Increased glycolysis and cellular crosstalk in eosinophilic chronic rhinosinusitis with nasal polyps.

Introduction: Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the sinonasal mucosa with distinct endotypes including type 2 (T2) high eosinophilic CRS with nasal polyps (eCRSwNP), T2 low non-eosinophilic CRS with nasal polyps (neCRSwNP), and CRS without nasal polyps (CRSsNP). Methods: Given the heterogeneity of disease, we hypothesized that assessment of single cell RNA sequencing (scRNA-seq) across this spectrum of disease would reveal connections between infiltrating and activated immune cells and the epithelial and stromal populations that reside in sinonasal tissue. Results: Here we find increased expression of genes encoding glycolytic enzymes in epithelial cells (EpCs), stromal cells, and memory T-cell subsets from patients with eCRSwNP, as compared to healthy controls. In basal EpCs, this is associated with a program of cell motility and Rho GTPase effector expression. Across both stromal and immune subsets, glycolytic programming was associated with extracellular matrix interactions, proteoglycan generation, and collagen formation. Furthermore, we report increased cell-cell interactions between EpCs and stromal/immune cells in eCRSwNP compared to healthy control tissue, and we nominate candidate receptor-ligand pairs that may drive tissue remodeling. Discussion: These findings support a role for glycolytic reprograming in T2-elicited tissue remodeling and implicate increased cellular crosstalk in eCRSwNP.

Rhinovirus infection of airway epithelial cells uncovers the non-ciliated subset as a likely driver of genetic susceptibility to childhood-onset asthma.

Asthma is a complex disease caused by genetic and environmental factors. Epidemiological studies have shown that in children, wheezing during rhinovirus infection (a cause of the common cold) is associated with asthma development during childhood. This has led scientists to hypothesize there could be a causal relationship between rhinovirus infection and asthma or that RV-induced wheezing identifies individuals at increased risk for asthma development. However, not all children who wheeze when they have a cold develop asthma. Genome-wide association studies (GWAS) have identified hundreds of genetic variants contributing to asthma susceptibility, with the vast majority of likely causal variants being non-coding. Integrative analyses with transcriptomic and epigenomic datasets have indicated that T cells drive asthma risk, which has been supported by mouse studies. However, the datasets ascertained in these integrative analyses lack airway epithelial cells. Furthermore, large-scale transcriptomic T cell studies have not identified the regulatory effects of most non-coding risk variants in asthma GWAS, indicating there could be additional cell types harboring these "missing regulatory effects". Given that airway epithelial cells are the first line of defense against rhinovirus, we hypothesized they could be mediators of genetic susceptibility to asthma. Here we integrate GWAS data with transcriptomic datasets of airway epithelial cells subject to stimuli that could induce activation states relevant to asthma. We demonstrate that epithelial cultures infected with rhinovirus significantly upregulate childhood-onset asthma-associated genes. We show that this upregulation occurs specifically in non-ciliated epithelial cells. This enrichment for genes in asthma risk loci, or 'asthma heritability enrichment' is also significant for epithelial genes upregulated with influenza infection, but not with SARS-CoV-2 infection or cytokine activation. Additionally, cells from patients with asthma showed a stronger heritability enrichment compared to cells from healthy individuals. Overall, our results suggest that rhinovirus infection is an environmental factor that interacts with genetic risk factors through non-ciliated airway epithelial cells to drive childhood-onset asthma.

Development of a Pipeline for Removing Allergy Labels in Patients Undergoing Hematopoietic Stem Cell Transplantation.

