RBL). Selected were 276 patients over 19 years of age to complete the questionnaire for oral health habit and take QLF images, periapical and panoramic radiographs. Oral health habit score, age, and sex showed a statistically significant correlation with FPI. FPI showed a lower value as the oral health habit score increased and the age decreased. Moreover, females showed lower FPI values than did males. RBL showed a statistically significant positive correlation with age but did not show any correlation with oral health habit scores and sex. There was no correlation between FPI and RBL. The results of this study suggest that the clinical use of QLF allows plaque detection by non-invasive procedures and can aid in a more objective estimation for oral hygiene status.

Efficient Selection of Recombinant Fluorescent Vaccinia Virus Strains and Rapid Virus Titer Determination by Using a Multi-Well Plate Imaging System.

Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte®S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer.

A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging.

Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.

Macrophage targeted theranostic strategy for accurate detection and rapid stabilization of the inflamed high-risk plaque.

Rationale: Inflammation plays a pivotal role in the pathogenesis of the acute coronary syndrome. Detecting plaques with high inflammatory activity and specifically treating those lesions can be crucial to prevent life-threatening cardiovascular events.Methods: Here, we developed a macrophage mannose receptor (MMR)-targeted theranostic nanodrug (mannose-polyethylene glycol-glycol chitosan-deoxycholic acid-cyanine 7-lobeglitazone; MMR-Lobe-Cy) designed to identify inflammatory activity as well as to deliver peroxisome proliferator-activated gamma (PPARγ) agonist, lobeglitazone, specifically to high-risk plaques based on the high mannose receptor specificity. The MMR-Lobe-Cy was intravenously injected into balloon-injured atheromatous rabbits and serial in vivo optical coherence tomography (OCT)-near-infrared fluorescence (NIRF) structural-molecular imaging was performed.Results: One week after MMR-Lobe-Cy administration, the inflammatory NIRF signals in the plaques notably decreased compared to the baseline whereas the signals in saline controls even increased over time. In accordance with in vivo imaging findings, ex vivo NIRF signals on fluorescence reflectance imaging (FRI) and plaque inflammation by immunostainings significantly decreased compared to oral lobeglitazone group or saline controls. The anti-inflammatory effect of MMR-Lobe-Cy was mediated by inhibition of TLR4/NF-κB pathway. Furthermore, acute resolution of inflammation altered the inflamed plaque into a stable phenotype with less macrophages and collagen-rich matrix.Conclusion: Macrophage targeted PPARγ activator labeled with NIRF rapidly stabilized the inflamed plaques in coronary sized artery, which could be quantitatively assessed using intravascular OCT-NIRF imaging. This novel theranostic approach provides a promising theranostic strategy for high-risk coronary plaques.

Clinical assessment of an automated fluorescent plaque index scoring with quantitative light-induced fluorescence.

The aims of this study were to evaluate the clinical applicability of a new fluorescent plaque index scoring (FPI) with the Turesky modified Quigley-Hein plaque index (mQH) and to evaluate its relationship with plaque maturity. In total 69 subjects participated in this study. White-light and fluorescent images of anterior teeth were acquired using a Qraycam (AIOBIO, Seoul, Korea). FPI was obtained from fluorescent images using the proprietary software (Q-Ray v.1.39, Inspektor Research System BV, Amsterdam, The Netherlands). Teeth were stained with a two-tone disclosing agent. mQH was used to manually score the combined red and blue disclosed plaque (Combi-mQH) and blue disclosed plaque (Blue-mQH) with the white-light images. Linear relationships between FPI and Combi-mQH (or Blue-mQH) were evaluated by using simple linear regression analysis. Differences of Combi-mQH (or Blue-mQH) with respect to FPI scores were statistically evaluated by using ANOVA with Duncan post hoc correction. FPI showed a moderate positive correlation with Combi-mQH (r = 0.66, P < 0.001) and a high positive correlation with Blue-mQH (r = 0.78, P < 0.001). The model explanatory power (R2) between FPI and Blue-mQH was 60.8 %, which is 16.8 % higher than the explanatory power observed with Combi-mQH (44.0 %). Both Combi-mQH and Blue-mQH increased significantly with increasing FPI score (P < 0.001). In this study we found that the FPI scoring system can be used to detect plaque and quantitatively distinguish plaque levels. In addition, FPI was determined to be useful in clinic because of its ability to detect and distinguish old and mature plaque.

