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B. C. Behera, Pune, India
T. Chen, Stanford, CA/USA
E. Corsini, Milan, Italy
P. Diel, Cologne, Germany
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P. B. Farmer, Leicester/UK
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M. Lotti, Bonn, Germany / Padova, Italy
P. Micke, Uppsala, Sweden
A. N. Misra, Ranchi, Jharkhand State, India
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K. Renganathan, Johnstown, PA/USA
S. D. Ray, Fort Wayne, IN/USA
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A. Winterpacht, Erlangen, Germany
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Effect of glucose and insulin supplementation on the isolation of primary human hepatocytes
(2019-11-18) Damm, Georg; Schicht, Gerda; Zimmermann, Andrea; Rennert, Christiane; Fischer, Nicolas; Kießig, Melanie; Wagner, Tristan; Kegel, Victoria; Seehofer, Daniel
Primary human hepatocytes (PHHs) remain the gold standard for in vitro investigations of xenobiotic metabolism and hepatotoxicity. However, scarcity of liver tissue and novel developments in liver surgery has limited the availability and quality of tissue samples. In particular, warm ischemia shifts the intracellular metabolism from aerobic to anaerobic conditions, which increases glycogenolysis, glucose depletion and energy deficiency. Therefore, the aim of the present study was to investigate whether supplementation with glucose and insulin during PHH isolation could reconstitute intracellular glycogen storage and beneficially affect viability and functionality. Furthermore, the study elucidated whether the susceptibility of the tissue’s energy status correlates with body mass index (BMI). PHHs from 12 donors were isolated from human liver tissue obtained from partial liver resections using a two-step EDTA/collagenase perfusion technique. For a direct comparison of the influence of glucose/insulin supplementation, we modified the setup, enabling the parallel isolation of two pieces of one tissue sample with varying perfusate. Independent of the BMI of the patient, the glycogen content in liver tissue was notably low in the majority of samples. Furthermore, supplementation with glucose and insulin had no beneficial effect on the glycogen concentration of isolated PHHs. However, an indirect improvement of the availability of energy was shown by increased viability, plating efficiency and partial cellular activity after supplementation. The plating efficiency showed a striking inverse correlation with increasing lipid content of PHHs. However, 60 h of cultivation time revealed no significant impact on the maintenance of albumin and urea synthesis or xenobiotic metabolism after supplementation. In conclusion, surgical procedures and tissue handling may decrease hepatic energy resources and lead to cell stress and death. Consequently, PHHs with low energy resources die during the isolation process without supplementation of glucose/insulin or early cell culture, while their survival rates are improved with glucose/insulin supplementation.
E-selectin as a prognostic factor of patients hospitalized due to acute inflammatory respiratory diseases
(2019-11-11) Nakano, Hiroshi; Inoue, Sumito; Shibata, Yoko; Abe, Koya; Murano, Hiroaki; Yang, Sujeong; Machida, Hiroyoshi; Sato, Kento; Sato, Chisa; Nemoto, Takako; Nishiwaki, Michiko; Kimura, Tomomi; Yamauchi, Keiko; Sato, Masamichi; Igarashi, Akira; Tokairin, Yoshikane; Watanabe, Masafumi
When examining patients with acute inflammatory respiratory diseases, it is difficult to distinguish between infectious pneumonia and interstitial pneumonia and predict patient prognosis at the beginning of treatment. In this study, we assessed whether endothelial selectin (E-selectin) predicts the outcome of patients with acute inflammatory respiratory diseases. We measured E-selectin serum levels in 101 patients who were admitted to our respiratory care unit between January 2013 and December 2013 because of acute inflammatory respiratory diseases that were eventually diagnosed as interstitial pneumonia (n = 38) and lower respiratory tract infection (n = 63). Seven of these patients (n = 101) died. The pneumonia severity score was significantly higher and the oxygen saturation of arterial blood measured by pulse oximeter (SpO2)/fraction of inspiratory oxygen (FiO2) was significantly lower in the deceased patients than in the surviving patients. There were significantly fewer peripheral lymphocytes and significantly higher E-selectin serum levels in the deceased patients than in the surviving patients. In the multiple logistic regression analysis, the E-selectin serum levels and SpO2/FiO2 ratio were independent predictive factors of prognosis. The risk of death during acute respiratory disease was determined using a receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.871 as calculated from the ES, and the cutoff value was 6453.04 pg/ml, with a sensitivity of 1.00 and a specificity of 0.72 (p = 0.0027). E-selectin may be a useful biomarker for predicting the prognosis of patients with acute inflammatory respiratory diseases.
