PRR16/Largen Induces Epithelial-Mesenchymal Transition through the Interaction with ABI2 Leading to the Activation of ABL1 Kinase

Advanced or metastatic breast cancer affects multiple organs and is a leading cause of cancer-related death. Cancer metastasis is associated with epithelial-mesenchymal metastasis (EMT). However, the specific signals that induce and regulate EMT in carcinoma cells remain unclear. PRR16/Largen is a cell size regulator that is independent of mTOR and Hippo signalling pathways. However, little is known about the role PRR16 plays in the EMT process. We found that the expression of PRR16 was increased in mesenchymal breast cancer cell lines. PRR16 overexpression induced EMT in MCF7 breast cancer cells and enhances migration and invasion. To determine how PRR16 induces EMT, the binding proteins for PRR16 were screened, revealing that PRR16 binds to Abl interactor 2 (ABI2). We then investigated whether ABI2 is involved in EMT. Gene silencing of ABI2 induces EMT, leading to enhanced migration and invasion. ABI2 is a gene that codes for a protein that interacts with ABL proto-oncogene 1 (ABL1) kinase. Therefore, we investigated whether the change in ABI2 expression affected the activation of ABL1 kinase. The knockdown of ABI2 and PRR16 overexpression increased the phosphorylation of Y412 in ABL1 kinase. Our results suggest that PRR16 may be involved in EMT by binding to ABI2 and interfering with its inhibition of ABL1 kinase. This indicates that ABL1 kinase inhibitors may be potential therapeutic agents for the treatment of PRR16-related breast cancer.

Loss of EMP2 Inhibits Melanogenesis of MNT1 Melanoma Cells via Regulation of TRP-2

Melanogenesis is the production of melanin from tyrosine by a series of enzyme-catalyzed reactions, in which tyrosinase and DOPA oxidase play key roles. The melanin content in the skin determines skin pigmentation. Abnormalities in skin pigmentation lead to various skin pigmentation disorders. Recent research has shown that the expression of EMP2 is much lower in melanoma than in normal melanocytes, but its role in melanogenesis has not yet been elucidated. Therefore, we investigated the role of EMP2 in the melanogenesis of MNT1 human melanoma cells. We examined TRP-1, TRP-2, and TYR expression levels during melanogenesis in MNT1 melanoma cells by gene silencing of EMP2. Western blot and RT-PCR results confirmed that the expression levels of TYR and TRP-2 were decreased when EMP2 expression was knocked down by EMP2 siRNA in MNT1 cells, and these changes were reversed when EMP2 was overexpressed. We verified the EMP2 gene was knocked out of the cell line (EMP2 CRISPR/Cas9) by using a CRISPR/Cas9 system and found that the expression levels of TRP-2 and TYR were significantly lower in the EMP2 CRISPR/Cas9 cell lines. Loss of EMP2 also reduced migration and invasion of MNT1 melanoma cells. In addition, the melanosome transfer from the melanocytes to keratinocytes in the EMP2 KO cells cocultured with keratinocytes was reduced compared to the cells in the control coculture group. In conclusion, these results suggest that EMP2 is involved in melanogenesis via the regulation of TRP-2 expression.

Fungal Community Analyses of Endophytic Fungi from Two Oak Species, Quercus mongolica and Quercus serrata, in Korea

Fungal endophytes have been recorded in various plant species with a richness of diversity, and their presence plays an essential role in host plant protection against biotic and abiotic stresses. This study applied the Illumina MiSeq sequencing platform based on the amplification of fungal ribosomal ITS2 region to analyze fungal endophytic communities of two oak species (Quercus mongolica and Q. serrata) with different oak wilt disease susceptibilities in Korea. The results showed a total of 230,768 sequencing reads were obtained and clustered at a 97% similarity threshold into 709 operational taxonomic units (OTUs). The OTUs of Q. serrata were higher than that of Q. mongolica with the number of 617 OTUs and 512 OTUs, respectively. Shannon index also showed that Q. serrata had a significantly higher level of fungal diversity than Q. mongolica. Total of OTUs were assigned into 5 fungal phyla, 17 classes, 60 orders, 133 families, 195 genera, and 280 species. Ascomycota was the dominant phylum with 75.11% relative abundance, followed by Basidiomycota with 5.28%. Leptosillia, Aureobasidium and Acanthostigma were the most abundant genera detected in Q. serrata with the average relative abundance of 2.85, 2.76, and 2.19%, respectively. On the other hand, Peltaster, Cladosporium and Monochaetia were the most common genera detected in Q. mongolica with the average relative abundance of 4.83, 3.03, and 2.87%, respectively. Our results indicated that fungal endophytic communities were significantly different between two oak species and these differences could influence responses of host trees to oak wilt disease caused by Raffaelea quercus-mongolicae.

