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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 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Unbiased compound screening with a reporter gene assay highlights the role of p13 in the cardiac cellular stress response

Highlights: • Unbiased screening with 2320 known compounds identified 12 chemical probes for p13. • > 80% screened agents increased or decreased p13 mRNA and affected cardiac function. • Four cardiotonic agents decreased p13 levels and induced oxidative stress. • In vivo profile of p13 expression shows its importance in heart pathophysiology. • PGC1-alpha, a mitochondrial master regulator, markedly elevated p13 expression. We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Long non-coding RNA LUCAT1 modulates methotrexate resistance in osteosarcoma via miR-200c/ABCB1 axis

Highlights: • LncRNA LUCAT1 and ABCB1 protein are both up-regulated in osteosarcoma methotrexate-resistant cells. • LncRNA LUCAT1 knockdown suppress the expression levels drug resistance related genes, proliferation, invasion and tumor growth of osteosarcoma cells in vitro and vivo. • Bioinformatics predictive tools and luciferase assay reveal the LUCAT1/miR-200c/ABCB1 pathway. • Rescue experiments confirm the combined role of LUCAT1, miR-200c and ABCB1 on osteosarcoma methotrexate resistance. Long non-coding RNAs (lncRNAs) have been verified to participate in the tumorigenesis of multiple cancers. Nevertheless, the deepgoing role molecular mechanisms of lncRNAs on osteosarcoma chemoresistance remain unclear. In present study, we investigate the function of lncRNA LUCAT1 on osteosarcoma methotrexate (MTX) resistant phenotype and discover the potential regulatory mechanism. Results showed that LUCAT1 was up-regulated in MTX-resistant cells (MG63/MTX, HOS/MTX) compared to that in parental cells. LncRNA LUCAT1 and ABCB1 protein expression levels were both up-regulated when induced by different concentration of methotrexate. In vitro and vivo, LUCAT1 knockdown decreased the expression levels drug resistance related genes (MDR1, MRP5, LRP1), proliferation, invasion and tumor growth of osteosarcoma cells. Bioinformatics tools and luciferase assay reveled that miR-200c both targeted the 3′-UTR of LUCAT1 and ABCB1 mRNA, suggesting the modulation of LUCAT1 on ABCB1 through sponging miR-200c. Rescue experiments confirmed the combined role of LUCAT1, miR-200c and ABCB1 on osteosarcoma proliferation, invasion and methotrexate resistance. Overall, results indicate the vital role of LUCAT1 in the methotrexate resistance regulation through miR-200c/ABCB1 pathway, providing a novel insight and treatment strategy for osteosarcoma drug resistance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Inhibition of CIP2A attenuates tumor progression by inducing cell cycle arrest and promoting cellular senescence in hepatocellular carcinoma

Highlights: • High expression level of CIP2A was frequently observed in hepatocellular carcinoma (HCC). • CIP2A expression level was associated with unfavorable overall and recurrence-free survival in HCC patients. • Inhibition of CIP2A attenuated HCC progression via inducing cell cycle arrest and promoting cellular senescence. CIP2A is a recent identified oncogene that inhibits protein phosphatase 2A (PP2A) and stabilizes c-Myc in cancer cells. To investigate the potential oncogenic role and prognostic value of CIP2A, we comprehensively analyzed the CIP2A expression levels in pan-cancer and observed high expression level of CIP2A in majority cancer types, including hepatocellular carcinoma (HCC). Based on a validation cohort including 60 HCC and 20 non-tumorous tissue samples, we further confirmed the high mRNA and protein expression levels of CIP2A in HCC, and found high CIP2A mRNA expression level was associated with unfavorable overall and recurrence-free survival in patients with HCC. Mechanistic investigations revealed that inhibition of CIP2A significantly attenuated cellular proliferation in vitro and tumourigenicity in vivo. Bioinformatic analysis suggested that CIP2A might be involved in regulating cell cycle. Our experimental data further confirmed CIP2A knockdown induced cell cycle arrest at G1 phase. We found accumulated cellular senescence in HCC cells with CIP2A knockdown, companying expression changes of senescence associated proteins (p21, CDK2, CDK4, cyclin D1, MCM7 and FoxM1). Mechanistically, CIP2A knockdown repressed FoxM1 expression and induced FoxM1 dephosphorylation. Moreover, inhibition of PP2A by phosphatase inhibitor rescued the repression of FoxM1. Taken together, our results showed that CIP2A was highly expressed in HCC. Inhibition of CIP2A induced cell cycle arrest and promoted cellular senescence via repressing FoxM1 transcriptional activity, suggesting a potential anti-cancer target for patients with HCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Rbfox family proteins make the homo- and hetero-oligomeric complexes