A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus-host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus-host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

Detection of dental plaque and its potential pathogenicity using quantitative light-induced fluorescence.

Quantitative light-induced fluorescence (QLF) technology can detect some dental plaque as red fluorescence. This in vivo study aimed to identify the microbial characteristics of red fluorescent (RF) dental plaque using 16S rRNA gene sequencing and evaluate the correlations between RF plaque and the clinical symptoms of dental diseases. Paired supragingival plaque samples collected from each 10 subjects and consisted of RF and non-RF dental plaques as observed by QLF technology using a 405 nm blue light source for excitation. The characteristics of the bacterial communities in the RF and non-RF plaque samples were compared by sequencing analysis. An increase in microbial diversity was observed in RF plaque compared with the non-RF plaque. There were significant differences in the community compositions between the 2 types of dental plaque. Periodontopathic bacteria were significantly more abundant in the RF plaque than non-RF plaque. The fluorescence intensity of RF plaque was significantly related to the proportion of the periodontopathic bacterial community and the presence of gingival inflammation. In conclusion, the plaque red fluorescence is associated with changes in the microbial composition and enrichment of periodontopathic pathogens, which suggests that RF plaque detected by QLF technology could be used as a risk indicator for gingival inflammation.

COMPARISON OF DENTAL PLAQUE DETECTION USING VARIOUS WAVELENGTH OF LIGHT EMITTING DIODE (LED) (Analysis based on digital imaging techniques)

Background: Dental plaque indicates the grade of dental hygiene. Bacterial dental plaque will emit reddish fluorescence, when irradiated by compatible rays. Fluorescence is produced by an object that absorbs appropriate light spectrum photons. Advances technology has developed Light Emitting Diode (LED) that can emit visible light with low energy. Objective: The purpose of this research is to observe the wavelength of LED light that can be used as dental plaque detector. Methods: A quasi experimental study was done on 44 maxillary and mandibular central incisor teeth, Muallimin Yogyakarta boarders. Teeth were exposed using LED colour ring lamp with 400nm wavelength (UV), 420nm (violet), and 450nm (blue). Teeth were photographed using DSLR camera. As control, teeth were applied with disclosing agent. Image result observed by determining the reddish dental plaque fluorescence and counting surface with software design by SST-Laboratory, Faculty of Engineering, UGM, based on digital imaging technique. The comparison of fluorescence surface plaque area between UV light detection, violet, and blue by using disclosing agent which had been undertaken. Data were analyzed by using nonparametric Wilcoxon test. Result: Detection using UV LED 400nm showed reddish fluorescent dental plaque surface (25,7632+20,8247), violet 420nm and blue 450nm showed no fluorescence area, and as control group (29,9177 + 22,1266). Nonparametric Wilcoxon test results showed that there were no significant difference between detection of 400nm UV LED and disclosing agent (p> 0.05). Conclusion: The conclusion of this study is the wavelength of the LED light be able to used as a dental plaque detector is UV LED wavelength 400nm.

Red fluorescence imaging for dental plaque detection and quantification: pilot study.

The red fluorescence of dental plaque originating from porphyrins in oral bacteria may allow visualization, detection, and scoring of plaque without disclosing agents. Two studies were conducted. The first included 24 healthy participants who abstained from oral hygiene for 24h. Dental plaque was collected from tooth surfaces, and a 10% solution was prepared. These were scanned by a molecular spectrometer to identify the optimum excitation and emission wavelengths of plaque for developing a red fluorescence imaging system. Fourteen healthy subjects completed the second study. After a washout period (1 week), participants had a prophylaxis at baseline and abstained from oral hygiene during the study. They were monitored using the fluorescence imaging system at baseline, 24h, and 48h. A dentist clinically assessed plaque after disclosing and on red fluorescence images. Three descriptors were extracted from images and a RUSBoost classifier derived computer fluorescence scores through cross-validation. Red fluorescence plaque levels increased during the 48-h accumulation. Plaque progression was identified by dentist assessment and computer analysis, presenting significant differences between visits at tooth and subject levels (p<0.05). Moderate correlations showed between clinical plaque and red fluorescence plaque (r=0.62 dentist, r=0.55 computer). The best agreement was observed when disclosing plaque threshold at level 2, for both dentist evaluation (sensitivity 71.1%, specificity 67.7%, accuracy 70.2%) and computer classification (sensitivity 68.4%, specificity 62.9%, accuracy 67.1%). Given the correlation with clinical diagnosis, red fluorescence imaging shows its potential for providing an objective and promising method for proper oral hygiene assessment.