Fungal Community Analyses of Endophytic Fungi from Two Oak Species, Quercus mongolica and Quercus serrata, in Korea

Fungal endophytes have been recorded in various plant species with a richness of diversity, and their presence plays an essential role in host plant protection against biotic and abiotic stresses. This study applied the Illumina MiSeq sequencing platform based on the amplification of fungal ribosomal ITS2 region to analyze fungal endophytic communities of two oak species (Quercus mongolica and Q. serrata) with different oak wilt disease susceptibilities in Korea. The results showed a total of 230,768 sequencing reads were obtained and clustered at a 97% similarity threshold into 709 operational taxonomic units (OTUs). The OTUs of Q. serrata were higher than that of Q. mongolica with the number of 617 OTUs and 512 OTUs, respectively. Shannon index also showed that Q. serrata had a significantly higher level of fungal diversity than Q. mongolica. Total of OTUs were assigned into 5 fungal phyla, 17 classes, 60 orders, 133 families, 195 genera, and 280 species. Ascomycota was the dominant phylum with 75.11% relative abundance, followed by Basidiomycota with 5.28%. Leptosillia, Aureobasidium and Acanthostigma were the most abundant genera detected in Q. serrata with the average relative abundance of 2.85, 2.76, and 2.19%, respectively. On the other hand, Peltaster, Cladosporium and Monochaetia were the most common genera detected in Q. mongolica with the average relative abundance of 4.83, 3.03, and 2.87%, respectively. Our results indicated that fungal endophytic communities were significantly different between two oak species and these differences could influence responses of host trees to oak wilt disease caused by Raffaelea quercus-mongolicae.

Ethyl acetate extract of Smilax glabra Roxb roots and its major active compound astilbin promote osteoblastogenesis in vitro by upregulating bone cell differentiation- associated genes

Objective: To investigate the osteoblastogenic activity of the ethyl acetate (EtOAc) extract of Smilax glabra Roxb roots and its major active compound astilbin. Methods: Astilbin was isolated from EtOAc extract using silica gel chromatography combined with fraction crystallization. Chemical structure of astilbin was determined by analysis of the spectroscopic data in comparison with the literature. MTT method was used to detect the toxicity. Alkaline phosphatase (ALP) activity was determined by the spectrophotometric method at 405 nm using p-nitrophenyl phosphate as a substrate. Calcium deposition was stained with alizarin red-S, distained with cetylpyridium chloride, and quantified at 562 nm. In silico model for astilbin-ALP interaction was analyzed using AutoDock 4.2.6. The changes in expression of osteoblast differentiation related genes were determined using quantitative real-time PCR. Results: Both the EtOAc extract and astilbin had no toxicity toward osteoblast MC3T3-E1 cells at 5.0, 10, 25, and 50 μg/mL. At 25 μg/ mL, they enhanced ALP activity and mineralization of osteoblasts up to 30% and 55% for the EtOAc extract and 22% and 41% for astilbin, respectively. Molecular docking analysis of astilbin-ALP interaction revealed Arg167, Asp320, His324, and His437 were key residues participating in hydrophobic interaction; meanwhile, His434 and Thr436 residues were involved in hydrogen bond formation in the active site of human tissue-nonspecific ALP. Moreover, the expression level of genes opn, col1, osx, and runx2 were up-regulated in astilbin treated samples with the fold changes as 2.2; 3.7; 4.1; 2.3, respectively at 10 μg/mL (P<0.05). Conclusions: The EtOAc extract and its major compound astilbin exhibit osteoblastogenic activity by up-regulating important markers for bone cell differentiation. It could be a new and promising osteogenic agent with dual actions for therapeutic applications.

