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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 361 records · Page 20
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Downregulation of cytochrome c oxidase subunit 7A1 expression is important in enhancing cell proliferation in adenocarcinoma cells

Mitochondrial Dysfunction has been implicated in multiple human diseases, including cancer. Among all cancer, lung cancer is the most common type of cancer worldwide with low survival rates. Mammals possess multiple subunits of the mitochondrial enzyme Cytochrome C oxidase (COX). The COX subunits are expressed in a tissue specific manner and have been implicated in cancer cell metabolism although their molecular and regulatory mechanisms are not clearly understood. In this study, we aimed at identifying novel gene signatures in lung cancer. We performed extensive analysis of seven different Gene Expression Omnibus (GEO) datasets pertaining to different stages of lung adenocarcinoma and identified that multiple subunits of COX genes are differentially expressed in these patients. Amongst all COX genes, the expression of COX7A1 gene was observed to be highly down regulated in these patients. In order to validate the GEO datasets, we looked at the expression of multiple COX genes using quantitative real time PCR (qPCR) using human lung adenocarcinoma cell line A549. Our results confirmed that COX 7A1 gene expression was indeed highly reduced in these cells. Overexpression of COX7A1 in human lung cancer cells led to inhibition of cell proliferation and increase in cell death via apoptosis. These results indicated that low level of COX7A1 gene expression is essential to regulate cell viability and inhibit cell death in lung adenocarcinoma. Our study has identified COX7A1 as a novel gene that might play a crucial role in the etiology of lung adenocarcinoma and can serve as a biomarker for lung cancer disease progression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

CNPY2 promoted the proliferation of renal cell carcinoma cells and increased the expression of TP53

Renal cell carcinoma (RCC) is the most common type of kidney cancer. However, the mechanisms underlying the progression of the disease are not well understood. The data in this report suggest that canopy FGF signaling regulator 2 (CNPY2) is a promoter of RCC progression. We found that CNPY2 significantly promoted growth of RCC cells and upregulated TP53 gene expression. Although TP53 is widely known as a tumor suppressor, in RCC TP53 promoted tumor cell growth. A typical p53 target gene, CDKN1A, was upregulated by both p53 and CNPY2 in RCC cells, suggesting that CNPY2 increased the expression level of TP53. Consistent with these results, CNPY2 and TP53 expression levels were positively correlated in RCC patients. These findings suggested that CNPY2 promoted cancer cell growth in RCC through regulating TP53 gene expression. - Highlights: • CNPY2 promoted growth of renal cell carcinoma (RCC) cells. • TP53 expression levels were increased by CNPY2 in RCC cells. • Growth of RCC cells was promoted by TP53. • CNPY2 expression positively correlated with TP53 expression in RCC patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Increased expression of microRNA-31-5p inhibits cell proliferation, migration, and invasion via regulating Sp1 transcription factor in HepG2 hepatocellular carcinoma cell line

Accumulating evidence has suggested that microRNA-31-5p (miR-31-5p) is dysfunctional in hepatocellular carcinoma (HCC). However, the molecular mechanism of HCC remains unclear. In this study, we investigated the role of miR-31-5p in tumor formation and development of HCC. The expression of miR-31-5p was detected in HCC tissues, corresponding adjacent tissues, normal liver tissues, and HCC cell lines. miR-31-5p mimics and an inhibitor were transfected into HepG2 cells to assess the effects of miR-31-5p on cell proliferation, apoptosis, cell cycle, migration, and invasion assays. Western blotting was used to detect the expression of Sp1 transcription factor (SP1), cyclin D1, and survivin in transfected HCC cells and control cells. The expression of miR-31-5p was significantly decreased in HCC cells and HCC tissues. Overexpression of miR-31-5p inhibited HCC cell growth, migration, and invasion. Overexpression of miR-31-5p reduced the expression of SP1 and cyclin D1, and knockdown of SP1 decreased cyclin D1 expression. The dual luciferase assay showed that miR-31-5p directly targeted SP1 in HepG2. Together, the results suggested that miR-31-5p acted as a tumor suppressor to regulate SP1, and that miR-31-5p could be used as a therapeutic target for the treatment of HCC. - Highlights: • The expression of miR-31-5p was significantly up-regulated in HCC tissues. • Increased expression of miR-31-5p inhibits cell proliferation, migration, and invasion. • Sp1 transcription factor is the target gene for miR-31-5p.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Dual expression of constitutively active Gαs-protein-coupled receptors differentially establishes the resting activity of the cAMP-gated HCN2 channel in a single compartment