MicroRNA-9-5p functions as a tumor suppressor in prostate cancer via targeting UTRN
(2019-11-07) Guo, Zhenyu; Huang, Shuchang; Zhou, Yihong; Cheng, Wenjie; Chu, Xi; Zheng, Xiaoqing; Zheng, Hao
Accumulating evidence indicates that miR-9-5p plays an important role in several diseases, especially tumor progression. In this study, we investigated the clinical significance and biological function of miR-9-5p in prostate cancer (PCa). Using quantitative real time PCR (qRT-PCR) analysis, we found miR-9-5p level was significantly down-regulated in PCa tissues and cell lines. The decreased miR-9-5p expression was associated with tumor size, preoperative PSA, Gleason score and lymph node metastasis. Kaplan-Meier survival analysis showed patients with low level of miR-9-5p had significantly decreased rates of overall survival (OS). Multivariate analyses showed that miR-9-5p was an independent predictor of PCa patients’ prognosis. Through CCK-8 and Transwell assays, miR-9-5p overexpression by miR-9-5p mimics transfection was demonstrated to suppress the proliferation, migration and invasion of PCa cells. Mechanistically, luciferase reporter assay, qRT-PCR and Western blot demonstrated that Utrophin (UTRN) is a direct target of miR-9-5p in PCa cells. The status of UTRN protein in PCa tissues was much higher than that in adjacent tissues by immunohistochemical staining and its mRNA levels were inversely correlated with miR-9-5p in PCa tissues. Importantly, UTRN knockdown by siUTRN imitated the suppressive effects of miR-9-5p on cell proliferation, migration and invasion in PCa. In summary, miR-9-5p might novel prognostic biomarker in and targeting UTRN by miR-9-5p could be potential therapeutic candidates for PCa.
Antiviral activity of flavonoids present in aerial parts of Marcetia taxifolia against Hepatitis B virus, Poliovirus, and Herpes Simplex Virus in vitro
(2019-11-05) Ortega, Joseph Thomas; Serrano, María Luisa; Suárez, Alírica Isabel; Baptista, Jani; Pujol, Flor Helene; Cavallaro, Lucía Vicenta; Campos, Héctor Rodolfo; Rangel, Héctor Rafael
Marcetia taxifolia is a neotropical plant present in South America and it has been evaluated in several biological models due to the presence of active metabolites. Nevertheless, there is a limited quantity of studies related to the antiviral activity of the compounds present in this genus. In our work, the antiviral effect of the compounds isolated from the aerial parts of Marcetia taxifolia was evaluated against Hepatitis B virus (HBV), Herpes Simplex Virus type 1 (HSV-1), and Poliovirus type 1 (PV-1). The cytopathic effect and viral quantification by qPCR were determined as indicative of antiviral activity. Our data show that myricetin rhamnoside (MyrG), myricetin-3-α-O-ramnosil (1→6)-α-galactoside (MyrGG), 5,3'-dihydroxy-3,6,7,8,4’-pentamethoxyflavone (PMF), 5-hydroxy-3,6,7,3',4'pentamethoxyflavone (PMF-OH) had antiviral activity without cytotoxic effects. The methoxyflavones PMF and PMF-OH were the most active compounds, showing an antiviral effect against all the evaluated viruses. Computational studies showed that these compounds could interact with the Reverse Transcriptase. Altogether, these results suggest that the flavonoids (related to myricetin and methoxyflavones) are the main antiviral compounds present in the aerial parts of Marcetia taxifolia. Furthermore, our results showed that the methoxyflavones have a broad antiviral activity, which represents an opportunity to evaluate these flavonoids as lead molecules to develop new antiviral compounds.
Transgenerational influence of parental morphine exposure on pain perception, anxiety-like behavior and passive avoidance memory among male and female offspring of Wistar rats
(2019-11-05) Ahmadian-Moghadam, Hamid; Sadat-Shirazi, Mitra-Sadat; Seifi, Fereshteh; Niknamfar, Saba; Akbarabadi, Ardeshir; Toolee, Heidar; Zarrindast, Mohammad-Reza
Accumulating evidence suggests that epigenetic mechanisms play an important role in the formation and maintenance of memory within the brain. Moreover, the effect of parental drug-exposure before gestation on behavioral state of offspring has been little studied. The main objective of the current study is to evaluate the effect of parental morphine exposure on avoidance memory, morphine preference and anxiety-like behavior of offspring. The total of 32 males and 32 females were used for mating. The animals were treated with morphine. The offspring according to their parental morphine treatment was divided into four groups (n=16) including paternally treated, maternally treated, both of parents treated and naïve animals. The pain perception, anxiety-like behavior, and avoidance memory were evaluated in the offspring. In the current study, the total of 256 offspring was used for the experiments (4 tasks × 4 groups of offspring × 8 female offspring × 8 male offspring). The finding revealed that the avoidance memory and visceral pain were reduced significantly in male and female offspring with at least one morphine-treated parent. Moreover, anxiety-like behavior was reduced significantly in the male offspring with at least one morphine-treated parent. While anxiety-like behavior was increased significantly in female offspring that were treated by morphine either maternally or both of parents. The data revealed that the endogenous opioid system may be altered in the offspring of morphine-treated parent(s), and epigenetic role could be important. However, analysis of variance signified the important role of maternal inheritance.