Ethyl acetate extract of Smilax glabra Roxb roots and its major active compound astilbin promote osteoblastogenesis in vitro by upregulating bone cell differentiation- associated genes

Objective: To investigate the osteoblastogenic activity of the ethyl acetate (EtOAc) extract of Smilax glabra Roxb roots and its major active compound astilbin. Methods: Astilbin was isolated from EtOAc extract using silica gel chromatography combined with fraction crystallization. Chemical structure of astilbin was determined by analysis of the spectroscopic data in comparison with the literature. MTT method was used to detect the toxicity. Alkaline phosphatase (ALP) activity was determined by the spectrophotometric method at 405 nm using p-nitrophenyl phosphate as a substrate. Calcium deposition was stained with alizarin red-S, distained with cetylpyridium chloride, and quantified at 562 nm. In silico model for astilbin-ALP interaction was analyzed using AutoDock 4.2.6. The changes in expression of osteoblast differentiation related genes were determined using quantitative real-time PCR. Results: Both the EtOAc extract and astilbin had no toxicity toward osteoblast MC3T3-E1 cells at 5.0, 10, 25, and 50 μg/mL. At 25 μg/ mL, they enhanced ALP activity and mineralization of osteoblasts up to 30% and 55% for the EtOAc extract and 22% and 41% for astilbin, respectively. Molecular docking analysis of astilbin-ALP interaction revealed Arg167, Asp320, His324, and His437 were key residues participating in hydrophobic interaction; meanwhile, His434 and Thr436 residues were involved in hydrogen bond formation in the active site of human tissue-nonspecific ALP. Moreover, the expression level of genes opn, col1, osx, and runx2 were up-regulated in astilbin treated samples with the fold changes as 2.2; 3.7; 4.1; 2.3, respectively at 10 μg/mL (P<0.05). Conclusions: The EtOAc extract and its major compound astilbin exhibit osteoblastogenic activity by up-regulating important markers for bone cell differentiation. It could be a new and promising osteogenic agent with dual actions for therapeutic applications.

Screening of Endophytic Fungal Isolates Against Raffaelea quercus-mongolicae Causing Oak Wilt Disease in Korea

Oak wilt disease caused by Raffaelea quercus-mongolicae has emerged obviously in Korea. We selected antifungal isolates against R. quercus-mongolicae among 368 endophytic fungal isolates from different parts of oak and pine trees. The experiment was conducted in the primary and secondary screenings by dual culture test. The antifungal activity of the selected isolates was assessed in culture filtrate test based on the inhibition rates in mycelial growth, sporulation, and spore germination of oak wilt fungus. Five isolates, E089, E199, E282, E409 and E415, showed strong antifungal activity in culture filtrate test, and their antifungal activity decreased on the culture media supplemented with heated culture filtrate. Higher mycelial growth inhibitions on the unheated media were recorded in E409 (Colletotrichum acutatum), E089 (Daldinia childiae), E415 (Alternaria alternata) and E199 (Daldinia childiae) with the inhibition rates of 79.0%, 70.1%, 68.9% and 64.5%, respectively. These isolates also had the higher sporulation inhibitions on unheated media with the rates of 96.8%, 84.2%, 82.8% and 80.5%, respectively. The spore germination of the oak wilt fungus was completely inhibited by E282 (Nectria balsamea) on both unheated and heated media. These results showed that a higher number of potent antifungal isolates against oak wilt fungus was isolated from the petiole compared to the other parts. This study could contribute to the development of biological control approaches for the management of oak wilt disease caused by R. quercus-mongolicae.