Penicillin allergy is reported by 10% to 20 % of patients, but when evaluated only 1% to 2% may have a true allergy. Patients undergoing hematopoietic stem cell transplantation (HSCT) have a high likelihood of requiring beta-lactam antibiotics due to increased infection risk, which can be limited by a penicillin allergy label. When a penicillin allergy is recorded, alternatives are needed, including more expensive broader-spectrum antibiotics, with increases in drug-resistant bacteria, longer hospital stays, higher expenditures, and increases in nosocomial infections, such as Clostridium difficile colitis. This group of patients already undergoes extensive pretreatment testing and would especially benefit from allergy delabeling. This study aimed to develop a self-sustaining, low-cost pipeline between an HSCT clinic and an allergy clinic to identify and successfully delabel low-risk patients who endorse an allergy to penicillin, amoxicillin, amoxicillin-clavulanate, piperacillin-tazobactam, or ampicillin before admission to the hospital. We developed a survey to triage allergy risk, identified key stakeholders in building the pipeline, and underwent 4 plan, do, study, act (PDSA) cycles. Changes were made in each of the PDSA cycles to minimize cost and uncompensated provider time, as well as to increase patient retention throughout the pipeline by increasing appointment availability and decreasing reliance on patients to independently progress through the pathway. Of the 410 patients with planned HSCT who were screened over 11 months, 89 (21.7%) were listed as having a penicillin and/or beta lactam allergy. All but 1 (66 of 67; 98.5%) of the participants completed the survey accurately when confirmed by an allergist, and the survey was 100% accurate in predicting delabeling success in low-risk patients. Of eligible patients, 43.8% (n = 39) were successfully delabeled before their transplant date, and 97.4% of these (n = 38) have undergone HSCT to date. This pipeline is maintained by approximately 5 hours of work per week (1 hour of allergy physician time, 4 hours of nurse and/or clinical coordinator time), with no other direct costs. There is an estimated direct savings of at least $1914.93 per patient delabeled. We successfully designed and implemented a pipeline between the HSCT clinic and the allergy clinic as a quality improvement initiative to identify and address high rates of reported beta-lactam allergies. We identified and addressed patient-based factors, logistical, temporal, and financial barriers that impacted patient retention and sustainability. This model is expected to yield significant and sustained cost savings for the healthcare system as well as to improve patient outcomes, and this hypothesis is currently undergoing formal analysis. We anticipate that this model can be used to create a similar pipeline in other healthcare systems for HSCT recipients, as well as patients in other clinical settings, such as oncology and chimeric antigen receptor T cell therapy.

Increased epithelial mTORC1 activity in chronic rhinosinusitis with nasal polyps.

Background: The airway epithelium plays a central role in the pathogenesis of chronic respiratory diseases such as asthma and chronic rhinosinusitis with nasal polyps (CRSwNP), but the mechanisms by which airway epithelial cells (EpCs) maintain inflammation are poorly understood. Objective: We hypothesized that transcriptomic assessment of sorted airway EpCs across the spectrum of differentiation would allow us to define mechanisms by which EpCs perpetuate airway inflammation. Methods: Ethmoid sinus EpCs from adult patients with CRS were sorted into 3 subsets, bulk RNA sequenced, and analyzed for differentially expressed genes and pathways. Single cell RNA-seq (scRNA-seq) datasets from eosinophilic and non-eosinophilic CRSwNP and bulk RNA-seq of EpCs from mild/moderate and severe asthma were assessed. Immunofluorescent staining and ex vivo functional analysis of sinus EpCs were used to validate our findings. Results: Analysis within and across purified EpC subsets revealed an enrichment in glycolytic programming in CRSwNP vs CRSsNP. Correlation analysis identified mammalian target of rapamycin complex 1 (mTORC1) as a potential regulator of the glycolytic program and identified EpC expression of cytokines and wound healing genes as potential sequelae. mTORC1 activity was upregulated in CRSwNP, and ex vivo inhibition demonstrated that mTOR is critical for EpC generation of CXCL8, IL-33, and CXCL2. Across patient samples, the degree of glycolytic activity was associated with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in severe asthma. Conclusions: Together, these findings highlight a metabolic axis required to support epithelial generation of cytokines critical to both chronic T2 and non-T2 inflammation in CRSwNP and asthma.

mScarlet and split fluorophore mScarlet resources for plasmid-based CRISPR/Cas9 knock-in in C. elegans.

