A Mechanistic Analysis of the Neural Modulation of the Inflammatory System Through Vagus Nerve Stimulation: A Systematic Review and Meta-analysis.

OBJECTIVE: We aimed to conduct a systematic review and meta-analysis assessing the antiinflammatory effects of various VNS methods while exploring multiple antiinflammatory pathways. MATERIALS AND METHODS: We included clinical trials that used electrical stimulation of the vagus nerve and assessed inflammatory markers up to October 2022. We excluded studies lacking control groups, those with combined interventions, or abstracts without full text. We adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and the Cochrane Handbook for Systematic Reviews. For each inflammatory marker, a random-effects meta-analysis using the inverse variance method was performed. Methods used include transcutaneous auricular VNS (taVNS), transcutaneous cervical VNS (tcVNS), invasive cervical VNS (iVNS), and electroacupuncture VNS (eaVNS). Main reported outcomes included tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1ß, C-reactive protein (CRP), and IL-10. Risk of bias was evaluated using the Cochrane Collaboration Tool (RoB 2.0). RESULTS: This review included 15 studies, involving 597 patients. No statistically significant general VNS effect was observed on TNF-α, IL-6, and IL-1ß. However, CRP, IL-10, and interferon (IFN)-γ were significantly modulated by VNS across all methods. Subgroup analysis revealed specific stimulation techniques producing significant results, such as taVNS effects in IL-1ß and IL-10, and iVNS in IL-6, whereas tcVNS and eaVNS did not convey significant pooled results individually. Cumulative exposure to VNS, higher risk of bias, study design, and pulse width were identified as effect size predictors in our meta-regression models. CONCLUSIONS: Pooling all VNS techniques indicated the ability of VNS to modulate inflammatory markers such as CRP, IL-10, and IFN-γ. Individually, methods such as taVNS were effective in modulating IL-1ß and IL-10, whereas iVNS modulated IL-6. However, different VNS techniques should be separately analyzed in larger, homogeneous, and powerful studies to achieve a clearer and more consistent understanding of the effect of each VNS method on the inflammatory system.

Consensus Statements on the Definition, Classification, and Diagnostic Tests for Tinnitus: A Delphi Study Conducted by the Korean Tinnitus Study Group.

BACKGROUND: Tinnitus is a bothersome condition associated with various symptoms. However, the mechanisms of tinnitus are still uncertain, and a standardized assessment of the diagnostic criteria for tinnitus is required. We aimed to reach a consensus on diagnosing tinnitus with professional experts by conducting a Delphi study with systematic review of the literature. METHODS: Twenty-six experts in managing tinnitus in Korea were recruited, and a two-round modified Delphi study was performed online. The experts evaluated the level of agreement of potential criteria for tinnitus using a scale of 1-9. After the survey, a consensus meeting was held to establish agreement on the results obtained from the Delphi process. Consensus was defined when over 70% of the participants scored 7-9 (agreement) and fewer than 15% scored 1-3 (disagreement). To analyze the responses of the Delphi survey, the content validity ratio and Kendall's coefficient of concordance were evaluated. RESULTS: Consensus was reached for 22 of the 38 statements. For the definition of tinnitus, 10 out of 17 statements reached consensus, with three statements achieving complete agreement including; 1) Tinnitus is a conscious perception of an auditory sensation in the absence of a corresponding external stimulus, 2) Tinnitus can affect one's quality of life, and 3) Tinnitus can be associated with hearing disorders including sensorineural hearing loss, vestibular schwannoma, Meniere's disease, otosclerosis, and others. For the classification of tinnitus, 11 out of 18 statements reached consensus. The participants highly agreed with statements such as; 1) Vascular origin is expected in pulse-synchronous tinnitus, and 2) Tinnitus can be divided into acute or chronic tinnitus. Among three statements on the diagnostic tests for tinnitus only Statement 3, "There are no reliable biomarkers for sensory or emotional factors of tinnitus." reached consensus. All participants agreed to perform pure-tone audiometry and tinnitus questionnaires, including the Tinnitus Handicap Inventory and Tinnitus Questionnaire. CONCLUSION: We used a modified Delphi method to establish a consensus-based definition, a classification, and diagnostic tests for tinnitus. The expert panel reached agreement for several statements, with a high level of consensus. This may provide practical information for clinicians in managing tinnitus.

