Anti-Periodontitis Effects of Dendropanax morbiferus H.Lév Leaf Extract on Ligature-Induced Periodontitis in Rats.

Periodontitis is caused by pathogens in the oral cavity. It is a chronic infectious disease that causes symptoms including gingival bleeding and tooth loss resulting from the destruction of periodontal tissues coupled with inflammation. Dendropanax morbiferus H.Lév (DM) is a natural product that exhibits various biological activities with few side effects. In this study, the potential of DM leaf hot-water extracts (DMWE) as a treatment for periodontitis was determined and its anti-oxidant and anti-inflammatory effects were evaluated. Compounds in DMWE were identified by high-performance liquid chromatography (HPLC) and nitric oxide (NO) and prostaglandin E2 (PGE2) production was measured in RAW 264.7 cells. We measured the gingival index and gingival sulcus depth, and micro-CT was performed in vivo using a ligature-induced periodontitis rat model, which is similar to human periodontitis. The DMWE-treated group exhibited a decrease in cytokine concentration and relieved the gingival index and gingival sulcus depth compared with the periodontitis-induced control group. In addition, micro-CT and histological analysis revealed that DMWE exhibited anti-inflammatory effects and improved alveolar bone loss in periodontitis-induced rats. These findings suggest that DMWE has excellent anti-oxidant and anti-inflammatory effects that protect and prevent periodontal tissue damage and tooth loss caused by the inflammatory response.

Antihyperuricemic Effect of Dendropanax morbifera Leaf Extract in Rodent Models.

Dendropanax morbifera is a well-known traditional medicine used in China and Korea to treat intestinal disorders, urosis, diuresis, and chronic glomerulonephritis. Hyperuricemia is a metabolic disorder characterized by a high uric acid level in serum due to an imbalance between uric acid production and excretion and causes gout. Recently, the prevalence of hyperuricemia worldwide has been continuously increasing. Xanthine oxidase (XOD) inhibitors (allopurinol (ALP) and febuxostat) and uricosuric agents (benzbromarone and probenecid) are used to treat hyperuricemia clinically. However, because these drugs are poorly tolerated and cause side effects, such as kidney diseases, hepatotoxicity, gastrointestinal symptoms, and hypersensitivity syndrome, only a limited number of drugs are available. We investigated the antihyperuricemic effects of Dendropanax morbifera leaf ethanol extract (DMLE) and its underlying mechanisms of action through in vitro and in vivo studies. We evaluated uric acid levels in serum and urine, and xanthine oxidase (XOD) inhibition activity in the serum and liver tissue of a hyperuricemic rat model of potassium oxonate (PO)-induced hyperuricemic rats. In vitro study, XOD-inhibitory activity was the lowest among the test substances at the IC50 of ALP. However, the IC50 of DMLE-70 was significantly low compared with that of other DMLEs (p < 0.05). In PO-induced hyperuricemic rats, uric acid (UA) levels in serum and urine were significantly reduced in all DMLE-70 and allopurinol-treated (ALT) groups than in the PC group (p < 0.05). UA levels in urine were lower than those in serum in all DME groups. In PO-induced hyperuricemic rats, DMEE-200 reduced UA concentration in serum and increased UA excretion in the urine. These findings suggest that DMLE exerts antihyperuricemic and uricosuric effects on promoting UA excretion by enhanced secretion and inhibition of UA reabsorption in the kidneys. Thus, DMLE may be a potential treatment for hyperuricemia and gout.

Development and Validation of a Next-Generation Sequencing-Based Multigene Assay to Predict the Prognosis of Estrogen Receptor-Positive, HER2-Negative Breast Cancer.

