Sustainable Development Goals' health-related indicators for Brazil and Ecuador: an analysis for the period of 1990-2019.

OBJECTIVE: This article aims to analyse the evolution of 40 Sustainable Development Goals' (SDGs) health-related indicators in Brazil and Ecuador from 1990 to 2019. STUDY DESIGN: Epidemiological study of long-term trends in 40 SDGs' health-related indicators for Brazil and Ecuador from 1990 to 2019, using estimates from the Global Burden of Disease Study. METHODS: Forty SDGs' health-related indicators and an index from 1990 to 2017 for Brazil and Ecuador, and their projections up to 2030 were extracted from the Institute for Health Metrics and Evaluation's Global Burden of Disease website and analysed. The percent annual change (PC) between 1990 and 2019 was calculated for both countries. RESULTS: Both countries have made progress on child stunting (Brazil: PC = -38%; Ecuador: PC = -43%) and child wasting prevalences (Brazil: PC = -42%; Ecuador: PC = -41%), percent of vaccine coverage (Brazil: PC = +215%; Ecuador: PC = +175%), under-5 (Brazil: PC = -75%; Ecuador: PC = -60%) and neonatal mortality rates (Brazil: PC = -69%; Ecuador: PC = -51%), health worker density per 1000 population (Brazil: PC = +153%; Ecuador: PC = +175%), reduction of neglected diseases prevalences (Brazil: PC = -40%; Ecuador: PC = -58%), tuberculosis (Brazil: PC = -27%; Ecuador: PC = -55%) and malaria incidences (Brazil: PC = -97%; Ecuador: PC = -100%), water, sanitation and hygiene mortality rates (Brazil and Ecuador: PC = -89%). However, both countries did not show sufficient improvement in maternal mortality ratio to meet SDGs targets (Brazil: PC = -37%; Ecuador: PC = -40%). Worsening of indicators were found for violence, such as non-intimate partner violence for both countries (Brazil: PC = +26%; Ecuador: PC = +18%) and suicide mortality rate for Ecuador (PC = +66%), child overweight indicator for Brazil (PC = -67%), disaster mortality rates (Brazil: PC = +100%; Ecuador: PC = +325%) and alcohol consumption (Brazil: PC = +46%; Ecuador: PC = +35%). CONCLUSIONS: Significant improvements are necessary in both countries requiring the strengthening of health and other policies, particularly concerning the prevention and management of violence and alcohol consumption, and preparedness for dealing with environmental disasters.

Electrokinetic Active Particles for Motion-Based Biomolecule Detection.

Detection of biomolecules is essential for patient diagnosis, disease management, and numerous other applications. Recently, nano- and microparticle-based detection has been explored for improving traditional assays by reducing required sample volumes and assay times as well as enhancing tunability. Among these approaches, active particle-based assays that couple particle motion to biomolecule concentration expand assay accessibility through simplified signal outputs. However, most of these approaches require secondary labeling, which complicates workflows and introduces additional points of error. Here, we show a proof-of-concept for a label-free, motion-based biomolecule detection system using electrokinetic active particles. We prepare induced-charge electrophoretic microsensors (ICEMs) for the capture of two model biomolecules, streptavidin and ovalbumin, and show that the specific capture of the biomolecules leads to direct signal transduction through ICEM speed suppression at concentrations as low as 0.1 nM. This work lays the foundation for a new paradigm of rapid, simple, and label-free biomolecule detection using active particles.

Bubble-Based Microrobots with Rapid Circular Motions for Epithelial Pinning and Drug Delivery.

Remotely powered microrobots are proposed as next-generation vehicles for drug delivery. However, most microrobots swim with linear trajectories and lack the capacity to robustly adhere to soft tissues. This limits their ability to navigate complex biological environments and sustainably release drugs at target sites. In this work, bubble-based microrobots with complex geometries are shown to efficiently swim with non-linear trajectories in a mouse bladder, robustly pin to the epithelium, and slowly release therapeutic drugs. The asymmetric fins on the exterior bodies of the microrobots induce a rapid rotational component to their swimming motions of up to ≈150 body lengths per second. Due to their fast speeds and sharp fins, the microrobots can mechanically pin themselves to the bladder epithelium and endure shear stresses commensurate with urination. Dexamethasone, a small molecule drug used for inflammatory diseases, is encapsulated within the polymeric bodies of the microrobots. The sustained release of the drug is shown to temper inflammation in a manner that surpasses the performance of free drug controls. This system provides a potential strategy to use microrobots to efficiently navigate large volumes, pin at soft tissue boundaries, and release drugs over several days for a range of diseases.

