Resveratrol inhibits arecoline-induced fibrotic properties of buccal mucosal fibroblasts via miR-200a activation.

Background/purpose: Oral submucous fibrosis (OSF) is a precancerous lesion in the oral cavity, commonly results from the Areca nut chewing habit. Arecoline, the main component of Areca nut, is known to stimulate the activation of myofibroblasts, which can lead to abnormal collagen I deposition. Meanwhile, Resveratrol is a non-flavonoid phenolic substance that can be naturally obtained from various berries and foods. Given that resveratrol has significant anti-fibrosis traits in other organs, but little is known about its effect on OSF, this study aimed to investigate the therapeutic impact of resveratrol on OSF and its underlying mechanism. Materials and methods: The cytotoxicity of resveratrol was tested using normal buccal mucosal fibroblasts (BMFs). Myofibroblast phenotypes such as collagen contractile, enhanced migration, and wound healing capacities in dose-dependently resveratrol-treated fBMFs were examined. Results: Current results showed that arecoline induced cell migration and contractile activity in BMFs as well as upregulated the expressions of α-SMA, type I collagen, and ZEB1 markers. Resveratrol intervention, on the other hand, was shown to inhibit arecoline-induced myofibroblast activation and reduce myofibroblast hallmarks and EMT markers. Additionally, resveratrol was also demonstrated to restore the downregulated miR-200a in the arecoline-stimulated cells. Conclusion: In a nutshell, these findings implicate that resveratrol may have an inhibitory influence on arecoline-induced fibrosis via the regulation of miR-200a. Hence, resveratrol may be used as a therapeutic strategy for OSF intervention.

LINC01296 promotes cancer stemness traits in oral carcinomas by sponging miR-143.

Background/purpose: Emerging evidence has shown that various failures in cancer therapy, such as drug resistance, metastasis, and cancer relapse are attributed to cancer stem cells (CSCs). Also, growing attention has been paid to the regulation of non-coding RNAs in cancer stemness. Here, we aimed to investigate the contribution of LINC01296 in the modulation of oral CSCs. Materials and methods: The phenotypic assays including migration, invasion, and colony-forming abilities were carried out in CSCs of two types of oral cancer cells (SAS and GNM) following the knockdown of LINC01296. In addition, the percentage of cells expressing stemness marker, ALDH1, and drug resistance marker, ABCG2, was examined as well as the self-renewal capacity after silencing of LINC01296. Moreover, a luciferase reporter was used to validate the direct interaction between LINC01296 and miR-143. Results: Our results showed that LINC01296 was significantly overexpressed in oral cancer tissues and positively correlated with stemness markers. The phenotypic and flow cytometry assays demonstrated that suppression of LINC01296 reduced the aggressiveness, cancer stemness features, and colony-forming and self-renewal abilities in oral CSCs. Furthermore, we demonstrated that LINC01296 may enhance cancer stemness features through suppression of the effect of miR-143. Conclusion: Silencing of LINC01296 may be a promising direction for oral cancer therapy by reducing cancer stemness via regulation of miR-143.

Down-regulation of miR-29c promotes the progression of oral submucous fibrosis through targeting tropomyosin-1.

BACKGROUND/PURPOSE: Various microRNAs (miRs) have been found to be associated with the development of the precancerous condition of the oral cavity, oral submucous fibrosis (OSF). The expression of miR-29c is dysregulated in oral cancer, but its role in OSF has not been investigated. The purpose of the study is to investigate the functional role of miR-29c and its target in OSF. METHODS: The expression levels of miR-29c in OSF tissues and fibrotic buccal mucosal fibroblasts (fBMFs) were assessed using next-generation sequencing and real-time Polymerase Chain Reaction (PCR) analysis. MiR-29c mimic and inhibitors were employed to examine its functional role of myofibroblast transdifferentiation. In addition, several myofibroblast phenotypes, such as collagen gel contraction and migration were tested, and a luciferase reporter assay was conducted to confirm the relationship between miR-29c and its predicted target, tropomyosin-1 (TPM1). RESULTS: We observed that miR-29c expression was downregulated in fBMFs. fBMFs transfected with miR-29c mimics exhibited reduced migration ability and collagen gel contractility, whereas inhibition of miR-29c in normal BMFs induced the myofibroblast phenotypes. Results from the luciferase reporter assay showed that TPM1 was a direct target of miR-29c and the expression of TPM1 was suppressed in the fBMFs transfected with miR-29c mimics. Besides, we confirmed that the expression of miR-29c was indeed downregulated in OSF specimens. CONCLUSION: MiR-29c seems to exert an inhibitory effect on myofibroblast activation, such as collagen gel contractility and migration ability, via suppressing TPM1. These results suggested that approaches to upregulate miR-29c may be able to ameliorate the progression of OSF.

