Transcriptional Analysis of Mice Melanoma B16-F10 Cells in Response to Directed Current Electric Fields.

The endogenous electric field (EF) is widely observed among tissues. It is supposed to be an important environmental factor in tumor metastasis. To explore the role of endogenous EFs in tumor metastasis, the migration of mouse melanoma B16-F10 cells in directed current EFs (dcEFs) was investigated. The transcriptome of melanoma B16-F10 cells in response to EF stimulation was analyzed using RNA sequencing. The results demonstrated that the mouse melanoma B16-F10 cells migrated toward the cathode in applied dcEFs. Directional migration occurred in a voltage-dependent manner. Approximately 3000 upregulated and 2613 downregulated genes were identified under dcEF. Some genes correlated with cell migration, such as Serpine1, Ctgf, Fosb, and Fos, were upregulated. The signaling pathways involved in cell motility were significantly altered. Some genes, highly related to tumorigenesis, invasion, and metastasis, are upregulated in response to EF stimulation. Endogenous EFs may play a role in tumorigenesis and metastasis in vivo. © 2022 Bioelectromagnetics Society.

Applied electric fields suppress osimertinib-induced cytotoxicity via inhibiting FOXO3a nuclear translocation through AKT activation.

Osimertinib is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor against T790M-mutant non-small cell lung cancer (NSCLC). Acquired resistance to osimertinib is a growing clinical challenge that is not fully understood. Endogenous electric fields (EFs), components of the tumor microenvironment, are associated with cancer cell migration and proliferation. However, the impact of EFs on drug efficiency has not been studied. In this study, we observed that EFs counteracted the effects of osimertinib. EFs of 100 mV/mm suppressed osimertinib-induced cell death and promoted cell proliferation. Transcriptional analysis revealed that the expression pattern induced by osimertinib was altered by EFs stimulation. KEGG analysis showed that differential expression genes were mostly enriched in PI3K-AKT pathway. Then, we found that osimertinib inhibited AKT phosphorylation, while EFs stimulation resulted in significant activation of AKT, which could override the effects generated by osimertinib. Importantly, pharmacological inhibition of PI3K/AKT by LY294002 diminished EF-induced activation of AKT and restored the cytotoxicity of osimertinib suppressed by EFs, which proved that AKT activation was essential for EFs to attenuate the efficacy of osimertinib. Furthermore, activation of AKT by EFs led to phosphorylation of forkhead box O3a (FOXO3a), and reduction in nuclear translocation of FOXO3a induced by osimertinib, resulting in decreased expression of Bim and attenuated cytotoxicity of osimertinib. Taken together, we demonstrated that EFs suppressed the antitumor activity of osimertinib through AKT/FOXO3a/Bim pathway, and combination of PI3K/AKT inhibitor with osimertinib counteracted the effects of EFs. Our findings provided preliminary data for therapeutic strategies to enhance osimertinib efficacy in NSCLC patients.

Health-related quality of life after mandibular resection for oral cancer: reconstruction with free fibula flap.

OBJECTIVES: Mandibular resection for oral cancer is often necessary to achieve an adequate margin of tumor clearance.Mandibular resection has been associated with a poor health-related quality of life (HRQOL), particularly before free fibula flap to reconstruct the defect. The aim of this study was to evaluate health-related quality of life in patients who have had mandibular resections of oral cancer and reconstruction with free fibula flap. STUDY DESIGNS: There were 115 consecutive patients between 2008 and 2011 who were treated by primary surgery for oral squamous cell carcinoma, 34 patients had a mandibular resection. HRQOL was assessed by means of the 14-item Oral Health Impact Profile (OHIP-14) and University of Washington Quality of Life (UW-QOL) questionnaires after 12 months postoperatively. RESULTS: In the UW-QOL the best-scoring domain was mood, whereas the lowest scores were for chewing and saliva. In the OHIP-14 the lowest-scoring domain was social disability, followed by handicap, and psychological disability. CONCLUSION: Mandible reconstruction with free fibula flap would have significantly influenced on patients'quality of life and oral functions.The socio-cultural data show a fairly low level of education for the majority of patients.

3D arrays for high throughput assay of cell migration and electrotaxis.