Search for a Nonrelativistic Boson in Two-Body Antimuon Decay.

We demonstrate the feasibility of probing the charged lepton-flavor-violating decay µ^{+}→e^{+}X^{0} for the presence of a slow-moving neutral boson X^{0} capable of undergoing gravitational binding to large structures and, as such, able to participate in some cosmological scenarios. A short exposure to surface antimuons from beam line M20 at TRIUMF generates a branching ratio limit of ≲10^{-5}. This is comparable to or better than previous searches for this channel, although in a thus-far-unexplored region of X^{0} phase space very close to the kinematic limit of the decay, where m_{X^{0}} approaches m_{µ^{+}}. The future improved sensitivity of the method using a customized p-type point-contact germanium detector is described.

Antimicrobial Efficacy of Different Irrigant Solutions Using a Novel Biofilm Model: An In Vitro Confocal Laser Scanning Microscopy Experiment.

AIM: To determine the ability of different irrigation solutions to biomechanically remove Enterococcus faecalis biofilm from a novel artificial root canal model during chemomechanical preparation. METHODS: High resolution micro-computer-tomography scans of a mandibular molar's mesial root were used to produce 50 identical 3D-printed resin root canal models. These were cultured with E.faecalis over seven days to generate biofilm and subjected to chemomechanical preparation using: saline; 17% ethylenediaminetetraacetic acid (EDTA) or 2% sodium hypochlorite (NaOCl) alongside positive/negative controls (n = 10). Canals were prepared to 40/.06 taper, with 1 mL irrigation between instruments, followed by 5 mL penultimate rinse, 30 s ultrasonic activation and 5 mL final rinse. Residual biofilm volume (pixels) was determined following immunofluorescent staining and confocal-laser-scanning-microscopy imaging. Statistical comparisons were made using Kruskal-Wallis with post-hoc Dunn's tests (α ⟨0.05). RESULTS: In all canal thirds, the greatest biofilm removal was observed with NaOCl, followed by EDTA and saline. The latter had significantly higher E.faecalis counts than NaOCl and EDTA (P ⟨0.01). However, no statistical differences were found between EDTA and NaOCl or saline and positive controls (P ⟩0.05). CONCLUSIONS: Within limitations of this model, 17% EDTA was found to be as effective as 2% NaOCl at eradicating E.faecalis biofilm following chemomechanical preparation. Further investigations with multi-species biofilms are encouraged.

Search for Lepton Number and Flavor Violation in K

Searches for the lepton number violating K^{+}→π^{-}µ^{+}e^{+} decay and the lepton flavor violating K^{+}→π^{+}µ^{-}e^{+} and π^{0}→µ^{-}e^{+} decays are reported using data collected by the NA62 experiment at CERN in 2017-2018. No evidence for these decays is found and upper limits of the branching ratios are obtained at 90% confidence level: B(K^{+}→π^{-}µ^{+}e^{+})<4.2×10^{-11}, B(K^{+}→π^{+}µ^{-}e^{+})<6.6×10^{-11} and B(π^{0}→µ^{-}e^{+})<3.2×10^{-10}. These results improve by 1 order of magnitude over previous results for these decay modes.

Potential application of immunotherapy for modulation of pulp inflammation: opportunities for vital pulp treatment.