Rbfox family of proteins that consists of Rbfox1, Rbfox2, and Rbfox3 in mammals regulates alternative pre-mRNA splicing in various tissues via direct binding to their RNA binding element. Although many studies have indicated the splicing activity of each member of the Rbfox family, the interactions of Rbfox family proteins are largely unknown. Here, we have investigated interactions among Rbfox family proteins. Co-immunoprecipitation (Co-IP) and GST-pull down assays confirmed that Rbfox proteins form homo and hetero complexes. Moreover, in vivo crosslinking using disuccinimidyl suberate treatment indicated that the Rbfox proteins form a dimer which then assembles with other proteins to form a large multiprotein complex. Duolink in situ proximity ligation (PLA) assay revealed that neuron specific Rbfox3 protein interacts with other Rbfox family proteins. This study is the first to provide an evidence that Rbfox family proteins form homo- and hetero-oligomeric complexes in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Activation of AMP-activated protein kinase by compound 991 protects osteoblasts from dexamethasone

Highlights: • Compound 991 (C991) activates AMPK signaling in osteoblast cells. • C991 attenuates dexamethasone (Dex)-induced osteoblast cell death. • AMPK activation is required for C991-mediated osteoblast protection against Dex. • C991 inhibits Dex-induced oxidative stress in osteoblasts. • Genetic AMPK inhibition reverses C991's anti-oxidant actions in osteoblasts. Dexamethasone (Dex) induces direct cytotoxicity to cultured osteoblasts. The benzimidazole derivative compound 991 (“C991”) is a novel and highly-efficient AMP-activated protein kinase (AMPK) activator. Here, in both MC3T3-E1 osteoblastic cells and primary murine osteoblasts, treatment with C991 activated AMPK signaling, and significantly attenuated Dex-induced apoptotic and non-apoptotic cell death. AMPKα1 knockdown (by shRNA), complete knockout (by CRISPR/Cas9 method) or dominant negative mutation (T172A) not only blocked C991-mediated AMPK activation, but also abolished its pro-survival effect against Dex in osteoblasts. Further studies showed that C991 boosted nicotinamide adenine dinucleotide phosphate (NADPH) activity and induced mRNA expression of NF-E2-related factor 2 (Nrf2)-regulated genes (heme oxygenase-1 and NADPH quinone oxidoreductase 1). Additionally, C991 alleviated Dex-induced reactive oxygen species (ROS) production in osteoblasts. Notably, genetic AMPK inhibition reversed the anti-oxidant actions by C991 in Dex-treated osteoblasts. Together, we conclude that C991 activates AMPK signaling to protect osteoblasts from Dex.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Tyrosine kinase Fyn regulates iNOS expression in LPS-stimulated astrocytes via modulation of ERK phosphorylation

Highlights: • Fyn regulates LPS-induced iNOS expression in the cortices. • Fyn modulates iNOS expression and nitrite levels in LPS-stimulated astrocytes. • Fyn regulates iNOS expression in LPS-stimulated astrocytes via ERK phosphorylation. The high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in activated glial cells in response to neuroinflammatory stimuli have neurotoxic effects on the brain. At basal levels, iNOS expression is low, and proinflammatory stimuli induce iNOS expression in astrocytes, microglia, and oligodendrocytes. Fyn, a non-receptor tyrosine kinase, regulates iNOS expression in several types of immune cells. However, its role in stimulated astrocytes is less clear. In this study, we investigated the role of Fyn in the regulation of lipopolysaccharide (LPS)-induced iNOS expression in astrocytes from mice and rats. Intracerebroventricular LPS injections in cortical regions enhanced iNOS mRNA and protein levels, which were increased in Fyn-deficient mice. Accordingly, LPS-induced nitrite production was enhanced in primary astrocytes cultured from Fyn-deficient mice or rats. Similar results were observed in cultured astrocytes after the siRNA-induced knockdown of Fyn expression. Finally, we observed increased LPS-induced extracellular signal-regulated protein kinase (ERK) activation in Fyn-deficient astrocytes. These results suggested that Fyn has a regulatory role in iNOS expression in astrocytes during neuroinflammatory responses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Identification and characterization of human bone-derived cells

