Next-generation sequencing analysis of exosomal microRNAs: Fusobacterium nucleatum regulates the expression profiling of exosomal microRNAs in human colorectal cancer cells

Colon cancer is one of the most common malignant tumors, but there are still a few validated biomarkers of colon cancer. Exosome-mediated microRNAs (miRNAs) have been recognized as potential biomarkers in cancers, and miRNAs can regulate a variety of genes. Recently, Fusobacterium nucleatum was discovered in the tissues of human colon cancer patients. Its role in colon cancer was highlighted. F. nucleatum may contribute to the progression of colon cancer through the mechanism of exosome-mediated miRNAs transfer. However, the exosomal miRNAs regulation mechanism by F. nucleatum in colon cancer is not well known. Thus, we performed next-generation sequencing to investigate the overall pattern of exosomal miRNAs expression in the colon cancer cell culture supernatant. We have confirmed the alterations of various exosomal miRNAs. In addition, to investigate the function of exosomal miRNAs, a Kyoto Encyclopedia of Genes and Genomes analysis was performed on the target genes of changed miRNAs. Potential target genes were associated with a variety of signaling pathways, and one of these pathways was related to colorectal cancer. These findings suggested that F. nucleatum can alter exosomal miRNAs released from colorectal cancer cells. Furthermore, exosomal miRNAs altered by F. nucleatum could be potential biomarkers for the diagnosis and therapy of colon cancer.

Local Injection of Growth Hormone for Temporomandibular Joint Osteoarthritis

PURPOSE: Osteoarthritis (OA) of the temporomandibular joint (TMJ) elicits cartilage and subchondral bone defects. Growth hormone (GH) promotes chondrocyte growth. The aim of this study was to evaluate the efficacy of intra-articular injections of GH to treat TMJ-OA.MATERIALS AND METHODS: Monosodium iodoacetate (MIA) was used to induce OA in the TMJs of rats. After confirming the induction of OA, recombinant human GH was injected into the articular cavities of rats. Concentrations of GH and IGF-1 were measured in the blood and synovial fluid, and OA grades of cartilage and subchondral bone degradation were recorded by histological examination and micro-computed tomography.RESULTS: MIA-induced OA in the rat TMJ upregulated insulin-like growth factor-1 (IGF-1) rather than GH levels. GH and IGF-1 concentrations were increased after local injection of GH, compared with controls. Locally injected GH lowered osteoarthritic scores in the cartilage and subchondral bone of the TMJ.CONCLUSION: Intra-articular injection of GH improved OA scores in rat TMJs in both cartilage and subchondral bone of the condyles without affecting condylar bone growth. These results suggest that intra-articular injection of human GH could be a suitable treatment option for TMJ-OA patients in the future.

Langerhans cell histiocytosis of the mandible: two case reports and literature review

Langerhans cell histiocytosis (LCH) is a rare disorder characterized by the proliferation of dendritic cells resulting in local or systemic symptoms. The clinical symptoms of patients with Langerhans cell histiocytosis depend on the site and the degree of involvement. This article describes two case histories of unifocal bony Langerhans cell histiocytosis with mandibular involvement and further discusses the appropriate management of such via a review of the literature.

Langerhans cell histiocytosis of the mandible: two case reports and literature review

Langerhans cell histiocytosis (LCH) is a rare disorder characterized by the proliferation of dendritic cells resulting in local or systemic symptoms. The clinical symptoms of patients with Langerhans cell histiocytosis depend on the site and the degree of involvement. This article describes two case histories of unifocal bony Langerhans cell histiocytosis with mandibular involvement and further discusses the appropriate management of such via a review of the literature.