Caries results in the demineralization and destruction of enamel and dentine, and as the disease progresses, irreversible pulpitis can occur. Vital pulp therapy (VPT) is directed towards pulp preservation and the prevention of the progression of inflammation. The outcomes of VPT are not always predictable, and there is often a poor correlation between clinical signs and symptoms, and the events occurring at a molecular level. The inflamed pulp expresses increased levels of cytokines, including tumour necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1ß, IL-4, IL-6, IL-8, IL-17 and IL-23, which recruit and drive a complex cellular immune response. Chronic inflammation and sustained cytokine release can result in irreversible pulp damage and a decreased capacity for tissue healing. Other chronic inflammatory diseases, such as psoriasis, inflammatory bowel diseases and rheumatoid arthritis, are also characterized by an dysregulated immune response composed of relatively high cytokine levels and increased numbers of immune cells along with microbial and hard-soft tissue destructive pathologies. Whilst anti-cytokine therapies have been successfully applied in the treatment of these diseases, this approach is yet to be attempted in cases of pulp inflammation. This review therefore focuses on the similarities in the aetiology between chronic inflammatory diseases and pulpitis, and explores how anti-cytokine therapies could be applied to manage an inflamed pulp and facilitate healing. Further proof-of-concept studies and clinical trials are justified to determine the effectiveness of these treatments to enable more predictable outcomes in VPT.

Investigation of microbial profile, levels of endotoxin and lipoteichoic acid in teeth with symptomatic irreversible pulpitis: a clinical study.

AIM: To investigate the microbial profile, and levels of endotoxin (LPS) and lipoteichoic acid (LTA), in infected dentine (ID) and root canals (RC) at different phases of root canal treatment in teeth with symptomatic irreversible pulpitis. METHODOLOGY: Ten volunteers were included, and samples were collected from infected dentine (ID) and the root canal lumen (RC) using sterile excavators and paper points, respectively. RC samples were taken before (S1) and after (S2) chemo-mechanical canal preparation (CMP), and after intracanal medication (ICM; S3). Checkerboard DNA-DNA hybridization was used for microbial analysis. The levels of LPS and LTA were evaluated using the limulus amebocyte lysate assay and ELISA, respectively. Shapiro-Wilk's test was used to verify data normality. Friedman's test was used to evaluate statistical differences using checkerboard DNA-DNA hybridization in the ID and RC at the different phases of the RC treatment. Post hoc Dunn's multiple comparison test was used to verify significant differences recorded at the different time-points. The levels of LPS and LTA were analysed statistically by using repeated measures anova and Tukey's post hoc test to evaluate differences in both sites. The significance level was set at 5% (P < 0.05). RESULTS: A total of 40 DNA probes were used for microbial investigation of ID and RC samples using checkerboard DNA-DNA hybridization. The levels and complexity of bacteria were similar in the ID and initial RC samples. The levels of LPS and LTA in ID were significantly higher than the initial RC samples (S1; P < 0.05). Canal preparation was effective in significantly decreasing the levels of bacteria, LPS and LTA (P < 0.05). ICM did not provide additional reduction in the levels of bacteria and LPS (P > 0.05). However, a significant reduction in the levels of LTA was observed after ICM (P < 0.05). CONCLUSION: The microbial profile of infected dentine and root canals of teeth with irreversible pulpitis was complex, harbouring different species including Gram-positive and Gram-negative, cocci and bacilli, and facultative and strict anaerobes. Root canal preparation was effective in reducing the levels of bacteria, LPS and LTA from the root canals of teeth with pulpitis.

Photobiomodulation of oral fibroblasts stimulated with periodontal pathogens.

Photobiomodulation (PBM) utilises light energy to treat oral disease, periodontitis. However, there remains inconsistency in the reporting of treatment parameters and a lack of knowledge as to how PBM elicits its molecular effects in vitro. Therefore, this study aimed to establish the potential immunomodulatory effects of blue and near infra-red light irradiation on gingival fibroblasts (GFs), a key cell involved in the pathogenesis of periodontitis. GFs were seeded in 96-well plates in media + / - Escherichia coli lipopolysaccharide (LPS 1 µg/ml), or heat-killed Fusobacterium nucleatum (F. nucleatum, 100:1MOI) or Porphyromonas gingivalis (P. gingivalis, 500:1MOI). Cultures were incubated overnight and subsequently irradiated using a bespoke radiometrically calibrated LED array (400-830 nm, irradiance: 24 mW/cm2 dose: 5.76 J/cm2). Effects of PBM on mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assays, total reactive oxygen species production (ROS assay) and pro-inflammatory/cytokine response (interleukin-8 (IL-8) and tumour growth factor-ß1 (TGFß1)) were assessed 24 h post-irradiation. Data were analysed using one-way ANOVA followed by the Tukey test. Irradiation of untreated (no inflammatory stimulus) cultures at 400 nm induced 15%, 27% and 13% increases in MTT, ROS and IL-8 levels, respectively (p < 0.05). Exposure with 450 nm light following application of P. gingivalis, F. nucleatum or LPS induced significant decreases in TGFß1 secretion relative to their bacterially stimulated controls (p < 0.001). Following stimulation with P. gingivalis, 400 nm irradiation induced 14% increases in MTT, respectively, relative to bacteria-stimulated controls (p < 0.05). These findings could identify important irradiation parameters to enable management of the hyper-inflammatory response characteristic of periodontitis.