Highlights: • In non-osteogenic condition, BdCs are more correlated to MSCs than FOBs in bioinformatics result. • BdCs sustained not only high ALP, OPN and osterix expression, but differentiated to mature osteoblast. • In osteogenic condition, BdCs are similar with FOBs than MSCs in physiological changes results. • We suggest BdCs are osteoprogenitor cells or undifferentiated preosteoblasts. This study was designed to identify and characterize primary bone-derived cells (BdCs) and investigate the potential role of osteoblast differentiation. Primary BdCs were isolated from surgical bone for comparative analysis with mesenchymal stem cells (MSCs) and fetal osteoblasts (FOBs) and for potential differentiation to mature osteoblasts. Using three different cells, we successfully cultivated human osteoblast differentiation and activity which were evaluated using microarray and biochemical methods. BdCs are more correlated to MSCs in bioinformatics result and similar with FOBs in gene expression. In particular, Osterix, osteoprogenitor marker, was high expressed in BdCs, while the expression in MSCs and FOBs were very low. Furthermore, BdCs exhibited a marked alkaline phosphatase (ALP) expression, early stage of osteogenic marker, and retained osteogenic properties and physiological changes into maturation as in FOBs. BdCs also showed an increase in bone morphogenic protein 2 (BMP2), osteopontin (OPN), and osteocalcin (OCN) mRNA expressions during differentiation. This study suggests that BdCs may be osteoprogenitor cells or undifferentiated preosteoblasts with strong capacity to differentiate toward mature osteoblasts.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Effect of erythropoietin production induced by hypoxia on autophagy in HepG2 cells

Highlights: • Erythropoietin production by hypoxia restrained autophagy induction. • Autophagy induction by hypoxia did not affect expression of erythropoietin mRNA. • Increase of phosphorylated STAT3 by erythropoietin restrained autophagy induction. • Restraint of autophagy induction by erythropoietin did not affect cell viability. The induction of hypoxia inducible factor (HIF) and autophagy are important cellular responses to hypoxia. The production of erythropoietin (EPO) regulated by HIF is increased by hypoxia and participates in cell protection in various organs and tissues. Signal Transducers and Activator of Transcription 3 (STAT3) is a regulatory factor that is common to autophagy induction and EPO-EPO receptor signaling. In this study, we analyzed the promotion of EPO production and autophagy, and the participation of STAT3, in HepG2 cells under hypoxia. Treatment with EPO siRNA (si-EPO) significantly increased autophagy induction by hypoxia, while treatment with recombinant EPO inhibited the effect of si-EPO. NSC74859, an inhibitor of the phosphorylation of STAT3, increased autophagy induction to the same extent as si-EPO treatment. Even when 3-Methyladenine, an inhibitor of autophagy, was added, the increase of EPO mRNA expression due to hypoxia was not affected. Hypoxia-induced EPO restrained autophagy induction through the EPO receptor and phosphorylation of STAT3. Because cell viability with treatment of si-EPO under hypoxia did not increase over the control, our results suggested that EPO produced by hypoxia prevented excess autophagy induction.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Overexpression of microRNA-26a protects against deficient β-cell function via targeting phosphatase with tensin homology in mouse models of type 2 diabetes

The prevalence of type 2 diabetes mellitus (T2DM) increased rapidly in the world. The development of β-cell dysfunction is the quintessential defects in T2DM patients However, the pathogenesis of β-cell dysfunction is still unclear. MicroRNAs are short non-coding RNAs and has been reported to be involved in pathogenesis of β-cell dysfunction and T2DM. Here, we investigated the mechanisms by which miR-26a regulate β-cell function and insulin signaling pathway in high fat diet (HFD) fed and db/db T2DM mice model. The expression of miR-26a was down-regulated dramatically in the serum and islets of both HFD and db/db mice model. miR-26a overexpression protected against HFD-induced diabetes and maintained prolonged normoglycemic time in HFD fed mice. Overexpression of miR-26a improved β-cell dysfunction in T2DM mice. Further, we identified that PTEN is a direct target gene of miR-26a. Overexpression of miR-26a significantly inhibited the luciferase activity of hPTEN 3′-UTR, while the effect of miR-26a disappeared when the miR-26a potential binding site within the PTEN 3′-UTR was mutated. Overexpression of miR-26a reduced both the mRNA and protein levels of PTEN in vitro and in vivo. We also found that miR-26a overexpression increased the expression of p-Akt and p-FoxO-1, while the effect of miR-26a was blocked by PTEN overexpression. In conclusion, our data indicated that miR-26a potentially contributes to the β-cell dysfunction in T2DM, and miR-26a may be a new therapeutic strategy against T2DM.