Paeonol inhibits profibrotic signaling and HOTAIR expression in fibrotic buccal mucosal fibroblasts.

BACKGROUND/PURPOSE: Betel nut chewing is the major risk factor of oral submucous fibrosis (OSF). Various studies have sought to discover alternative strategies to alleviate oral fibrogenesis. In the present study, we aimed to evaluate the anti-fibrosis effect of paeonol, a phenolic component derived from Paeonia Suffruticosa. METHODS: The cytotoxicity of paeonol was tested using normal and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues. Collagen gel contraction, Transwell migration, invasion, and wound healing capacities were examined. Besides, the activation of TGF-ß/Smad2 signaling and expression levels of type I collagen, α-SMA, and long non-coding RNA HOTAIR were measured as well. RESULTS: Paeonol exerted a higher cytotoxic effect on fBMFs compared to normal BMFs. The arecoline-induced myofibroblast activities, including collagen gel contractility, cell motility, and wound healing ability were all suppressed by paeonol treatment. In addition, the activation of the TGF-ß/Smad2 pathway was inhibited along with a lower expression of α-SMA and type I collagen in paeonol-treated cells. Also, the administration of paeonol decreased the mRNA expression of HOTAIR in fBMFs. CONCLUSION: Our results indicate that paeonol may be a promising compound to attenuate the progression of oral fibrogenesis in OSF patients.

Ex Vivo Expanded Circulating Tumor Cells for Clinical Anti-Cancer Drug Prediction in Patients with Head and Neck Cancer.

The advanced-stage head and neck cancer (HNC) patients respond poorly to platinum-based treatments. Thus, a reliable pretreatment method for evaluating platinum treatment response would improve therapeutic efficiency and outcomes. This study describes a novel strategy to predict clinical drug responses in HNC patients by using eSelect, a lab-developed biomimetic cell culture system, which enables us to perform ex vivo expansion and drug sensitivity profiling of circulating tumor cells (CTCs). Forty liquid biopsies were collected from HNC patients, and the CTCs were expanded ex vivo using the eSelect system within four weeks. Immunofluorescence staining confirmed that the CTC-derived organoids were positive for EpCAM and negative for CD45. Two illustrative cases present the potential of this strategy for evaluating treatment response. The statistical analysis confirmed that drug sensitivity in CTC-derived organoids was associated with a clinical response. The multivariant logistic regression model predicted that the treatment accuracy of chemotherapy responses achieved 93.75%, and the area under the curves (AUCs) of prediction models was 0.8841 in the whole dataset and 0.9167 in cisplatin specific dataset. In summary, cisplatin sensitivity profiles of patient-derived CTCs expanded ex vivo correlate with a clinical response to cisplatin treatment, and this can potentially underpin predictive assays to guide HNC treatments.

Honokiol inhibits arecoline-induced oral fibrogenesis through transforming growth factor-ß/Smad2/3 signaling inhibition.

BACKGROUND/PURPOSE: The habit of areca nut chewing has been regarded as an etiological factor of precancerous oral submucous fibrosis (OSF). In the present study, we aimed to evaluate the anti-fibrosis effect of honokiol, a polyphenolic component derived from Magnolia officinalis. METHODS: The cytotoxicity of honokiol was tested using normal and fibrotic buccal mucosal fibroblasts (fBMFs) derived from OSF tissues. Collagen gel contraction, Transwell migration, invasion, and wound healing capacities were examined. Besides, the expression of TGF-ß/Smad2 signaling as well as α-SMA and type I collagen were measured as well. RESULTS: Honokiol exerted higher cytotoxicity of fBMFs compared to normal cells. The arecoline-induced myofibroblast activities, including collagen gel contractility, cell motility and wound healing capacities were all suppressed by honokiol treatment. In addition, the expression of the TGF-ß/Smad2 pathway was downregulated along with a lower expression of α-SMA and type I collagen in honokiol-receiving cells. CONCLUSION: Our data suggest that honokiol may be a promising compound to alleviate the progression of oral fibrogenesis and prevent the transformation of OSF oral epithelium into cancer.

