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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 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Vasostatin-2 inhibits cell proliferation and adhesion in vascular smooth muscle cells, which are associated with the progression of atherosclerosis

Recently, the serum expression level of vasostatin-2 was found to be reduced and is being studied as an important indicator to assess the presence and severity of coronary artery disease; the functional properties of vasostatin-2 and its relationship with the development of atherosclerosis remains unclear. In this study, we attempted to detect the expression of vasostatin-2 and its impact on human vascular smooth muscle cells (VSMCs). Quantitative real-time PCR (qRT-PCR) and western blot were used to assess the expression level of vasostatin-2 in VSMCs between those from atherosclerosis and disease-free donors; we found that vasostatin-2 was significantly down-regulated in atherosclerosis patient tissues and cell lines. In addition, the over-expression of vasostatin-2 apparently inhibits cell proliferation and migration in VSMCs. Gain-of-function in vitro experiments further show that vasostatin-2 over-expression significantly inhibits inflammatory cytokines release in VSMCs. In addition, cell adhesion experimental analysis showed that soluble adhesion molecules (sICAM-1, sVCAM-1) had decreased expression when vasostatin-2 was over-expressed in VSMCs. Therefore, our results indicate that vasostatin-2 is an atherosclerosis-related factor that can inhibit cell proliferation, inflammatory response and cell adhesion in VSMCs. Taken together, our results indicate that vasostatin-2 could serve as a potential diagnostic biomarker and therapeutic option for human atherosclerosis in the near future. - Highlights: • Vasostatin-2 levels were down-regulated in atherosclerosis patient tissues and VSMCs. • Ectopic expression of vasostatin-2 directly affects cell proliferation and migration in vitro. • Ectopic expression of vasostatin-2 protein affects pro-inflammatory cytokines release in VSMCs. • Ectopic expression of vasostatin-2 protein affects cell adhesion in VSMCs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

MicroRNA-367 is a potential diagnostic biomarker for patients with esophageal squamous cell carcinoma

Purpose: In this study, we investigated whether microRNA-367 (miR-367) may serve as a circulating biomarker and tumor oncogene in esophageal squamous carcinoma (ESCC). Methods: Circulating serum miR-367 was compared by quantitative RT-PCR (qRT-PCR) between 35 ESCC patients and 35 normal control patients, as well paired ESCC tumor tissues and adjacent non-tumor esophageal epithelial tissues in 46 patients. The correlation between serum miR-367 and clinicopathological properties of ESCC patients was assessed. The overall survival (OS) was assessed by Kaplan–Meier method and compared by log-rank test between patients with high serum miR-367 and low serum miR-367. The possibility of miR-367 being independent prognostic factor for ESCC was also assessed. Furthermore, lentivirus-mediated miR-367 downregulation was conducted in ESCC cell lines Kyse30 and TE-1 cells to assess the possible oncogenic effect of miR-367 on ESCC proliferation and cell cycle transition in vitro. Results: MiR-367 was aberrantly upregulated in sera and tumors of ESCC patients, whereas downregulated in ESCC patients after the treatments of esophagectomy and chemotherapy. Serum miR-367 was found to be closely correlated with the clinicopathological properties of differentiation grades, clinical stage and tumor metastasis in ESCC patients. Serum miR-367 was also confirmed to be associated with OS, as well as serving independent prognostic factor in ESCC patients. Moreover, lentivirus-induced miR-367 downregulation inhibited cancer growth and cell cycle transition in Kyse30 and TE-1 cells. Conclusion: MiR-367 is a potential biomarker for ESCC and may act as an oncogene in regulating ESCC development. - Highlights: • MiR-367 was aberrantly upregulated in sera and tumors of ESCC patients. • MiR-367 was downregulated in ESCC patients after esophagectomy or chemotherapy. • Serum miR-367 was correlated with the clinicopathological properties of ESCC patients. • Serum miR-367 was associated with OS in ESCC patients. • lentivirus-induced miR-367 downregulation inhibited ESCC growth and cell cycle transition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis in osteosarcoma cells