Mucosa-associated Lymphoid Tissue Misdiagnosis as Glomus Tympanicum.

Middle ear tumors are diverse, but relatively uncommon. The most frequent tumor in the middle ear is glomus tumor, followed by others such as schwannoma and cholesteatoma. We experienced a case of Mucosa-associated lymphoid tissue hyperplasia as a middle ear tumor. The mass behind tympanic membrane appeared a hypervascular tumor, mimicking a glomus tumor, but the form of multiple separate masses in middle ear and mastoid cavity was the distinguishing feature that set it apart from a glomus tumor. Additionally, another characteristic was its tendency to easily shrink under pressure. This characteristic should be considered when encounter a hypervascular looking middle ear mass. Laryngoscope, 134:1894-1896, 2024.

Inhibition of lipopolysaccharide-induced inflammation by trophoblast-conditioned medium and trophoblast-derived extracellular vesicles in human middle ear epithelial cells.

Otitis media is a common disease but can cause severe inner ear inflammation and hearing loss if it persists for more than two weeks. This study elucidates the inflammation-inhibiting efficacy of conditioned medium (CM) and extracellular vesicles (EVs) derived from human trophoblast (TB) cells in lipopolysaccharide (LPS)-induced human middle ear epithelial cells (HMEECs). TB-conditioned medium (TB-CM) reduced the inflammatory response and regulated mucin and epithelial sodium channel genes in LPS-induced HMEECs. The underlying mechanism of cell migration during inflammatory healing in LPS-induced HMEECs treated with TB-CM was determined by RNA-sequencing analysis. Specifically, the NF-κB pathway related to the copper metabolism MURR1 domain protein was studied and verified through siRNA. This elucidation of the anti-inflammatory effect of TB-CM and TB-derived EVs demonstrates their clinical potential to treat chronic inflammation.

Osteogenic Efficacy of Human Trophoblasts-Derived Conditioned Medium on Mesenchymal Stem Cells.

Trophoblasts play an important role in the regulation of the development and function of the placenta. Our recent study demonstrated the skin regeneration capacity of trophoblast-derived extracellular vesicles (EV). Here, we aimed to determine the potential of trophoblast-derived conditioned medium (TB-CM) in enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We found that TB-CM promoted the osteogenic differentiation of MSCs in a dose-dependent manner. Furthermore, it inhibited adipogenesis of MSCs. We also found that the primary trophoblast-derived conditioned medium (PTB-CM) significantly enhanced the proliferation and osteogenic differentiation of human MSCs. Our study demonstrated the regulatory mechanisms underlying the TB-CM-induced osteogenesis in MSCs. An upregulation of genes associated with cytokines/chemokines was observed. The treatment of MSCs with TB-CM stimulated osteogenesis by activating several biological processes, such as mitogen-activated protein kinase (MAPK) and bone morphogenetic protein 2 (BMP2) signaling. This study demonstrated the proliferative and osteogenic efficacies of the trophoblast-derived secretomes, suggesting their potential for use in clinical interventions for bone regeneration and treatment.

Comprehensive analysis of human chorionic membrane extracts regulating mesenchymal stem cells during osteogenesis.

OBJECTIVE: Human chorionic membrane extracts (CMEs) from placenta are known to be a natural biomaterial for bone regeneration, with their excellent osteogenic efficacy on osteoblasts. However, little is known about the regulatory mechanism involved. METHODS AND RESULTS: We have shown the in vitro and in vivo bone-forming ability of CME using human osteoblasts and bone defect animal models, suggesting that CME greatly enhances osteogenesis by providing an osteoconductive environment for the osteogenesis of osteoblasts. Proteomic analysis revealed that CME contained several osteogenesis-related stimulators such as osteopontin, osteomodulin, Thy-1, netrin 4, retinol-binding protein and DJ-1. Additionally, 23 growth factors/growth factor-related proteins were found in CME, which may trigger mitogen-activated protein kinase (MAPK) signalling as a specific cellular signalling pathway for osteogenic differentiation. Microarray analysis showed four interaction networks (chemokine, Wnt signalling, angiogenesis and ossification), indicating the possibility that CME can promote osteogenic differentiation through a non-canonical Wnt-mediated CXCL signalling-dependent pathway. CONCLUSIONS: The results of this study showed the function and mechanism of action of CME during the osteogenesis of osteoblasts and highlighted a novel strategy for the use of CME as a biocompatible therapeutic material for bone regeneration.