Cell behaviour in 3D environments can be significantly different from those in 2D cultures. With many different 3D matrices being developed and many experimental modalities used to modulate cell behaviour in 3D, it is necessary to develop high throughput techniques to study behaviour in 3D. We report on a 3D array on slide and have adapted this to our electrotaxis chamber, thereby offering a novel approach to quantify cellular responses to electric fields (EFs) in 3D conditions, in different matrices, with different strains of cells, under various field strengths. These developments used Dictyostelium cells to illustrate possible applications and limitations.

A new fixation method for stick-shaped specimens in microtensile tests: laboratory tests and FEA.

PURPOSE: To introduce a new fixation method for stick-shaped specimens for use in microtensile tests and to evaluate the effect of this new method on microtensile bond strength, failure modes, and stress distribution. MATERIALS AND METHODS: Flat mid-coronal dentin surfaces were prepared on 12 caries-free human third molars and randomly divided into two groups for testing with two dental adhesives (Adper Single Bond 2 [SB2] and Clearfil SE Bond [SEB]). Following adhesive application and composite buildups, the bonded teeth were sectioned into beams. Sticks from each tooth were then equally divided into two subgroups for microtensile bond testing according to the utilized gripping devices (a flat Ciucchi's jig and the experimental setup). Failure modes were examined with a field-emission scanning electron microscope (FESEM). Three-dimensional models of each gripping device and specimen were developed, and stress distributions were analyzed by finite element analysis (FEA). Statistical significance was set at α = 0.05 RESULTS: Compared to those fixed using a flat Ciucchi's jig, sticks fixed with the experimental setup yielded lower bond strength values (p = 0.021 for SB2 and p = 0.007 for SEB) and more mixed failure patterns (p = 0.036 for both SB2 and SEB). In addition, the experimental setup guaranteed a uniaxial tensile force that was perpendicular to the bonding interface and produced a more uniform stress distribution at the bonding interface. CONCLUSION: An experimental setup for fixing microtensile sticks was proposed that was designed to provide a uniform stress distribution at the adhesive interface. FEA and failure mode analysis confirmed such uniform distribution, thus supporting the validity of the bond strength results obtained with this new fixture design.

Essential oil of lemon myrtle (Backhousia citriodora) induces S-phase cell cycle arrest and apoptosis in HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Lemon myrtle (Backhousia citriodora F.Muell.) leaves, whether fresh or dried, are used traditionally in folk medicine to treat wounds, cancers, skin infections, and other infectious conditions. However, the targets and mechanisms related to anti-cancer effect of lemon myrtle are unavailable. In our study, we found that the essential oil of lemon myrtle (LMEO) showed anti-cancer activity in vitro, and we initially explored its mechanism of action. MATERIALS AND METHODS: We analyzed the chemical compositions of LMEO by GC-MS. We tested the cytotoxicity of LMEO on various cancer cell lines using the MTT assay. Network pharmacology was used also to analyze the targets of LMEO. Moreover, the mechanisms of LMEO were investigated through scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. RESULTS: LMEO showed cytotoxicity on various cancer cell lines with values of IC50 40.90 ± 2.23 (liver cancer HepG2 cell line), 58.60 ± 6.76 (human neuroblastoma SH-SY5Y cell line), 68.91 ± 4.62 (human colon cancer HT-29 cell line) and 57.57 ± 7.61 µg/mL (human non-small cell lung cancer A549 cell line), respectively. The major cytotoxic chemical constituent in LMEO was identified as citrals, which accounted for 74.9% of the content. Network pharmacological analysis suggested that apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), androgen receptor (AR), cyclin-dependent kinases 1 (CDK1), nuclear factor erythroid 2-related factor 2 (Nrf-2), fatty acid synthase (FASN), epithelial growth factor receptor (EGFR), estrogen receptor 1 (ERα) and cyclin-dependent kinases 4 (CDK4) are potential cytotoxic targets of LMEO. These targets are closely related to cell migration, cycle and apoptosis. Notley, the p53 protein had the highest confidence to co-associate with the eight common targets, which was further confirmed by scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. LMEO significantly inhibited the migration of HepG2 cells in time-dependent and dose-dependent manner. Moreover, LMEO caused a S-phase blocking on HepG2 cells and promoted apoptosis in the meanwhile. Western blot results indicated that p53 protein, Cyclin A2 and Bax proteins were up-regulated, while Cyclin E1 and Bcl-2 proteins were down-regulated. CONCLUSION: LMEO showed cytotoxicity in various cancer cell lines in vitro. Pharmacological networks showed LMEO to have multi-component and multi-targeting effects that are related to inhibit migration of HepG2 cells, and affect cell cycle S-phase arrest and apoptosis through modulation of p53 protein.