The influence of irrigant activation, concentration and contact time on sodium hypochlorite penetration into root dentine: an ex vivo experiment.

AIM: To establish whether irrigant activation techniques, namely manual dynamic activation (MDA), passive ultrasonic irrigation (PUI) and sonic irrigation (SI), improve the tubular penetration of sodium hypochlorite (NaOCl) into root dentine when compared with conventional needle irrigation (CNI). Secondly, investigate if increasing NaOCl concentration and/or contact time improves the performance of these techniques. METHODOLOGY: A total of 83 extracted human maxillary permanent canines were decoronated to 15 mm, and root canals prepared to a size 40, .10 taper. Root dentine was stained with crystal violet for 72 h and embedded in silicone. Eighty specimens were randomly distributed into 16 groups (n = 5) according to the irrigant activation technique, NaOCl concentration (2%; 5.25%) and irrigant contact time (10 min; 20 min). All activation techniques were used for 60 s in the last minute of irrigation. Additionally, three teeth were not exposed to NaOCl to confirm adequate dentine staining had occurred (i.e. negative control). All specimens were subsequently dissected, observed under a light microscope and NaOCl penetration depth (µm) determined by measuring the average width of bleached dentine using ImageJ software. Statistical comparisons were made with paired and unpaired t-tests, anovas followed by post hoc Tukey's and Dunnett's tests, and a general linear model (α < 0.05). RESULTS: Overall, NaOCl penetration ranged from 38.8 to 411.0 µm with MDA, PUI and SI consistently resulting in significantly greater tubular infiltration than CNI (P < 0.05). The deepest measurements in the coronal, middle and apical segments were all recorded in the MDA; 5.25%; 20 min group and the least in the CNI; 2%; 10 min group. Increasing either irrigant concentration or contact time resulted in significantly greater NaOCl penetration depths for all techniques and segments of the canal (P < 0.05). However, when irrigant concentration and contact time were increased together, a significant interaction effect between these two independent variables was observed on overall NaOCl penetration (P < 0.05). CONCLUSIONS: Agitating irrigants with MDA, PUI or SI, as well as using greater irrigant concentrations or contact times, potentiated NaOCl penetration into root dentine. However, longer durations of NaOCl exposure at lower concentrations resulted in similar depths of tubular penetration as those achieved at higher concentrations.

Violet-Blue Light Arrays at 405 Nanometers Exert Enhanced Antimicrobial Activity for Photodisinfection of Monomicrobial Nosocomial Biofilms.

Light-emitting diodes (LEDs) demonstrate therapeutic effects for a range of biomedical applications, including photodisinfection. Bands of specific wavelengths (centered at 405 nm) are reported to be the most antimicrobial; however, there remains no consensus on the most effective irradiation parameters for optimal photodisinfection. The aim of this study was to assess decontamination efficiency by direct photodisinfection of monomicrobial biofilms using a violet-blue light (VBL) single-wavelength array (SWA) and multiwavelength array (MWA). Mature biofilms of nosocomial bacteria (Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) were grown on 96-well polypropylene PCR plates. The biofilms were then exposed to VBL for 2,700 s (SWA) and 1,170 s (MWA) to deliver 0 to 670 J/cm2, and the antibacterial activity of VBL was assessed by comparing the seeding of the irradiated and the nonirradiated biofilms. Nonirradiated groups were used as controls. The VBL arrays were characterized optically (spectral irradiance and beam profile) and thermally. The SWA delivered 401-nm VBL and the MWA delivered between 379-nm and 452-nm VBL, albeit at different irradiances and with different beam profiles. In both arrays, the irradiated groups were exposed to increased temperatures compared to the nonirradiated controls. All bacterial isolates were susceptible to VBL and demonstrated reductions in the seeding of exposed biofilms compared with the nonirradiated controls. VBL at 405 nm exerted the most antimicrobial activity, exhibiting reductions in seeding of up to 94%. Decontamination efficiency is dependent on the irradiation parameters, bacterial species and strain, and experimental conditions. Controlled experiments that ameliorate the heating effects and improve the optical properties are required to optimize the dosing parameters to advance the successful clinical translation of this technology.IMPORTANCE This study reports the efficacy of VBL and blue light (BL) and their antimicrobial activity against mature biofilms of a range of important nosocomial pathogens. While this study investigated the antibacterial activity of a range of wavelengths of between 375 and 450 nm and identified a specific wavelength region (∼405 nm) with increased antibacterial activity, decontamination was dependent on the bacterial species, strain, irradiation parameters, and experimental conditions. Further research with controlled experiments that ameliorate the heating effects and improve the optical properties are required to optimize the dosing parameters to advance the successful clinical translation of this technology.