Screening of Endophytic Fungal Isolates Against Raffaelea quercus-mongolicae Causing Oak Wilt Disease in Korea

Oak wilt disease caused by Raffaelea quercus-mongolicae has emerged obviously in Korea. We selected antifungal isolates against R. quercus-mongolicae among 368 endophytic fungal isolates from different parts of oak and pine trees. The experiment was conducted in the primary and secondary screenings by dual culture test. The antifungal activity of the selected isolates was assessed in culture filtrate test based on the inhibition rates in mycelial growth, sporulation, and spore germination of oak wilt fungus. Five isolates, E089, E199, E282, E409 and E415, showed strong antifungal activity in culture filtrate test, and their antifungal activity decreased on the culture media supplemented with heated culture filtrate. Higher mycelial growth inhibitions on the unheated media were recorded in E409 (Colletotrichum acutatum), E089 (Daldinia childiae), E415 (Alternaria alternata) and E199 (Daldinia childiae) with the inhibition rates of 79.0%, 70.1%, 68.9% and 64.5%, respectively. These isolates also had the higher sporulation inhibitions on unheated media with the rates of 96.8%, 84.2%, 82.8% and 80.5%, respectively. The spore germination of the oak wilt fungus was completely inhibited by E282 (Nectria balsamea) on both unheated and heated media. These results showed that a higher number of potent antifungal isolates against oak wilt fungus was isolated from the petiole compared to the other parts. This study could contribute to the development of biological control approaches for the management of oak wilt disease caused by R. quercus-mongolicae.

MS-5, a Naphthalene Derivative, Induces the Apoptosis of an Ovarian Cancer Cell CAOV-3 by Interfering with the Reactive Oxygen Species Generation

Reactive oxygen species (ROS) are widely generated in biological processes such as normal metabolism and response to xenobiotic exposure. While ROS can be beneficial or harmful to cells and tissues, generation of ROS by diverse anti-cancer drugs or phytochemicals plays an important role in the induction of apoptosis. We recently identified a derivative of naphthalene, MS-5, that induces apoptosis of an ovarian cell, CAOV-3. Interestingly, MS-5 induced apoptosis by down-regulating the ROS. Cell viability was evaluated by water-soluble tetrazolium salt (WST-1) assay. Apoptosis was evaluated by flow cytometry analysis. Intracellular ROS (H₂O₂), mitochondrial superoxide, mitochondrial membrane potential (MMP) and effect on cycle were determined by flow cytometry. Protein expression was assessed by western blotting. The level of ATP was measured using ATP Colorimetric/Fluorometric Assay kit. MS-5 inhibited growth of ovarian cancer cell lines, CAOV-3, in a concentration- and time-dependent manner. MS-5 also induced G1 cell cycle arrest in CAOV-3 cells, while MS-5 decreased intracellular ROS generation. In addition, cells treated with MS-5 showed the decrease in MMP and ATP production. In this study, we found that treatment with MS-5 in CAOV-3 cells induced apoptosis but decreased ROS level. We suspect that MS-5 might interfere with the minimum requirements of ROS for survival. These perturbations appear to be concentration-dependent, suggesting that MS-5 may induce apoptosis by interfering with ROS generation. We propose that MS-5 may be a potent therapeutic agent for inducing apoptosis in ovarian cancer cell through regulation of ROS.

Elevated Ki-67 (MIB-1) Expression as an Independent Predictor for Poor Prognosis After Radical Cystectomy for Bladder Cancer

PURPOSE: We tried to investigate the clinical impact of Ki-67 (MIB-1) expression on the oncological and survival outcomes in patients with bladder cancer (BCa) after the radical cystectomy. MATERIALS AND METHODS: We retrospectively analyzed the data of 230 patients who were treated by radical cystectomy for BCa. Multivariate Cox-proportional hazards models and logistic regression tests were performed to evaluate the prognostic value of each variable. RESULTS: The patients with positive Ki-67 expression showed significant worse clinical characteristics and pathologic outcomes than negative Ki-67 group. Furthermore, the patients with Ki-67 expression showed significant worse recurrence (p=0.018) and cancer-specific mortality free survival (p=0.019) than negative Ki-67 group. The overall survival was also revealed to be inferior in Ki-67 positive group than Ki-67 negative group but the statistical significance was marginal (p=0.062). Subsequent multivariate Cox analyses showed that Ki-67 was independent predictor for disease recurrence after surgery (hazard ratio, 3.142; 95% CI, 1.287–7.671; p=0.012). CONCLUSIONS: In our study, high Ki-67 expression was significantly related with worse clinical outcomes after radical cystectomy in the patients with BCa. Further prospective and basic researches are needed to validate the true prognostic value of Ki-67.