Macrophage migration inhibitory factor is involved in endovascular trophoblast cell function in vitro
(2019-10-31) Vilotic, Aleksandra; Jovanovic Krivokuca, Milica; Stefanoska, Ivana; Vrzic Petronijevic, Svetlana; Petronijevic, Milos; Vicovac, Ljiljana
Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine abundantly present at the feto-maternal interface proposed to play a role in establishment of pregnancy. We have previously shown that pharmacological inhibition of enzymatic activity of MIF decreases extravillous trophoblast invasion and migration in vitro. This study aimed to further elucidate potential role of endogenous trophoblast MIF, and to assess its importance for endovascular trophoblast cell function in particular. Attenuation of MIF by siRNA reduced HTR-8/SVneo cell invasion through Matrigel (59 % of control), expression of integrin α1 (86 % of control) and levels of MMP2 and MMP9 (87 % and 57 % of control, respectively). MIF specific siRNA reduced the ability of HTR-8/SVneo to differentiate in to endothelial-like phenotype, as determined by Matrigel tube formation assay. The total tube length was decreased to 68.6 %, while the number of branching points was reduced to 57.8 % of control. HTR-8/SVneo cell capacity to integrate into HUVEC monolayers was reduced by knock-down of MIF. This could be partly caused by reduced N-cadherin expression to 63 % of control, which decreased with knock-down of MIF, as the expression of this protein was recently shown essential for trophoblast-endothelial interaction. These novel findings indicate a novel role for trophoblast MIF in spiral artery remodeling process.
[(7-chloroquinolin-4-yl)amino]acetophenones and their copper(II) derivatives
(2019-10-28) Parra, Yonathan de Jesús; Andueza L, Felix D; Ferrer M, Rosa E; Bruno Colmenarez, Julia; Acosta, María E; Charris, Jaime; Rosenthal, Philip J; Gut, Jiri
The synthesis of the compounds [(7-chloroquinolin-4-yl)amino]acetophenones (4, 5) and their copper(II) complexes (4a, 5a) is reported. The compounds were characterized using a wide range of spectroscopic and spectrometric techniques, such as FTIR, UV-vis, NMR, EPR, ESI-CID-MS2. The spectral results suggested that the ligand acted as chelating species coordinating the metal through the endocyclic nitrogen of the quinoline ring in both complexes, with general formulae ex pressed in two ways, according to the phase in which they are: [Cu(L)2Cl2] for solid phase and [Cu(L)2][2Cl] for liquid phase. The EPR study of the Cu (II) complexes indicated a probable distorted tetrahedral coordination geometry. This result was confirmed by the calculated optimized structures at the DFT/B3LYP method with the 6-31G (d,p) basis set. The characterization of the fragmentation pattern of protonated free ligands was extended here to fragments as low as m/z 43, while for coordination complexes it extends to fragments at m/z 80 and m/z 111. The antimalarial activity of the compounds was determined through three different tests: inhibitory activity against in vitro growth of Plasmodium falciparum (W2), inhibition of hemozoin formation (β-hematin) and in vitro inhibitory activity against recombinant falcipain-2, where compound 5 showed considerable activity. However, the activity of free ligands against P. falciparum was increased by complexing with the Cu (II) metal ion. The values of the HOMO-LUMO energy gap of 3.847 eV (4a) and 3.932 eV (5a) were interpreted with high chemical activity and thus, could influence on biological activity. In both compounds, the total electron density surface mapped with electrostatic po tential clearly revealed the presence of high negative charge on the Cu atom. Also, this study reported the molecular docking of free ligands (4, 5) using software package ArgusLab 4.0.1. The results revealed the importance of water molecules as interaction bridges through hydrogen bonds between free ligands and β-hematin; at the same time, the hypothesis that π–π interaction between quinoline derivatives and the electronic system of hematin governs the formation of adducts was confirmed.