Highlights: • Constitutively active GPCRs were dually expressed in a single compartment. • HCN2 channels were differentially activated depending on the GPCR pairs. • Basal actions of GPCRs were abolished by an adenylate cyclase inhibitor. • Basal cAMP pools were specifically established by constitutively active GPCR pairs. The hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channel is a major subtype of the HCN channel family expressed in the nervous system that sets the membrane potential, regulates cell excitability and senses changes in the extracellular environment. Neurons express various Gα{sub s}-protein-coupled receptors (GPCRs), many of which show ligand-independent constitutive activity. These membrane-bound proteins are expressed in various subcellular compartments of neurons. Therefore, some proportion of HCN2 channels opens in response to the basal cAMP pool size produced by constitutively active GPCRs. Here, we employed an exogenous HEK293 expression system and voltage-clamp patch-clamp recordings to investigate basal HCN2 channel activity in the presence of two GPCRs with diverse basal activities in a single compartment. We utilized the β2-adrenoceptor (β2AR) together with odorant receptors (ORs), as both GPCR families are known to show strong basal activity. Consequently, β2AR alone strongly enhanced the activity of HCN2 channels, and co-expression of ORs further diversified the HCN2 channel activity, which was totally abolished by an adenylate cyclase inhibitor. Thus, we conclude that the dual expression of constitutively active GPCRs establishes the diverse range of the basal cAMP pool size in resting cells through mutual additive or suppressive interactions, even in the absence of external stimulation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Differential mGluR5 expression in response to the same stress causes individually adapted hippocampal network activity

Highlights: • Individuals show different changes in mGluR5 expression in response to the same stress. • Repetitive restraint stress causes theta EEG activity in the hippocampus. • Hippocampal mGluR5 expression determines the theta EEG response to repetitive restraint stress. Individual differences in stress vulnerability and resilience have been observed even within a single cohort of inbred rats or mice. Stress phenotypes are typically quantified as changes in the behavior of experimental animals, which is the outcome of altered electrical activity of the brain network. Although mGluR5 is associated with individual vulnerability to stress and can act as a sensitive biomarker of stress adaptation, our understanding of mGluR5-dependent modifications to neural network activities in vivo remains limited. Here, we examined individual rats for changes in hippocampal mGluR5 expression induced by restraint stress and found that these changes cause accompanying changes in hippocampal electroencephalography (EEG) activity. We found six days of restraint stress caused variable changes in hippocampal mGluR5 expression, ranging from 20.9% to 210.7% of the control group. The low mGluR5 protein group (LE) showed increased methylation of the mGluR5 CpG island, reduced mGluR5 mRNA levels, and unaltered basal EEG theta spectral power between stress day 1 and 6. In contrast, the high mGluR5 protein group (HE) showed reduced methylation of CpG sites, increased mGluR5 mRNA expression, and reduced basal theta spectral power on stress day 6. We also found that injection of lentiviruses expressing mGluR5-specific shRNAs into the hippocampus rescued this reduction in baseline theta power in HE rats. These data suggest a causal relationship between individual differences in the changes in hippocampal mGluR5 expression induced by repetitive restraint stress and the accompanying changes in ensemble neural activity in the hippocampus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Aberrant alternative splicing of RHOA is associated with loss of its expression and activity in diffuse-type gastric carcinoma cells