miR-130b was significantly up-regulated in osteosarcoma (OS) cells. Naked cuticle homolog 2 (NKD2) inhibited tumor growth and metastasis in OS by suppressing Wnt signaling. We used three miRNA target analysis tools to identify potential targets of miR-130b, and found that NKD2 is a potential target of miR-130b. Based on these findings, we hypothesize that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth. We detected the expression of miR-130b and NKD2 mRNA and protein by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found up-regulation of miR-130b and down-regulation of NKD2 mRNA and protein exist in OS cell lines. MTT and flow cytometry assays showed that miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells. Furthermore, we showed that NKD2 is a direct target of miR-130b, and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2. We further investigated whether miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting Wnt signaling, and the results confirmed our speculation that miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells. These findings will offer new clues for OS development and progression, and novel potential therapeutic targets for OS. - Highlights: • miR-130b is up-regulated and NKD2 is down-regulated in osteosarcoma cell lines. • Down-regulation of miR-130b inhibits proliferation of osteosarcoma cells. • Down-regulation of miR-130b promotes apoptosis of osteosarcoma cells. • miR-130b directly targets NKD2. • NKD2 regulates OS cell proliferation and apoptosis by inhibiting the Wnt signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

H{sup +}/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport

Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. -- Highlights: •PEPT2 is expressed in keratinocytes, which are more common than other skin cells. •Immunolocalization analysis using human skin revealed epidermal PEPT2 localization. •Keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. •Di- and tripeptide pass actively through the epidermis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and {sup 64}Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescence signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

The up-regulation of long non-coding RNA CCAT2 indicates a poor prognosis for prostate cancer and promotes metastasis by affecting epithelial-mesenchymal transition

Recently, long noncoding RNAs (lncRNAs) have been shown to have critical regulatory roles in human cancer biology. LncRNA CCAT2 is a novel identified lncRNA that was previously reported to be up-regulated in different cancers, however, its role in prostate cancer remains unclear. The aim of this study was to investigate the expression and role of lncRNA CCAT2 in prostate cancer. The expression levels of lncRNA CCAT2 in PCa tissues and cell lines (DU145 and 22RV1) were evaluated by quantitative real-time PCR (qRT-PCR), and its association with prognosis of patients was analyzed by statistical analysis. Furthermore, the effect of CCAT2 on proliferation, migration, and invasion was studied in PCa cells. We found that the expression level of CCAT2 was higher in PCa tissues and cells compared to adjacent non-tumor tissues and normal prostate stromal immortalized cells WPMY-1. Kaplan-Meier survival analysis revealed that patients with high CCAT2 expression level had poorer overall survival and progression-free survival than those with low CCAT2 expression. Furthermore, multivariate analysis showed that the status of CCAT2 expression was an independent prognostic indicator for this disease. We also found that knockdown of CCAT2 could inhibit cell growth, migration, and invasion in vitro. In addition, knockdown of CCAT2 stimulated epithelial-mesenchymal transition (EMT) through abrogating N-cadherin, vimentin expression and intensifing the expression levels of E-cadherin. In conclusion, our data suggested that lncRNA CCAT2 was a novel molecule involved in PCa progression, which provided a potential prognostic biomarker and therapeutic target for new therapies in patients with prostate cancer. - Highlights: • LncRNA CCAT2 is identified with overexpression in prostate cancer tissues and cell lines. • High expression of lncRNA CCAT2 can be an independent prognostic factor for overall survival and progression-free survival. • Knockdown of lncRNA CCAT2 can inhibit cell growth, migration, and invasion in vitro study. • Knockdown of lncRNA CCAT2 stimulates metastasis of prostate cancer cell through regulating EMT-associated gene expression including N-cadherin, Vimentin and E-cadherin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Overexpression of ZIC5 promotes proliferation in non-small cell lung cancer

Background: Non-small cell lung cancer (NSCLC) has become the leading cause of cancer-related deaths. It is therefore urgent that we identify new molecular targets to help cure NSCLC patients. Here, we identified ZIC5 as a potential novel oncogene. Methods: We detected the expression of ZIC5 in tumor and normal tissues of NSCLC patients using quantitative real-time PCR and explored its clinical appearance. We then knocked down ZIC5 to observe changes in NSCLC cell proliferation and metastasis. Nude mouse xenograft models were established to measure ZIC5's function in vivo. Results: Our results revealed that ZIC5 was expressed at dramatically higher levels in NSCLC tumor tissues than in normal tissues. High levels of ZIC5 expression were associated with a higher primary tumor grade. ZIC5 expression was significantly inhibited by small interfering RNA. After silencing ZIC5, the metastatic capacity of NSCLC cells was clearly lower. Knocking down ZIC5 significantly inhibited the proliferation of NSCLC cells, causing the cell cycle to be arrested in G2 phase. Xenograft tumor models showed that knocking down ZIC5 also inhibited tumor growth in vivo. Q-PCR and western blot analysis revealed that ZIC5 expression was closely associated with CCNB1 and CDK1 complex expression, while other cell cycle-related genes showed no significant correlation with ZIC5. Conclusions: Our experiment show that ZIC5 is highly upregulated in NSCLC tumor tissues and suggest that ZIC5 may act as an oncogene by influencing CCNB1 and CDK1 complex expression. ZIC5 may therefore be a potential biomarker and therapeutic target for NSCLC patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Cloning and expressions of peroxisome proliferator activated receptor alpha1 and alpha2 (PPARα1 and PPARα2) in loach (Misgurnus anguillicaudatus) and in response to different dietary fatty acids