Chair-side detection of Prevotella Intermedia in mature dental plaque by its fluorescence

BackgroundPrevotella intermedia/nigrescens is one of the well-known pathogens causing periodontal diseases, and the red florescence excited by the visible blue light caused by the protoporphyrin IX in the bacterial cells could be useful for the chair-side detection. The aim of this study was to evaluated levels of periodontal pathogen, especially P. intermedia in clinical samples of red fluorescent dental plaque. MethodsThirty two supra gingival plaque samples from six individuals were measured its fluorescence at 640nm wavelength excited by 409nm. Periodontopathic bacteria were counted by the Invader PLUS PCR assay. Co-relations the fluorescence intensity and bacterial counts were analyzed by Person’s correlation coefficient and simple and multiple regression analysis. Positive and negative predictive values of the fluorescence intensities for with or without P. intermedia in supragingival plaque was calculated. ResultsWhen relative fluorescence unit (RFU) were logarithmic transformed, statistically significant linear relations between RFU and bacterial counts were obtained for P. intermedia, Porphyromonas gingivalis and Tannerella forsythia. By the multiple regression analysis, only P. intermedia had statistically significant co-relation with fluorescence intensities. All of the fluorescent dental plaque contained P. intermedia m. In contrast, 28% of non-fluorescent plaques contained P. intermedia. ConclusionTo check the fluorescence dental plaque in the oral cavity could be the simple chair-side screening of the mature dental plaque before examining the periodontal pathogens especially P. intermedia by the PCR method.

Red fluorescence of dental plaque in children —A cross-sectional study

ObjectivesThe relation between the presence of red fluorescent plaque and the caries status in children was studied. In addition, the microbial composition of dental plaque from sites with red fluorescent plaque (RFP) and from sites with no red fluorescent plaque (NFP) was assessed. MethodsFluorescence photographs were taken from fifty children (6–14 years old) with overnight plaque. Full-mouth caries scores (ICDAS II) were obtained. The composition of a saliva sample and two plaque samples (RFP and NFP) was assessed using 16S rDNA sequencing. ResultsAt the site level, no clinically relevant correlations were found between the presence of RFP and the caries status. At the subject level, a weak correlation was found between RFP and the caries status when non-cavitated lesions were included (rs=0.37, p=0.007). The microbial composition of RFP differed significantly from NFP. RFP had more anaerobes and more Gram-negative bacterial taxa. The most discriminative operational taxonomic units (OTUs) for RFP were Corynebacterium, Leptotrichia, Porphyromonas and Selenomonas, while the most discriminative OTUs for NFP were Neisseria, Actinomyces, Streptococcus and Rothia. ConclusionsThere were no clinical relevant correlations in this cross-sectional study between the presence of RFP and (early) caries lesions. There were differences in the composition of these phenotypically different plaque samples: RFP contained more Gram-negative, anaerobic taxa and was more diverse than NFP.Clinical significance: The study outcomes provide more insight in the possibilities to use plaque fluorescence in oral health risk assessments.

Respiratory Syncytial Virus-Infected Mesenchymal Stem Cells Regulate Immunity via Interferon Beta and Indoleamine-2,3-Dioxygenase.