RhoA is a member of Rho family small GTPases that regulates diverse cellular functions. Recent large-scale sequencing studies have identified recurrent somatic mutations of RHOA in diffuse-type gastric carcinoma (DGC), indicating that RHOA is a driver of DGC. In this study, we investigated the possible abnormalities of RHOA in a panel of gastric carcinoma (GC) cell lines. Pulldown assay and immunoblot analysis showed that the activity and expression of RhoA were detectable in all GC cell lines tested, except for two DGC cell lines, HSC-59 and GSU. RHOA coding region sequencing revealed that aberrant alternative splicing of RHOA occurred in these cell lines. Quantitative real-time PCR analysis showed that the expression of wild-type RHOA was nearly undetectable, whereas splicing variants were almost exclusively expressed in HSC-59 and GSU cell lines. However, the expression levels of RHOA splicing variants were very low and the corresponding proteins were not detected by immunoblotting. Moreover, the splicing isoforms of RhoA protein were neither efficiently expressed nor activated even if ectopically expressed in cells. These results indicate that aberrant alternative splicing of RHOA results in the loss of its activity and expression in DGC cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Kinetic profiles of photocurrents in cells expressing two types of channelrhodopsin genes

Highlights: • Expression of an additional channelrhodopsin gene compensates ChR2 function. • Two channelrhodopsins can coexist in a cell without affecting their function. • Sensitivity to low light intensity is enhanced by expressing two channelrhodopsins. • Turning-ON and turning-OFF constants are prolonged in dual gene expression. Channelrhodopsin-2 (ChR2), a light-activated cation-selective ion channel, has been widely used as a tool in optogenetic research. ChR2 is specifically sensitive to wavelengths less than 550 nm. One of the methods to expand the sensitivity of a channelrhodopsin to a wider range of wavelengths is to express another channelrhodopsin in the cells by the transduction of an additional gene. Here, we report the characteristic features of cells expressing two types of channelrhodopsins, each having different wavelength sensitivities. In HEK293 cells stably expressing ChR2, photocurrents were elicited at stimuli of 400–550 nm, and the wavelength sensitivity range was expanded by the additional transduction of the modified Volvox channelrhodopsin-1 (mVChR1) gene, which has broad wavelength sensitivities, ranging from 400 to 600 nm. However, the photocurrent at 550 nm was lower than that of the mVChR1-expressing cell; moreover, the turning-on and turning-off constants were delayed, and the deactivation rates were decreased. Meanwhile, the response to lower light intensity was improved by the additional gene. Thus, the transduction of an additional gene is a useful method to improve the light and wavelength sensitivities, as well as photocurrent kinetic profiles, of channelrhodopsins.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The transcriptional factor Apt regulates neuroblast differentiation through activating CycE expression

Highlights: • Apt exclusively expresses in NB6-4T cells. • Apt controls NB6-4T differentiation. • Apt activates the expression of Cyc E in neuronal cells. • Gcm represses the expression of CycE through Apt in glial cells. In Drosophila, the thoracic neuroblast 6-4 (NB6-4T) divides asymmetrically into a glial precursor and a neuronal precursor, while the abdominal neuroblast 6-4 (NB6-4A) divides symmetrically to produce two glial cells. The underlying mechanism by which NB6-4T and NB6-4A undergo distinct differentiation is still elusive. Here, we find that the transcription factor Apontic (Apt) exclusively expresses in NB6-4T cells and is involved in regulating NB6-4T differentiation. Loss of Apt results in neuronal precursor loss. Epistasis analysis shows that Apt controls NB6-4T differentiation through activating CycE expression. On the other hand, Gcm suppresses Apt expression in the NB6-4A cell, thus inhibiting CycE expression. Taken together, our findings reveal a Gcm-Apt-CycE axis that regulates neuroblast and glia cell differentiation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MicroRNA-155 inversely correlates with esophageal cancer progression through regulating tumor-associated macrophage FGF2 expression