Peroxisome proliferator activated receptor alpha1 and alpha2 (PPARα1 and PPARα2) were investigated in loach (Misgurnus anguillicaudatus) by RACE (rapid amplification of cDNA ends) and qPCR (real-time quantitative PCR) for the first time. The cDNA sequences of PPARα1 and PPARα2 were 2042bp and 2407bp, respectively encoding 467 and 465 amino acids. Sequence alignments of deduced amino acids showed significant homology between the two subtypes of PPARα, indicating 70% identity. The two genes revealed sensible changes in transcriptions during early life stages of the loach, and the highest transcriptions of the two genes both appeared at some day after hatching. PPARα1 predominantly expressed in liver, while PPARα2 markedly expressed in heart. The expression regulation of PPARα1 and PPARα2 in response to dietary fatty acids was determined in livers of loaches fed with diets containing fish oil (FO group) and soybean oil (SO group) for 75 days. The expression level of PPARα1 in FO group was significantly higher than those in SO group (P < 0.01), while the expression level of PPARα2 in FO group was also significantly higher than those in SO group (P < 0.05). There was no significant difference in the expression level between PPARα1 and PPARα2 in SO group, whereas significant difference in FO group. These indicated that lipid resources could regulate the expressions of these two genes in the loach. Our results will provide opportunities to better understand the functional characterization of PPARα1 and PPARα2 in further studies. - Highlights: • The full-length cDNAs of loach PPARα1 and PPARα2 were obtained by a RACE PCR method. • Phylogenetic and protein characterizations of these two genes were predicted. • These two genes differentially expressed at different early life stages and tissues indicating their different functions. • n-3 PUFA may regulate the activation of PPARα in the loach.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

MiR-33a suppresses proliferation of NSCLC cells via targeting METTL3 mRNA

Methyltransferase like 3 (METTL3) was incipiently known as a methyltransferase which was responsible for N{sup 6}-methyladenosine (m{sup 6}A) methylation. METTL3 can promote the expression of several crucial oncoproteins and its high expression enhanced proliferation, survival, and invasion of human lung cancer cells. However, how METTL3 was regulated is seldom understood in non-small-cell lung carcinoma (NSCLC). In the present study, miR-33a was suspicious to target to the 3'-untranslated region (3'UTR) of METTL3 mRNA via in silico prediction. Besides, the expressions of METTL3 were higher in NSCLC tissues than those in adjacent tissues, and METTL3 expressions were positively related to the expressions of miR-33a in NSCLC tissues which confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). MiR-33a can directly target to the 3'UTR of METTL3 mRNA which examined by luciferase reporter gene assay. Moreover, we found that miR-33a can reduce the expression of METTL3 at both mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Functionally, miR-33a can reduce the proliferation of A549 and NCI-H460 cells. Conversely, inhibition of miR-33a by anti-miR-33a can rescue that using 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and 5-ethynyl-2-deoxyuridine (EdU) assay. Similarly, miR-33a can reduce cellular anchorage-independent growth of A549 cells. Additionally, the negative influences of miR-33a on the downstream genes of METTL3 were examined by Western blot analysis. Thus, we concluded that miR-33a can attenuate NSCLC cells proliferation via targeting to the 3'UTR of METTL3 mRNA. Our findings provide new insights into the mechanism of METTL3 regulation by micro RNA, and supports METTL3 as a therapeutic target in NSCLC. - Highlights: • METTL3 expression is negatively correlated with miR-33a. • MiR-33a can directly target to the 3'UTR of METTL3 mRNA. • MiR-33a inhibits NSCLC cells proliferation through decreasing METTL3. • MiR-33a induces the down-regulation of EGFR through targeting METTL3.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