Fluorescent proteins allow the expression of a gene and the behavior of its protein product to be observed in living animals. The ability to create endogenous fluorescent protein tags via CRISPR genome engineering has revolutionized the authenticity of this expression, and mScarlet is currently our first-choice red fluorescent protein (RFP) for visualizing gene expression in vivo . Here, we have cloned versions of mScarlet and split fluorophore mScarlet previously optimized for C. elegans into the SEC-based system of plasmids for CRISPR/Cas9 knock-in. Ideally, an endogenous tag will be easily visible while not interfering with the normal expression and function of the targeted protein. For low molecular weight proteins that are a fraction of the size of a fluorescent protein tag (e.g. GFP or mCherry) and/or proteins known to be non-functional when tagged in this way, split fluorophore tagging could be an alternative. Here, we used CRISPR/Cas9 knock-in to tag three such proteins with split-fluorophore wrmScarlet: HIS-72, EGL-1, and PTL-1. Although we find that split fluorophore tagging does not disrupt the function of any of these proteins, we were unfortunately unable to observe the expression of most of these tags with epifluorescence, suggesting that split fluorophore tags are often very limited as endogenous reporters. Nevertheless, our plasmid toolkit provides a new resource that enables straightforward knock-in of either mScarlet or split mScarlet in C. elegans.

Internet of Everything and Digital Twin enabled Service Platform for Cold Chain Logistics.

The proliferation of the e-commerce market has posed challenges to staff safety, product quality, and operational efficiency, especially for cold chain logistics (CCL). Recently, the logistics of vaccine supply under the worldwide COVID-19 pandemic rearouses public attention and calls for innovative solutions to tackle the challenges remaining in CCL. Accordingly, this study proposes a cyber-physical platform framework applying the Internet of Everything (IoE) and Digital Twin (DT) technologies to promote information integration and provide smart services for different stakeholders in the CCL. In the platform, reams of data are generated, gathered, and leveraged to interconnect and digitalize physical things, people, and processes in cyberspace, paving the way for digital servitization. Deep learning techniques are used for accident identification and indoor localization based on Bluetooth Low Energy (BLE) to actualize real-time staff safety supervision in the cold warehouse. Both algorithms are designed to take advantage of the IoE infrastructure to achieve online self-adapting in response to surrounding evolutions. Besides, with the help of mobile and desktop applications, paperless operation for shipment, remote temperature and humidity (T&H) monitoring, anomaly detection and warning, and customer interaction are enabled. Thus, information traceability and visibility are highly fortified in this way. Finally, a real-life case study is conducted in a pharmaceutical distribution center to demonstrate the feasibility and practicality of the proposed platform and methods. The dedicated hardware and software are developed and deployed on site. As a result, the effectiveness of staff safety management, operational informatization, product quality assurance, and stakeholder loyalty maintenance shows a noticeable improvement. The insights and lessons harvested in this study may spark new ideas for researchers and inspire practitioners to meet similar needs in the industry.

An IL-12-Based Nanocytokine Safely Potentiates Anticancer Immunity through Spatiotemporal Control of Inflammation to Eradicate Advanced Cold Tumors.

Treatment of immunologically cold tumors is a major challenge for immune checkpoint inhibitors (ICIs). Interleukin 12 (IL-12) can invigorate ICIs against cold tumors by establishing a robust antitumor immunity. However, its toxicity and systemic induction of counteracting immunosuppressive signals have hindered translation. Here, IL-12 activity is spatiotemporally controlled for safely boosting efficacy without the stimulation of interfering immune responses by generating a nanocytokine that remains inactive at physiological pH, but unleashes its full activity at acidic tumor pH. The IL-12-based nanocytokine (Nano-IL-12) accumulate and release IL-12 in tumor tissues, eliciting localized antitumoral inflammation, while preventing systemic immune response, counteractive immune reactions, and adverse toxicities even after repeated intravenous administration. The Nano-IL-12-mediated spatiotemporal control of inflammation prompt superior anticancer efficacy, and synergize with ICIs to profoundly inflame the tumor microenvironment and completely eradicate ICI-resistant primary and metastatic tumors. The strategy could be a promising approach toward safer and more effective immunotherapies.

Synchronization of Shop-Floor Logistics and Manufacturing Under IIoT and Digital Twin-Enabled Graduation Intelligent Manufacturing System.