Highlights: • miR-155 and FGF2 is associated with esophageal cancer progression. • miR-155 in TAMs suppressed ECA109 cell proliferation, migration and invasion, as well as reduction in angiogenesis. • miR-155-reduced cell growth, migration and invasion of ECA109 cells is associated with FGF2 suppression. Esophageal cancer (EC) is one of the most common malignancies with high incidence and mortality. Tumor-associated macrophages (TAMs) in the tumor microenvironment have been linked to the accelerated tumor progression. MicroRNAs (miR) are 19–25 nucleotide-long, noncoding RNA molecules, functioning as modulators of gene expression, and mediate a variety of biological functions, including tumor growth. In the present study, the effects and molecular mechanism of miR-155 in TAMs isolated from EC were explored. The expression of miR-155 and fibroblast growth factor-2 (FGF2) in EC tissues and cell lines were analyzed using reverse transcription-quantitative PCR (qRT-PCR) and western blot assays. TAMs were also transfected with the described constructs. Following, the culture medium from TAMs was collected for further analysis. The released FGF2, and inflammatory cytokines were quantified using ELISA. The cell viability, migrated and invaded levels were calculated through Cell Counting kit-8 (CCK8), and transwell analysis. Moreover, human umbilical vein endothelial cells (HUVEC) vasculature formation was determined using matrigel angiogenesis analysis. The results indicated that miR-155 expression was decreased in EC tissues and cell lines, while FGF2 expression was increased in comparison to those in the normal control group. Moreover, miR-155 mimics transfection up-regulated tumor necrosis factor α (TNF-α), interleukin (IL)-12 and inducible nitric oxide synthase (iNOS), while down-regulated IL-10, Arginase-1 (Arg-1) and IL-22 levels in the culture medium from TAMs. And enhancing miR-155 expression in TAMs suppressed the cell viability, migration and invasion of ECA109 cells and reduced the angiogenesis. Nevertheless, over-expressing FGF2 abolished the role of miR-155 in cancer cell survival, migration, invasion as well as angiogenesis. Our findings indicated that miR-155-regulated FGF2 expression from TAMs suppressed EC cell proliferation, migration, invasion and inhibited vasculature formation. Thus, miR-155-modulated FGF2 might be a potential therapeutic target to prevent EC progression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MicroRNA-139-5p suppresses myosin heavy chain I and IIa expression via inhibition of the calcineurin/NFAT signaling pathway

Highlights: • MiR-139-5p was higher in mouse slow-twitch than in fast-twitch muscle. • MiR-139-5p overexpression downregulated the expression of MyHC I and MyHC IIa. • CaN/NFAT signaling was notably inhibited by miR-139-5p overexpression. • MiR-139-5p suppressed MyHC I and IIa expression via inhibiting CaN/NFAT signaling. MicroRNAs (miRNAs) are a class of small non-coding RNAs that are widely involved in a variety of biological processes. Different skeletal muscle fiber type composition exhibits characteristic differences in functional properties and energy metabolism of skeletal muscle. However, the molecular mechanism by which miRNAs control the different type of muscle fiber formation is still not fully understood. In the present study, we characterized the role of microRNA-139-5p (miR-139-5p) in the regulation of myosin heavy chain (MyHC) isoform expression and its underlying mechanisms. Here we found that the expression of miR-139-5p was significantly higher in mouse slow-twitch muscle than in fast-twitch muscle. Overexpression of miR-139-5p downregulated the expression of MyHC I and MyHC IIa, whereas inhibition of miR-139-5p upregulated them. We also found that the levels of calcineurin (CaN), NFATc1, MEF2C and MCIP1.4, which are the components of CaN/NFAT signaling pathway that has shown to positively regulate slow fiber-selective gene expression, were notably inhibited by miR-139-5p overexpression. Furthermore, treatment of phenylephrine (PE), a α1-adrenoceptor agonist, abolished the inhibitory effect of miR-139-5p on MyHC I and MyHC IIa expression. Together, our findings indicated that the role of miR-139-5p in regulating the MyHC isoforms, especially MyHC I and MyHC IIa, may be achieved through inhibiting CaN/NFAT signaling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Over-expression of lysyl oxidase is associated with poor prognosis and response to therapy of patients with lower grade gliomas

Lower grade gliomas (LGGs) have highly diverse clinical phenotypes. The histological grade and type are insufficient to accurately predict the clinical outcomes of patients with LGGs. Therefore, identification of biomarkers that can facilitate the prediction of clinical outcomes in LGGs is urgently needed. Gene expression of LOX has been identified as a biomarker for various cancers. However, the clinical significance of LOX expression in LGGs has not been investigated. In this study, we analyzed the glioma RNA-seq dataset from TCGA (The Cancer Genome atlas) and identified lysyl oxidase (LOX) as a potential biomarker for LGGs. Kaplan-Meier survival analysis revealed that high LOX expression is associated with worse overall survival and recurrence free survival in LGG patients. Besides, high LOX expression is associated with poor response to primary therapy, follow-up treatment, targeted molecular therapy, and radiation therapy. Univariate and multivariate Cox regression analyses further confirmed LOX expression as an independent prognostic factor for LGG patients. Finally, we observed that LOX expression is significantly correlated with EMT (epithelial to mesenchymal transition) and IDH1 status in LGGs. In conclusion, our analyses suggest that LOX expression is a potential biomarker for prognosis and therapeutic response in LGGs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Leptin regulates the expression of angiopoietin-like 6