Reconstruction of cheek mucosal defect with a buccal fat pad flap in a squamous cell carcinoma patient: a case report and literature review / 대한악안면성형재건외과학회지

BACKGROUND: Squamous cell carcinoma (SCC) is the most commonly occurring malignant tumor in the oral cavity. In South Korea, it occurs most frequently in the mandible, tongue, maxilla, buccal mucosa, other areas of the oral cavity, and lips. Radial forearm free flap (RFFF) is the most widely used reconstruction method for the buccal mucosal defect. The scar of the forearm donor, however, is highly visible and unsightly, and a secondary surgical site is needed when such technique is applied. For these reasons, buccal fat pad (BFP) flap has been commonly used for closing post-surgical excision sites since the recent decades because of its reliability, ease of harvest, and low complication rate. CASE PRESENTATION: In the case reported herein, BFP flap was used to reconstruct a cheek mucosal defect after excision. The defect was completely covered by the BFP flap, without any complications. CONCLUSION: Discussed herein is the usefulness of BFP flap for the repair of the cheek mucosal defect. Also, further studies are needed to determine the possibility of using BFP flap when the defect is deep, and the maximum volume that can be harvested considering the changes in volume with age.

Drosophila Homolog of Human KIF22 at the Autism-Linked 16p11.2 Loci Influences Synaptic Connectivity at Larval Neuromuscular Junctions

Copy number variations at multiple chromosomal loci, including 16p11.2, have recently been implicated in the pathogenesis of autism spectrum disorder (ASD), a neurodevelopmental disease that affects 1~3% of children worldwide. The aim of this study was to investigate the roles of human genes at the 16p11.2 loci in synaptic development using Drosophila larval neuromuscular junctions (NMJ), a well-established model synapse with stereotypic innervation patterns. We conducted a preliminary genetic screen based on RNA interference in combination with the GAL4-UAS system, followed by mutational analyses. Our result indicated that disruption of klp68D, a gene closely related to human KIF22, caused ectopic innervations of axon branches forming type III boutons in muscle 13, along with less frequent re-routing of other axon branches. In addition, mutations in klp64D, of which gene product forms Kinesin-2 complex with KLP68D, led to similar targeting errors of type III axons. Mutant phenotypes were at least partially reproduced by knockdown of each gene via RNA interference. Taken together, our data suggest the roles of Kinesin-2 proteins, including KLP68D and KLP64D, in ensuring proper synaptic wiring.

Autophagy Inhibition Promotes Quercetin Induced Apoptosis in MG-63 Human Osteosarcoma cells

Quercetin is a natural flavonoid phytochemical that is extracted from various plants. Having an advantages due to its varied biological properties, such as anti-inflammatory, anti-viral, anti-oxidant, and anti-cancer effects, quercetin is used to treat many diseases. Recently, it has been reported that autophagy inhibition may play a key role in anti-cancer therapy. Therefore, in this study, we investigated the molecular mechanisms and anti-cancer effects of quercetin in human osteosarcoma cells via autophagy inhibition. We ascertained that quercetin inhibited cell proliferation and induced cell death, these process is demonstrated that apoptosis via the mitochondrial pathway and the caspase cascade. Quercetin also induced autophagy which was inhibited by 3-MA, autophagy inhibitor and the blockade of autophagy promoted the quercetin-induced apoptosis, confirming that autophagy is a pro-survival process. Thus, these findings demonstrate that quercetin is an effective anti-cancer agent, and the combination of quercetin and an autophagy inhibitor should enhance the effect of anti-cancer therapy.

NaF-induced Autophagy on SCC25 Human Tongue Squamous Cell Carcinoma Cells

Fluoride has been accepted as an important material for oral health and is widely used to prevent dental caries in dentistry. However, its safety is still questioned by some. Autophagy has been implicated in cancer cell survival and death, and may play an important role in oral cancer. This study was undertaken to examine whether sodium fluoride (NaF) modulates autophagy in SCC25 human tongue squamous cell carcinoma cells. NaF demonstrated anticancer activity via autophagic and apoptotic cell death. Autophagic vacuoles were detectable using observed to form by monodansylcadaverine (MDC) and acridine orange (AO). Analysis of NaF-treated SCC25 cells for the presence of biochemical markers revealed direct effects on the conversion of LC-3II, degradation of p62/SQSTM1, cleavage formation of ATG5 and Beclin-1, and caspase activation. NaF-induced cell death was suppressed by the autophagy inhibitor 3-methyladenine (3-MA). NaF-induced autophagy was confirmed as a pro-death signal in SCC25 cells. These results implicate NaF as a novel anticancer compound for oral cancer therapy.