7S,15R-Dihydroxy-16S,17S-Epoxy-Docosapentaenoic Acid, a Novel DHA Epoxy Derivative, Inhibits Colorectal Cancer Stemness through Repolarization of Tumor-Associated Macrophage Functions and the ROS/STAT3 Signaling Pathway.

Colorectal cancer is a highly malignant cancer that is inherently resistant to many chemotherapeutic drugs owing to the complicated tumor-supportive microenvironment (TME). Tumor-associated macrophages (TAM) are known to mediate colorectal cancer metastasis and relapse and are therefore a promising therapeutic target. In the current study, we first confirmed the anti-inflammatory effect of 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA), a novel DHA dihydroxy derivative synthesized in our previous work. We found that diHEP-DPA significantly reduced lipopolysaccharide (LPS)-induced inflammatory cytokines secretion of THP1 macrophages, IL-6, and TNF-α. As expected, diHEP-DPA also modulated TAM polarization, as evidenced by decreased gene and protein expression of the TAM markers, CD206, CD163, VEGF, and TGF-ß1. During the polarization process, diHEP-DPA treatment decreased the concentration of TGF-ß1, IL-1ß, IL-6, and TNF-α in culture supernatants via inhibiting the NF-κB pathway. Moreover, diHEP-DPA blocked immunosuppression by reducing the expression of SIRPα in TAMs and CD47 in colorectal cancer cells. Knowing that an inflammatory TME largely serves to support epithelial-mesenchymal transition (EMT) and cancer stemness, we tested whether diHEP-DPA acted through polarization of TAMs to regulate these processes. The intraperitoneally injected diHEP-DPA inhibited tumor growth when administered alone or in combination with 5-fluorouracil (5-FU) chemotherapy in vivo. We further found that diHEP-DPA effectively reversed TAM-conditioned medium (TCCM)-induced EMT and enhanced colorectal cancer stemness, as evidenced by its inhibition of colorectal cancer cell migration, invasion and expression of EMT markers, as well as cancer cell tumorspheres formation, without damaging colorectal cancer cells. DiHEP-DPA reduced the population of aldehyde dehydrogenase (ALDH)-positive cells and expression of colorectal stemness marker proteins (CD133, CD44, and Sox2) by modulating TAM polarization. Additionally, diHEP-DPA directly inhibited cancer stemness by inducing the production of reactive oxygen species (ROS), which, in turn, reduced the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). These data collectively suggest that diHEP-DPA has the potential for development as an anticancer agent against colorectal cancer.

Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea.

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Transcriptomic analysis of tobacco-flavored E-cigarette and menthol-flavored E-cigarette exposure in the human middle ear.

Electronic cigarettes (e-cigarettes) are the most widely used electronic nicotine delivery systems and are designed to imitate smoking and aid in smoking cessation. Although the number of e-cigarette users is increasing rapidly, especially among young adults and adolescents, the potential health impacts and biologic effects of e-cigarettes still need to be elucidated. Our previous study demonstrated the cytotoxic effects of electronic liquids (e-liquids) in a human middle ear epithelial cell (HMEEC-1) line, which were affected by the manufacturer and flavoring agents regardless of the presence of nicotine. In this study, we aimed to evaluate the gene expression profile and identify potential molecular modulator genes and pathways in HMEEC-1 exposed to two different e-liquids (tobacco- and menthol-flavored). HMEEC-1 was exposed to e-liquids, and RNA sequencing, functional analysis, and pathway analysis were conducted to identify the resultant transcriptomic changes. A total of 843 genes were differentially expressed following exposure to the tobacco-flavored e-liquid, among which 262 genes were upregulated and 581 were downregulated. Upon exposure to the menthol-flavored e-liquid, a total of 589 genes were differentially expressed, among which 228 genes were upregulated and 361 were downregulated. Among the signaling pathways associated with the differentially expressed genes mediated by tobacco-flavored e-liquid exposure, several key molecular genes were identified, including IL6 (interleukin 6), PTGS2 (prostaglandin-endoperoxide synthase 2), CXCL8 (C-X-C motif chemokine ligand 8), JUN (Jun proto-oncogene), FOS (Fos proto-oncogene), and TP53 (tumor protein 53). Under menthol-flavored e-liquid treatment, MMP9 (matrix metallopeptidase 9), PTGS2 (prostaglandin-endoperoxide synthase 2), MYC (MYC proto-oncogene, bHLH transcription factor), HMOX1 (heme oxygenase 1), NOS3 (nitric oxide synthase 3), and CAV1 (caveolin 1) were predicted as key genes. In addition, we identified related cellular processes, including inflammatory responses, oxidative stress and carcinogenesis, under exposure to tobacco- and menthol-flavored e-liquids. We identified differentially expressed genes and related cellular processes and gene signaling pathways after e-cigarette exposure in human middle ear cells. These findings may provide useful evidence for understanding the effect of e-cigarette exposure.