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

Collagen VI suppresses fibronectin-induced enteric neural crest cell migration by downregulation of focal adhesion proteins

Highlights: • We identified 1355 differentially expressed genes in intestine during development. • The identified differentially expressed genes were enriched extracellular proteins. • Collagen VI reduced fibronectin-induced enteric neural crest cell migration. • Collagen VI promoted the down regulation of fibronectin-induced p130{sup Cas} proteins. The enteric nervous system (ENS) is a network of neurons and glia that are derived from enteric neural crest cells (ENCCs) and essential for regulating peristaltic activity of the colon. ENCCs migrate along the gastrointestinal tract to form the ENS, and disruption of ENCC motility leads to ENS disorders, such as Hirschsprung's disease. Previous ENCC-transplant experiments show that ENCCs can invade into isolated mouse intestines by age E13.5, but not after E15.5. We hypothesized that altered age-specific micro-environments in the intestine are responsible for ENCC invasion/migration. Here, we compared gene expression in the intestine between at E11.5 and E15.5 and identified 1355 differentially expressed transcripts. Among these, we found that genes encoding extracellular matrix (ECM) proteins were enriched. Notably, collagen VI (ColVI) family members were upregulated in the E15.5 mouse intestine at the mRNA and protein levels, whereas fibronectin (FN) was downregulated; however, both proteins showed colocalization at E15.5. To understand the mechanisms of ColVI and FN in ENCC migration, we examined neurosphere or individual ENCC-adherence capabilities toward the ECM. ColVI suppressed FN-induced ENCC spreading/migration, whereas ColVI induced morphologically narrow ENCC spreading and weak stress-fiber formation as compared with those with FN. Additionally, in ENCCs cultured on plates containing ColVI, the expression and phosphorylation of p130{sup Cas}, a members of focal adhesion complexes, was reduced. These data indicated an inhibitory role of ColVI in ENCC migration and suggested that ColVI suppression in the intestine might represent a novel therapeutic strategy for aganglionic colonic diseases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

IL-13 regulates IL-17C expression by suppressing NF-κB-mediated transcriptional activation in airway epithelial cells

Highlights: • IL-1β markedly elevated IL-17C expression viathe NF-κB-signaling pathway in normal human bronchial epithelial cells. • IL-13 attenuated IL-1β-mediated IL-17C expression via a STAT6-signaling pathway. • IL-1β enhanced binding of the NF-κB p65 sub unit to two IL-17C promoter regions (−130/−120 and −157/−147). • IL-13 attenuated IL-1β-mediated p65 binding to IL-17C promoter regions, resulting in lower IL-17C expression. The cytokine interleukin (IL)-17C is highly expressed in epithelial tissues and involved in innate immune responses; however, the regulation of IL-17C expression in the airways remains poorly understood. Here, we show that IL-1β strongly induces both IL-17C mRNA and protein expression in primary normal human bronchial epithelial cells. Conversely, IL-13 significantly reduced the IL-1β-induced IL-17C expression. Attenuation of the nuclear factor (NF)-κB-signaling pathway using an NF-κB-subunit p65-specific small-interfering RNA (siRNA), reduced IL-1β-induced IL-17C expression, demonstrating the importance of NF-κB signaling in IL-17C regulation. The inhibitory effects of IL-13 on IL-17C expression were abolished when the Janus kinase (JAK)/signal transducer and activator of transcription 6 (STAT6)-signaling pathway was impaired, using either the JAK inhibitor ruxolitinib or a STAT6-specific siRNA. Western blot analysis demonstrated that IL-1β promoted both IκB-α phosphorylation and degradation, and p65 nuclear translocation. Although IL-13 induced STAT6 phosphorylation and nuclear translocation, it did not affect the activation of the IL-1β-mediated NF-κB-pathway. Using chromatin immunoprecipitation, we confirmed that IL-1β enhanced p65 binding to regions within the IL-17C promoter that flank putative NF-κB-binding sites (−130/−120 and −157/−147). Interestingly, IL-13 treatment reduced the IL-1β-mediated p65 binding to these regions. These findings demonstrate that NF-κB-mediated transcriptional mechanisms are critically involved in the IL-1β-mediated IL-17C induction, and that IL-13 negatively regulates this induction by suppressing NF-κB-based transcriptional activation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The splicing factor transformer2 (tra2) functions in the Drosophila fat body to regulate lipid storage