Dissecting dentine-pulp injury and wound healing responses: consequences for regenerative endodontics.

A thorough understanding of the biology of the dentine-pulp complex is essential to underpin new treatment approaches and maximize clinical impact for regenerative endodontics and minimally invasive vital pulp treatment (VPT) strategies. Following traumatic and carious injury to dentine-pulp, a complex interplay between infection, inflammation and the host defence responses will occur, which is critical to tissue outcomes. Diagnostic procedures aim to inform treatment planning; however, these remain clinically subjective and have considerable limitations. As a consequence, significant effort has focussed on identification of diagnostic biomarkers, although these are also problematic due to difficulties in identifying appropriate diagnostic fluid sources and selecting reproducible biomarkers. This is further compounded by the link between inflammation and repair as many of the molecules involved exhibit significant multifunctionality. The tertiary dentine formed in response to dental injury has been purposefully termed reactionary and reparative dentine to enable focus on associated biological processes. Whilst reactionary dentine produced in response to milder injury is generated from surviving primary odontoblasts, reparative dentine, in response to more intense injury, requires the differentiation of new odontoblast-like cells derived from progenitor/stem cells recruited to the injury site. These two diverse processes result in very different outcomes in terms of the tertiary dentine produced and reflect the intensity rather than specific nature (nonexposure versus exposure) of the injury. The subsequent identification of the odontoblast-like cell phenotype remains challenging due to lack of unique molecular or morphological markers. Furthermore, the cells ultimately lining the newly deposited dentine provide only a snapshot of events. The specific source and plasticity of the progenitor cells giving rise to the odontoblast-like cell phenotype are also of significant debate. It is likely that improved characterization of tertiary dentine may better clarify the influence of cell derivation for odontoblast-like cells and their diversity. The field of regenerative endodontics offers exciting new treatment opportunities, and to maximize outcomes, we propose that the term regenerative endodontics should embrace the repair, replacement and regeneration of dentine-pulp.

A systematic review of methods used to sample and analyse periradicular tissue fluid during root canal treatment.

AIM: The primary aim was to identify techniques used to sample and analyse periradicular tissue fluid (PTF) in permanent teeth diagnosed with apical disease during root canal treatment. Secondly, to identify the types of inflammatory mediators studied using this approach. METHODOLOGY: Data Sources: PubMed, EMBASE, Cochrane Library, Science Direct, Web of Science and OpenGrey. Eligibility Criteria: Clinical studies published until 1 June 2018 which utilized orthograde techniques to sample and analyse PTF were included. Cell culture, laboratory or animal studies and those concerned with investigating inflammatory mediator activity from within healthy or diseased pulp tissue, and not periradicular tissues, were excluded. Study appraisal and methods: In accordance with PRISMA guidelines, data were extracted on study characteristics, target mediators, sampling and assay techniques and the parameters associated with the PTF sampling and eluting protocol. A qualitative synthesis was conducted, and studies were critically appraised using a modified version of the Cochrane risk of bias tool. RESULTS: Study Characteristics: From 251 studies, 33 were eligible for inclusion. Sampling techniques included the use of paper points (n = 27), fine needle aspiration (n = 4) and filter strips (n = 2). Assay techniques included enzyme-linked immunosorbent assay (n = 18), quantitative polymerase chain reaction (n = 9), radioimmunoassay (n = 4), colorimetric assay (n = 2), immunofluorometric assay (n = 1) and cytometric bead array (n = 1). Forty-five different inflammatory mediators were targeted at the proteomic/metabolomic (n = 25) or transcriptomic level (n = 9). LIMITATIONS: Significant heterogeneity exists within the methodology, and only 5 studies disclosed unambiguous information about their PTF sampling and eluting protocols. CONCLUSIONS: Paper points and proteomic/metabolomic analysis are currently the preferred methods for studying and analysing PTF during root canal treatment. The most studied analytes were IL-1ß and TNF-α. IMPLICATIONS: Further research is required to develop an optimized PTF sampling and eluting protocol to overcome methodological heterogeneity, and future studies are advised to follow a standardized approach to reporting their methodology.

Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection.

Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a 'destructive' process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy-based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal. The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.

The effectiveness of hyperbaric oxygen therapy for healing chronic venous leg ulcers: A randomized, double-blind, placebo-controlled trial.

Over 30% of venous leg ulcers do not heal despite evidence-based treatment. This study aimed to determine the effectiveness of Hyperbaric Oxygen Therapy (HBOT) as an adjunct treatment for nonhealing venous leg ulcers. A randomized, double-blind, parallel group, placebo-controlled trial was undertaken in three hyperbaric medicine units. Adults with a venous leg ulcer, Transcutaneous Oxygen Measurement indicative of a hypoxic wound responsive to oxygen challenge, and without contraindications for HBOT; were eligible. Of 84 eligible patients, 10 refused and 74 enrolled. 43 participants achieved over 50% ulcer Percent Area Reduction (PAR) after four weeks of evidence-based care and were thus excluded from the intervention phase. Thirty-one participants were randomized to either 30 HBOT treatments (100% oxygen at 2.4 atmospheres absolute (ATA) for 80 minutes), or 30 "placebo" treatments, receiving a validated "sham" air protocol, initially pressurized to 1.2ATA, then cycled between 1.05-1.2ATA for eight minutes before settling at 1.05ATA. The primary outcome was numbers in each group completely healed. Secondary outcomes were ulcer PAR, pain and quality of life, 12 weeks after commencing interventions. The participants' mean age was 70 years (standard deviation (SD) 12.9) and median ulcer duration at enrolment was 62 weeks (range 4-3120). At 12 weeks, there was no significant difference between groups in the numbers completely healed. The HBOT intervention group had a mean of 95 (SD 6.53) ulcer PAR, compared to 54 (SD 67.8) mean PAR for the placebo group (t = -2.24, p = 0.042, mean difference -40.8, SE 18.2) at 12 weeks. HBOT may improve refractory healing in venous leg ulcers, however patient selection is important. In this study, HBOT as an adjunct treatment for nonhealing patients returned indolent ulcers to a healing trajectory.

Potential role of periodontal pathogens in compromising epithelial barrier function by inducing epithelial-mesenchymal transition.

BACKGROUND AND OBJECTIVE: Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal-like phenotype and this may be induced by exposure to gram-negative bacteria. It has been proposed that EMT is responsible for compromising epithelial barrier function in the pathogenesis of several diseases. However, the possible role of EMT in the pathogenesis of periodontitis has not previously been investigated. The aim of this study therefore was to investigate whether gram-negative, anaerobic periodontal pathogens could trigger EMT in primary oral keratinocytes in vitro. MATERIAL AND METHODS: Primary oral keratinocytes were harvested from labial mandibular mucosa of Wistar Han rats. Cells were exposed to heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis (100 bacteria/epithelial cell) and to 20 µg/mL of Escherichia coli lipopolysaccharide over an 8-day period. Exposure to bacteria did not significantly change epithelial cell number or vitality in comparison with unstimulated controls at the majority of time-points examined. Expression of EMT marker genes was determined by semiquantitative RT-PCR at 1, 5, and 8 days following stimulation. The expression of EMT markers was also assessed by immunofluorescence (E-cadherin and vimentin) and using immunocytochemistry to determine Snail activation. The loss of epithelial monolayer coherence, in response to bacterial challenge, was determined by measuring trans-epithelial electrical resistance. The induction of a migratory phenotype was investigated using scratch-wound and transwell migration assays. RESULTS: Exposure of primary epithelial cell cultures to periodontal pathogens was associated with a significant decrease in transcription (~3-fold) of E-cadherin and the upregulation of N-cadherin, vimentin, Snail, matrix metalloproteinase-2 (~3-5 fold) and toll-like receptor 4. Bacterial stimulation (for 8 days) also resulted in an increased percentage of vimentin-positive cells (an increase of 20% after stimulation with P. gingivalis and an increase of 30% after stimulation with F. nucleatum, compared with controls). Furthermore, periodontal pathogens significantly increased the activation of Snail (60%) and cultures exhibited a decrease in electrical impedance (P < .001) in comparison with unexposed controls. The migratory ability of the cells increased significantly in response to bacterial stimulation, as shown by both the number of migrated cells and scratch-wound closure rates. CONCLUSION: Prolonged exposure of primary rat oral keratinocyte cultures to periodontal pathogens generated EMT-like features, which introduces the possibility that this process may be involved in loss of epithelial integrity during periodontitis.