Highlights: • High-fat diet results in increased hepatic ANGPTL6 expression. • Leptin treatment upregulates ANGPTL6 expression in hepatocytes. • Exercise training decreases serum ANGPTL6 as well as leptin. Angiopoietin-like 6 (ANGPTL6) is a hepatokine that antagonizes obesity and insulin resistance by increasing energy expenditure. Despite its beneficial effects on metabolism, human studies have shown a paradoxical increase in ANGPTL6 level in the serum of patients with metabolic diseases, which has been interpreted as a compensatory upregulation. However, the regulatory mechanism of ANGPTL6 remains unclear. Since upregulation of ANGPTL6 is induced on metabolic stress, we investigated the hepatic expression of ANGPTL6 by leptin, a representative adipokine of obesity. Mice on a high-fat diet showed increased serum leptin levels and hepatic Angptl6 expression, which were attenuated by exercise training. A single leptin injection also induced hepatic ANGPTL6 expression and increased serum ANGPTL6 levels. In an in vitro model using primary hepatocytes, leptin treatment significantly upregulated ANGPTL6 expression at the mRNA and protein levels, as well as the amount of secreted ANGPTL6 protein in conditioned media. Similarly, exercise training on human participants also showed diminished serum levels of leptin and ANGPTL6. Altogether, these results strongly indicated that hepatic ANGPTL6 expression was determined by leptin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Octanoic acid potentiates glucose-stimulated insulin secretion and expression of glucokinase through the olfactory receptor in pancreatic β-cells

Highlights: • Olfactory receptor Olfr15 is expressed on the plasma membranes of mouse pancreatic β-cells. • Octanoic acid potentiates glucose-stimulated insulin secretion via Olfr15. • Long-term octanoic acid treatment increases the expression of glucokinase via Olfr15. • Olfr15 expression is decreased and octanoic acid-induced insulin secretion is impaired in islets of diabetic model mice. Olfactory receptors (ORs) are G protein-coupled receptors that mediate olfactory chemosensation, leading to the perception of smell. ORs are expressed in many tissues, but their functions are largely unknown. Here, we show that the olfactory receptor Olfr15 is highly and selectively expressed in both mouse pancreatic β-cells and MIN6 cells. In addition, octanoic acid (OA), a medium-chain fatty acid, potentiates glucose-stimulated insulin secretion (GSIS). The OA-induced enhancement of GSIS was inhibited by Olfr15 knockdown. Treatment with a PLC inhibitor or an Ins(1,4,5)P{sub 3} receptor (IP{sub 3}R) antagonist also blocked the OA-induced enhancement of GSIS. These results suggest that OA potentiates GSIS via Olfr15 though the PLC-IP{sub 3} pathway. Furthermore, long-term treatment with OA increased cellular glucose uptake in MIN6 cells by up-regulating the expression of glucokinase (GK). Moreover, this process was blocked by an IP{sub 3}R antagonist and a Ca{sup 2+}/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor. Similarly, OA stimulated GK promoter activity, while either Olfr15 or CaMKIV knockdown blocked the stimulatory effect of OA on GK promoter activity. These results suggest that long-term treatment of OA induces GK promoter activity via Olfr15 through the IP{sub 3}-CaMKK/CaMKIV pathway. In islets from type 2 diabetic mice, the expression level of Olfr15 and the OA-induced enhancement of GSIS were strongly reduced. Collectively, our results highlight the crucial role of the olfactory receptor Olfr15 in potentiating GSIS in pancreatic β-cells, suggesting that Olfr15 may be an important therapeutic target in type 2 diabetes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Allethrin and prallethrin stimulates MUC5AC expression through oxidative stress in human airway epithelial cells