MiR-509-5p suppresses the proliferation, migration, and invasion of non-small cell lung cancer by targeting YWHAG

Dysregulation of microRNAs (miRNAs) expression is prevalent in human malignancy development and progression. As previously reported, miR-509-5p has been identified as a tumor suppressor in various cancers, but its role and functional mechanism in non-small lung cancer (NSCLC) remains unknown. In the present study, we found that miR-509-5p was significantly downregulated in NSCLC tissues and cell lines using Quantitative real-time PCR (qRT-PCR). In addition, overexpression of miR-509-5p markedly inhibited the proliferation, migration, invasion and phosphorylation of Akt of NSCLC cells. Moreover, we identified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) as a direct target of miR-509-5p. Knockdown of YWHAG mimicked the inhibitory effects of miR-509-5p on NSCLC cells for proliferation and motility. Finally, the negative correlation between miR-509-5p and YWHAG expression in NSCLC specimens was demonstrated. Taken together, our present study revealed the tumor suppressive role of miR-509-5p in NSCLC by targeting YWHAG, suggesting that miR-509-5p/YWHAG axis might be considered as a novel and potential target for clinical diagnosis and therapeutics of NSCLC. - Highlights: • The tumor suppressive role of miRNA-509-5p in non-small cell lung cancer is revealed. • MiR-509-5p exerts its influence on NSCLC cells by targeting YWHAG. • MiR-509-5p inhibits the phosphorylation of Akt in NSCLC cells. • MiR-509-5p can be deemed as a potential diagnostic and therapeutic target for NSCLC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

SNHG16 contributes to breast cancer cell migration by competitively binding miR-98 with E2F5

Long noncoding RNAs (lncRNAs) have been proved to play important roles in cellular processes of cancer, including the development, proliferation, and migration of cancer cells. In the present study, we demonstrated small nucleolar RNA host gene 16 (SNHG16) as an oncogene on cell migration in breast cancer. Expression levels of SNHG16 were found to be frequently higher in breast cancer tissues than in the paired noncancerous tissues. Gain- and loss-of-function studies proved that SNHG16 significantly promoted breast cancer cell migration. We predicted SNHG16 as a competitive endogenous RNA (ceRNA) of E2F transcription factor 5 protein (E2F5) via competition for the shared miR-98 through bioinformatics analysis, and proved this regulation using relative quantitative real-time PCR (qRT-PCR), western blot, RNA immunoprecipitation (RIP) assay and luciferase reporter assay. In addition, we identified a positive correlation between SNHG16 and E2F5 in breast cancer tissues. Furthermore, we demonstrated that forced expression of miR-98 could partially abrogate SNHG16-mediated increase of breast cancer cells migration, suggesting that SNHG16 promoted cell migration in a miR-98 dependent manner. Taken together, our findings indicated that SNHG16 induces breast cancer cell migration by competitively binding miR-98 with E2F5, and SNHG16 can serve as a potential therapeutic target for breast cancer treatment. - Highlights: • SNHG16 was first shown to work as a ceRNA by competitively binding miR-98 with E2F5. • SNHG16 promotes breast cancer cell migration in a miR98-dependent manner. • Direct combination of SNHG16 with miR-98 was proved using RIP and luciferase assay. • The positive regulation of E2F5 by SNHG16 in breast cancer was revealed.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

MicroRNA-29 regulates myocardial microvascular endothelial cells proliferation and migration in association with IGF1 in type 2 diabetes

Background: In our study, we investigated the expression and function of microRNA-29 in myocardial microvascular endothelial cells (MMEVC) in type 2 diabetic Goto-Kakizaki (GK) rats. Methods: MiR-29 gene expression was compared, by qRT-PCR between diabetic GK rat MMEVC and non-diabetic Wistar rat MMEVC. MiR-29 was downregulated in GK MMEVC and its effect on angiogenic properties of proliferation and migration was examined. Potential downstream target gene of miR-29, insulin growth factor 1 (IGF1), was assessed by dual-luciferase reporter assay, qRT-PCR and western blot in GK MMEVC. IGF1 was also downregulated by siRNA in miR-29-downregulated GK MMEVC. Its effect on miR-29-associated angiogenic regulation on MMEVC proliferation and migration was further investigated. Results: MiR-29 was substantially upregulated in GK MMEVC than in Wistar MMEVC. Transfection of synthetic miR-29 inhibitor successfully downregulate endogenous miR-29 in GK MMEVC, and subsequently promoted angiogenesis by increasing cell proliferation and migration. IGF1 was confirmed to be downstream target gene of miR-29 in GK MMEVC, with its gene and protein expressions both upregulated in miR-29-downregualted GK MMEVC. Conversely, siRNA-mediated IGF1 downregulation reversed the pro-angiogenic effect of miR-29 downregulation in GK MMEVC, as it decreased cell proliferation and migration. Conclusion: Our study suggests that miR-29 downregulation, through its inverse regulation on downstream target of IGF1 gene, is a pro-angiogenic factor in MMEVC in type 2 diabetic rats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Taxifolin enhances osteogenic differentiation of human bone marrow mesenchymal stem cells partially via NF-κB pathway