A bispecific antibody agonist of the IL-2 heterodimeric receptor preferentially promotes in vivo expansion of CD8 and NK cells.

The use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαßγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rßγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule's in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.

Economic and utilization outcomes of medication management at a large Medicaid plan with disease management pharmacists using a novel artificial intelligence platform from 2018 to 2019: a retrospective observational study using regression methods.

BACKGROUND: Medication therapy management (MTM) and comprehensive medication management (CMM) have been practiced by clinical pharmacists as a predominantly manual activity with interventions documented in a record-keeping system. Program evaluations, largely based on estimations of projected savings and utilization reductions, have not accurately predicted actual claims and utilization changes, leading many to doubt the efficacy of medication management. OBJECTIVE: To assess the impact on actual medical claims of a novel artificial intelligence (AI) platform that identifies members and provides decision support to clinicians in performing telephonic interventions similar to MTM and CMM with high-risk Medicaid members. METHODS: This retrospective observational study used mixed-effects regression models that flexibly account for general trends in cost, as measured by actual claims, to identify the amount of savings and associated impact. To study the economics, total cost of care (TCoC), defined as all medication costs plus all noncapitated medical costs, was evaluated. Utilization was evaluated through the number of emergency department (ED) visits, hospital admissions, bed days, and readmissions. The study included 2,150 predominantly middle-aged (aged 40-64 years) Medicaid members with an average of 10 medications for chronic conditions among an average of 25 total medications. The analysis considered cost and utilization data from August 2017 through April 2019. Interventions occurred between January 2018 and February 2019. RESULTS: Statistically significant correlations were found between receiving interventions and decreased costs and utilization. The economic study found a 19.3% reduction in the TCoC (P < 0.001) that, applied to a preintervention monthly cost of $2,872, yielded a savings of $554 per member per month (PMPM). Medication costs showed a 17.4% reduction (P < 0.001), which, when applied to preintervention cost of $1,110, yielded a savings of $192 PMPM. The utilization study found a 15.1% reduction in ED visits (P = 0.002), a 9.4% reduction in hospital admissions (P = 0.008), and a 10.2% reduction in bed days (P = 0.01). Return on investment is 12.4:1 based on TCoC savings and program costs. CONCLUSIONS: This study evaluated the CMM-Wrap program, which used an advanced AI platform integrated with health plan data, clinical pharmacists trained in disease management, telephonic patient engagement, and closed-loop provider coordination. The results correlate cost and utilization savings with the program. The TCoC savings of $554 PMPM translates to approximately $1.2M a month and more than $14M annually for the 2,150 members in the study. We believe Medicaid and Medicare payment of AI enhanced telephonic CMM services would substantially decrease government health care expenditures, whereas improving health program expansion to Medicaid members with similar risks could save the Health Plan $109M annually. For instance, we estimate that California's Medicaid (Medi-Cal) program could save more than $1B annually by applying the program's observed impact to a similar high-risk cohort (about 1.6%) of Medi-Cal members. Additionally, benefits will accrue to nonmanaged health plans based on the savings themselves. DISCLOSURES: There was no external funding for this study. The program itself was funded by Inland Empire Health Plan. The retrospective study was a collaboration of the 3 partners (Surveyor Health, Inland Empire Health Plan, and Preveon Health) each of which funded its additional costs of preparing the study. Kessler, Mebine, E. Von Schweber, and L. Von Schweber are employed by Surveyor Health. McConnell and Jai are employed by Inland Empire Health Plan. Nguyen, Kiroyan, and Ho are employed by Preveon Health. Desai reports fees from Surveyor Health for work on this study. E. Von Schweber and L. Von Schweber have 2 patents licensed to Surveyor Health: Unified Evaluation, Presentation and Modification of Healthcare Regimens Method and Apparatus for Information Surveying.

miR-145 mediates the anti-cancer stemness effect of photodynamic therapy with 5-aminolevulinic acid (ALA) in oral cancer cells.

5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) has been used in the treatment of various precancerous and malignant lesions. Our previous work has demonstrated that ALA-PDT possesses the potential to serve as an adjuvant therapy against head and neck cancer via eliminating the cancer stem cells (CSCs) property. This study aimed to further investigate the possible molecular mechanism underlying the effect of ALA-PDT. Our results revealed that ALA-PDT upregulated the expression of microRNA-145 (miR-145) in two oral cancer cell lines. Overexpression of miR-145 in oral CSCs further enhanced the treatment effect of ALA-PDT with lower self-renewal, invasion capacities and reduced CD44 expression, while inhibition of miR-145 exhibited the opposite phenomena. These findings suggest that the anti-CSCs effect of ALA-PDT is due to an elevation of miR-145.