Increased liver injury in patients with chronic hepatitis and IgG directed against hepatitis E virus
(2019-10-25) Sévédé, Daouda; Doumbia, Moussa; Kouakou, Viviane; Djehiffe, Vicky; Pineau, Pascal; Dosso, Mireille
Type-E hepatitis is responsible for more than three million symptomatic cases and more than 40,000 deaths worldwide. The situation of this hepatitis is overall poorly known in sub-Saharan Africa. Notably, the baseline circulation of HEV outside sporadic outbreaks has been barely characterized in this large region. More specifically, the impact of superinfection by this virus on the health status of the large reservoir of patients chronically infected with other hepatitis viruses remains to be evaluated. We searched for anti-HEV immunoglobulins in a series of 200 pregnant women and 92 patients with persistent liver infections with hepatitis B or C viruses and subsequently tried to assess serological co-variations with demographical and clinical features. We observed that only 1.5 % of expectant mothers were seropositive of anti-HEV IgG while it was the case for 18.4 % of patients with chronic liver diseases (P=4.5E-07). The presence of anti-HEV was not linked to any of the collected demographical features (age, sex, education, pork meat consumption, water supply, …). By contrast, the presence of anti-HEV was significantly associated with increased levels (1.6-1.8-fold, P<0.0001) of blood aminotransferases (AST, ALT) in patients with persistent hepatitis B or C. Our work indicates that, in Ivory Coast, the presence of IgG directed against HEV might contribute to a deterioration of liver health in patients with already installed persistent liver infections. The mechanisms explaining such phenomenon at distance of acute phase of infection are still unknown but might be linked either to a residual persistence of HEV in a context of general immune exhaustion or to an inappropriate auto-immune reaction as already observed in the aftermath of other viral infection types.
Crocetin promotes angiogenesis in human endothelial cells through PI3K-Akt-eNOS signaling pathway
(2019-10-21) Nasirzadeh, Mahdieh; Rasmi, Yousef; Rahbarghazi, Reza; Kheradmand, Fatemeh; Karimipour, Mojtaba; Aramwit, Pornanong; Astinfeshan, Maryam; Gholinejad, Zafar; Daeihasani, Behrokh; Saboory, Ehsan; Shirpoor, Alireza; Rezabakhsh, Aysa; Zolali, Elmira; Khalaji, Naser
Previous studies proved the pro-angiogenic effect of Crocetin, a natural carotenoid dicarboxylic acid, in both in vivo and in vitro models. However, the exact mechanism of Crocetin action has not completely been elucidated yet. The current experiment was designed to find the activity of PI3K-Akt-eNOS axis after the treatment of endothelial cells with Crocetin in vitro. Human Umbilical Vein Endothelial Cells (HUVECs) were incubated with various concentrations of Crocetin (1, 5, 25, 50, and 100 μM) over a period of 72 h. Crocetin significantly increased HUVECs viability after 72 h as compared with the control group. We also found that Crocetin promoted the formation of the capillary-like structure compared to the control (p<0.05). Moreover, an improved migration rate and increased MMP-9 activity were observed in HUVECs that received 50 μM Crocetin (p<0.05). Crocetin enhanced the uptake of Ac-LDL which is correlated with increased lipid metabolism. Based on the data from thecurrent experiment, protein level of VEGFR-1, -2 and p-Akt/Akt, p-eNOS/eNOS ratios were increased 72 h after the treatment of HUVECs with Crocetin (p<0.05). In contrast, the transcription level of VEGF was reduced in Crocetin-treated cells. These data demonstrated that Crocetin promotes HUVECs angiogenesis potential by the modulation of VEGF signaling pathway and increased cell viability. The PI3K/Akt/eNOS axis is required for a Crocetin-associated activity in endothelial cells.
The effects of ovarian cancer cell-derived exosomes on vascular endothelial growth factor expression in endothelial cells
(2019-10-09) Ghorbanian, Mohammad; Babashah, Sadegh; Ataei, Farangis
Ovarian carcinoma is considered as a major clinical challenge worldwide. Exosomes, nano-sized intraluminal vesicles of multivesicular bodies, are secreted by most types of cells and play an important role in intercellular communication. Cancer cell-derived exosomes can develop cancer progression and metastasis by manipulating the local and distant biological environments. Angiogenesis is an important contributor to tumor progression. Vascular endothelial growth factor (VEGF) is the most potent pro-angiogenic protein and induces proliferation, sprouting, and vessel formation by endothelial cells. In this study, exosomes derived from ovarian epithelial cancer cells OVACAR-3 (exo-OVCAR-3) were successfully isolated and characterized by scanning electron microscopy in terms of size and morphology. Cellular internalization of exosomes labeled with PKH fluorescent dye was monitored by a fluorescence microscope. Our results elucidated that exosomes treatment (100 µg/ml) had a promoting effect on VEGF expression and secretion in endothelial cells. Furthermore, we demonstrated that exo-OVCAR-3 caused an increase in the proliferation and migration rate of endothelial cells. It seems that inducing VEGF by exo-OVCAR-3 can influence the vascular behavior of endothelial cells in vitro.