Cannabidiol enhances cytotoxicity of anti-cancer drugs in human head and neck squamous cell carcinoma.

Cannabidiol (CBD) has anti-tumorigenic activity. However, the anti-cancer effect of CBD on head and neck squamous cell carcinoma (HNSCC) remains unclear. The cytotoxicity of CBD on HNSCC was analyzed using cell survival and colony-forming assays in vitro. RNA-seq was used for determining the mechanism underlying CBD-induced cell death. Xenograft mouse models were used to determine CBD's effects in vivo. CBD treatment significantly reduced migration/invasion and viability of HNSCC cells in a dose- and time-dependent manner. HNSCC mouse xenograft models revealed anti-tumor effects of CBD. Furthermore, combinational treatment with CBD enhanced the efficacy of chemotherapy drugs. Apoptosis and autophagy processes were involved in CBD-induced cytotoxicity of HNSCCs. RNA-seq identified decreased expression of genes associated with DNA repair, cell division, and cell proliferation, which were involved in CBD-mediated cytotoxicity toward HNSCCs. We identified CBD as a new potential anti-cancer compound for single or combination therapy of HNSCC.

Genomic approach to explore altered signaling networks of olfaction in response to diesel exhaust particles in mice.

Airborne pollutants have detrimental effect on the human body and the environment. Diesel exhaust particles (DEPs) are known to be major component of particulate matter (PM) and cause respiratory diseases and neurotoxicity. However, the effects of air pollutants on the sensory nervous system, especially on the olfactory sense, have not been well studied. Herein, we aimed to explore DEP-induced changes in the olfactory perception process. Olfactory sensitivity test was performed after DEP inhalation in mice. Microarray was conducted to determine the differentially expressed genes, which were then utilized to build a network focused on neurotoxicity. Exposure to DEPs significantly reduced sniffing in mice, indicating a disturbance in the olfactory perception process. Through network analysis, we proposed five genes (Cfap69, Cyp26b1, Il1b, Il6, and Synpr) as biomarker candidates for DEP-mediated olfactory dysfunction. Changes in their expression might provoke malfunction of sensory transduction by inhibiting olfactory receptors, neurite outgrowth, and axonal guidance as well as lead to failure of recovery from neuroinflammatory damage through inhibition of nerve regeneration. Thus, we suggest the potential mechanism underlying DEPs-mediated olfactory disorders using genomic approach. Our study will be helpful to future researchers to assess an individual's olfactory vulnerability following exposure to inhalational environmental hazards.

Large and Externally Positioned Ligand-Coated Nanopatches Facilitate the Adhesion-Dependent Regenerative Polarization of Host Macrophages.

Macrophages can associate with extracellular matrix (ECM) demonstrating nanosequenced cell-adhesive RGD ligand. In this study, we devised barcoded materials composed of RGD-coated gold and RGD-absent iron nanopatches to show various frequencies and position of RGD-coated nanopatches with similar areas of iron and RGD-gold nanopatches that maintain macroscale and nanoscale RGD density invariant. Iron patches were used for substrate coupling. Both large (low frequency) and externally positioned RGD-coated nanopatches stimulated robust attachment in macrophages, compared with small (high frequency) and internally positioned RGD-coated nanopatches, respectively, which mediate their regenerative/anti-inflammatory M2 polarization. The nanobarcodes exhibited stability in vivo. We shed light into designing ligand-engineered nanostructures in an external position to facilitate host cell attachment, thereby eliciting regenerative host responses.