Highlights: • Allethrin and prallethrin are implicated in modulation of airway mucin secretion. • Allethrin and prallethrin induce MUC5AC expression. • Allethrin and prallethrin induce MUC5AC expression via ROS. Pyrethroids, including allethrin and prallethrin, have been widely used as major components of the common commercial insecticides. The toxicity of allethrin and prallethrin were well established that it interfered with the way that the nerves and brain function. However, limited information was available regarding respiratory effects in humans following inhalation exposure to allethrin and prallethrin. Therefore, we demonstrated effect of allethrin and prallethrin, and the mechanism involved, on the mucin expressions in human airway epithelial cells. In human airway NCI-H292 epithelial cells, the effects of allethrin and prallethrin and its signaling pathway for airway mucin, especially MUC5AC, were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, and enzyme-linked immunosorbent assay (ELISA). The mechanism of allethrin and prallethrin-induced MUC5AC expression in airway epithelial cells was studied in terms of reactive oxygen species (ROS) by flow cytometry analysis. Allethrin and prallethrin significant increased MUC5AC expression in human airway NCI-H292 epithelial cells. We also demonstrated allethrin and prallethrin induced a marked rise of ROS production. In addition, NAC (ROS scavenger) and DPI (NADPH oxidase inhibitor) inhibited allethrin and prallethrin-induced MUC5AC expression. These results are first to describe that allethrin and prallethrin-induced MUC5AC expression through ROS in human airway epithelial cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

High expression of fibroblast growth factor-21 in liposarcoma patients indicated better prognosis and less recurrence

Liposarcoma is the most common soft tissue malignancy. We investigated the relationship between the expression of fibroblast growth factor −21 protein and recurrence in the liposarcoma tissues from 40 patients. The patients were divided into two groups (low/no- and high-expressing) for further survival analysis according to fibroblast growth factor −21 expression in their tumor tissue. Immunohistochemical staining showed that fibroblast growth factor −21 protein was located in the cytoplasm. The fibroblast growth factor −21 protein was significantly less expressed in liposarcoma than in normal tissue (p < 0.05). Fibroblast growth factor −21 protein expression was related to gender, but not age, cell differentiation or tumor size. The patients in the low/no fibroblast growth factor 21 expression group were more likely to relapse and die in a shorter period of time. The patients in the high-expression group had a better prognosis and less recurrence. fibroblast growth factor −21 has the potential to act both as a biomarker for liposarcoma prognosis as well as a marker for the response to therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Glucocorticoid-induced CREB activation and myostatin expression in C2C12 myotubes involves phosphodiesterase-3/4 signaling

Muscle atrophy in metabolic conditions like chronic kidney disease (CKD) and diabetes are associated with glucocorticoid production, dysfunctional insulin/Akt/FoxO3 signaling and increased myostatin expression. We recently found that CREB, a transcription factor proposed to regulate myostatin expression, is highly phosphorylated in some wasting conditions. Based on a novel Akt-PDE3/4 signaling paradigm, we hypothesized that reduced Akt signaling contributes to CREB activation and myostatin expression. C2C12 myotubes were incubated with dexamethasone (Dex), an atrophy-inducing synthetic glucocorticoid. Akt/CREB signaling and myostatin expression were evaluated by immunoblot and qPCR analyses. Inhibitors of Akt, phosphodiesterase (PDE)-3/4, and protein kinase A (PKA) signaling were used to test our hypothesis. Incubating myotubes with Dex for 3–24 h inhibited Akt phosphorylation and enhanced CREB phosphorylation as well as myostatin mRNA and protein. Inhibition of PI3K/Akt signaling with LY294002 similarly increased CREB phosphorylation. Isobutyl-methylxanthine (IBMX, a pan PDE inhibitor), milrinone (PDE3 inhibitor) and rolipram (PDE4 inhibitor) augmented CREB phosphorylation and myostatin expression. Inhibition of protein kinase A by PKI reverted Dex- or IBMX-induced CREB phosphorylation and myostatin expression. Our study provides evidence supporting a newly identified mechanism by which a glucocorticoid-related reduction in Akt signaling contributes to myostatin expression via CREB activation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Dietary omega-3 and -6 fatty acids affect the expression of prostaglandin E2 synthesis enzymes and receptors in mice uteri during the window of pre-implantation