The Inhibition of Oxidative Stress by Chios Gum Mastic is Associated with Autophagy

Chios Gum Mastic (CGM) is a natural resin extracted from the leaves of Pistacia lentiscus, a plant endemic to the Greek island of Chios. It has been used by traditional healers, and it has antibacterial, antifungal properties, and therapeutic benefits for the skin. The CGM reduces the formation of dental plaque and bacterial growth in oral saliva, and recent studies have demonstrated the role of antioxidant activity of CGM. Although CGM has been widely investigated, its protective effect against oxidative-damage to keratinocytes, as well as the relationship between CGM and autophagy, has not been investigated. The aim of this study was to assess the protective effect of CGM against H2O2-induced oxidative stress and to evaluate the autophagic features induced by CGM in human keratinocytes. The pretreatment with CGM significantly reduced apoptosis in H2O2-exposed HaCaT cells. It promoted the degradation of caspase-3, caspase-8, and caspase-9; and it induced the formation of the processed PARP. The treatment with CGM caused an increase in vesicle formation compared to control group. The level of p62 was reduced and the conversion of LC3-I to LC3-II was increased in CGM treated HaCaT cells. Also, the treatment with CGM increased cleavage of ATG5-ATG12 complex. In summary, CGM helps the cells to survive under stressful conditions by preventing apoptosis and enhancing autophagy. Besides, the present investigation provides evidence to support the antioxidant potential of CGM in vitro and opens up a new horizon for future experiments.

The Role of HS-1200 Induced Autophagy in Oral Cancer Cells

Bile acids and synthetic bile acid derivatives induce apoptosis in various kinds of cancer cells and thus have anticancer properties. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, few data are available regarding the role of autophagy in oral cancers and there have been no reports of autophagic cell death in OSCCs (oral squamous cell carcinoma cells) induced by HS-1200, a synthetic bile acid derivative. We thus examine whether HS-1200 modulates autophagy in OSCCs. Our findings indicate that HS-1200 has anticancer effects in OSCCs, and we observed in these cells that autophagic vacuoles were visible by monodansylcadaverine (MDC)and acridine orange staining. When we analyzed HS-1200-treated OSCC cells for the presence of biochemical markers, we observed that this treatment directly affects the conversion of LC-3II, degradation of p62/SQSTM1 and full-length beclin-1, cleavage of ATG5-12 and the activation of caspase. An autophagy inhibitor suppressed HS-1200-induced cell death in OSCCs, confirming that autophagy acts as a pro-death signal in these cells. Furthermore, HS-1200 shows anticancer activity against OSCCs via both autophagy and apoptosis. Our current findings suggest that HS-1200 may potentially contribute to oral cancer treatment and thus provide useful information for the future development of a new therapeutic agent.

Partial sublingual glandectomy with ranula excision: a new conservative method for treatment

OBJECTIVES: This study evaluated the clinical results of partial sublingual glandectomy accompanying the excision of ranula as new treatment modality. MATERIALS AND METHODS: A total of 43 patients who were treated between 1999 and 2007 for oral or plunging ranula were reviewed. All patients were treated surgically by various methods with a total of 55 different procedures performed. Ten cases of partial sublingual glandectomy with excision of the ranula were conducted. All excised specimens were examined. We compared the clinical outcomes resulting from each treatment method. RESULTS: The recurrence rates for marsupialization, excision of ranula, marsupialization with gauze packing, total excision of sublingual gland and ranula, and partial sublingual glandectomy with excision of ranula were 50%, 25%, 25%, 0% and 10%, respectively. Of the 10 patients treated by partial sublingual glandectomy with ranula excision, only one experienced recurrence (10%), i.e., plunging ranula. None of the ranulas contained an epithelial lining, and the excised portion of the feeding sublingual glands showed degenerative changes. CONCLUSION: In removal of ranulas, we found that excision of the attached sublingual gland, which removed the feeding portion and degenerative acinar cells, yielded good outcomes. Thus, as a new conservative method for treatment, we recommend partial sublingual glandectomy to accompany excision of the ranula.