PURPOSE: Multigene assays provide useful prognostic information regarding hormone receptor (HR)-positive breast cancer. Next-generation sequencing (NGS)-based platforms have numerous advantages including reproducibility and adaptability in local laboratories. This study aimed to develop and validate an NGS-based multigene assay to predict the distant recurrence risk. EXPERIMENTAL DESIGN: In total, 179 genes including 30 reference genes highly correlated with the 21-gene recurrence score (RS) algorithm were selected from public databases. Targeted RNA-sequencing was performed using 250 and 93 archived breast cancer samples with a known RS in the training and verification sets, respectively, to develop the algorithm and NGS-Prognostic Score (NGS-PS). The assay was validated in 413 independent samples with long-term follow-up data on distant metastasis. RESULTS: In the verification set, the NGS-PS and 21-gene RS displayed 91.4% concurrence (85/93 samples). In the validation cohort of 413 samples, area under the receiver operating characteristic curve plotted using NGS-PS values classified for distant recurrence was 0.76. The best NGS-PS cut-off value predicting distant metastasis was 20. Furthermore, 269 and 144 patients were classified as low- and high-risk patients in accordance with the cut-off. Five- and 10-year estimates of distant metastasis-free survival (DMFS) for low- versus high-risk groups were 97.0% versus 77.8% and 93.2% versus 64.4%, respectively. The age-related HR for distant recurrence without chemotherapy was 9.73 (95% CI, 3.59-26.40) and 3.19 (95% CI, 1.40-7.29) for patients aged ≤50 and >50 years, respectively. CONCLUSIONS: The newly developed and validated NGS-based multigene assay can predict the distant recurrence risk in ER-positive, HER2-negative breast cancer.

Examination of the Biomark assay as an alternative to Oncotype DX for defining chemotherapy benefit.

Currently the 21-gene recurrence score (RS) assay called Oncotype DX is recommended by the National Comprehensive Cancer Network guideline for defining the benefit of chemotherapy. To overcome the cost disadvantages of the Oncotype DX assay and the turnaround time, a multigene assay was examined to compare the correlation of the RS and the predicted score (PS) of the present study. Paraffin-embedded tissues of 50 cases with early-stage estrogen receptor (ER)-positive breast cancer, who underwent the Oncotype DX test were used. A total of 149 candidate genes with high correlation to the RS were identified, in another project (Lee et al, unpublished data). Reverse transcription-quantitative polymerase chain reaction biomark assays were conducted using the dynamic array integrated fluidic circuit and the correlation analysis was performed with BRB ArrayTools. A predictive model was developed by the coefficient and gene expression, and 41 genes were identified. If the cut-off was ≥18, the predicted model was 18/50 cases, and the RS was 19, indicating that the differential rate of predicted response against RS was 2%. If the cutoff was ≥11, the predicted model was 38/50 cases and the RS was 34, indicating a difference of 8%. Genes common to the Oncotype DX and the Biomark assay include marker of proliferation Ki-67, aurora kinase A, Erb-B2 receptor tyrosine kinase 2, glutathione S-transferase Mu 1, estrogen receptor 1, progesterone receptor, B-cell lymphoma 2, signal peptide CUB domain EGF-like 2 and 5 reference genes. The remaining 28 genes are involved in various pathways and functions. This result indicates that there is a significant correlation between PS and RS scores, although validation of results is required to accurately determine the risk of distant recurrence. The Biomark assay is an easy and inexpensive way to measure mRNA expression. The present study demonstrates the possibility of the Biomark assay as an alternative for defining chemotherapy benefit in individual patients with ER-positive early-stage breast cancer.

A retrospective analysis on the relationship between intraoperative hypothermia and postoperative ileus after laparoscopic colorectal surgery.

Postoperative ileus (POI) is an important factor prolonging the length of hospital stay following colorectal surgery. We retrospectively explored whether there is a clinically relevant association between intraoperative hypothermia and POI in patients who underwent laparoscopic colorectal surgery for malignancy within the setting of an enhanced recovery after surgery (ERAS) program between April 2016 and January 2017 at our institution. In total, 637 patients were analyzed, of whom 122 (19.2%) developed clinically and radiologically diagnosed POI. Overall, 530 (83.2%) patients experienced intraoperative hypothermia. Although the mean lowest core temperature was lower in patients with POI than those without POI (35.3 ± 0.5°C vs. 35.5 ± 0.5°C, P = 0.004), the independence of intraoperative hypothermia was not confirmed based on multivariate logistic regression analysis. In addition to three variables (high age-adjusted Charlson comorbidity index score, long duration of surgery, high maximum pain score during the first 3 days postoperatively), cumulative dose of rescue opioids used during the first 3 days postoperatively was identified as an independent risk factor of POI (odds ratio = 1.027 for each 1-morphine equivalent [mg] increase, 95% confidence interval = 1.014-1.040, P <0.001). Patients with hypothermia showed significant delays in both progression to a soft diet and discharge from hospital. In conclusion, intraoperative hypothermia was not independently associated with POI within an ERAS pathway, in which items other than thermal measures might offset its negative impact on POI. However, as it was associated with delayed discharge from the hospital, intraoperative maintenance of normothermia is still needed.