A hnRNPA2B1 agonist effectively inhibits HBV and SARS-CoV-2 omicron in vivo.

The twenty-first century has already recorded more than ten major epidemics or pandemics of viral disease, including the devastating COVID-19. Novel effective antivirals with broad-spectrum coverage are urgently needed. Herein, we reported a novel broad-spectrum antiviral compound PAC5. Oral administration of PAC5 eliminated HBV cccDNA and reduced the large antigen load in distinct mouse models of HBV infection. Strikingly, oral administration of PAC5 in a hamster model of SARS-CoV-2 omicron (BA.1) infection significantly decreases viral loads and attenuates lung inflammation. Mechanistically, PAC5 binds to a pocket near Asp49 in the RNA recognition motif of hnRNPA2B1. PAC5-bound hnRNPA2B1 is extensively activated and translocated to the cytoplasm where it initiates the TBK1-IRF3 pathway, leading to the production of type I IFNs with antiviral activity. Our results indicate that PAC5 is a novel small-molecule agonist of hnRNPA2B1, which may have a role in dealing with emerging infectious diseases now and in the future.

Gene expression profiles of HLA-G1 overexpressed in hES cells.

The goals of this study were to analyze the change in the global gene expression profile of exogenous human leukocyte antigen-G1 (HLA-G1) overexpressed in human embryonic stem (hES) cells and to explore the molecular mechanism by which the overexpression of HLA-G1 modifies immunologic pathways. Microarray and quantitative real-time PCR analyses were performed to quantify the differential expression pattern of HLA-G1 + H1 hES cells. The results showed that HLA-G1 differentially regulated the expression of 425 genes with at least a twofold increase or decrease. These differentially expressed genes were classified into 13 functional groups, including cellular components, biological processes, and molecular functions. The pathways of focal adhesion, the TGF-ß signaling pathway, and the immune response were the most predominantly affected. The synergism of these genes could explain the mechanism of the immunosuppression of HLA-G1 + H1 hES cells. Thus, the expression pattern reflected a broad spectrum of roles of HLA-G1 in hES cells.

The effect of transient proanthocyanidins preconditioning on the cross-linking and mechanical properties of demineralized dentin.

Proanthocyanidin-based preconditioners were prepared by adding powdered proanthocyanidins-rich grape seed extract to various solvents at different concentrations. Demineralized dentin specimens were preconditioned for 20, 30, 60 or 120 s, followed by the evaluation of their cross-linking degree, mechanical properties and micromorphology. The cross-linking degree of the demineralized dentin collagen exhibited concentration- and time- dependent increase after preconditioning treatment, irrespective of the preconditioner and the solvent. When treated for the same exposure time, specimens after 15% proanthocyanidins preconditioning resulted in the highest mean ultimate tensile strength compared with all the other groups tested. Five percent glutaraldehyde control group produced the highest cross-linking degree, but the ultimate tensile strength was lower than that of 15% proanthocyanidins group. The field emission scanning electron microscopy confirmed that the demineralized dentin collagen was in a homogeneous and regular arrangement after preconditioning and maintained expanding, regardless of the surface moisture conditions.

Nanoleakage and microtensile bond strength at the adhesive-dentin interface after different etching times.