Roselle attenuates cardiac hypertrophy after myocardial infarction in vivo and in vitro
(2019-09-26) Si, Lislivia Yiang-Nee; Ramalingam, Anand; Ali, Shafreena Shaukat; Aminuddin, Amnani; Ng, Pei-Yuen; Latip, Jalifah; Kamisah, Yusof; Budin, Siti Balkis; Zainalabidin, Satirah
Roselle (Hibiscus sabdariffa Linn) has been traditionally used as folk medicine for hypertension and maintaining cardiovascular health, with therapeutic potential in protecting against numerous cardiovascular diseases. However, it remains unclear whether roselle can be used for management of cardiac hypertrophy seen after myocardial in- farction (MI). This study therefore investigated the effects of aqueous roselle extract on cardiac hypertrophy aris- ing from myocardial infarction both in vivo and in vitro. For in vivo study, male Sprague-Dawley rats were divided into control or MI groups (receiving 85 mg/kg isoproterenol s.c. for 2 days) and were given roselle extract (100 mg/kg, p.o daily) for 28 days. Cardiac structure and functional changes were evaluated at study end-point using histology, Langendorff analysis and gene expression analysis. In vitro effects of roselle were also assessed on ANG II-induced cardiomyocytes hypertrophy using H9c2 cells, simulating cardiac hypertrophy evident after MI. Roselle significantly ameliorated MI-induced cardiac systolic and diastolic dysfunction, as seen across improve- ment in left ventricular developed pressure (LVDP) and its derivative (LVdP/dtmax) and isovolumic relaxation (Tau). Oxidative stress evident across elevated pro-oxidant markers (NOX2 subunit of NADPH oxidase and 8- isoprostane) as well as reduced antioxidant markers (superoxide dismutase and glutathione) were also significantly attenuated by roselle. Furthermore, roselle treatment markedly reduced markers of cardiac remodeling (cardiac hypertrophy and fibrosis) compared to the untreated MI rats. On in vitro analysis, roselle significantly attenuated ANG II-induced cardiomyoycte hypertrophy in dose-dependent manner. This study demonstrated that roselle at- nd dysfunction seen after MI both in vivo and in vitro, and these effects are likely mediated by phenolic compounds found in roselle extract.
Δ-Aminolevulinate dehydratase and glutathione peroxidase activity in Alzheimer's disease
(2019-09-25) Garlet, Quelen Iane; Haskel, Maria Vaitsa Losh; Pereira, Romaiana Picada; da Silva, Weber Cláudio Francisco Nunes; da Rocha, João B. Teixeira; Oliveira, Cláudia Sirlene; Bonini, Juliana Sartori
Alzheimer’s disease (AD) is a neurodegenerative pathology that affects elderly people all over the world. Several studies have demonstrated that oxidative stress is an aggravating factor for AD development and progression. Therefore, this study aimed to evaluate the activity of two oxidative stress markers, glutathione peroxidase (GPx) and δ -aminolevulinate dehydratase ( δ -ALA-D), as well as correlate them with blood metal levels and AD progression. For this purpose, 88 elderly individuals were divided in two groups: AD group (34 patients diagnosed with AD) and control group (34 subjects paired by age with the AD group). The Mini-Mental State Examination and the Clinical Dementia Rating (CDR ) were used as tools to classify the AD progression. GPx and δ -ALA-D activities were measured in all subjects through blood tests. Both enzymes’ activities were decreased in AD patients when compared to the age-matched control group, regardless of the CDR. Moreover, GPx activity was positively correlated with selenium levels in the blood; and the δ -ALA-D activity was negatively correlated with blood cop- per levels. Taken together, our results indicated that, for the first time, blood δ -ALA-D activity was significantly inhibited in AD patients. While literature reports conflicting data regarding GPx activity in AD patients, the δ - ALA-D activity seems to be a more consistent tool to be applied as an earlier AD marker.