Toxicogenomic study to identify potential signaling alterations related to nasal inflammatory damages induced by diesel exhaust particles in primary human nasal epithelial cells.

In this study, we aimed to identify signaling alteration caused by exposure to diesel exhaust particles (DEPs) using primary human nasal epithelial cells (PHNECs). Global gene expression profiles in PHNECs following 50 and 200 µg/ml of DEP exposure were identified using microarray analysis. To cover the limitation of array-based mRNA expression analysis, text-mining-based software was used to analyze the integrative biological networks and relevant disease-focused functions among identified DEP-responsive genes. The confidence was valued based on the connectivity between the analyzed pathway and marker candidates. Through a literature-based pathway analysis, the stimulation of inflammation- and immune response-related processes mediated by TNF were predicted as major signaling alterations in PHNECs caused by DEP exposure. CSF3, CXCL8, MMP1, and VEGFA were identified as key hub genes in the predicted pathway. Significant expression level changes in the five key genes following DEP exposure were validated in terms of protein and mRNA expression. Although further studies are required, our toxicogenomic investigation provides key clues to the exact mechanism underlying DEP-induced nasal inflammatory damage. It also suggests an efficient approach for other research on adverse effects occurring in the upper respiratory tract following DEP exposure.

Association Between Hearing Loss and Postural Instability in Older Korean Adults.

Importance: Hearing loss is one of the most frequently occurring sensory disabilities worldwide. The association between hearing loss and postural instability in an older population remains to be studied. Objective: To determine whether the odds of postural instability increase with the degree of hearing loss and to suggest criteria for hearing rehabilitation for postural instability. Design, Setting, and Participants: This cross-sectional population-based study of 3864 participants 40 years and older with hearing loss used data from 2010 through 2012 in the fifth edition of the Korea National Health and Nutrition Examination Survey, a nationwide survey of South Korea. Pure tone audiometry was performed on both the left and right sides and each hearing grade of each side was classified into 1 of 3 subcategories: normal hearing, mild hearing loss, or moderate hearing loss (moderate hearing loss or worse). Postural instability was defined as the failure to remain standing on a foam pad surface with feet 10 cm apart with arms folded and hands cupping the elbows and eyes closed. A binomial logistic regression model was used to assess the association between hearing loss and postural instability. Data were analyzed from August 18, 2019, to September 2, 2019. Exposures: Age-related hearing loss defined as a threshold of 26 dB or more. Main Outcomes and Measures: The degree of hearing loss and postural instability. Results: Of the 3864 participants included in the study, the mean (SD) age was 57.8 (11.3) years and 2135 (55.2%) were women. Female sex was associated with higher odds of postural instability compared with male sex (odds ratio [OR], 1.65; 95% CI, 1.12-2.42, adjusted for age and hearing status), and the odds of postural instability increased with every 1 year increase in age (OR, 1.13; 95% CI, 1.10-1.16, adjusted for sex and hearing status). Unilateral or bilateral mild hearing loss was not associated with an increase in the odds of postural instability, but moderate hearing loss present on at least 1 side was associated with an increase in the odds of postural instability: unilateral moderate hearing loss group (OR, 2.71; 95% CI, 1.12-6.10, adjusted for age and sex), one mild and the other moderate hearing loss group (OR, 2.18; 95% CI, 1.16-4.09, adjusted for age and sex), and bilateral moderate hearing loss group (OR, 2.34; 95% CI, 1.27-4.33, adjusted for age and sex). Conclusions and Relevance: Aging, female sex, and having hearing loss were associated with postural instability in this analysis. Moderate or worse hearing loss present on at least 1 side was associated with increased odds of postural instability. Future research in this area is warranted to identify more precise interrelationships and preventive measures.

Effects of Amniotic Membrane Extract on the Hyperplastic Response of the Middle Ear Mucosa in a Bacterially-Induced Otitis Media Rat Model: A Preliminary Study.