Optimization of RNA Extraction from Formalin-Fixed Paraffin-Embedded Blocks for Targeted Next-Generation Sequencing.

PURPOSE: Breast cancer has a high prevalence in Korea. To achieve personalized therapy for breast cancer, long-term follow-up specimens are needed for next-generation sequencing (NGS) and multigene analysis. Formalin-fixed paraffin-embedded (FFPE) samples are easier to store than fresh frozen (FF) samples. The objective of this study was to optimize RNA extraction from FFPE blocks for NGS. METHODS: RNA quality from FF and FFPE tissues (n=5), expected RNA amount per unit area, the relationship between archiving time and quantity/quality of FFPE-extracted RNA (n=14), differences in quantitative real-time polymerase chain reaction (qRT-PCR) and NGS results, and comparisons of both techniques with tissue processing at different institutions (n=96) were determined in this study. RESULTS: The quality of RNA did not show any statistically significant difference between paired FF and FFPE specimens (p=0.49). Analysis of tumor cellularity gave an expected RNA amount of 33.25 ng/mm2. Archiving time affected RNA quality, showing a negative correlation with RNA integrity number and a positive correlation with threshold cycle. However, RNA from samples as old as 10 years showed a 100% success rate in qRT-PCR using short primers, showing that the effect of archiving time can be overcome by proper experiment design. NGS showed a higher success rate than qRT-PCR. Specimens from institution B (n=46), which were often stored in a refrigerator for more than 6 hours and fixed without slicing, showed lower success rates and worse results than specimens from the other institutes. CONCLUSION: Archived FFPE tissues can be used to extract RNA for NGS if they are properly processed before fixation. The expected amount of RNA per unit size calculated in this study will be useful for other researchers.

Whole-lung lavage complicated with pneumothorax: a case report.

A patient with pulmonary alveolar proteinosis underwent whole lung lavage of the right lung. Lavage of the left lung was not immediately possible because of severe hypoxemia. Three days later, after correction of hypoxemia, we re-attempted the left lung lavage. However, the patient had severe hypoxemia (SpO2 < 80%) within a few minutes of performing right one lung ventilation (OLV). On bronchoscopic examination, proper tube location was confirmed. Bronchodilator nebulization and steroid injection were attempted with no effect. While searching for the cause of the hypoxemia, we found that the breath sound from the right lung had become very weak and distant compared with that from initial auscultation. Right pneumothorax was diagnosed on chest X-ray and a chest tube was inserted. After confirming pneumothorax resolution, we re-tried right OLV and were able to proceed with the left lung lavage without signs of aggravating air leak, loss of tidal volume, or severe hypoxemia.

EGF induces epithelial-mesenchymal transition through phospho-Smad2/3-Snail signaling pathway in breast cancer cells.

Epithelial-mesenchymal transition (EMT) can contribute to tumor invasion, metastasis, and resistance to chemotherapy or hormone therapy. EMT may be induced by a variety of growth factors, such as epidermal growth factor (EGF). Most studies regarding EMT have focused on TGF-ß-Smads signaling. The mechanism of EGF-induced EMT via activation of the Smad2/3 in breast cancer cells, MCF-7 and MDA-MB-231, remains unclear. The expression levels of Snail, vimentin, and fibronectin were increased by EGF treatment in a time-dependent manner, while the expression level of E-cadherin was decreased. EGF-induced nuclear co-localization of phospho-Smad2/3 and Snail and cancer cell migration were inhibited by pretreatment with an ERK1/2 inhibitor, PD98059 and a phospho-Smad2 inhibitor, SB203580. Knockdown of Smad2/3 expression suppressed EGF-induced expressions of Snail, vimentin, fibronectin, and cancer cell invasion, suggesting an acquisition of the mesenchymal and migratory phenotype in less aggressive MCF-7 cells. Moreover, MDA-MB-231 cells were shown that EGF-induced EMT, and cell invasion through ERK1/2-phospho-Smad2/3-Snail signaling pathway. We have discovered that EGF-stimulated activation of Smad2/3 upregulated several key EMT markers, inhibited E-cadherin expression, promoted EMT, enhanced migration and invasion in MCF-7 and MDA-MB-231 breast cancer cells. Identification of this molecular mechanism may provide new molecular targets for the development of therapies for metastatic breast cancer.