Highlights: • Transformer2 (tra2) is a splicing protein whose metabolic functions are unknown. • Tra2 functions in the Drosophila fat body to modulate triglyceride levels. • Decreasing tra2 in the fat body promotes starvation resistance. • Tra2 alters CPT1 splicing in the fly fat body. Excess nutrients are stored as triglycerides mainly in the adipose tissue of an animal and these triglycerides are located in structures called lipid droplets. Previous genome-wide RNAi screens in Drosophila cells identified splicing factors as playing a role in lipid droplet formation. Our lab has recently identified the SR protein, 9G8, as an important factor in fat storage as decreasing its levels results in augmented triglyceride storage in the fat body. Previous in vitro studies have implicated 9G8 in the regulation of splicing of the sex determination gene doublesex (dsx) by binding to transformer (tra) and transformer2 (tra2); however, any function of these sex determination proteins in regulating metabolism is unknown. In this study, we have uncovered a role of tra2 to regulate fat storage in vivo. Inducing tra2{sup dsRNA} in the adult fat body resulted in an increase in triglyceride levels but had no effect on glycogen storage. Consistent with the triglyceride phenotype, tra2 knockdown flies lived longer under starvation conditions. In addition, this increase in triglycerides is due to more fat storage per cell and not an increase in the number of fat cells. Interestingly, the splicing of CPT1, an enzyme involved in the breakdown of lipids, was altered in flies with decreased tra2. The less-catalytically active isoform of CPT1 accumulated in tra2{sup dsRNA} flies suggesting a decrease in lipid breakdown, which is consistent with the increased triglyceride levels observed in these flies. Together, these results suggest a link between mRNA splicing, sex determination and lipid metabolism and may provide insight into the mechanisms underlying tissue-specific splicing and nutrient storage.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Adenovirus infection induces HuR relocalization to facilitate virus replication

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3′-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

High glucose up-regulates microRNA-34a-5p to aggravate fibrosis by targeting SIRT1 in HK-2 cells

Highlights: • The expression of SIRT1 is declined and the level of miR-34a-5p is up-regulated in the kidneys of DN mice. • SIRT1 prevents TGF-β1-induced pro-fibrotic gene expression. • SIRT1 expression was declined time-dependently under high glucose in HK-2 cells. • MiR-34a-5p aggravate fibrosis through directly suppressing SIRT1. • MiR-34a-5p inhibitor reverse the expressions of downstream genes through SIRT1 in HK-2 cells. Tubulointerstitial fibrosis (TIF) is crucial in the development of renal fibrosis in diabetic nephropathy(DN). Previous data shows that SIRT1 plays an important role on fibrosis, but the effect on TIF in DN and underlying mechanisms remains uncertain. In this study, we evaluated the vital role of SIRT1 and identified SIRT1 as a downstream target gene of microRNA-34a-5p (miR-34a-5p) in TIF of DN. The result revealed that expression of miR-34a-5p, fibronectin(FN),collagen type I (COL1) and transforming growth factor β1 (TGF-β1) were up-regulated accompanied by the corresponding down-regulation of SIRT1 in renal tissues of high fat diet and streptozotocin(HFD/STZ)induced diabetic mice with DN, and that the SIRT1 mRNA level was negatively correlated with miR-34a-5p expression in high glucose stimulated human proximal tubule cell line(HK-2) cells. We then demonstrated that overexpression of SIRT1 reduced, whereas small interfering RNA targeting SIRT1 enhanced the expressions of TGF-β1 and fibrosis-related genes including FN and COL1 in HK-2 cells. Furthermore, we identified that miR-34a-5p directly suppressed SIRT1 to increase the profibrogenic effects of TGFβ1 through targeting the 3'untranslated region of SIRT1. The functional correlation of miR-34a-5p induced SIRT1 decrease was supported by overexpression and inhibition of miR-34a-5p in HK-2 cells. All the results reveal that SIRT1 which is vital in the evolution of renal TIF in DN can be directly suppressed by miR-34a-5p, and suggest that miR-34a-5p is a new target for DN treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Effects of dietary glucose and sodium chloride on intestinal glucose absorption of common carp (Cyprinus carpio L.)