Logistics interfaces with manufacturing throughout the entire production process need synchronous operations. For achieving integrated organization and operations between manufacturing and logistics, this article introduces the concept of shop-floor logistics and manufacturing synchronization with four principles, including: 1) synchronization-oriented manufacturing system; 2) synchronized information sharing; 3) synchronized decision making; and 4) synchronized operations. The marriage of the principles rendered the development of an overall framework of the Industrial Internet of Things (IIoT) and digital twin-enabled graduation intelligent manufacturing system (GiMS). A mixed-integer programming-based synchronization mechanism is proposed under GiMS. To meet the requirement of fast decision making in real-life shop-floor logistics and manufacturing synchronization problems, an equivalent constraint programming model is developed and tested. The observation and analysis of the case company show the advantage of the proposed concept and approach with the best performance regarding key performance indicators. The concept of synchronization provides an insight for understanding the interaction of logistics and manufacturing at the operational level. This article potentially enables manufacturers to reevaluate and develop their manufacturing planning and control strategies in the IIoT and digital twin-based manufacturing environment.

The Four Pillars for Successful Regenerative Therapy in Endodontics: Stem Cells, Biomaterials, Growth Factors, and Their Synergistic Interactions.

Endodontics has made significant progress in regenerative approaches in recent years, thanks to advances in biologically based procedures or regenerative endodontic therapy (RET). In recent years, our profession has witnessed a clear conceptual shift in this therapy. RET was initially based on a blood clot induced by apical bleeding without harvesting the patient's cells or cell-free RET. Later, the RET encompassed the three principles of tissue engineering, stromal/stem cells, scaffolds, and growth factors, aiming for the regeneration of a functional dentin pulp complex. The regenerated dental pulp will recover the protective mechanisms including innate immunity, tertiary dentin formation, and pain sensitivity. This comprehensive review covers the basic knowledge and practical information for translational applications of stem cell-based RET and tissue engineering procedures for the regeneration of dental pulp. It will also provide overall information on the emerging technologies in biological and synthetic matrices, biomaterials, and signaling molecules, recent advances in stem cell therapy, and updated experimental results. This review brings useful and timely clinical evidence for practitioners to understand the challenges faced for a successful cell-based RET and the importance of preserving or reestablishing tooth vitality. The clinical translation of these current bioengineering approaches will undoubtedly be beneficial to the future practice of endodontics.

A new direction in managing avulsed teeth: stem cell-based de novo PDL regeneration.

Management of avulsed teeth after replantation often leads to an unfavorable outcome. Damage to the thin and vulnerable periodontal ligament is the key reason for failure. Cell- or stem cell-based regenerative medicine has emerged in the past two decades as a promising clinical treatment modality to improve treatment outcomes. This concept has also been tested for the management of avulsed teeth in animal models. This review focuses on the discussion of limitation of current management protocols for avulsed teeth, cell-based therapy for periodontal ligament (PDL) regeneration in small and large animals, the challenges of de novo regeneration of PDL on denuded root in the edentulous region using a mini-swine model, and establishing a prospective new clinical protocol to manage avulsed teeth based on the current progress of cell-based PDL regeneration studies.

Cell-derived Extracellular Matrix Proteins in Colloidal Microgel as a Self-Assembly Hydrogel for Regenerative Endodontics.

INTRODUCTION: This study investigated a colloidal microgel for angiogenic and odontogenic differentiation of cells in the presence of cell-derived extracellular matrix (ECM) proteins using a 3-dimensional culture model. METHODS: Viscoelastic properties of human dental pulp were determined to understand the native ECM environment. ECM proteins were extracted from dental pulp stem cell (DPSC) cultures, and MaxGel (Millipore Sigma, Burlington, MA) was used as a commercially available ECM protein. DPSCs were incubated in colloidal microgels in the presence of ECM proteins or gelatin methacryloyl (GelMA) as a bulk hydrogel (n = 9/group). The viability and odontogenic differentiation of DPSCs within hydrogels was determined using viability assays, mineralization staining, calcium and alkaline phosphatase assays, and quantitative polymerase chain reaction for odontogenic gene expression. Angiogenic properties of endothelial cells were determined using tubule formation assays and quantitative polymerase chain reaction to detect angiogenic gene expression. RESULTS: Dental pulp had a higher elastic modulus than the viscous modulus, showing a solidlike response similar to hydrogels. DPSC-derived ECM showed higher collagen and GAG than MaxGel (P < .05). The viability of DPSCs was similar in colloidal microgels, whereas higher cell viability, calcium deposition, and alkaline phosphatase activity were observed in GelMA (P < .05). Colloidal microgels allowed tubule-like structures by endothelial cells, whereas no tubular formation was observed in GelMA. DPSC-derived ECM in colloidal microgel up-regulated odontogenic gene expression, whereas MaxGel up-regulated angiogenic gene expression (P < .05). CONCLUSIONS: Colloidal microgels allowed cellular organization that can improve penetration and nutritional supply in a full-length root canal system. The bioactivity of cell-derived ECM proteins can be modified depending on the external stimulus.