Highlights: • Dietary omega-3 and -6 could affect uterine expression of PGE pathway especially mPGES and EP2 during pre-implantation period. • Uterine expression of mPGES and EP2 on the day of implantation has a positive correlation with implantation rate. • One of the important mechanisms of PUFA in affecting female pregnancy is through mPGES and EP2. Considering possible effects of poly-unsaturated fatty acids (PUFA) on embryo implantation more likely through PGs, we investigated effects of dietary omega-3 and -6 PUFA on prostaglandin E2 (PGE2) signaling in mice uterus during pre-implantation period. The mRNA expressions of microsomal- and cytosolic- PGE synthase (mPGES and cPGES) and protein expressions of PGE receptor 2 and 4 (EP2 and EP4) were evaluated in uterus tissues of control as well as omega 3 and omega 6 received mice at days 1–5 of pregnancy. Expression of cPGES gene was not significantly different between groups but the mPGES expression on days 4 and 5 of pregnancy in supplemented groups was higher than controls. Omega-3 significantly decreased EP2 levels on days 3 and 4, while omega-6 caused an increase on days 3–5 of pregnancy. The levels of EP4 were significantly higher in the omega-6 group than other groups on days 4 and 5 of pregnancy. Also the implantation rate was higher in omega -6 compared to omega-3 group (p = 0.006). Moreover, there were significant correlations between implantation rate with expression levels of mPGES and EP2. Our results showed negative and positive effects of respectively dietary omega-3 and -6 PUFA on PGE2 signaling and implantation rate.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

E3317 promotes cholesterol efflux in macrophage cells via enhancing ABCA1 expression

Highlights: • E3317 was first found as an ABCA1 upregulator in an ABCA1 transcriptional activity assay. • E3317 increased ABCA1 mRNA and protein expression in both L02 and RAW264.7 cells. • E3317 promoted cholesterol efflux and reduced cellular lipid accumulation in RAW264.7 macrophages dependent on ABCA1. • E3317 upregulated ABCA1 expression through activating PPARγ. • Y327 and F363 are the key PPARγ binding epitopes of E3317. Reverse cholesterol transport (RCT) plays an important role in cholesterol and lipid metabolism. Regulating the activities of key transporters and receptors in RCT, such as ATP-binding cassette transporter A1 (ABCA1), helps to prevent atherosclerotic cardiovascular disease. In this study, we used an ABCA1 promoter luciferase reporter assay to screen 20,000 compounds for ABCA1 upregulators. Compound E3317 (N-(6-butylbenzo[d]thiazol-2(3H)-ylidene)-3-(N-(2-cyanoethyl)sulfamoyl)benzamide)) was identified as a positive hit with an EC{sub 50} value of 0.2 μM in ABCA1p-LUC HepG2 cells. Thus, we hypothesized that E3317 might have cholesterol- and lipid metabolism-regulating effects through ABCA1 upregulation. E3317 significantly increased ABCA1 mRNA and protein expression in hepatic L02 cells and RAW264.7 macrophages. E3317 promoted cholesterol efflux to apolipoprotein A-I in RAW264.7 macrophages and significantly decreased lipid accumulation in oxidized low-density lipoprotein-induced murine RAW264.7 macrophages. Further studies using ABCA1 siRNA showed that the promotion of cholesterol efflux and decrease of lipid accumulation by E3317 depended on ABCA1 expression. Mechanistic studies indicated that E3317 regulated ABCA1 expression via activating nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), which plays an important role in the regulation of glucose homeostasis and lipid metabolism. The structure of E3317 was docked in the ligand-binding domain of PPARγ (PBD code: 4EMA) to find the key binding amino acids. Site mutation assays confirmed that Y327 and F363 were the key PPARγ binding epitopes of E3317. Our results revealed that E3317 upregulates ABCA1 expression and thereby promotes cholesterol efflux. E3317 may regulate ABCA1 expression through PPARγ. Our findings provide a new compound, E3317, which may have beneficial cardiovascular effects.

60 APPLIED LIFE SCIENCES↗