Respiratory syncytial virus (RSV) has been reported to infect human mesenchymal stem cells (MSCs) but the consequences are poorly understood. MSCs are present in nearly every organ including the nasal mucosa and the lung and play a role in regulating immune responses and mediating tissue repair. We sought to determine whether RSV infection of MSCs enhances their immune regulatory functions and contributes to RSV-associated lung disease. RSV was shown to replicate in human MSCs by fluorescence microscopy, plaque assay, and expression of RSV transcripts. RSV-infected MSCs showed differentially altered expression of cytokines and chemokines such as IL-1β, IL6, IL-8 and SDF-1 compared to epithelial cells. Notably, RSV-infected MSCs exhibited significantly increased expression of IFN-β (~100-fold) and indoleamine-2,3-dioxygenase (IDO) (~70-fold) than in mock-infected MSCs. IDO was identified in cytosolic protein of infected cells by Western blots and enzymatic activity was detected by tryptophan catabolism assay. Treatment of PBMCs with culture supernatants from RSV-infected MSCs reduced their proliferation in a dose dependent manner. This effect on PBMC activation was reversed by treatment of MSCs with the IDO inhibitors 1-methyltryptophan and vitamin K3 during RSV infection, a result we confirmed by CRISPR/Cas9-mediated knockout of IDO in MSCs. Neutralizing IFN-β prevented IDO expression and activity. Treatment of MSCs with an endosomal TLR inhibitor, as well as a specific inhibitor of the TLR3/dsRNA complex, prevented IFN-β and IDO expression. Together, these results suggest that RSV infection of MSCs alters their immune regulatory function by upregulating IFN-β and IDO, affecting immune cell proliferation, which may account for the lack of protective RSV immunity and for chronicity of RSV-associated lung diseases such as asthma and COPD.

Dynamics of red fluorescent dental plaque during experimental gingivitis—A cohort study

ObjectivesThe dynamics of red fluorescent plaque (RFP) in comparison to clinical plaque and bleeding scores were studied during an experimental gingivitis protocol in a cohort of healthy participants. MethodsForty-one participants were monitored for RFP before (24h plaque), during 14 days plaque accumulation (days 2, 5, 9, 14) and after 7 days recovery (24h plaque). RFP was assessed on fluorescence photographs of the vestibular aspect of the anterior teeth (cuspid to cuspid) in the upper and lower jaw. Clinical plaque and bleeding were assessed at days −14, 0, 14 and 21. ResultsRFP of 24h plaque was reproducible (days −14, 0), then increased during 14 days plaque accumulation and returned to baseline after 7 days recovery. Groups of low, moderate and high RFP formers were statistically significantly different at all times even already at baseline. The individual RFP response during 14 days plaque accumulation correlated well with RFP of 24h plaque (days −14, 0). RFP correlated moderate to well with clinical plaque at days −14, 0, 14 and 21. From day 2 of the gingivitis challenge RFP correlated with bleeding at day 14. ConclusionsRFP provided an objective measure of oral hygiene status. Given the correlation with clinical parameters found, the amount of RFP after 24h plaque accumulation was indicatory for the inflammatory response during a prolonged period of no oral hygiene. This trial was registered at the public trial register ​of the Central Committee on Research Involving Human Subjects (CCMO) under number NL51111.029.14 Clinical significanceThis paper shows the association between RFP after 24h plaque accumulation and inflammatory response after a prolonged period of no oral hygiene. Red plaque fluorescence can be used to identify subjects at risk for developing gingival inflammation.

Seven major genomic deletions of vaccinia virus Tiantan strain are sufficient to decrease pathogenicity

Attenuated strain TTVAC7, as a multi-gene-deleted vaccinia virus Tiantan strain (VTT), was constructed by knocking out parts of non-essential genes related to virulence, host range and immunomodulation of VTT, and by combining double marker screening with exogenous selectable marker knockout techniques. In this study, shuttle vector plasmids pTC-EGFP, pTA35-EGFP, pTA66-EGFP, pTE-EGFP, pTB-EGFP, pTI-EGFP and pTJ-EGFP were constructed, which contained seven pairs of recombinant arms linked to the early and late strong promoter pE/L, as well as to enhanced green fluorescent protein (EGFP) as an exogenous selectable marker. BHK cells were co-transfected/infected successively with the above plasmids and VTT or gene-deleted VTT, and homologous recombination and fluorescence plaque screening methods were used to knock out the gene fragments (TC: TC7L ∼ TK2L; TA35: TA35L; TA66: TA66R; TE: TE3L ∼ TE4L; TB: TB13R; TI: TI4L; TJ: TJ2R). The Cre/LoxP system was then applied to knock out the exogenous selectable marker, and ultimately the gene-deleted attenuated strain TTVAC7 was obtained. A series of in vivo and in vitro experiments demonstrated that not only the host range of TTVAC7 could be narrowed and its toxicity weakened significantly, but its high immunogenicity was maintained at the same time. These results support the potential of TTVAC7 to be developed as a safe viral vector or vaccine.