Effects of intentionally treated water on the growth of mesenchymal stem cells: An exploratory study.

OBJECTIVE: This study explored if human primary mesenchymal stem cells (MSCs), derived from two donors and cultivated in a medium made with intentionally treated water, would exhibit more growth and pluripotency than MSCs from the same source but grown in untreated (control) water. DESIGN: To create the treated water, three Buddhist monks directed their attention toward commercially bottled water while holding the intention that the water would enhance the growth of MSCs. Under double-blind conditions, cell culture growth mediums were prepared with the treated and untreated water, which was in turn used to grow the primary MSCs. Primary cells obtained from two donors were designated as Cells #1 and Cells #2. The prediction was that treated water would result in increased cell proliferation, that more cells would enter the cell cycle growth phase, and that there would be increased expression of genes (NANOG, OCT4 and SOX2) associated with improved cell growth and decreased expression of genes (p16, p21, and p53) associated with a decline in cell growth. The improved growth hypothesis was directional, thus one-tailed p-values were used to evaluate the results. RESULTS: Proliferation averaged across Cells #1 and #2 showed overall increased growth in treated as compared to control water (p = 0.0008). Cells #1 and #2 considered separately had differences in the same direction but only Cells #2 showed a significant difference on day 6 (p = 0.01). For cell cycle, there was a significantly greater percentage of Cells #2 in the S interphase with treated vs. control water (p = 0.04). For the gene expression analysis, when considering the average across the two donor cells, only the NANOG gene expression was in the predicted direction (p = 0.01); by contrast, the p16 gene expression was significantly opposite to the predicted direction (p = 0.005, one-tailed, post-hoc). For Cells #1 considered separately, no differences were significant except for p16, which resulted in an effect opposite to the predicted outcome (p = 0.05). For Cells #2, three genes were significantly in the predicted directions: NANOG (p = 0.0008), OCT4 (p = 0.005), and P53 (p = 0.05); p16 was significantly opposite to the prediction (p = 0.001). CONCLUSION: Intentionally treated water appeared to have some biological effects on the growth, pluripotency and senescence of human MSCs. This was especially the case in one of the two donor cells tested, but the effects were not consistently in the predicted direction. As an exploratory study, caution is warranted in interpreting these outcomes, and adjustment for multiple testing would likely reduce some of the weaker effects to nonsignificant. But given the double-blind protocol, as well as several more significant outcomes in the predicted directions, further research is warranted.

Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans.

Streamlined, selection-based CRISPR knock-in protocols for C. elegans were first introduced six years ago (Dickinson et al. 2015; Schwartz and Jorgensen 2016). Though these selection-based approaches are powerful, one drawback has been the requirement to inject large numbers of P0 worms (~30-60 per gene target). We have found that a combination of high-purity DNA and a lower concentration of Cas9/sgRNA plasmid dramatically improves efficiency, often resulting in multiple independent CRISPR knock-ins via as few as 10 injected worms, comparable to the efficiency of melted dsDNA templates and purified Cas9 protein (Dokshin et al. 2018; Ghanta and Mello 2020).

Potential of tailored amorphous multiporous calcium silicate glass for pulp capping regenerative endodontics-A preliminary assessment.