Hornerin Is Involved in Breast Cancer Progression.

PURPOSE: The S100 gene family, which comprises over 20 members, including S100A1, S100A2, S100A8, S100A9, profilaggrin, and hornerin encodes low molecular weight calcium-binding proteins with physiological and pathological roles in keratinization. Recent studies have suggested a link between S100 proteins and human cancer progression. The purpose of the present study was to determine the expression levels of hornerin, S100A8, and S100A9 and evaluate their roles in the progression of invasive ductal carcinoma (IDC). METHODS: Seventy cases of ductal carcinoma in situ (DCIS), IDC, and metastatic carcinoma in lymph nodes (MCN) were included. Tissue microarrays were constructed from lesions of DCIS, IDC, and MCN from the same patients. Expression of hornerin, S100A8, and S100A9 was analyzed using immunohistochemistry. RESULTS: The expression of hornerin was associated with the estrogen receptor-negative (p=0.003) and the human epidermal growth factor receptor 2-positive (p=0.002) groups. The expression of S100A8 was associated with a higher pT stage (p=0.017). A significant (p<0.001) correlation between the expression of S100A9 and S100A8 was also found. The mean percentages of hornerin-positive tumor cells in DCIS, IDC, and MCN were 1.0%±3.3% (mean±standard deviation), 12.0%±24.0%, and 75.3%± 27.6%, respectively. The expression of hornerin significantly (p<0.001) increased with the progression of carcinoma. The mean levels of S100A8 and S100A9 in DCIS, IDC, and MCN were not significantly (p>0.050) different. The expression of hornerin increased in a stepwise manner (DCIS

Anticancer effect of metformin on estrogen receptor-positive and tamoxifen-resistant breast cancer cell lines.

Acquisition of tamoxifen resistance (TR) during anti-estrogenic therapy using tamoxifen is a major obstacle in the treatment of estrogen receptor (ER)-positive breast cancer. As a biguanide derivative, metformin is commonly used to treat type II diabetes. It has recently emerged as a potential anticancer agent. The objective of the present study was to investigate the anticancer activity of metformin in relation to ERα expression and its signaling pathway in ERα-positive MCF-7 and MDA-MB-361 breast cancer cells as well as TR MCF-7 breast cancer cells. Metformin inhibited both protein and mRNA levels of ERα in the presence or absence of estrogen (E2) in the MCF-7, TR MCF-7 and MDA-MB-361 cells. Metformin repressed E2-inducible estrogen response element (ERE) luciferase activity, protein levels and mRNA levels of E2/ERα-regulated genes [including c-Myc, cyclin D1, progesterone receptor (PR) and pS2] to a greater degree than tamoxifen, resulting in inhibition of cell proliferation of MCF-7, TR MCF-7 and MDA-MB-361 cells. Collectively, our results suggest that one of the anticancer mechanisms of metformin could be attributable to the repression of expression and transcriptional activity of ERα. Metformin may be a good therapeutic agent for treating ERα-positive breast cancer by inhibiting the expression and function of ERα. In addition, metformin may be useful to treat tamoxifen-resistant breast cancer.

Anti-cancer effect of metformin by suppressing signaling pathway of HER2 and HER3 in tamoxifen-resistant breast cancer cells.

