Cell-free supernatant of Lactococcus lactis IDCC 2301 exerts anti-inflammatory effects in LPS-induced macrophages via NF-κB and MAPK signaling pathways.

This study investigated the anti-inflammatory effects of cell-free supernatant of Lactococcus lactis IDCC 2301 on lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Expression of inflammatory mediators and cytokines, and the production of nitric oxide (NO) and prostaglandin E2 (PGE2) were qualitatively analysed. The expression of signal transductors in inflammatory cascades was quantified by western blot. Treatment with cell-free supernatant of L. lactis IDCC 2301 significantly decreased the mRNA expression levels of tumour necrosis factor (TNF-α) and interleukins including IL-1ß and IL-6. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) were also remarkably reduced in LPS-induced macrophages after the treatment. Furthermore, L. lactis IDCC 2301 reduced the levels of both dephosphorylated and phosphorylated forms of nuclear factor-kappa B (NF-κB), IκB-α, extracellular signal-regulated kinases (ERK), c-Jun amino-terminal kinases (JNK), and p38 in LPS-induced RAW 264.7 cells. Therefore, L. lactis IDCC 2301 shows anti-inflammatory activity by suppressing the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways.

MRI-Based Assessment of the Pharyngeal Constrictor Muscle as a Predictor of Surgical Margin after Transoral Robotic Surgery in HPV-Positive Tonsillar Cancer.

BACKGROUND AND PURPOSE: Transoral robotic surgery is an emerging strategy for treating human papillomavirus-positive cancers, but the role of MR imaging in predicting the surgical outcome has not been established. We aimed to identify preoperative MR imaging characteristics that predispose the outcome of transoral robotic surgery toward an insecure (positive or close) surgical margin in human papillomavirus-positive tonsillar squamous cell carcinoma. MATERIALS AND METHODS: Between December 2012 and May 2019, sixty-nine patients underwent transoral robotic surgery at our institution. Among these, 29 who were diagnosed with human papillomavirus-positive tonsillar squamous cell carcinoma, did not receive neoadjuvant treatment, underwent preoperative 3T MR imaging, and had postoperative pathologic reports and were included in this retrospective study. Two neuroradiologists evaluated the preoperative MR imaging scans to determine the tumor spread through the pharyngeal constrictor muscle using a 5-point scale: 1, normal constrictor; 2, bulging constrictor; 3, thinning constrictor; 4, obscured constrictor; and 5, tumor protrusion into the parapharyngeal fat. The risk of an insecure surgical margin (involved or <1 mm) according to the MR imaging scores was predicted using logistic regression with the Firth correction. RESULTS: The interobserver agreement for the MR imaging scores was excellent (κ = 0.955, P < .001). A score of ≥4 could predict an insecure margin with 87.5% sensitivity and 92.3% specificity (area under the curve = 0.899) and was the only significant factor associated with an insecure margin in the multivariable analysis (OR, 6.59; 95% CI, 3.11-22.28; P < .001). CONCLUSIONS: The pre-transoral robotic surgery MR imaging scoring system for the pharyngeal constrictor muscle is a promising predictor of the surgical margin in human papillomavirus-positive tonsillar squamous cell carcinoma.

Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation.

Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked ß-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation.

Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells.

Mucin is an important physical barrier against enteric pathogens. VvpE is an elastase encoded by Gram-negative bacterium Vibrio vulnificus; however, the functional role of VvpE in intestinal mucin (Muc) production is yet to be elucidated. The recombinant protein (r) VvpE significantly reduced the level of Muc2 in human mucus-secreting HT29-MTX cells. The repression of Muc2 induced by rVvpE was highly susceptible to the knockdown of intelectin-1b (ITLN) and sequestration of cholesterol by methyl-ß-cyclodextrin. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into the membrane lipid rafts coupled with ITLN to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of ERK, which was downregulated by the silencing of the PKCδ. Moreover, rVvpE induced region-specific methylation in the Muc2 promoter to promote the transcriptional repression of Muc2. In two mouse models of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus exhibited an increased survival rate and maintained the level of Muc2 expression in intestine. These results demonstrate that VvpE inhibits Muc2 expression by hypermethylation via lipid raft-mediated ROS signaling in the intestinal epithelial cells.

Arachidonic acid promotes skin wound healing through induction of human MSC migration by MT3-MMP-mediated fibronectin degradation.