PURPOSE: To evaluate the effect of etching time on the nanoleakage and microtensile bond strength (microTBS) at the interface between etch-and-rise adhesives and dentin. METHODS: Eighty molars were sectioned to expose the superficial dentin and randomly divided into four groups according to the adhesives used: OptiBond Solo (OB), Single Bond (SB), One-Step (OS), and Prime&Bond NT (PB). Within each group, a total-etching technique was applied, and the dentin surfaces were etched for 0, 15, 30 or 60 seconds. Each treated tooth was then sectioned into sticks. A non-trimming microTBS test and TEM observation for nanoleakage were sequentially performed. RESULTS: The degree of nanoleakage increased as etching time increased (P < 0.05). For the OS and PB groups, the highest microTBS values were achieved with 15 seconds of etching, followed by 30, 60 and 0 seconds. For the OB and SB groups, the microTBS values for 15- and 30-second etching times were similar and were both significantly higher than those of 0 and 60 seconds (P < 0.05).

Clinical outcomes of implant-retained mandibular overdentures using the bar and magnetic attachment systems: an up to 5-year retrospective study.

BACKGROUND: Implant-retained mandibular overdentures (IODs) represent an effective and reliable treatment modality for edentulous patients. The present retrospective study compared the clinical outcomes of IODs using bar attachment (BA) system with those using magnetic attachment (MA) system after functioning for up to 5 years. METHODS: Human subjects treated with IODs between 01-01-2010 and 12-31-2014 were identified from patient records. Of the 54 subjects who met the inclusion criteria, 48 subjects including 26 treated with BA-IODs and 22 with MA-IODs (96 mandibular implants) were recruited for the study. The implant units and prostheses were evaluated individually for peri-implant health. Prosthetic complications and maintenance during follow-up were recorded. The subjects responded to the visual analog scale (VAS) and the Oral Health Impact Profile questionnaires for evaluation of patient satisfaction and oral health-related quality of life (OHRQoL). RESULTS: The survival rates of the implants and prostheses were 96.9% and 95.8%, respectively, over a mean observation period of 48±11.3 (range, 13-64) months. Peri-implant probing depth (PPD) and plaque index (PI) were significantly better for the MA group compared with the BA group (P<0.05), while marginal bone loss (MBL) and sulcus bleeding index (SBI) showed no significant differences (P>0.05). Prosthetic complications and maintenance were attachment-dependent. Most recruited subjects were satisfied with their prostheses. There was no statistically significant difference regarding general patient satisfaction or OHRQoL between the two groups (P>0.05). Nevertheless, patients complained that the BA-IODs were significantly more difficult to clean than the MA-IODs (P<0.05). CONCLUSIONS: IODs have an ideal medium-term outcome irrespective of the attachment design. It is recommended that oral hygiene instructions and regular clinical examination be given to subjects wearing IODs.

Characterization of a homogeneous taxonomic group of marine magnetotactic cocci within a low tide zone in the China Sea.

Magnetotactic bacteria are a heterologous group of motile prokaryotes, ubiquitous in aquatic habitats and cosmopolitan in distribution. Here, we studied the diversity of magnetotactic bacteria in a seawater pond within an intertidal zone at Huiquan Bay in the China Sea. The pond is composed of a permanently submerged part and a low tide subregion. The magnetotactic bacteria collected from the permanently submerged part display diversity in morphology and taxonomy. In contrast, we found a virtually homogenous population of ovoid-coccoid magnetotactic bacteria in the low tide subregion of the pond. They were bilophotrichously flagellated and exhibited polar magnetotactic behaviour. Almost all cells contained two chains of magnetosomes composed of magnetite crystals. Intriguingly, the combination of restriction fragment length polymorphism analysis (RFLP) and sequencing of cloned 16S rDNA genes from the low tide subregion samples as well as fluorescence in situ hybridization (FISH) revealed the presence of a homogenous population. Moreover, phylogenetic analysis indicated that the Qingdao Huiquan low tide magnetotactic bacteria belong to a new genus affiliated with the alpha-subclass of Proteobacteria. This finding suggests the adaptation of the magnetotactic bacterial population to the marine tide.

Correction: Caveolin-1-mediated STAT3 activation determines electrotaxis of human lung cancer cells.

[This corrects the article DOI: 10.18632/oncotarget.21306.].

Antibacterial functionalization of an experimental self-etching primer by inorganic agents: microbiological and biocompatibility evaluations.