Taxifolin, a flavonoid compound, has been reported to stimulate osteogenic differentiation in osteoblasts. The present study investigated whether taxifolin affects the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and the molecular mechanisms involved. The proliferation and osteogenic differentiation of hBMSCs in the presence of taxifolin were examined by CCK-8 assay, alkaline phosphatase (ALP) activity, ALP staining and Alizarin red staining. The expression of osteogenic differentiation markers were detected by real-time quantitative PCR (RT-PCR) analysis and western blot assay. The activation of potential related pathways was examined by luciferase reporter assay, immunofluorescence and western blot analysis. Taxifolin treatment increased osteogenic differentiation of hBMSCs without cytotoxicity. Luciferase reporter assay showed that taxifolin could not activate estrogen receptor pathway, but inhibit TNF-α-induced NF-κB signaling pathway activation in osteogenic induction condition. Moreover, the nucleus translocation of NF-κB under TNF-α treatment was inhibited by taxifolin treatment. The taxifolin-induced osteogenic differentiation effects of hBMSCs were abolished by TNF-α treatment. In conclusion, our results suggested that taxifolin could promote osteogenesis of hBMSCs, partially through antagonism of NF-κB signaling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

KLF2 attenuates bleomycin-induced pulmonary fibrosis and inflammation with regulation of AP-1

Highlights: • KLF2 is downregulated in BLM-treated rats. • KLF2 alleviates BLM-induced lung fibrosis. • KLF2 suppresses the production of pro-inflammatory cytokines. • KLF2 prevents TGF-β1 induced lung fibrosis in vitro. • Effects of KLF2 on AP-1. Pulmonary fibrosis (PF) is a chronic, fibrosing interstitial pneumonia and devastating disease. Here we investigated the potential roles of Kruppel-like factor 2 (KLF2) on pulmonary fibrosis and inflammation response. A mouse model of pulmonary fibrosis was established by intratracheal injection of bleomycin (BLM). The mRNA and protein levels of KLF2 were assayed by RT-PCR and Western blotting respectively. The extent of lung fibrosis was determined using hematoxylin and eosin (HE) staining and Masson's trichrome staining, and the hydroxyproline content was quantified. RT-PCR was used to evaluate the mRNA expression of collagen type 1a1 (col1a1), col3a1, α-SMA, TNF-α, IL-1β and IL-6. The concentrations of TNF-α, IL-1β, and IL-6 in bronchoalveolar lavage fluid (BALF) and lung tissue were examined by ELISA. Also, the effects of KLF2 on activator protein-1 (AP-1) were evaluated by measuring the c-Jun and c-Fos protein levels. We found that KLF2 was remarkably downregulated in BLM-treated rats, both in mRNA and protein levels. Additionally, overexpression of KLF2 attenuated the destruction of the alveolar space and pulmonary interstitial collagen hyperplasia, and deposition reduced the expression of col1a1, col3a1, and α-SMA, and blocked the production of TNF-α, IL-1β, and IL-6 in BALF and lung tissue in vivo. Moreover, adenoviral transduction of KLF2 inhibited TGF-β1-induced expression of col1a1, col3a1, and α-SMA in vitro. Mechanically, BLM up-regulated c-Jun and c-Fos expression, which was impeded by KLF2 overexpression. Taken together, our data indicate that KLF2 attenuates pulmonary fibrosis and inflammation, possibly through the regulation of AP-1.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens

Highlights: • We cloned a fat-taste receptor candidate gene, CD36, from chickens. • Fat-taste receptor genes, GPR120 and CD36, were expressed in chicken oral tissues. • Several lipase genes were expressed in chicken oral tissues. • Chicken oral tissues contained functional lipases. It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Angiotensin-converting enzyme 2 regulates mitochondrial function in pancreatic β-cells

Highlights: • ACE2 improved glucose stimulated insulin secretion (GSIS) and mitochondrial membrane potential in INS-1 cells. • PCR array demonstrated that ACE2 up-regulated 67 mitochondria-related genes in INS-1 cells. • ACE2 deletion suppressed mitochondrial oxidation and calcium influx in islets. • ACE2 over-expression in db/db mice promoted mitochondrial oxidation and calcium influx in islets. Mitochondrial metabolism plays an essential role in the regulation of insulin release and glucose homeostasis. Evidence demonstrated that the angiotensin-converting enzyme 2 (ACE2) participates in the regulation of glucose metabolism, however, its role in mitochondrial metabolism remains unclear. The purpose of our study was to determine if ACE2 can regulate mitochondrial function in pancreatic β-cells. We found that ACE2 over-expression restored glucose-stimulated insulin secretion (GSIS) and mitochondrial membrane potential (MMP) in the presence of H{sub 2}O{sub 2} in INS-1 cells. PCR array demonstrated that ACE2 over-expression up-regulated 67 mitochondria-related genes in INS-1 cells. In pancreatic islets, ACE2 ablation attenuated intracellular calcium influx with a decrease in GSIS. Ace2{sup −/y} mice islets exhibited impaired mitochondrial respiration and lower production of ATP, along with decreased expression of genes involved in mitochondrial oxidation. In islets from db/db mice, ACE2 over-expression increased intracellular calcium influx and restored impaired mitochondrial oxidation, potentially causing an increase in GSIS. These results shed light on the potential roles of ACE2 in mitochondrial metabolism, moreover, may improve our understanding of diabetes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Elovl4 can effectively elongate C18 polyunsaturated fatty acids in loach Misgurnus anguillicaudatus

Highlights: • The full-length cDNAs of loach elovl4a and elovl4b were obtained by a RACE PCR method. • Phylogenetic and protein characterizations of these two genes were predicted. • Elovl4a differentially expressed at different tissues in loach, so did elovl4b. • Loach elovl4 could effectively elongate C18 PUFAs, which provided an alternative pathway for C20 biosynthesis in fish. In this study, full-length cDNA sequences of elovl4a and elovl4b from loach Misgurnus anguillicaudatus were cloned. The full-length cDNAs of loach elovl4a and elovl4b were 2423 and 2054bp, encoding 315 and 300 amino acids, respectively. The deduced amino acid sequences of elovl4a and elovl4b in loach both shared the highest identity with those of Danio rerio, whereas lower identity score between loach elovl4a and elovl4b was present. Temporal expression and tissue expression of loach elovl4a and elovl4b were studied by reverse transcriptase PCR. Results of the tissue expression analyses suggested different functions of loach elovl4a and elovl4b. Functional characterizations of loach elovl4a and elovl4b on synthesis of fatty acids, especially elongating C18 polyunsaturated fatty acids (PUFAs) to longer-chain fatty acids, were studied by heterologous expression in Saccharomyces cerevisiae. Loach elovl4a and elvol4b enzymes were able to elongate all fatty acids tested including 18:2n-6, 18:3n-3, 18:3n-6, 20:4n-6 and 20:5n-3. At last, expression levels of the two elovl4 genes of loach fin cells incubated with 18:2n-6 and 18:3n-3 of different concentrations were measured. Expressions of elovl4a and elovl4b of loach fin cells were significantly up-regulated by 18:2n-6 and 18:3n-3. The results obtained here indicated that loach elovl4 could effectively elongate C18 PUFAs. This was a systematic report of elovl4's elongating functions towards C18 and provided an alternative pathway for C20 biosynthesis in fish species.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Comparison of two hyoscyamine 6β-hydroxylases in engineering scopolamine biosynthesis in root cultures of Scopolia lurida

Highlights: • Overexpression of SlH6H or HnH6H significantly promoted the anisodamine production at similar level. • Overexpression of SlH6H or HnH6H markedly enhanced the scopolamine biosynthesis at different level. • HnH6H exhibited better effects on the enhancement of scopolamine biosynthesis than SlH6H. • Overexpression of HnH6H led to the increased production of total tropane alkaloids. Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6β-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production.

60 APPLIED LIFE SCIENCES↗