Cigarette smoke modifies neutrophil chemotaxis, neutrophil extracellular trap formation and inflammatory response-related gene expression.

BACKGROUND AND OBJECTIVE: Cigarette smoking is a major risk factor for periodontitis, and smoking perturbs neutrophil reactive oxygen species production. This study tested the hypothesis that cigarette smoke extract (CSE) and its components/metabolites nicotine, cotinine and thiocyanate (SCN-), may influence neutrophil functions. MATERIAL AND METHODS: Chemotaxis was assessed in neutrophils pre-treated with CSE using real-time video microscopy. Neutrophil extracellular trap (NET) release in response to CSE, nicotine, cotinine, SCN- as well as to phorbol 12-myristate-13-acetate and hypochlorous acid following pre-treatment with CSE, nicotine, cotinine or SCN- was assessed using fluorescence-based assays. The impact of CSE and SCN- treatment on neutrophil respiratory burst- and inflammation-related gene expression (NFKBIE, DNAJB1, CXCL8, NCF1, NCF2, CYBB) was determined by real-time polymerase chain reaction. RESULTS: Both CSE and SCN- pre-treatment inhibited phorbol 12-myristate-13-acetate-stimulated NET release. Additionally, SCN- inhibited hypochlorous acid-stimulated NET formation, while SCN- alone stimulated NET release. Overall, neutrophils pre-treated with CSE exhibited reduced speed, velocity and directionality relative to untreated neutrophils. Although CSE and SCN- promoted DNAJB1 expression, increased redox-related gene expression was only detected in response to SCN-. CONCLUSION: These results suggest that CSE can alter ex vivo neutrophil activation by mechanisms independent of SCN- and nicotine, and SCN- may contribute to the perturbed innate immune responses observed in smokers.

Effects of histone deacetylase inhibitors on regenerative cell responses in human dental pulp cells.

AIM: To investigate the growth, migratory and adhesive effects of trichostatin A (TSA) and valproic acid (VPA), two histone deacetylase inhibitors (HDACis), on human dental pulp stem cells (hDPSCs). METHODOLOGY: To verify that TSA or VPA functions as an HDAC inhibitor, the expressions of histones H3 and H4 were examined using Western blotting analysis. hDPSC growth and metabolic activity was evaluated by MTT viability analysis at different time-points and by cell count experiments. The expression of cell cycle regulatory proteins and apoptosis-associated proteins was examined by Western blot analysis. Migration effects were investigated using wound healing and transwell migration assays. An adhesion assay was also performed in the presence and absence of HDACis. The levels of chemokines and adhesion molecules relevant to repair in hDPSCs were also assessed by qRT-PCR and Western blot analysis. The data were analysed, where appropriate, using Student's t-test or one-way anova followed by the Student-Newman-Keuls test using SPSS software. RESULTS: Trichostatin A and VPA enhanced acetylation of histones H3 and H4 (P < 0.05). Significant increases (P < 0.05) in MTT levels in hDPSCs were observed after treatment with TSA (2 and 20 nmol L-1 ) or VPA (1 and 10 mmol L-1 ). Cell numbers were not significantly affected at the concentration of TSA (0.2-10 nmol L-1 ) or VPA (0.01-100 mmol L-1 ) applied compared with the control at 3, 5 or 7 days (P > 0.05). At the same time, the expression of Cdx2 and cyclin A was upregulated by 2 nmol L-1 TSA and 1 mmol L-1 VPA (P < 0.05). Higher TSA or VPA concentrations induced apoptosis in hDPSCs in the cell count and apoptosis experiments (P < 0.05). Moreover, TSA and VPA significantly depressed the expression of Cdx2 and cyclin A (P < 0.05), whilst it significantly improved the level of p21 (P < 0.05). TSA and VPA promoted migration and adhesion of hDPSCs (P < 0.05). The levels of chemokines and adhesion molecules were significantly upregulated after exposure of hDPSCs to 20 nmol L-1 TSA or 1 mmol L-1 VPA (P < 0.05). CONCLUSIONS: Histone deacetylase inhibitors at specific concentrations promoted proliferation, migration and adhesion of hDPSCs, which may contribute to novel regenerative therapies for pulpal disease treatment.