Highlights: • Glucose absorption was primarily occurred in proximal and mid intestine in carp. • Food intake promoted SGLT1 and GLUT2 expressions, glucose transport in carp. • Dietary glucose promoted SGLT1 and GLUT2 expressions, glucose transport in carp. • Dietary NaCl enhanced SGLT1 and GLUT2 expressions, glucose transport in carp. • High-glucose diet increased while high-NaCl decreased intestinal villus height. The co-transport of sodium and glucose is the first step for intestinal glucose absorption. Dietary glucose and sodium chloride (NaCl) may facilitate this physiological process in common carp (Cyprinus carpio L.). To test this hypothesis, we first investigated the feeding rhythm of intestinal glucose absorption. Carps were fed to satiety once a day (09:00 a.m.) for 1 month. Intestinal samples were collected at 01:00, 05:00, 09:00, 13:00, 17:00 and 21:00. Result showed that food intake greatly enhanced sodium/glucose cotransporter 1 (SGLT1) and glucose transporter type 2 (GLUT2) expressions, and improved glucose absorption, with highest levels at 09:00 a.m.. Then we designed iso-nitrogenous and iso-energetic diets with graded levels of glucose (10%, 20%, 30%, 40% and 50%) and NaCl (0%, 1%, 3% and 5%), and submitted to feeding trial for 10 weeks. The expressions of SGLT1 and GLUT2, brush border membrane vesicles (BBMVs) glucose transport and intestinal villus height were determined after the feeding trial. Increasing levels of dietary glucose and NaCl up-regulated mRNA and protein levels of SGLT1 and GLUT2, enhanced BBMVs glucose transport in the proximal, mid and distal intestine. As for histological adaptive response, however, high-glucose diet prolonged while high-NaCl diet shrank intestinal villus height. Furthermore, we also found that higher mRNA levels of SGLT1 and GLUT2, higher glucose transport capacity of BBMVs, and higher intestinal villus were detected in the proximal and mid intestine, compared to the distal part. Taken together, our study indicated that intestinal glucose absorption in carp was primarily occurred in the proximal and mid intestine, and increasing levels of dietary glucose and NaCl enhanced intestinal glucose absorption in carp.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Integrative analysis of rewired central metabolism in temozolomide resistant cells

Highlights: • Temozolomide (TMZ) resistant Neurospheroids (NSP) identified in U87MG cell line. • NSP shows varied dose response, two fold higher IC{sub 50} and 30% lower growth rate. • Altered Glucose and Glutamine uptake in U87MG and NSP shapes metabolic dynamics. • Differential mRNA levels of CAN genes & ABC transporters drive respiration/growth. • Integrative paradigm identifies potential metabolic drivers for TMZ resistance. An authenticated U87MG clonal glioblastoma cell line was investigated to identify a sub-population of neurospheroidal (NSP) cells within the main epithelial population (U87MG). The NSP cells sorted using Fluorescence Assisted Cell Sorting (FACS) showed varied morphology, 30% lower growth rates, 40% higher IC{sub 50} values for temozolomide drug and could differentiate into the glial cell type (NDx). Metabolite profiling using HR-LCMS identified glucose, glutamine and serine in both populations and tryptophan only in U87MG as growth limiting substrates. Glycine, alanine, glutamate and proline were secreted by U87MG, however proline and glycine were re-utilized in NSP. Exo-metabolite profiling and phenotypic microarrays identified differential metabolism of primary carbon sources glucose and derived pyruvate for U87MG; glutamine and derived glutamate metabolism in NSP. Differential mRNA abundance of AKT1, PTEN, PIK3CA controlling metabolism, drug efflux, nutrient transport and epigenetic control MDM2 are potentially critical in shaping DNA methylation effects of temozolomide. Our study provides a new insight into the combined effect of these factors leading to temozolomide resistance in NSP.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Inhibition of Notch signaling pathway using γ-secretase inhibitor delivered by a low dose of Triton-X100 in cultured oral cancer cells

Highlights: • A novel method is developed to deliver γ-secretase inhibitor DAPT using a low dose of Triton-X100. • This approach effectively blocks the cleavage of Notch1 receptor. • The elimination of Notch1 intracellular domain from cell nuclei is confirmed. • Notch1 signaling blockade in mRNA level is demonstrated upon applying this delivery system. How to effectively delivering therapeutic agents, including γ-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notch1 intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of γ-secretase inhibitor, DAPT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAPT alone could not block the γ-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notch1, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hes1). Our data suggest that Notch1/Hes1 signaling pathway is deactivated using DAPT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notch1 could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver γ-secretase inhibitor DAPT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases.

60 APPLIED LIFE SCIENCES↗