Impaired immunomodulatory ability of type 2 diabetic adipose-derived mesenchymal stem cells in regulation of inflammatory condition in mixed leukocyte reaction
(2019-09-23) Aliakbari, Sara; Mohammadi, Mobin; Rezaee, Mohammad Ali; Amini, Abbas Ali; Fakhari, Shohreh; Rahmani, Mohammad Reza
The immunomodulatory properties of type 2 diabetic patients’ adipose-derived mesenchymal stem cells (D-ASCs) has not been extensively studied. In this study, we compared the immunomodulatory properties of D-ASCs and non-diabetic subjects mesenchymal stem cells (ND-ASCs) in co-culture with mixed leukocyte reaction (MLR). ASCs were isolated from adipose tissue samples of type 2 diabetic and non-diabetic subjects (age: 40-55). D-ASCs and ND-ASCs were co-cultured with two-way MLR. Peri pheral blood mononuclear ce lls (PBMCs) proliferation ratio, protein levels of IFN- γ and IL-10, mRNA expression of COX-2 , TNF- α , TGF- β 1 and IL-6 genes in MLR, D-ASCs and ND-ASCs co-cultures were assessed using XTT, ELISA and Real-time qRT-PCR, respectively. PBMCs proliferation on days 2 and 4 of D-ASCs co-culture was higher than ND-ASCs co-culture of the same days ( p < 0.001). IFN- γ level decreased on day 4 compared to day 2 of ND-ASCs co-culture, but its level had not changed in D-ASCs co-culture. COX-2 expression on days 2 and 4 of D-ASCs co-culture was lower than ND- ASCs co-culture of the same days ( p < 0.05). The expression of TNF- α and IL-6 on days 2 and 4 of D-ASCs co- culture were higher than ND-ASCs co-culture of the same days ( p < 0.001). TGF- β 1 on day 4 of ND-ASCs co- culture showed a slightly higher expression than D-ASCs co-culture of the same day. Lower suppression of PBMCs proliferation, declined expre ssion of anti-inflammatory and upregulated expression of pro-inflammatory factors in D-ASCs co-culture, indicated an impairment of these cells in modulation of the inflammatory condition.
MicroRNA-494 induces breast cancer cell apoptosis and reduces cell viability by inhibition of nicotinamide phosphoribosyltransferase expression and activity
(2019-09-12) Ghorbanhosseini, Seyedeh Sara; Nourbakhsh, Mitra; Zangooei, Mohammad; Abdolvahabi, Zohreh; Bolandghamtpour, Zahra; Hesari, Zahra; Yousefi, Zeynab; Panahi, Ghodratollah; Meshkani, Reza
Breast cancer (BC) is the most preval ent cause of cancer-related death in women worldwide. BC is frequently associated with elevated levels of nicotinamide phosphoribos yltransferase (NAMPT) in blood and tumor tissue. MicroRNA-494 (miR-494) has been described to play key anti-tumor roles in human cancers. The aim of the present study was to investigate the inhibitory effect of miR-494 on NAMPT-mediated viability of BC cells. In this experimental study, MCF-7 and MDA-MB-231 cells were cultured and then transfec ted with miR-494 mimic, miR-494 inhibitor and their negative controls. The mRNA and protein expression of NAMPT were assessed using real-time PCR and Western blotting, respectively. Subseq uently, intracellular NAD levels were determined by a colorimetric method. Finally, cell apoptosis was examined by fl ow cytometry. Bioinformatics evaluations pre- dicted NAMPT as a miR-494 target gene which was confirmed by luciferase reporter assay. Our results showed an inverse relationship between the expression of miR-494 and NAMPT in both MCF-7 and MDA-MB-231 cell lines. miR-494 significantly down-regulated NAMPT mRNA and protein expression and was also able to reduce the cellular NAD content. Cell viability was decreased fo llowing miR-494 up-regulation. In addition, apoptosis was induced in MCF-7 and MDA-MB-231 cells by miR-494 mimic. Our findings indicate that miR-494 acts as a tumor suppressor and has an important effect in suppressing the growth of BC cells through NAMPT. Therefore, miR-494 might be considered as a novel therapeutic target for the management of human breast cancer.
Long non-coding RNA RPPH1 promotes the proliferation, invasion and migration of human acute myeloid leukemia cells through down-regulating miR-330-5p expression
(2019-09-11) Lei, Bo; He, Aili; Chen, Yinxia; Cao, Xinmei; Zhang, Pengyu; Liu, Jie; Ma, Xiaorong; Qian, Lu; Zhang, Wanggang
Multiple studies have revealed that the long non-coding RNA RPPH1 (Ribonuclease P RNA Component H1) is involved in disease progression of solid tumors and neurodegenerative diseases. We aimed to explore the functions of RPPH1 in the pathogenesis of acute myeloid leukemia (AML) and the underlying molecular mechanisms. The expression of RPPH1 was examined in blood samples of AML patients and human AML cell lines including THP-1 and HL-60. The microRNAs (miRNAs) targets of RPPH1 were predicted with online tools and validated with the dual luciferase reporter assay. The malignant behaviors of AML cells with lentivirus medicated knockdown of RPPH1 and/or administration of miR-330-5p inhibitor were assessed. Cell proliferation was determined by the CCK-8 and EdU incorporation methods, and cell invasion and migration were assayed with transwell experiments. The effects of RPPH1 knockdown on in vivo tumor growth were evaluated in nude mice with xenografted THP-1 cells. RPPH1 was expressed in the AML tissues and cell lines and its high expression predicted worse overall survival in AML patients. miR-330-5p was validated to be a direct target of RPPH1. Knockdown of RPPH1 suppressed the proliferation, invasion and migration ability of human AML cells, which was partially reversed by additional administration with miR-330-5p inhibitor. RPPH1 knockdown significantly inhibited the growth of xenografted THP-1 tumor in nude mice. Our work highlights the contributions of RPPH1 in promoting AML progression through targeting miR-330-5p, and suggests that the RPPH1/miR-330-5p axis is a potential target for AML treatments.