Assessing the use of Quantitative Light-induced Fluorescence-Digital as a clinical plaque assessment

BackgroundThe aims of this study were to compare the relationship between red fluorescent plaque (RF plaque) area by Quantitative Light-induced Fluorescence-Digital (QLF-D) and disclosed plaque area by two-tone disclosure, and to assess the bacterial composition of the RF plaque by real time-PCR. MethodsFifty healthy subjects were included and 600 facial surfaces of their anterior teeth were examined. QLF-D was taken on two separate occasions (before and after disclosing), and the RF plaque area was calculated based on Plaque Percent Index (PPI). After disclosing, the stained plaque area was analyzed to investigate the relationship with the RF plaque area. The relationship was evaluated using Pearson correlation and paired t-test. Then, the RF and non-red fluorescent (non-RF) plaque samples were obtained from the same subject for real-time PCR test. Total 10 plaque samples were compared the ratio of the 6 of bacteria using Wilcoxon signed rank test. ResultsRegarding the paired t-test, the blue-staining plaque area (9.3±9.2) showed significantly similarity with the RF plaque area (9.1±14.9, p=0.80) at ΔR20, however, the red-staining plaque area (31.6±20.9) presented difference from the RF plaque area (p<0.0001). In addition, bacterial composition of Prevotella intermedia and Streptococcus anginosus was associated with substantially more the RF plaque than the non-RF plaque (p<0.05). ConclusionsThe plaque assessment method using QLF-D has potential to detect mature plaque, and the plaque area was associated with the blue-staining area using two-tone disclosure.

Phage-mediated counting by the naked eye of miRNA molecules at attomolar concentrations in a Petri dish.

The ability to count biomolecules such as cancer-biomarker miRNAs with the naked eye is seemingly impossible in molecular diagnostics. Here, we show an ultrasensitive naked-eye-counting strategy for quantifying miRNAs by employing T7 phage-a bacteria-specific virus nanoparticle-as a surrogate. The phage is genetically engineered to become fluorescent and capable of binding a miRNA-capturing gold nanoparticle (GNP) in a one-to-one manner. Target miRNAs crosslink the resultant phage-GNP couple and miRNA-capturing magnetic microparticles, forming a sandwich complex containing equimolar phage and miRNA. The phage is then released from the complex and developed into one macroscopic fluorescent plaque in a Petri dish by plating it in a host bacterial medium. Counting the plaques by the naked eye enables the quantification of miRNAs with detection limits of ∼3 and ∼5 aM for single-target and two-target miRNAs, respectively. This approach offers ultrasensitive and convenient quantification of disease biomarkers by the naked eye.

Evaluation of the co-existence of the red fluorescent plaque bacteria P. gingivalis with S. gordonii and S. mutans in white spot lesion formation during orthodontic treatment

BackgroundEarly detection of white spot lesions (WSLs) around brackets during orthodontic treatment is important for treatment and prevention. But it is unclear whether red fluorescent plaque (RFP) bacteria Porphyromonas gingivalis and its co-existence with Streptococcus mutans and Streptococcus gordonii has any significant influence on this. Therefore the role of this bacterial co existence and WSLs formation during one year of fixed orthodontic therapy was evaluated. MethodsFourteen 12 to 22 year old (mean 15±3 years) consecutive patients attending the University of Liverpool dental hospital were recruited for this study. Quantitative Light-induced Fluorescence (QLF) was used to identify RFP and enamel demineralisation, respectively, on anterior labial surfaces before and after placement of fixed orthodontic appliances. Bacterial composition was determined by denaturing gradient gel electrophoresis (DGGE) following nested PCR amplification of the 16S rRNA V2-V3 hypervariable region. ResultsWSLs were recorded on 4.2% of tooth surfaces and WSL development was not associated with RFP bacteria P. gingivalis presence. Differences in RFP bacteria P. gingivalis presence with S. mutans and S. gordonii, were observed before and after appliance placement. Intra subject changes in plaque flora between visits were not significantly associated with WSL development (p>0.05). However, DGGE profiles indicated that apart from S. mutans, S. gordonii might also have a role in human enamel demineralisation. ConclusionsFixed orthodontic brackets in adolescents may play a role in altering bacterial composition around brackets during orthodontic treatment and it is plausible that S. gordonii also have a role in human enamel demineralisation. Combinatorial approach of QLF technology and DGGE may be useful in determining bacterial composition during orthodontic therapy which could inform clinical interventions.