Development of new therapeutic strategies is becoming increasingly important to overcome tamoxifen resistance. Recently, much interest has been focused on anti-tumor effects of metformin commonly used to treat type II diabetes. Increased protein expression and signaling of epidermal growth factor receptor (EGFR) family is a possible mechanism involved in tamoxifen resistance. Since HER2/HER3 heterodimers are able to induce strong downstream signaling and activate various biological responses such as cellular proliferation and growth, we investigated the anti-cancer effect of metformin by inhibition of signaling pathway via downregulation of HER2 and HER3 using tamoxifen-resistant MCF-7 (TR MCF-7) cells. Compared to MCF-7 cells, TR MCF-7 cells showed increased expression of EGFR, HER2, and HER3, and metformin inhibited the expression of these proteins in a dose- and time-dependent manner. Metformin inhibited activation of HER2 (Tyr1248)/HER3 (Tyr1289)/Akt (Ser473) as well as cell proliferation and colony formation by estrogenic promotion in MCF-7 and TR MCF-7 cells. Known as a HER3 ligand, heregulin (HRG)-ß1-induced phosphorylation of HER2, HER3 and Akt, and protein interaction of HER2/HER3 and colony formation were inhibited by metformin in both cells. Consistent with the results in the two cell lines, we identified that metformin inhibited HER2/HER3/Akt signaling axis activated by HRG-ß1 using the HER2 and HER3-overexpressing breast cancer cell line SK-BR-3. Lastly, lapatinib-induced HER3 upregulation was significantly inhibited by treatment of metformin in HER3 siRNA-transfected TR MCF-7 cells. These data suggest that metformin might overcome tamoxifen resistance through the inhibition of expression and signaling of receptor tyrosine kinase HER2 and HER3.

Molecular dynamics simulation strategies for protein-micelle complexes.

The structure and stability of membrane proteins can vary widely in different detergents and this variability has great practical consequences for working with membrane proteins. Nevertheless, the mechanisms that operate to alter the behavior of proteins in micelles are poorly understood and not predictable. Atomic simulations could provide considerable insight into these mechanisms. Building protein-micelle complexes for simulation is fraught with uncertainty, however, in part because it is often unknown how many detergent molecules are present in the complex. Here, we describe several convenient ways to employ Micelle Builder in CHARMM-GUI to rapidly construct protein-micelle complexes and performed simulations of the isolated voltage-sensor domain of voltage-dependent potassium-selective channel and an antimicrobial peptide papiliocin with varying numbers of detergents. We found that once the detergent number exceeds a threshold, protein-detergent interactions change very little and remain very consistent with experimental observations. Our results provide a platform for future studies of the interplays between protein structure and detergent properties at the atomic level. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

6-shogaol attenuates H2O2-induced oxidative stress via upregulation of Nrf2-mediated γ-glutamylcysteine synthetase and heme oxygenase expression in HepG2 cells.

The signaling pathway by which 6-shogaol protects HepG2 cells against H2O2-induced oxidative stress was investigated. Cellular anti-oxidant activities, the GSH level, and anti-oxidant response element (ARE) promoter activity were analyzed. Activated protein kinases and nuclear transcription factor-erythroid 2-related factor 2 (Nrf2) accumulation in the nucleus, and phase II detoxification and anti-oxidant enzymes were analyzed using western blotting. 6-Shogaol enhanced cellular anti-oxidant activities, the GSH level, and ARE promoter activities. Nrf2 accumulation in the nucleus, c-jun N-terminal kinase (JNK) activation, and γ-glutamylcysteine synthetase (GCS) and heme oxygenase-1 (HO-1) expressions were increased by 6-shogaol. Blockage of the JNK signaling pathway removed the elicitation effect of 6-shogaol on JNK activation, Nrf2 accumulation in nucleus, and GCS and HO-1 expression, but partially suppressed cellular anti-oxidant activities and ARE promoter activities. 6-shogaol exerts an indirect cellular anti-oxidant activity based on up-regulation of GCS and HO-1 via a JNK-mediated Nrf2 signaling pathway.

Functional Roles of Aromatic Residues and Helices of Papiliocin in its Antimicrobial and Anti-inflammatory Activities.