A clinically validated prediction method for facial soft-tissue changes following double-jaw surgery.

PURPOSE: It is clinically important to accurately predict facial soft-tissue changes prior to orthognathic surgery. However, the current simulation methods are problematic, especially in anatomic regions of clinical significance, e.g., the nose, lips, and chin. We developed a new 3-stage finite element method (FEM) approach that incorporates realistic tissue sliding to improve such prediction. METHODS: In Stage One, soft-tissue change was simulated, using FEM with patient-specific mesh models generated from our previously developed eFace template. Postoperative bone movement was applied on the patient mesh model with standard FEM boundary conditions. In Stage Two, the simulation was improved by implementing sliding effects between gum tissue and teeth using a nodal force constraint scheme. In Stage Three, the result of the tissue sliding effect was further enhanced by reassigning the soft-tissue-bone mapping and boundary conditions using nodal spatial constraint. Finally, our methods have been quantitatively and qualitatively validated using 40 retrospectively evaluated patient cases by comparing it to the traditional FEM method and the FEM with sliding effect, using a nodal force constraint method. RESULTS: The results showed that our method was better than the other two methods. Using our method, the quantitative distance errors between predicted and actual patient surfaces for the entire face and any subregions thereof were below 1.5 mm. The overall soft-tissue change prediction was accurate to within 1.1 ± 0.3 mm, with the accuracy around the upper and lower lip regions of 1.2 ± 0.7 mm and 1.5 ± 0.7 mm, respectively. The results of qualitative evaluation completed by clinical experts showed an improvement of 46% in acceptance rate compared to the traditional FEM simulation. More than 80% of the result of our approach was considered acceptable in comparison with 55% and 50% following the other two methods. CONCLUSION: The FEM simulation method with improved sliding effect showed significant accuracy improvement in the whole face and the clinically significant regions (i.e., nose and lips) in comparison with the other published FEM methods, with or without sliding effect using a nodal force constraint. The qualitative validation also proved the clinical feasibility of the developed approach.

Design, development and clinical validation of computer-aided surgical simulation system for streamlined orthognathic surgical planning.

PURPOSE: There are many proven problems associated with traditional surgical planning methods for orthognathic surgery. To address these problems, we developed a computer-aided surgical simulation (CASS) system, the AnatomicAligner, to plan orthognathic surgery following our streamlined clinical protocol. METHODS: The system includes six modules: image segmentation and three-dimensional (3D) reconstruction, registration and reorientation of models to neutral head posture, 3D cephalometric analysis, virtual osteotomy, surgical simulation, and surgical splint generation. The accuracy of the system was validated in a stepwise fashion: first to evaluate the accuracy of AnatomicAligner using 30 sets of patient data, then to evaluate the fitting of splints generated by AnatomicAligner using 10 sets of patient data. The industrial gold standard system, Mimics, was used as the reference. RESULT: When comparing the results of segmentation, virtual osteotomy and transformation achieved with AnatomicAligner to the ones achieved with Mimics, the absolute deviation between the two systems was clinically insignificant. The average surface deviation between the two models after 3D model reconstruction in AnatomicAligner and Mimics was 0.3 mm with a standard deviation (SD) of 0.03 mm. All the average surface deviations between the two models after virtual osteotomy and transformations were smaller than 0.01 mm with a SD of 0.01 mm. In addition, the fitting of splints generated by AnatomicAligner was at least as good as the ones generated by Mimics. CONCLUSION: We successfully developed a CASS system, the AnatomicAligner, for planning orthognathic surgery following the streamlined planning protocol. The system has been proven accurate. AnatomicAligner will soon be available freely to the boarder clinical and research communities.

Checklist usage decreases critical task omissions when training residents to separate from simulated cardiopulmonary bypass.