Antibacterial activities have been demonstrated on oral bacteria with inorganic antibacterial agents (ABAs) after their incorporations into an experimental self-etching primer (ESP) before curing. This study was to assess their biocompatibility and antibacterial activity after curing. Six ABAs were incorporated respectively into ESP for treating specimens. After curing, their bactericidal activities on Streptococcus mutans and influences to the early bacterial colonization were assessed by direct contact and viable count. Systemic toxicity in rats after short-term oral exposure and direct contact cytotoxicity with NIH3T3 fibroblasts were tested. Incorporation of ZnOw AT-83, Longbei antibiotic, Antim-AMS2 or IONPURE-H significantly enhanced the antibacterial effect of ESP after curing, even after 1 month aging. Specimens treated by ESP with ZnOw AT-83, Longbei antibiotic or Antim-AMS2 showed slightly less bacterial adhesion than control. Animal experiments revealed neither toxic signs nor significant differences in body weight gain between control and other groups. Cell vitality or proliferation rates were ranged from 76% to 100% with respect to controls. Basic magnesium hypochlorite, ZnOw AT-83 and ZnOw AT-88 were less toxic. Toxicity only observed in areas beneath the specimens and/or in the direct vicinity of the specimen edge. From microbiological and biocompatibility aspects, the tested ABAs can be effectively incorporated in ESP to provide antibacterial activity against S. mutans. ZnOw AT-83 was the most promising one.

Occlusion and Disocclusion Time Changes in Single Unit Crowns Designed by Functional Generated Path Technique: A Randomised Clinical Trial.

Although it is believed that implementation of the functional generated path (FGP) technique can facilitate occlusal surface design for restorations, it has not been objectively compared in situ with the conventional fabrication yet. Therefore, in the present study, a single-blind crossover clinical trial was conducted using T-scan to compare changes in occlusion time (OT) and disocclusion time (DT) of single posterior artificial crowns designed differently using FGP technique (FGP), average-value FGP technique (AVR) and conventional fabrication (CON). Each of the 10 participants took part in the study tried three artificial crowns in different sequences according to a computer generated randomization list. The results objectively revealed that changes in OT and DT were significantly smaller for FGP than CON (P < 0.05) and considerably smaller for AVR than CON, respectively. The subjective feedback and the occlusal adjusting time were better and shorter for FGP and AVR than CON (P < 0.05). No harm to the participants occurred. Overall, FGP is an efficient technique showing more physiological harmonious relationship with the articulating system.

Caveolin-1-mediated STAT3 activation determines electrotaxis of human lung cancer cells.

Migration of cancer cells leads to the invasion of distant organs by primary tumors. Further, endogenous electric fields (EFs) in the tumor microenvironment direct the migration of lung cancer cells by a process referred to as electrotaxis - although the precise mechanism remains unclear. Caveolin-1 (Cav-1) is a multifunctional scaffolding protein that is associated with directional cell migration and lung cancer invasion; however, its precise role in lung cancer electrotaxis is unknown. In the present study, we first detected outward electric currents on the tumor body surface in lung cancer xenografts using a highly-sensitive vibrating probe. Next, we found that highly-metastatic H1650-M3 cells migrated directionally to the cathode. In addition, reversal of the EF polarity reversed the direction of migration. Mechanistically, EFs activated Cav-1 and the downstream signaling molecule STAT3. RNA interference of Cav-1 reduced directional cell migration, which was accompanied by dampened STAT3 activation. Furthermore, pharmacological inhibition of STAT3 significantly reduced the electrotactic response, while rescue of STAT3 activation in Cav-1 knock-down cells restored electrotaxis. Taken together, these results suggest that endogenous EFs in the tumor micro-environment might play an important role in lung cancer metastasis by guiding cell migration through a Cav-1/STAT3-mediated signaling pathway.

Chemical Composition and Acetylcholinesterase Inhibitory Activity of Essential Oils from Piper Species.