Metformin restores the mitochondrial membrane potentials in association with a reduction in TIMM23 and NDUFS3 in MPP+-induced neurotoxicity in SH-SY5Y cells
(2019-09-10) Chanthammachat, Pitak; Dharmasaroja, Permphan
SH - SY5Y cells exposed to l - methyl - 4 - phenylpyridinium (MPP + ) develop mitochondrial dys- function and other cellular responses similar to th ose that occur in the dopaminergic neurons of patients with Parkinson ’ s disease (PD) . It has been shown in animal models of PD that neuronal death can be prevented by metformin, an anti - diabetic drug . Both MPP + and metformin inhibit complex I of the mitochondrial resp iratory chain. It has been reported that decreased levels of the mitochondrial inner membrane proteins TI MM23 and NDUFS3 are associated with the in- creased generation of reactive oxygen species and mitochondrial de polarization. In the present study, we investigated the e ffects of metformin on MPP + - induced neurotoxicity using differen- tiated human SH - SY5Y neuroblastoma cells . The results showed that pretreatment with met- formin increased the viability of MPP + - treated SH - SY5Y cells. Pretreatment with metformin decreased the expr ession of TIMM23 and NDUFS3 in MPP + - treated SH - SY5Y cells. This was correlated with reduced mitochondrial fragmenta tion and an improvement in the mitochondrial membrane potential . These results suggest that metformin pretreatment protects against MPP + - induced neurotoxicity, and offer insights into th e potential role of me tformin in protecting against toxin - induced parkinsonism
Implications of the presence of yeasts in tracheobronchial secretions of critically ill intubated patients
(2019-09-09) Ferreira, Elenice Gomes; Yatsuda, Fabrício; Pini, Marcio; Jarros, Isabele Carrilho; Veiga, Flávia Franco; de Oliveira, Admilton Gonçalves; Negri, Melyssa; Svidzinski, Terezinha Inez Estivalet
The presence of some microorganisms in the respiratory tract is a known risk factor for the infection of air passages; however, it is not clear whether this holds true for Candida spp. Thus, our objective was to determine the frequency of yeast colonization in the tracheobronchial secretions of critically ill intubated patients and to assess the presence of these yeasts in the infra-cuff region of the endotracheal tube (ET). Patients aged 18 years or older who had been using an endotracheal tube for 48 hours were recruited. Tracheal secretions were collected; after extubation, the ETs were cut into two fragments in the infra-cuff region. One of these fragments was placed in a solution containing antibiotics and sent to the lab for cultu re and identification of yeasts. The remaining fragment was fixed and subjected to scanning electron microscopy (SEM). In total, 20 patients with an average age of 73.3 years (± 13.1) participated in this study. These patients remained under endotracheal intubation and invasive mechanical ventilation for an average of 6. 4 (± 1.8) and 13.5 days (± 15), respectively. Of these patients, 45 % showed respiratory tract colonization by yeasts of the Candida genus, with C. albicans being the most frequently isolated species (66.7 %). Moreover, in almost 90 % of these patients, blastoconidia of the same yeast were found in the infra-cuff portion of the ET, as evidenced by SEM, strongly fixed on the ET surface. Yeasts isolated from both the infra-cuff region and the tracheobronchial secretions were susceptible to amphotericin B and fluconazole. In conclusion, our results show that the frequency of colonization by yeasts of the Candida genus in the tracheobronchial secretions of intubated patients within 48 hours is high, and that these species can also be found as a biofilm on the ET surface.