A cecropin-like peptide, papiliocin, isolated from the swallowtail butterfly Papilio xuthus, possesses high selectivity against gram-negative bacteria. Since Trp(2) and Phe(5) are highly conserved residues in cecropin-like peptides, we investigated the role of Trp(2) and Phe(5) in antibacterial activity. Substitution of Trp(2) and Phe(5) in papiliocin with Ala (papiliocin-2A and papiliocin-5A) revealed that Trp(2) is a key residue in its antibacterial activities. In order to understand the structural requirements for papiliocin function and to design shorter, but more potent, peptide antibiotics, we designed papiliocin constructs, PapN (residues Arg(1)-Ala(22) from the N-terminal amphipathic helix). PapN exhibited significant broad-spectrum antibacterial activities without cytotoxicity. Bactericidal kinetics of peptides against E.coli showed that papiliocin completely and rapidly killed E.coli in less than 10 minutes at 2× MIC concentration, while papiliocin-2A and papiliocin-5A killed four times more slowly than papiliocin. The PapN series peptides permeabilized bacterial membranes less effectively than papiliocin, showing no antibacterial activities in an hour. The results imply that the Trp(2) and Phe(5) in the amphipathic N-terminal helix are important in the rapid permeabilization of the gram-negative bacterial membrane. The hydrophobic C-terminal residues permeabilize the hydrophobic bacterial cell membrane synergistically with these aromatic residues, providing selectivity against gram-negative bacteria.

Profiling of viral proteins expressed from the genomic RNA of Japanese encephalitis virus using a panel of 15 region-specific polyclonal rabbit antisera: implications for viral gene expression.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is closely related to West Nile (WN), yellow fever (YF), and dengue (DEN) viruses. Its plus-strand genomic RNA carries a single open reading frame encoding a polyprotein that is cleaved into three structural (C, prM/M, and E) and at least seven nonstructural (NS1/NS1', NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins, based on previous work with WNV, YFV, and DENV. Here, we aimed to profile experimentally all the viral proteins found in JEV-infected cells. We generated a collection of 15 JEV-specific polyclonal antisera covering all parts of the viral protein-coding regions, by immunizing rabbits with 14 bacterially expressed glutathione-S-transferase fusion proteins (for all nine viral proteins except NS2B) or with a chemically synthesized oligopeptide (for NS2B). In total lysates of JEV-infected BHK-21 cells, immunoblotting with these antisera revealed: (i) three mature structural proteins (~12-kDa C, ~8-kDa M, and ~53-kDa E), a precursor of M (~24-kDa prM) and three other M-related proteins (~10-14 kDa); (ii) the predicted ~45-kDa NS1 and its frameshift product, ~58-kDa NS1', with no evidence of the predicted ~25-kDa NS2A; (iii) the predicted but hardly detectable ~14-kDa NS2B and an unexpected but predominant ~12-kDa NS2B-related protein; (iv) the predicted ~69-kDa NS3 plus two major cleavage products (~34-kDa NS3N-term and ~35-kDa NS3C-term), together with at least nine minor proteins of ~16-52 kDa; (v) the predicted ~14-kDa NS4A; (vi) two NS4B-related proteins (~27-kDa NS4B and ~25-kDa NS4B'); and (vii) the predicted ~103-kDa NS5 plus at least three other NS5-related proteins (~15 kDa, ~27 kDa, and ~90 kDa). Combining these data with confocal microscopic imaging of the proteins' intracellular localization, our study is the first to provide a solid foundation for the study of JEV gene expression, which is crucial for elucidating the regulatory mechanisms of JEV genome replication and pathobiology.

Risk factors of emergence agitation in adults undergoing general anesthesia for nasal surgery.