The essential oils (EOs) derived from aromatic plants such as Piper species are considered to play a role in alleviating neuronal ailments that are associated with inhibition of acetylcholinesterase (AChE). The chemical compositions of 23 EOs prepared from 16 Piper spp. were analyzed by both gas chromatography with a flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 76 compounds were identified in the EOs from the leaves and stems of 19 samples, while 30 compounds were detected in the EOs from the fruits of four samples. Sesquiterpenes and phenylpropanoids were found to be rich in these EOs, of which asaricin, caryophyllene, caryophyllene oxide, isospathulenol, (+)-spathulenol, and ß-bisabolene are the major constituents. The EOs from the leaves and stems of Piper austrosinense, P. puberulum, P. flaviflorum, P. betle, and P. hispidimervium showed strong AChE inhibitory activity with IC50 values in the range of 1.51 to 13.9 mg/mL. A thin-layer chromatography (TLC) bioautography assay was employed to identify active compound(s) in the most active EO from P. hispidimervium. The active compound was isolated and identified as asaricin, which gave an IC50 value of 0.44 ± 0.02 mg/mL against AChE, comparable to galantamine with an IC50 0.15 ± 0.01 mg/mL.

Clinical evaluation of 546 tetracycline-stained teeth treated with porcelain laminate veneers.

OBJECTIVE: The purpose of this study was to evaluate the clinical result of 546 tetracycline-stained teeth restored with a porcelain laminate veneer system (Cerinate, Den-Mat, USA) for aesthetic reasons. METHODS: Tetracycline-stained teeth (546) were restored with a porcelain veneer system, and bonded with Ultra Bond resin cement. The restorations were recalled after 0.5, 1.5 and 2.5 years, respectively. Modified Ryge criteria were used to evaluate the veneers marginal adaptation, interfacial staining, secondary caries, postoperative sensitivity and the patients' satisfaction of the colour of the restorations. RESULTS: This study found that 99% veneers had excellent marginal adaptations; and less than 1% veneers required rebonding in the first 6 months; the colour of the veneers was stable and no evident staining was found. Almost all patients were satisfied with the colour match of their restorations 1 year after placement. CONCLUSIONS: The research indicated that the porcelain veneer restoration system under investigation provided a reliable and highly satisfactory choice for the aesthetic restoration of tetracycline-stained teeth.

[Analysis of type-I collagen fibrils and chondroitin sulfate distribution in human dentin by confocal laser scanning microscopy combined with dual immunofluorescent labeling technique].

OBJECTIVE: To introduce the method of dual immunofluorescence labeling of human dentin matrix without demineralization of the whole dentin fragments, and to analyze the distribution of type-I collagen fibrils and chondroitin sulfate in human dentin. METHODS: Forty 30 µm- thick middle coronal dentin sections were obtained from 8 freshly extracted human third molars and etched with 37% phosphoric acid(PA) gel for 15 s. After preconditioning with or without tosyl- phenylalanyl chloromethyl ketone(TPCK) treated trypsin digestion, sections were subjected to dual immunofluorescent labeling and scanned by confocal laser scanning microscopy to identify the type-I collagen fibrils and chondroitin sulfate. RESULTS: Chondroitin sulfate was localized in the lumens of the dentin tubules and peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin. After preconditioning with TPCK treated trypsin digestion, the red fluorescence was decreased or disappeared. CONCLUSIONS: The dual immunofluorescence labeling methodology can be used to study the human dentin matrix without demineralization of the whole dentin fragments. Chondroitin sulfate was localized in the lumens of the dentin tubules and peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin.

A large-scale screen reveals genes that mediate electrotaxis in Dictyostelium discoideum.

Directional cell migration in an electric field, a phenomenon called galvanotaxis or electrotaxis, occurs in many types of cells, and may play an important role in wound healing and development. Small extracellular electric fields can guide the migration of amoeboid cells, and we established a large-scale screening approach to search for mutants with electrotaxis phenotypes from a collection of 563 Dictyostelium discoideum strains with morphological defects. We identified 28 strains that were defective in electrotaxis and 10 strains with a slightly higher directional response. Using plasmid rescue followed by gene disruption, we identified some of the mutated genes, including some previously implicated in chemotaxis. Among these, we studied PiaA, which encodes a critical component of TORC2, a kinase protein complex that transduces changes in motility by activating the kinase PKB (also known as Akt). Furthermore, we found that electrotaxis was decreased in mutants lacking gefA, rasC, rip3, lst8, or pkbR1, genes that encode other components of the TORC2-PKB pathway. Thus, we have developed a high-throughput screening technique that will be a useful tool to elucidate the molecular mechanisms of electrotaxis.