Arachidonic acid (AA) is largely released during injury, but it has not been fully studied yet how AA modulates wound repair with stem cells. Therefore, we investigated skin wound-healing effect of AA-stimulated human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in vivo and its molecular mechanism in vitro. We found that transplantation of hUCB-MSCs pre-treated with AA enhanced wound filling, re-epithelization, and angiogenesis in a mouse skin excisional wound model. AA significantly promoted hUCB-MSCs migration after a 24 h incubation, which was inhibited by the knockdown of G-protein-coupled receptor 40 (GPR40). AA activated mammalian target of rapamycin complex 2 (mTORC2) and Aktser473 through the GPR40/phosphoinositide 3-kinase (PI3K) signaling, which was responsible for the stimulation of an atypical protein kinase C (PKC) isoform, PKCζ. Subsequently, AA stimulated phosphorylation of p38 MAPK and transcription factor Sp1, and induced membrane type 3-matrix metalloproteinase (MT3-MMP)-dependent fibronectin degradation in promoting hUCB-MSCs motility. Finally, the silencing of MT3-MMP in AA-stimulated hUCB-MSCs failed to promote the repair of skin wounds owing to impaired cell motility. In conclusion, AA enhances skin wound healing through induction of hUCB-MSCs motility by MT3-MMP-mediated fibronectin degradation, which relies on GPR40-dependent mTORC2 signaling pathways.

Vibrio vulnificus VvhA induces NF-κB-dependent mitochondrial cell death via lipid raft-mediated ROS production in intestinal epithelial cells.

The Gram-negative bacterium Vibrio vulnificus produces hemolysin (VvhA), which induces cytotoxicity in mammalian cells. However, our understanding of the cytotoxic mechanism and the modes of action of VvhA are still fragmentary and incomplete. The recombinant protein (r) VvhA (50 pg/ml) significantly induces necrotic cell death and apoptosis in human intestinal epithelial (INT-407) cells. The apoptotic cell death induced by rVvhA is highly susceptible to the sequestration of cholesterol by methyl-ß-cyclodextrin, whereas for necrotic cell death, this shows a marginal effect. We found that rVvhA induces the aggregation of lipid raft components coupled with NADPH oxidase enzymes, in which rVvhA increased the interaction of NADPH oxidase 2 (NOX2, gp91(phox)) with a cytosolic protein NCF1 (p47(phox)) to facilitate the production of reactive oxygen species (ROS). rVvhA uniquely stimulated a conventional PKC isoform PKCα and induced the phosphorylation of both ERK and JNK, which are responsible for the activation of transcription factor NF-κB. rVvhA induced an NF-κB-dependent imbalance of the Bcl-2/Bax ratio, the release of mitochondrial cytochrome c, and caspase-3/-9 activation during its promotion of apoptotic cell death. In addition, rVvhA has the ability to inhibit the expression of cell cycle-related proteins, such as CDK2, CDK4, cyclin D1, and cyclin E. These results demonstrate that rVvhA induces NF-κB-dependent mitochondrial cell death via lipid raft-mediated ROS production by the distinct activation of PKCα and ERK/JNK in intestinal epithelial cells.

Emergence agitation after orthognathic surgery: a randomised controlled comparison between sevoflurane and desflurane.

BACKGROUND: This study aimed to compare emergence agitation following sevoflurane and desflurane anaesthesia in adults undergoing orthognathic surgery. The hypothesis was that there might be a difference in the incidence of emergence agitation following sevoflurane and desflurane anaesthesia considering the difference in blood solubility and speed of recovery between the two. METHODS: In this prospective randomised double-blind study, 144 adults undergoing orthognathic surgery were randomly allocated to either sevoflurane or desflurane anaesthesia. The incidence of emergence agitation and possible causative factors were then compared between the groups. RESULTS: The incidence of emergence agitation was lower in the desflurane group than in the sevoflurane group (24% vs. 71%, P < 0.001). In addition, the time intervals between the discontinuation of anaesthetics and the first response, extubation, and discharge from post-anaesthesia care unit were significantly shorter in the desflurane group (P = 0.002, P < 0.001 and P = 0.003, respectively). The other variables were similar in the two groups. CONCLUSIONS: In adults undergoing orthognathic surgery, desflurane anaesthesia was associated with less emergence agitation than was sevoflurane anaesthesia.

Galectin-1 stimulates motility of human umbilical cord blood-derived mesenchymal stem cells by downregulation of smad2/3-dependent collagen 3/5 and upregulation of NF-κB-dependent fibronectin/laminin 5 expression.