OBJECTIVE: Separation from cardiopulmonary bypass (CPB) requires multiple preparatory steps, during which mistakes, omissions, and human errors may occur. Checklists have been used extensively in aviation to improve performance of complex, multistep tasks. The aim of this study was to (1) develop a checklist using a modified Delphi process to identify essential steps necessary to prepare for separation from CPB, and (2) compare the frequency of completed items with and without the use of a checklist in simulation. It was hypothesized that the use of a checklist would reduce the number of omissions. DESIGN: High-fidelity simulation study. SETTING: University-affiliated tertiary care facility. PARTICIPANTS: Seven cardiac anesthesiologists created a checklist using a modified Delphi process. Ten residents participated in 4 scenarios separating from CPB in simulation. INTERVENTIONS: Each scenario was performed first without a checklist and then again with a checklist. An observer graded participants' performance. MEASUREMENTS AND MAIN RESULTS: A pre-separation checklist containing 9 tasks was created using the Delphi process. Without using this checklist, 4 tasks were completed in at least 75% of scenarios, and 8 tasks were completed at least 75% of the time when using the checklist. There was a significant improvement in completion of 5 of the 9 items (p< 0.01). CONCLUSIONS: A modified Delphi process can be used to create a checklist of steps in preparing to separate from CPB. Using this checklist during simulation resulted in increased frequency of completing designated tasks in comparison to relying on memory alone. Checklists may reduce omission errors during complex periods of anesthesiologists' perioperative workflow.

Passenger deletions generate therapeutic vulnerabilities in cancer.

Inactivation of tumour-suppressor genes by homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighbouring genes. We propose that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities when the collaterally deleted gene is a member of a functionally redundant family of genes carrying out an essential function. The glycolytic gene enolase 1 (ENO1) in the 1p36 locus is deleted in glioblastoma (GBM), which is tolerated by the expression of ENO2. Here we show that short-hairpin-RNA-mediated silencing of ENO2 selectively inhibits growth, survival and the tumorigenic potential of ENO1-deleted GBM cells, and that the enolase inhibitor phosphonoacetohydroxamate is selectively toxic to ENO1-deleted GBM cells relative to ENO1-intact GBM cells or normal astrocytes. The principle of collateral vulnerability should be applicable to other passenger-deleted genes encoding functionally redundant essential activities and provide an effective treatment strategy for cancers containing such genomic events.

SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression.

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFß/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Magnetically responsive polymeric microparticles for oral delivery of protein drugs.

PURPOSE: Protein drugs cannot be delivered efficiently through oral routes. To address this challenge, we evaluated the effect of prolonged gastrointestinal transit on the bioavailability of insulin carried by magnetically responsive microparticles in the presence of an external magnetic field. METHODS: Magnetite nanocrystals and insulin were coencapsulated into poly(lactide-co-glycolide) (PLGA) microparticles and their effects on hypoglycemia were evaluated in mice in the presence of a circumferentially applied external magnetic field. RESULTS: A single administration of 100 U/kg of insulin-magnetite-PLGA microparticles to fasted mice resulted in a reduction of blood glucose levels of up to 43.8% in the presence of an external magnetic field for 20 h (bioavailability = 2.77 +/- 0.46 and 0.87 +/- 0.29% based on glucose and ELISA assay, respectively), significantly higher than similarly dosed mice without a magnetic field (bioavailability = 0.66 +/- 0.56 and 0.30 +/- 0.06%, based on glucose and ELISA assay, respectively). CONCLUSIONS: A substantially improved hypoglycemic effect was observed in mice that were orally administered with insulin-magnetite-PLGA microparticles in the presence of an external magnetic field, suggesting that magnetic force can be used to improve the efficiency of orally delivered protein therapeutics.

Cell type-specific effects of Rb deletion in the murine retina.

Certain cells of the human retina are extremely sensitive to loss of function of the retinoblastoma tumor suppressor gene RB. Retinoblastomas develop early in life and at high frequency in individuals heterozygous for a germ-line RB mutation, and sporadic retinoblastomas invariably have somatic mutation in the RB gene. In contrast, retinoblastomas do not develop in Rb+/- mice. Although retinoblastoma is thought to have developmental origins, the function of Rb in retinal development has not been fully characterized. Here we studied the role of Rb in normal retinal development and in retinoblastoma using conditional Rb mutations in the mouse. In late embryogenesis, Rb-deficient retinas exhibited ectopic S-phase and high levels of p53-independent apoptosis, particularly in the differentiating retinal ganglion cell layer. During postnatal retinal development, loss of Rb led to more widespread retinal apoptosis, and adults showed loss of photoreceptors and bipolar cells. Conditional Rb mutation in the retina did not result in retinoblastoma formation even in a p53-mutant background. However, on a p107- or p130-deficient background, Rb mutation in the retina caused retinal dysplasia or retinoblastoma.