OBJECTIVES: To identify the incidence and the risk factors of emergence agitation in adults undergoing general anesthesia for nasal surgery. METHODS: We retrospectively examined 792 patients aged ≥18 years who underwent general anesthesia for elective nasal surgery between July 2012 and August 2013. Patients in the postanesthesia care unit with a Richmond Agitation Sedation Scale≥+1 at any time were considered to have emergence agitation. RESULTS: The overall incidence of emergence agitation is 22.2%. From multivariate regression analysis, the following six variables were found to be significantly associated with emergence agitation (P<0.05): younger age, recent smoking, sevoflurane anesthesia, postoperative pain on the numerical rating scale (NRS)≥5, presence of a tracheal tube, and presence of a urinary catheter. Presence of a tracheal tube was the greatest risk factor, increasing the risk of developing emergence agitation by approximately fivefold (odds ratio, 5.448; 95% confidence interval, 2.973 to 9.982). Younger age was also a strong risk factor (odds ratio, 0.975 for each 1-year increase; 95% confidence interval, 0.964 to 0.987). Current smoking, sevoflurane anesthesia, postoperative pain of NRS≥5, and the presence of a urinary catheter nearly doubled the risk of emergence agitation. CONCLUSION: Emergence agitation following general anesthesia is a common complication in adult nasal surgery patients. To reduce the occurrence and consequences of agitation episodes, elimination of the associated risk factors is necessary, especially in at-risk patients.

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

Neuregulin-1 induces cancer stem cell characteristics in breast cancer cell lines.

In breast cancer, neuregulin-1 (NRG1) is known as a ligand for the HER3 receptor, which has no intrinsic tyrosine kinase activity. When activated by NRG1 binding, the HER3 receptor forms a heterodimer with other HER family receptors and mediates downstream signaling pathways, leading to multiple effects including growth, proliferation, decreased apoptosis, cellular migration and angiogenesis. Cancer stem cells (CSCs), a subgroup of cancer cells, are considered to have features of stem cells such as self-renewal ability and pluripotent differentiation into other types of mature cells. This study showed that NRG1 treatment induced CSC characteristics in breast cancer cell lines. Using breast cancer cell lines, MCF-7, SKBr-3 and MDA-MB 468, changes related to CSC characteristics were analyzed. Flow cytometry was used to analyze changes in CSC fractions in multiple cell lines after NRG1 treatment. Western blot analysis and immunofluorescence staining demonstrated the expression of CSC markers. To confirm that NRG1 treatment acts through the HER3 receptor, inhibition studies using small interfering RNA (siRNA) were performed. In MCF-7 and SKBr-3 cells, increases in the CSC fraction and expression of CSC markers were observed after NRG1 treatment. However, MDA-MB 468 cells showed high intrinsic expression of CSC markers and a high cellular fraction of CSCs, and in these cells, NRG1 treatment caused no significant change in CSC characteristics. Inhibition of the HER3 receptor blocked the NRG1-induced CSC characteristics, indicating that NRG1 functions through the HER3 receptor. The results imply the presence of a mechanism by which the HER receptors, activated by NRG1, contribute to the acquisition of CSC-like characteristics in some types of breast cancer.

Nrf2-mediated HO-1 induction coupled with the ERK signaling pathway contributes to indirect antioxidant capacity of caffeic acid phenethyl ester in HepG2 cells.

The objective of this study is to investigate the contributing effect of the nuclear transcription factor-erythroid 2-related factor 2 (Nrf2)-mediated signaling pathway on the indirect antioxidant capacity of caffeic acid phenethyl ester (CAPE) against oxidative stress in HepG2 cells. The result of an antioxidant response element (ARE)-luciferase assay showed that CAPE stimulated ARE promoter activity resulting in increased transcriptional and translational activities of heme oxygenase-1 (HO-1). In addition, CAPE treatment enhanced Nrf2 accumulation in the nucleus and the post-translational phosphorylation level of extracellular signal-regulated kinase (ERK) among several protein kinases tested. Treatment with ERK inhibitor U126 completely suppressed CAPE-induced ERK phosphorylation and HO-1 expression, but it only partly inhibited CAPE-induced Nrf2 accumulation and ARE promoter. Using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) method, the cellular antioxidant capacity of CAPE against 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)- or H2O2-induced oxidative stress also was shown to be partially suppressed by the ERK inhibitor. From the overall results it is proposed that the indirect antioxidant activity of CAPE against oxidative stress in HepG2 cells is partially attributed to induction of HO-1, which is regulated by Kelch-like erythroid-cell-derived protein with CNC homology (ECH)-associated protein 1 (Keap1)-independent Nrf2 activation relying on post-translational phosphorylation of ERK.