OBJECTIVES: Human amniotic membrane extract (AME) is known to contain numerous bioactive factors and anti-inflammatory substances. However, the anti-inflammatory effects of AME on the middle ear (ME) mucosa are unclear. This study assessed the effects of AME on the growth of the ME mucosa in response to bacterially-induced otitis media (OM). METHODS: OM was induced by inoculating nontypeable Haemophilus influenzae (NTHi) into the ME cavity of rats. ME mucosal explants were cultured in AME concentrations of 0, 5, 10, or 50 µg/mL. The area of explant outgrowth was measured in culture and analyzed at 1, 3, 5, and 7 days after explantation. The expression of Ki-67, mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the explants was also evaluated using quantitative polymerase chain reaction (PCR) and immunocytochemistry (ICC). RESULTS: The NTHi-induced ME mucosa growth increased gradually over the 7-day culture period in all explants at different AME concentrations. There was a trend for mucosal growth inhibition at higher concentrations of AME, although the growth was not significantly different among the groups until day 5. The ME mucosal explants treated with the 50 µg/mL concentration of AME showed significantly suppressed growth on postexplantation day 7 compared with other explants on the same day. PCR and ICC staining revealed that the expression of Ki-67, MUC5AC, TNF-α, and IL-10 further decreased in the explants with higher concentrations of AME than in those with lower concentrations of AME. CONCLUSION: Our results showed that higher concentrations of AME reduced the mucosal proliferative response in bacterial OM in rats. These findings provide evidence that AME has an influence on the inflammatory and proliferative responses to NTHi infection in ME mucosa.

Comparison between in vitro toxicities of tobacco- and menthol-flavored electronic cigarette liquids on human middle ear epithelial cells.

Since electronic-cigarettes (e-cigarettes) are considered less toxic than conventional tobacco smoking, the use of e-cigarettes has increased, and the market for e-cigarette liquids (e-liquids) is continuously increasing. However, many studies showed that e-cigarettes may cause various harmful effects in lung, oral and heart. In this study, we investigated the effects of e-liquids on otitis media (OM) using human middle ear epithelial cells (HMEECs). Menthol-flavored e-liquid induced significant cell death in HMEECs (IC50: 1.45 ± 0.14%) and tobacco-flavored e-liquid led to increase in inflammatory cytokine levels and higher mucin production. Flavored e-liquids decreased the mRNA levels of genes encoding epithelial sodium channels (ENaCs) in HMEECs. Apoptosis and autophagy reactions were induced by exposure of HMEECs to menthol- and tobacco-flavored e-liquids. Tobacco-flavored e-liquids caused a greater increase in the levels of autophagosome marker, LC3-II, compared to menthol-flavored e-liquids, which was followed by cell death. These results demonstrate that flavored e-liquids cause cytotoxicity via apoptosis, autophagy, inflammatory response, and mucin production in HMEECs. The flavors present in e-liquids might be a risk factor for the development of otitis media.

Ruthenium Chloride-Induced Oxidative Cyclization of Trans-Resveratrol to (±)-ε-Viniferin and Antimicrobial and Antibiofilm Activity Against Streptococcus pneumoniae.

Polyphenol ε-viniferin (2) is a protective phytochemical found in several plant families. Here, we report a simple and effective method for the synthesis of (±)-ε-viniferin (2) as major product and (±)-(E)-ω-viniferin (3) as a minor product. Synthesized viniferin compounds and standard viniferin were analyzed for antibacterial and antibiofilm activity against Gram-positive bacteria Streptococcus pneumoniae. The minimum inhibitory concentrations (MICs) of (±)-ε-viniferin (2) and standard viniferin were 20 µm. However, the MICs of (±)-(E)-ω-viniferin (3) and compound 8 were 40 µm. Although viniferin significantly (p < 0.05) reduced pre-established in vitro biofilms and killed bacteria within the biofilm, it was unable to prevent biofilm formation at sub-MIC concentrations. The time kill experiment revealed that viniferin killed bacteria and reduced 2.8 log10 bacteria at 2 × MIC concentration after 24 h. Scanning electron microscope (SEM) analysis and live/dead biofilm staining of pre-established biofilms revealed that viniferin treatment disrupts membrane integrity of biofilm bacteria. Crystal violet absorption, total protein, and DNA and RNA release revealed that viniferin alters bacterial cell permeability, eventually killing bacteria.

Engineering functional BMP-2 expressing teratoma-derived fibroblasts for enhancing osteogenesis.