Periodontopathogen LPSs Regulate MicroRNA Expression in Human Gingival Epithelial Cells

Periodontitis results from the activation of host immune and inflammatory defense responses to subgingival plaque bacteria, most of which are gram-negative rods with lipopolysaccharides (LPSs) in their cell walls. LPSs have been known to induce proinflammatory responses and recently it was reported also that they induce the expression of microRNAs (miRNAs) in host cells. In our current study therefore, we aimed to examine and compare the miRNA expression patterns induced by the LPSs of major periodontopathogens in the human gingival epithelial cell line, Ca9-22. The cells were treated with 1 microg/ml of E. coli (Ec) LPS or 5 microg/ml of an LPS preparations from four periodontopathogens Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn) for 24 h. After small RNA extraction from the treated cells, miRNA microarray analysis was carried out and characteristic expression profiles were observed. Fn LPS most actively induced miRNAs related to inflammation, followed by Aa LPS, Pi LPS, and Ec LPS. In contrast, Pg LPS only weakly activated miRNAs related to inflammation. Among the miRNAs induced by each LPS, miR-875-3p, miR-449b, and miR-520d-3p were found to be commonly up-regulated by all five LPS preparations, although at different levels. When we further compared the miRNA expression patterns induced by each LPS, Ec LPS and Pi LPS were the most similar although Fn LPS and Aa LPS also induced a similar miRNA expression pattern. In contrast, the miRNA profile induced by Pg LPS was quite distinctive compared with the other bacteria. In conclusion, miR-875-3p, miR-449b, and miR-520d-3p miRNAs are potential targets for the diagnosis and treatment of periodontal inflammation induced by subgingival plaque biofilms. Furthermore, the observations in our current study provide new insights into the inflammatory miRNA response to periodontitis.

Induction of Signal Transduction Pathway Genes in Dendritic Cells by Lipopolysaccharides from Porphyromonas gingivalis and Escherichia coli

Porphyromonas (P.) gingivalis lipopolysaccharide (Pg LPS) is the major pathogenic component of periodontal disease. In this study, we have attempted to determine the expression profiles of the signal transduction pathway genes induced by Pg LPS in comparison with Escherichia (E.) coli LPS (Ec LPS). DC2.4 cells were treated for two hours with 1 microg/ml of Pg LPS or 0.5 microg/ml of Ec LPS. The total RNA from these cells was then isolated and reverse-transcribed. Gene expression profiles were then analyzed with a signal transduction pathway finder GEArray Q series kit and significant changes in expression were confirmed by real-time PCR. The microarray results indicated that several genes, including Tnfrsf10b, Vcam1, Scyb9, Trim25, Klk6, and Stra6 were up-regulated in the DC2.4 cells in response to Pg LPS treatment, but were downregulated or unaffected by Ec LPS. Real-time PCR revealed that the expression of Trim25, Scyb9 and Tnfrsf10b was increased over the untreated control. Notably, Trim25 and Tnfrsf10b were more strongly induced by Pg LPS than by Ec LPS. These results provide greater insight into the signal transduction pathways that are altered by P. gingivalis LPS.

Comparison of Cytokine Gene Induction in RAW 264.7 Cells by Porphyromonas gingivalis and Escherichia coli Lipopolysaccharide