INTRODUCTION/OBJECTIVE: The tailored amorphous multi-porous (TAMP) material fabrication technology has led to a new class of bioactive materials possessing versatile characteristics. It has not been tested for dental applications. Thus, we aimed to assess its biocompatibility and ability to regenerate dental mineral tissue. METHODS: 30CaO-70SiO2 model TAMP discs were fabricated by a sol-gel method followed by in vitro biocompatibility testing with isolated human or mini-swine dental pulp stem cells (DPSCs). TAMP scaffolds were tested in vivo as a pulp exposure (pin-point, 1 mm, 2 mm, and entire pulp chamber roof) capping material in the molar teeth of mini-swine. RESULTS: The in vitro assays showed that DPSCs attached well onto the TAMP discs with comparable viability to those attached to culture plates. Pulp capping tests on mini-swine showed that after 4.5 months TAMP material was still present at the capping site, and mineral tissue (dentin bridge) had formed in all sizes of pulp exposure underneath the TAMP material. CONCLUSIONS: TAMP calcium silicate is biocompatible with both human and swine DPSCs in vitro and with pulp in vivo, it may help regenerate the dentin bridge after pulp exposure.

Pulp/Dentin Regeneration: It Should Be Complicated.

Stem cell-mediated regenerative endodontics has reached the human clinical trial phase; however, many issues still exist that prevent such technology to be a widely used clinical practice. These issues are not straightforward and are complicated. They should be because pulp regeneration is dealing with a small dead-end space. In addition, when regeneration is needed, the space is often heavily infected. The true standard of pulp regeneration should be everything except generation of some fibrous connective tissue and amorphous mineral deposit. As of now, we are still far short of reaching the standard of complete vascularized and innervated pulp regeneration with newly formed tubular dentin in all types of teeth. Thus, we need to go back to the bench and use established animal models or create new animal models to tackle those issues. This article will address several key issues including the possibility of pulp regeneration in small canals of molar teeth by enhancing the neovascularization, and whether the organized tubular dentin can be generated on the canal walls. Data from our semi-orthotopic tooth fragment mouse model have shown that complete pulp regeneration using dental pulp stem cells (DPSCs) in small canal has been inconsistent because of limited blood supply. This inconsistency is similar in our orthotopic miniature swine model, although in some cases vascularized pulp-like tissue can be formed throughout the canal space after DPSC transplantation. Furthermore, no tubular dentin was observed in the orthotopic pulp regeneration, despite the fact that DPSCs have the capacity to generate some tubular dentin-like structure in the hydroxyapatite/tricalcium phosphate-mediated ectopic pulp/dentin formation model in mice. Potential strategies to be tested to address these regeneration issues are discussed herein.

Effects of Cocooning on Coronavirus Disease Rates after Relaxing Social Distancing.

As coronavirus disease spreads throughout the United States, policymakers are contemplating reinstatement and relaxation of shelter-in-place orders. By using a model capturing high-risk populations and transmission rates estimated from hospitalization data, we found that postponing relaxation will only delay future disease waves. Cocooning vulnerable populations can prevent overwhelming medical surges.

Cocooning is essential to relaxing social distancing.

As the first wave of COVID-19 recedes, policymakers are contemplating the relaxation of shelter-in-place orders. Using a model capturing high-risk populations and transmission rates estimated from hospitalization data, we find that postponing relaxation will only delay a second wave and cocooning vulnerable populations is needed to prevent overwhelming medical surges.

Nanobubble-Enhanced Antimicrobial Agents: A Promising Approach for Regenerative Endodontics.