Bone morphogenetic protein 2 (BMP-2) is considered an effective growth factor for bone formation, and is used for making osteo-inductive scaffolds, but the related clinical investigations have shown low success rates. In this study, we genetically manipulated teratoma-derived fibroblast (TDF) cells by simultaneous introduction of BMP-2 and herpes simplex virus-thymidine kinase (HSV-tk) encoding genes. Self-production of BMP-2 in TDF cells strongly enhanced the alkaline phosphatase (ALP) activity, calcium content, and elevated the mRNA expression of osteogenic marker genes during in vitro osteogenesis. The bone formation volume was also remarkably enhanced in calvarial and femoral critical-size defect models. Ganciclovir (GCV) treatment induced apoptosis in TDF cells co-expressing HSV-tk and BMP-2, implying that HSV-tk suicide gene can modulate the side-effects of stem cell therapy, e.g., development of uncontrollable teratoma and tumor formation. Altogether, our findings revealed a safe and highly efficient technique with potential therapeutic applications for bone regeneration.

Anti-cancer effects of disulfiram in head and neck squamous cell carcinoma via autophagic cell death.

BACKGROUND: Disulfiram (DSF), which is used to treat alcohol dependence, has been reported to have anti-cancer effects in various malignant tumors. In this study, we investigated the anti-cancer effects and mechanism of DSF in HNSCC. METHODS: Head and neck squamous carcinoma cell lines (FaDu and Hep2) were used to analyze the anti-cancer effects of DSF. The anti-cancer effects of DSF were confirmed in vivo using a xenograft tumor model. RESULTS: The anti-cancer effects of DSF in HNSCC were found to be copper (Cu) dependent. Specifically, DSF/Cu markedly inhibited HNSCC at a concentration of 1 µM. After DSF/Cu administration, production of reactive oxygen species (ROS) was remarkable starting at 0.5 µM, suggesting that the inhibitory effects of DSF/Cu on HNSCC are mediated through the formation of ROS. The levels of phospho-JNK, phospho-cJun and phospho-p38 were increased after DSF/Cu treatment while levels of phospho-Akt were decreased. These results suggested that the inhibitory effects of DSF/Cu on HNSCC cells involve ROS formation and down-regulation of Akt-signaling. Through these molecular mechanisms, DSF ultimately induce the inhibitory effects on HNSCC cell lines mainly through autophagic cell death, not apoptotic cell death. Lastly, we investigated the clinical relevance of DSF/Cu using a HNSCC xenograft animal model, which showed that tumor growth was remarkably decreased by DSF (50 mg/kg injection). CONCLUSION: In treating patients with HNSCC, DSF may contribute to improved HNSCC patient's survival. The characteristic anti-cancer effects of DSF on HNSCC may suggest new therapeutic potential for this medication in HNSCC patients.

Effect of electronic cigarettes on human middle ear.

OBJECTIVE: Electronic cigarettes (e-cigarettes) are the most commonly used electronic nicotine delivery systems and are a relatively new product designed for smoking cessation. The market scale of electronic cigarettes is growing rapidly, but the potential impact of e-cigarettes on public health has not yet been verified. In this study, we examined the effect of e-liquids on a human middle ear epithelial cell (HMEEC) line. MATERIAL AND METHODS: The main components of e-liquids are propylene glycol, vegetable glycerin and flavoring agents with or without nicotine. We analyzed 73 bottles of e-liquids from 12 different manufacturers, evaluated the trace elements in e-liquids, and identified the cytotoxicity of e-liquids on HMEECs in the presence or absence of nicotine. RESULTS: In the trace elements analysis, nickel, arsenic, cadmium, and lead were detected in the e-liquids. E-liquids without nicotine decreased cell viability, and the average IC 50 value of total e-liquids (n = 73) was 2.48 ±â€¯0.93%. Among the different flavors, menthol-flavored e-liquids significantly reduced cell viability, and their average IC 50 value (n = 28) was 1.85 ±â€¯0.80%. The average IC 50 values were distinct among manufacturers and the proportion of the solvents. CONCLUSION: The present study provides evidence that e-cigarettes influence and reduce human middle ear cell viability even without the application of nicotine. Additionally, the cytotoxicity of e-liquids was affected by the flavoring agents.