The mTOR kinase inhibitor rapamycin enhances the expression and release of pro-inflammatory cytokine interleukin 6 modulating the activation of human microglial cells
(2019-09-06) Cappoli, Natalia; Mezzogori, Daniele; Tabolacci, Elisabetta; Coletta, Isabella; Navarra, Pierluigi; Pani, Giovambattista; Dello Russo, Cinzia
Emerging evidence suggests the potential use of rapamycin in treatment of several neurological disorders. The drug readily crosses the blood brain barrier and may exert direct immunomodulatory effects within the brain. Microglia are the main innate immune cells of the brain, thus critically involved in the initiation and development of inflammatory processes at this level. However, there are conflicting data from rodent studies about the pharmacological effects of rapamycin on microglial inflammatory responses. Considering that rodent microglia display relevant biochemical and harmacological differences compared to human microglia, in the present study we studied the effects of rapamycin in an experimental model of human microglia, the human microglial clone 3 (HMC3) cell line. Rapamycin was tested in the nM range both under basal conditions and in cells activated with a pro-nflammatory cytokine cocktail, consisting in a mixture of interferon-γ and interleukin-1β (II). The drug significantly increased II stimulatory effect on interleukin-6 (IL-6) expression and release in the HMC3 cells, while reducing the production of free oxygen radicals (ROS) both under basal conditions and in cells activated with II. Consistently with its known molecular mechanism of action, rapamycin reduced the extent of activation of the so-called ‘mechanistic’ target of rapamycin complex 1 (mTORC1) kinase and the total amount of intracellular proteins. In contrast to rodent cells, rapamycin did not alter human microglial cell viability nor inhibited cell proliferation. Moreover, rapamycin did not exert any significant effect on the morphology of the HMC3 cells. All together these data suggest that the inhibition of mTORC1 in human microglia by rapamycin results in complex immunomodulatory effects, including a significant increase in the expression and release of the pro-inflammatory IL-6.
Senescent HUVECs-secreted exosomes trigger endothelial barrier dysfunction in young endothelial cells
(2019-09-03) Wong, Pooi-Fong; Tong, King-Leng; Jamal, Juliana; Khor, Eng-Soon; Lai, Siew-Li; Mustafa, Mohd Rais
Accumulation of senescent endothelial cells can cause endothelium dysfunction which eventually leads to age-related vascular disorders. The senescent-associated secretory phenotype (SASP) cells secrete a plethora of soluble factors that negatively influence the surrounding tissue microenvironment. The present study sought to investigate the effects of exosomes, which are nano-sized extracellular vesicl es known for intercellular communications secreted by SASP cells on young endothelial cells. Exosomes were isolated from the condition media of senescent human umbilical vein endothelial cells (HUVECs) and then confirmed by the detection of exosome specific CD63 and CD9 expressions, electron microscopy and acetylcholinesterase assay. The purified exosomes were used to treat young HUVECs. Exposure to exosomes repressed the expression of adherens junction proteins including vascular endothelial (VE)-cadherin and beta-catenin, decreased cell growth kinetics and impaired endothelial migration potential of young endothelial cells. These findings suggest that senescent HUVECs-secreted exosomes could disrupt barrier integrity that underpins endothelial barrier dysfunction in healthy young endothelial cells.
Up-regulation of miR-381 inhibits NAD+ salvage pathway and promotes apoptosis in breast cancer cells
(2019-08-27) Bolandghamat Pour, Zahra; Nourbakhsh, Mitra; Mousavidzadeh, Kazem; Madjd, Zahra; Ghorbanhosseini, Seyedeh Sara; Abdolvahabi, Zohreh; Hesari, Zahra; Mobaser, Samira Ezzati
Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme involved in nicotinamide adenine di- nucleotide (NAD) salvage pathway, is overexpressed in many human malignancies such as breast cancer. This enzyme plays a critical role in survival and growth of cancer cells. MicroRNAs (miRNAs) are among the most important regulators of gene expression, and serve as potential targets for diagnosis, prognosis, and therapy of breast cancer. Therefore, the aim of this study was to asse ss the effect of NAMPT inhibition by miR-381 on breast cancer cell survival. MCF-7 and MDA-MB-2 31 cancer cell lines were transfected with miR-381 mimic, inhibitor, and their corresponding negative controls (NCs). Subsequently, the level of NAMPT and NAD was assessed using real-time PCR, immuno-blotting, and enzymatic methods, resp ectively. In order to evalua te apoptosis, cells were labelled with Annexin V-FITC and propidium iodide and analyzed by flow cytometry. Bioinformatics analysis was performed to recognize whether NAMPT 3 ′ -untranslated region (UTR) is a direct target of miR-381 and the results were authenticated by the luciferase re porter assay using a vector containing the 3 ′ -UTR sequence of NAMPT. Our results revealed that the 3 ′ -UTR of NAMPT was a direct target of miR-381 and its up-regulation decreased NAMPT gene and protein expression, leading to a notable reduction in intracellular NAD and subse- quently cell survival and induction of apoptosis. It can be concluded that miR-381 has a vital role in tumor sup- pression by down-regulation of NAMPT, and it can be a promising candidate for breast cancer therapy.

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