Porphyromonas gingivalis lipopolysaccharide (Pg LPS) is an important virulence factor in chronic periodontitis. The aim of this study was to compare the expression of inflammatory cytokine genes in Escherichia coli LPS (Ec LPS) and Pg LPS-stimulated mouse macrophage RAW 264.7 cells. Cells were treated with Ec LPS and Pg LPS for 18 hours, and the cytokine gene expression profile was assessed using microarrays and confirmed by real-time PCR. Microarray analysis showed that both types of LPS induced a significant increase in the expression of IL-17beta, IL-2, Ccl4, Cxcl2 and TNFalpha compared with the control. However, LT-b was up-regulated by Pg LPS but not by Ec LPS. Real-time PCR analysis of these genes showed similar results for LT-b, Ccl4, Cxcl2, and TNF-alpha but found that IL-17beta and IL-2 were upregulated by Pg LPS but not by Ec LPS. These data indicate that Pg LPS stimulates the transcription of IL-17beta, IL-2, Ccl4, Cxcl2, LT-b, and TNFalpha, all of which may be involved in the pathogenesis of chronic periodontitis.

Chios Gum Mastic Induces Cell Cycle Arrest and Apoptosis in YD9 Human Oral Squamous Carcinoma Cells / 체질인류학회지

Chios gum mastic (CGM) is obtained from the stem and leaves of Pistacia lentiscus trees and has been extensively used for centuries in Mediterranean and Middle Eastern countries, both as a dietary supplement and herbal remedy. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying modulation of cell cycle and induction of apoptosis in YD9 human oral squamous carcinoma cell line treated with CGM. The viability of YD9 cells and human normal keratinocyes (HaCaT cells), and the growth inhibition of YD9 cells were assessed by the MTT assay and clonogenic assay respectively. The hoechst staining and DNA electrophoresis were conducted to observe the YD9 cells undergoing apoptosis. YD9 cells were treated with CGM, and Western blotting, immunocytochemistry, confocal microscopy and FACScan flow cytometry were conducted. Mitochondrial membrane potential change and proteasome activity were measured. CGM treatment on YD9 cells resulted in a does-dependent inhibition of cell growth and induced apoptotic cell death. And tested YD9 cells showed several lines of apoptotic manifestation. Flow cytometric analysis revealed that CGM resulted in G1 arrest in cell cycle progression which was associated with decrease in the protein expression of cyclin D1, cyclin D3, Cdk2 and Cdk4, and increase in the protein expression of p21(WAF1/CIP1) and p53. These results demonstrate that CGM induces G1 the cell cycle arrest via the modulation of cell cycle-related proteins, and apoptosis via mitochondria and caspase pathway in YD9 cells, suggesting that CGM can be considered as a novel therapeutic strategy for human oral squamous cell carcinoma from its strong cell cycle arrest and apoptosis-inducing activity.

Apoptotic Effect of Co-Treatment with a Natural Product, Chios Gum Mastic, and a Synthetic Chenodeoxycholic Acid Derivative, HS-1200, on Human Osteosarcoma Cells / 체질인류학회지

Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. Recently it reported that CGM induced apoptosis in a few cancer cells in vitro. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM and a CDCA derivative, HS-1200 on human osteosarcoma (HOS) cells. To investigate whether the co-treatment of CGM and HS-1200 compared with each single treatment efficiently reduced the viability of HOS cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and DNA hypoploidy, Westen blot analysis and immunofluorescent staining were performed to study the alterations of the expression level and translocation of apoptosis-related proteins in co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. In this study, HOS cells co-treated with CGM and HS-1200 showed several lines of apoptotic manifestation whereas each single treated HOS cells did not. Although the single treatment of 40 microgram/mL CGM or 25 micrometer HS-1200 for 24 h did not induce apoptosis, the cotreatment of them induced prominently apoptosis. Therefore our data provide the possibility that combination therapy of CGM and HS-1200 could be considered as a novel therapeutic strategy for human osteosarcoma.