Identification of Enterovirus C105 for the first time in New Zealand

In this report we describe a new approach in HIV sentinel surveillance that was piloted in Viet Nam in 2009 and is currently being rolled out in all provinces. It comprises a brief behavioural questionnaire added to the HIV sentinel surveillance surveys conducted routinely among people who inject drugs, female sex workers and men who have sex with men. Timely reporting of data from this system has resulted in improvements to HIV prevention efforts for most at-risk populations.

The presence of high level soluble herpes virus entry mediator in sera of gastric cancer patients

The development of gastric cancer (GC) is closely related to chronic inflammation caused by Helicobacter pylori infection, and herpes virus entry mediator (HVEM) is a receptor expressed on the surface of leukocytes that mediates potent inflammatory responses in animal models. However, the role of HVEM in human GC has not been studied. Previously, we showed that the interaction of HVEM on human leukocytes with its ligand LIGHT induces intracellular calcium mobilization, which results in inflammatory responses including induction of proinflammatory cytokine production and anti-bacterial activities. In this study, we report that leukocytes from GC patients express lower levels of membrane HVEM (mHVEM) and have lower LIGHT-induced bactericidal activities than those from healthy controls (HC). In contrast, levels of soluble HVEM (sHVEM) in the sera of GC patients were significantly higher than in those of HC. We found that monocyte membrane-bound HVEM is released into the medium when cells are activated by proinflammatory cytokines such as TNF-alpha and IL-8, which are elevated in the sera of GC patients. mHVEM level dropped in parallel with the release of sHVEM, and release was completely blocked by the metalloprotease inhibitor, GM6001. We also found that the low level of mHVEM on GC patient leukocytes was correlated with low LIGHT-induced bactericidal activities against H. pylori and S. aureus and production of reactive oxygen species. Our results indicate that mHVEM on leukocytes and sHVEM in sera may contribute to the development and/or progression of GC.

Study complication of aplastic anemia following chemotherapy of acute myelogenous leukemia

Background: Aplastic anemia following chemotherapy of acute leukemia is a common complication, which may lead to severe consequences. Objective: To study characteristics of aplastic anemia occurred in ccute myelogenous leukemia (AML) patients, following chemotherapy. Subjects and methods: A prospective study was carried out in 50 AML patients treated at National Institute of Hematology and Blood Transfusion from Aug 2005 to Dec 2006. These patients were treated by induction chemotherapy with "3+7" regime. Result: Aplastic anemia had been seen in 100% patients. Characteristics of this condition were poor marrow cells (average marrow cell count was 15.1\xb112.6 G/l) and strongly decreased counts of hemoglobin, white blood cells and platelets. Hemoglobin, white blood cell and platelet counts at the lowest level were 83.32 g/l; 0.96 G/l; 30.18 G/l; respectively. This situation prolonged for 3-4 weeks and changed into the most severe condition at the end of second week after chemotherapy. Infection frequency was 92%. Conclusion: Aplastic anemia following chemotherapy of AML patients is a common complication with severe consequences such as significant decrease of WBC and platelet counts, which may lead to opportunistic infection. Hence, this complication must be monitored, detected and treated promptly. \r\n', u'\r\n', u'

Study the changes in clinical and in blood cell indices after hydroxyurea treatment for chronic myelo-granulocytic leukemia

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of the primitive hematopoietic stem cell. The research is performed to examine the effectiveness of hydroxyurea in treating patients with CML in chronic stage. The results showed that hydroxyurea dramatically improved clinical and hematological condition. All patients reached complete or partial remission