Cleaning lateral morphological features of the root canal: the role of streaming and cavitation.

AIM: To investigate the effects of ultrasonic activation file type, lateral canal location and irrigant on the removal of a biofilm-mimicking hydrogel from a fabricated lateral canal. Additionally, the amount of cavitation and streaming was quantified for these parameters. METHODOLOGY: An intracanal sonochemical dosimetry method was used to quantify the cavitation generated by an IrriSafe 25 mm length, size 25 file inside a root canal model filled with filtered degassed/saturated water or three different concentrations of NaOCl. Removal of a hydrogel, demonstrated previously to be an appropriate biofilm mimic, was recorded to measure the lateral canal cleaning rate from two different instruments (IrriSafe 25 mm length, size 25 and K 21 mm length, size 15) activated with a P5 Suprasson (Satelec) at power P8.5 in degassed/saturated water or NaOCl. Removal rates were compared for significant differences using nonparametric Kruskal-Wallis and/or Mann-Whitney U-tests. Streaming was measured using high-speed particle imaging velocimetry at 250 kfps, analysing both the oscillatory and steady flow inside the lateral canals. RESULTS: There was no significant difference in amount of cavitation between tap water and oversaturated water (P = 0.538), although more cavitation was observed than in degassed water. The highest cavitation signal was generated with NaOCl solutions (1.0%, 4.5%, 9.0%) (P < 0.007) and increased with concentration (P < 0.014). The IrriSafe file outperformed significantly the K-file in removing hydrogel (P < 0.05). Up to 64% of the total hydrogel volume was removed after 20 s. The IrriSafe file typically outperformed the K-file in generating streaming. The oscillatory velocities were higher inside the lateral canal 3 mm compared to 6 mm from WL and were higher for NaOCl than for saturated water, which in turn was higher than for degassed water. CONCLUSIONS: Measurements of cavitation and acoustic streaming have provided insight into their contribution to cleaning. Significant differences in cleaning, cavitation and streaming were found depending on the file type and size, lateral canal location and irrigant used. In general, the IrriSafe file outperformed the K-file, and NaOCl performed better than the other irrigants tested. The cavitation and streaming measurements revealed that both contributed to hydrogel removal and both play a significant role in root canal cleaning.

Dark Matter Search Results from the PICO-60 C_{3}F_{8} Bubble Chamber.

New results are reported from the operation of the PICO-60 dark matter detector, a bubble chamber filled with 52 kg of C_{3}F_{8} located in the SNOLAB underground laboratory. As in previous PICO bubble chambers, PICO-60 C_{3}F_{8} exhibits excellent electron recoil and alpha decay rejection, and the observed multiple-scattering neutron rate indicates a single-scatter neutron background of less than one event per month. A blind analysis of an efficiency-corrected 1167-kg day exposure at a 3.3-keV thermodynamic threshold reveals no single-scattering nuclear recoil candidates, consistent with the predicted background. These results set the most stringent direct-detection constraint to date on the weakly interacting massive particle (WIMP)-proton spin-dependent cross section at 3.4×10^{-41} cm^{2} for a 30-GeV c^{-2} WIMP, more than 1 order of magnitude improvement from previous PICO results.