INTRODUCTION: In this study, we investigated the properties of nanobubble (NB) water and its effect on smear layer removal and strengthening the efficiency of disinfecting agents used in regenerative endodontic treatment. METHODS: NB water was generated in a NB Generator. The NB size, concentration, and pH were measured. Porcine teeth were enlarged to size 60 by using hand-files and irrigated with either NB water or 17% EDTA or received no further irrigation. The ability of irrigants to remove the smear layer was evaluated by using a scanning electron microscope (9 roots/group). Other samples (6 roots/group) were subjected to Vickers hardness test to determine the dentin microhardness. Autofluorescent tetracycline mixed with distilled water or NB water was placed inside the root canal space of porcine teeth, and the depth of medicament penetration into the dentinal tubules was visualized by using fluorescent stereomicroscope (5 roots/group). For the disinfection experiment, human roots were prepared, autoclaved, and inoculated with Enterococcus faecalis for 3 weeks. Canals were then disinfected by (1) standard needle irrigation (SNI) with 5.25% NaOCl, (2) 5.25% NaOCl with ultrasonication (US), (3) 5.25% NaOCl + XP finisher (XP), (4) SNI with 1.5% NaOCl, or (5) SNI with 1.5% NaOCl in NB water (5 roots/group). Teeth were split open and stained with LIVE/DEAD BackLight and visualized by using confocal laser scanning microscope (CLSM) at the coronal, middle, and apical thirds of the canal. The ratio of dead/total bacteria in the dentinal tubules at various depth levels (50, 100, and 150 µm) was calculated. RESULTS: NB water was more effective in removing smear layer than 17% EDTA and could allow infiltration of tetracycline into the dentinal tubule more than 1 mm. NB water did not alter the dentin microhardness compared with 17% EDTA (P < .05). At 50-µm depth, CLSM analysis showed no statistically significant difference between 1.5% NaOCl in NB water and 5.25% NaOCl with or without irrigation activation at the coronal, middle, and apical root segments (P > .05), ie, these groups had stronger bacterial killing than 1.5% NaOCl (P < .05). At deeper levels (100 and 150 µm), higher concentrations of NaOCl were more effective than 1.5% NaOCl with or without NB water. No statistically significant difference was noted between 5.25% NaOCl with and without irrigation activation at most depth levels (P > .05). CONCLUSIONS: NB water can allow smear layer removal and enhance tubular penetration of medicaments without changing dentin microhardness. In large canal models, NB water appears to improve the tubular disinfection capacity of lower concentration of NaOCl up to 50 µm. On the other hand, the use of irrigation activation (US or XP) did not provide any added disinfection into the dentinal tubules compared with SNI. These results suggest that NB water may be a promising adjunct to endodontic irrigants and medicaments.

Dens evaginatus: Current treatment options.

BACKGROUND: Teeth with dens evaginatus (DE) are more commonly observed in Western countries than previously. This is due to the increase in populations of patients of Asian origin, in whom DE is more common than in people of European origin. The interest in DE has also increased with the introduction of a procedure called regenerative endodontics. CASE DESCRIPTION: A narrative review of treatment options for teeth with DE is presented, based on pulpal conditions and maturity of the teeth. PRACTICAL IMPLICATIONS: Early recognition of teeth with DE allows for treatment choices that generally lead to good outcomes and can aid in preserving developing teeth in young patients.

Polymeric Micelles Loading Proteins through Concurrent Ion Complexation and pH-Cleavable Covalent Bonding for In Vivo Delivery.

Protein drugs have great potential as targeted therapies, yet their application suffers from several drawbacks, such as instability, short half-life, and adverse immune responses. Thus, protein delivery approaches based on stimuli-responsive nanocarriers can provide effective strategies for selectively enhancing the availability and activation of proteins in targeted tissues. Herein, polymeric micelles with the ability of encapsulating proteins are developed via concurrent ion complexation and pH-cleavable covalent bonding between proteins and block copolymers directed to pH-triggered release of the protein payload. Carboxydimethylmaleic anhydride (CDM) is selected as the pH-sensitive moiety, since the CDMamide bond is stable at physiological pH (pH 7.4), while it cleaves at pH 6.5, that is, the pathophysiological pH of tumors and inflammatory tissues. By using poly(ethylene glycol)-poly(l-lysine) block copolymers having 45% CDM addition, different proteins with various sizes and isoelectric points are loaded successfully. By using myoglobin-loaded micelles (myo/m) as a model, the stability of the micelles in physiological conditions and the dissociation and release of functional myoglobin at pH 6.5 are successfully confirmed. Moreover, myo/m shows extended half-life in blood compared to free myoglobin and micelles assembled solely by polyion complex, indicating the potential of this system for in vivo delivery of proteins.