Blood uric acid levels in patients with chronic granulocytic leukemia

The study aimed to investigate the changes in blood uric acid level. Participants were 61 patients with chronic granulocytic leukemia who were treated at the Central Institute of Hematology and Blood Transfusion between 9/1999 and 12/2000. Main findings: Blood uric acid level has increased in a significant percentage of patients: 35.9% of males and 36.4% of females have blood uric acid level of 529 and 423Mmol/l, respectively. Mean level of blood uric acid for whole group was 356 +/- 132Mmol/l for males and 281+/-129 Mmol/l for females. Blood uric acid level was not changed consistently with white blood cell count. However, there was a tendency of having higher blood uric acid level in the patients with higher WBC count. The symptoms of secondary gout were not observed in patients of this study

Cytological features of peripheral blood in patients with chronic myelo-granulocytic leukemia

The study was carried out on 61 chronic myelo-granulocytic leukemia (CML) patients aimed to investigate the cytological features of peripheral blood. The results showed that all patients had laboratory features of normocytic and normochromic anemia (RBC counts: 2.8T/L +/- SD 0.59). All patients have increased WBC and platelet counts (WBC count: 210.5G/L +/- SD 101.0, platelet count: 624.3G/L +/- SD 431.9). The differentiated white cells showed all types of granulocytic cell (myeloblast 1.5%, promyelocytes 4%, myelocytes 15.5%, metamyelocytes 14.8%, band forms 7.1%, segmented neutrophils 44.9%, basophils 3.3%, eosinophils 2.4%, lymphocytes 5.4%, monocytes 0.1%) with the increase on absolute number and percentage of basophil and eosinophil. However, there was no elevation in myelocyte count compared with metamyelocyte count

Some opinions of the clinical progress of the patients with chronic granular leukemia

The aim of this study is to examine the effectiveness of hydroxyurea in treating CML patients in chronic phase. The result of the research shows that the induction treatment with hydroxyurea (initial dose: 40-60mg/kg body weight daily) radically improved clinical condition for CML patients: the size of spleen was reduced in the majority of patients (91.7% and 63.9% patients have palpable spleen (P<0.05), 83.3% and 5.6% patients have enlarged spleen third and forth degree before and after induction treatment respectively, percentage of patients with symptoms of high viscosity syndrome decreased from 36.1% to 0% after treatment with P<0.001, symptoms of anemia were decreased in the majority of patients. The follow up during the time of induction treatment shows that main clinical symptoms (enlarged spleen, high viscosity syndrome, anemia) have improved gradually in accordance with the decrease of WBC

Investigating the clinical and hematological features of chronic myelogenous leukemia at early initial hospitalization

From January 1997 to January 1998, there were 21 patients with chronic myelogenous leukemia who admitted to the Clinical Department, National Institute of Hematology and Blood Transfusion, B¹ch Mai Hospital. Mean age of patients was 42.85 for men and 38.12 for women. Among these patients, splenomegaly was observed in 100% of patients. Hyperleukocytosis, hyperthrombocytemia, erythrocytopenia, and hyperuricemia all were measured. 94% of patients were positive with Philadelphia chromosome

Hematological changes during the aggressive treatment of chronic myeloid leukemia (CML) in the chronic duration by hydroxyurea

The aim of this study is to examine the effectiveness of hydroxyurea in treating CML patients in chronic phase. The result of the research shows that the induction treatment with hydroxyurea with initial dose of 40-60mg/kg body weight daily radically improves hematological condition for CML patients in chronic phase. All patients (42 patients) reached hematological remission (33.3% patients reached complete remission), 66.7% patients reached partial remission WBC decrease from 193.9117.7 g/l to 10.16.0 g/l. Percentage of myeloblast, promyelocyte, myelocyte and metamyelocyte in peripheral blood decrease from 35.08.0% to 6.96.6%. Hemoglobin concentration increase from 86.119.0 g/l to 96.013.3 g/l, RBC increase from 2.90.7 T/l to 3.20.5 T/l. The follow up during the time of induction treatment shows that main hematological indicators have changed gradually when approaching normal physiological values