The Role of CD44 in the Adhesion of Human Osteosarcoma to Hyaluronic Acid / 체질인류학회지

Osteoblast expresses a sequence of extracellular matrix during differentiation, suggest that multiple adhesion mechanisms regulate osteoblast differentiation and bone development. Hyaluronic acid (HA) is a glycosaminoglycan (GAG) which mainly distribute in cartilage and extracellular matrix (ECM). HA has very high molecular weights and their structures occupy large solvent domains which give solution of high viscosity. During early development and before tissue differentiation, HA can constitute the major structural macromolecule in the ECM, where it can promote both cell proliferation and migration. CD44 is a cell surface receptor for HA. The polymorphic family of integral membrane glycoproteins CD44 is found on a wide variety of cells. CD44's function in the cell membrane is transmembrane signalling between extracellular matrix and acin filament. In present study, HOS was used as a model to examine whether HOS adhere to HA, CD44 and GAGs on cell surface participate in the adhesion to HA and there is difference in CD44 expression at that time. HOS adhered to HA in the manner of time -dependent. After incubation for 180 minutes, about 90% of cells adhered to HA. When HOS was pretreated with anti -CD44 antibody, hyaluronidase and genistein, the adhesion rate was significantly decreased. CD44 was more expressed in HOS plated on HA -coated wells than BSA -coated wells. Taken together, HOS has a adhesive affinity to HA. CD44, GAGs on cell surface and tyrosine kinases play an important role in the adhesion of HOS to HA.

The Preparation of Frozen Red Blood Cells and a Procedure for Deglycerolizing Frozen RBCs using COBE 2991 Blood Cell Processor / 대한수혈학회지

BACKGROUND: The cryopreservation of red blood cells (RBCs) has not ever been applied to the clinical services in Korea. The aim of this study was designed to supply the frozen-thawed RBCs as a routine service through estimation of efficiency and safety after freezing, thawing and washing. METHODS: Fifteen fresh packed RBCs were frozen with 40 percent(wt/vol) glycerol. After frozen storage at -70degrees for at least one month, the RBCs were thawed and washed in the COBE 2991 blood cell processor. We measured the blood cell count, RBC recovery rate, K+, LDH, specific gravity, osmolarity, and the percentage of hemolysis in the supernatant after deglycerolization. Autologous transfusions were done to the four voluntary donors with deglycerolized autologous blood for clinical assessment. RESULTS: The freeze-thaw-wash recovery rate of RBC was 76.8+/-10.0%, which is not enough to pass the AABB standard. But the recovery rate was increased up to 87.0+/-2.1% with the 4 stepwise predilution technique. The supernatant plasma specific gravity, osmolarity, and K+ were 1.006+/-0.001, 292+/-3 mOsm/KgH20, and 1.1+/-0.2mEq/L, respectively. The Hb ATP and 2,3-DPG were 3.6+/-0.8nmol/g and 13.4+/-4.5nmol/g. In simulated study, the free hemoglobin was 2.8+/-1.1mg/dL and 0.4+/-0.2% of total hemoglobin. In four autologous transfusion cases, plasma haptoglobin level was 96.0+/-40.8 mg/dL (reference range 30~200 mg/dL) and urine hemoglobin was not observed after 2~6 hours later after transfusion. CONCLUSION: The results of this study indicated that technical experiences for freezing, thawing and washing were established for clinical use of frozen RBCs in Korea.

Osteoblastoma-like Osteosarcoma occurred in the Mandible: Report of A Case

A Case Report of Multiple odontogenic keratocysts associated with basal cell nevus syndrome

The basal cell nevus syndrome is a well recognized entity, the major symptoms of which are basal cell nevi, multiple jaw cysts, skeletal anomalies, and ectopic calcification. The syndrome follows a hereditary pattern, which is characterized by a highly penetrant, autosomal dominant gene with multiple and variable effects. The patient often has a characteristic face, with frontal and temporoparietal bossing, which results in an increased cranial circumference. The eyes may appear widely separated, and 40 percent of patients have true ocular hypertelorism. Jaw cysts are one of the most constant features of the syndrome and are present in at least 75 percent of the patients. The cysts are odontogenic keratocysts and frequently multiple. Radiographically, the cysts in patients with basal cell nevus syndrome do not differ significantly from isolated keratocysts. The cysts in patients with this syndrome are often associated with the crowns of unerupted teeth; on radiographs they may mimic dentigerous cysts. We report a case of multiple odontogenic keratocysts associated with basal cell nevus syndrome with the literature of review.