Galectin-1 (Gal-1) belongs to a family of endogenous lectins with conserved carbohydrate recognition domains binding ß-galactosidase sugars and plays a vital role in regulating stem cell functions including determination of cell fate. However, our understanding of the functional roles of Gal-1 in human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) is still fragmentary and incomplete. Gal-1 significantly increased motility after a 24-h incubation, and this effect was inhibited by ß-lactose. We analyzed 17 extracellular matrix (ECM) genes in UCB-MSCs. Gal-1 decreased the expression of collagen genes COL3A1 (COL-3) and COL5A1 (COL-5) but increased the expression of fibronectin (FN) and laminin 5 (LM-5), that were reversed by ß-lactose. Gal-1 increased protein kinase C (PKC), c-Src, and caveolin-1 (Cav-1) phosphorylation that was attenuated by ß-lactose and the Src inhibitor PP2. In addition, pretreatment with the lipid raft disruptor Mß-CD and the PKC inhibitors inhibited Gal-1-induced UCB-MSC motility. In addition, Gal-1 reduced smad2/3 phosphorylation and induced nuclear factor (NF)-κB phosphorylation. Pretreatment with Mß-CD attenuated Gal-1-reduced smad2/3 phosphorylation, COL-3, and COL-5 expression but did not affect NF-κB phosphorylation, FN, or LM-5 expression. In contrast, PKC inhibitors only attenuated NF-κB phosphorylation, FN, and LM-5 expression. Reconstructing Gal-1-induced genetic changes by replacing it with siRNA specific for COL-3 or COL-5, or treatment of the cells with FN and LM-5 proteins, increased motility and its related proteins such as focal adhesion kinase, Akt, Erk, integrins, and matrix metalloproteinase-2. A combined treatment with COL-3/COL-5 siRNA or FN/LM-5 compared with that of single treatments was synergistic. However, a single Gal-1 treatment maximally stimulated motility and related protein phosphorylation/expression. These results demonstrate that Gal-1 stimulated human UCB-MSC motility by decreasing COL-3/COL-5 expression and increasing FN/LM-5 expression through a PKC-dependent NF-κB and c-Src/Cav-1-dependent smad2/3 pathway that was critical for governing the activation of FAK, Akt, Erk, integrins, and MMP2.

The assessment of the short- and long-term changes in the condylar position following sagittal split ramus osteotomy (SSRO) with rigid fixation.

The purpose of this study was to compare the short- and long-term changes in condylar position related to the glenoid fossa, and skeletal and occlusal stability after orthognathic surgery. All of the study patients were assessed by cone-beam computed tomography images for condylar rotational changes and anteroposterior position in the pre-surgery, post-surgery and post-retention period. The condylar positions were evaluated on three planes: axial, coronal and sagittal. In the skeletal and occlusal measurements, there was no significant difference between the post-surgery group and the post-retention group. After sagittal split ramus osteotomy (SSRO), the condyle on the axial plane rotated inward (P < 0.05) and maintained during the post-retention period. In the anteroposterior condylar position related to the glenoid fossa, the condyles had changed from the anterior position in the pre-surgery group to a concentric position in the post-surgery group and then returned to the anterior position in the post-retention groups. These results suggested that the changed anteroposterior condylar position in the glenoid fossa after SSRO with rigid fixation had moved from a concentric to anterior position for post-retention period.

The effect on the recovery profile of a change from enflurane to desflurane during the latter part of anaesthesia.

This study compared emergence and recovery characteristics after either enflurane anaesthesia or crossover from enflurane to desflurane anaesthesia. At an estimated 1 h prior to the end of operation, enflurane was either reduced (group E, n = 23) or replaced with desflurane (group X, n = 23). At the end of the operation, emergence and recovery characteristics of the two groups were compared. The crossover technique accelerated recovery compared with enflurane anaesthesia. The time taken for the eyes to open in response to painful pinching or a verbal command, and to regain awareness of age and name, were significantly shorter after crossover anaesthesia than after enflurane anaesthesia. The digit symbol substitution test and serial seven test scores were significantly better in patients subjected to crossover anaesthesia than in those subjected to enflurane anaesthesia. We conclude that, during surgery, the substitution of enflurane with desflurane in the latter part of anaesthesia can improve recovery.

Metachronous colorectal cancer.

OBJECTIVE: Although the diagnosis of metachronous colorectal cancer have increased, due primarily to improvements in diagnostic modalities, the potential risk factors for these tumours are not well known. We compared the characteristics of patients with metachronous and sporadic primary colorectal cancer to determine risk factors for its occurrence. PATIENTS AND METHODS: We reviewed the records of 5447 patients with colorectal cancer, who had been treated at Asan Medical Centre between July 1989 and January 2004. A metachronous cancer was defined as a secondary colorectal cancer occurring more than 6 months after the index cancer. RESULTS: Metachronous colorectal cancer occurred in 39 (0.7%) patients. Their average age was 53 years, somewhat younger than the average age of sporadic colorectal cancer patients (58 years). In patients with metachronous cancer, the cancer was more likely to be located in the right colon (P < 0.03), and the incidence of synchronous polyps or cancer was significantly higher (P < 0.001). The relative distributions of histological grades and clinicopathological characteristics were similar in index and metachronous cancers. Metachronous cancers were diagnosed more frequently at an early stage. The time interval between index and metachronous cancer ranged from 6 to 215 months (mean 39 months), with 13 (33.3%) patients diagnosed with metachronous cancer after 5 years. CONCLUSION: We found that in patients aged < 50 years, existence of synchronous polyps or cancer influence on the development of metachronous colorectal cancer. Regular follow-up is necessary for early detection, even after 5 years, for these patients.