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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 1,063 records · Page 59
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DOE OSTI2014

Insights on the antitumor effects of kahweol on human breast cancer: Decreased survival and increased production of reactive oxygen species and cytotoxicity

Highlights: • Kahweol inhibits growth and attachment-independent proliferation of tumor cells. • Kahweol induces apoptosis in MDA-MB231 human breast cancer cells. • Kahweol-induced apoptosis involves caspase activation and cytochrome c release. • Kahweol does not protect against hydrogen peroxide cytotoxicity. • Kahweol increases hydrogen peroxide production by human breast cancer cells. - Abstract: The present study aims to identify the modulatory effects of kahweol, an antioxidant diterpene present in coffee beans, on a panel of human tumor cell lines. Kahweol inhibits tumor cell proliferation and clonogenicity and induces apoptosis in several kinds of human tumor cells. In the estrogen receptor-negative MDA-MB231 human breast cancer, the mentioned effects are accompanied by caspases 3/7 and 9 activation and cytochrome c release. On the other hand, kahweol increases the production of reactive oxygen species and their cytotoxicity in human breast cancer cells but not in normal cells. Taken together, our data suggest that kahweol is an antitumor compound with inhibitory effects on tumor cell growth and survival, especially against MDA-MB231 breast cancer cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

MiR-224 expression increases radiation sensitivity of glioblastoma cells

Highlights: • MiR-224 expression in established glioblastoma cell lines and sporadic tumor tissues is low. • Exogenous miR-224 expression was found to increase radiation sensitivity of glioblastoma cells. • MiR-224 expression brought about 55–60% reduction in API5 expression levels. • Transfection with API5 siRNA increased radiation sensitivity of glioblastoma cells. • Low miR-224 and high API5 expression correlated with worse survival of GBM patients. - Abstract: Glioblastoma (GBM) is the most common and highly aggressive primary malignant brain tumor. The intrinsic resistance of this brain tumor limits the efficacy of administered treatment like radiation therapy. In the present study, effect of miR-224 expression on growth characteristics of established GBM cell lines was analyzed. MiR-224 expression in the cell lines as well as in primary GBM tumor tissues was found to be low. Exogenous transient expression of miR-224 using either synthetic mimics or stable inducible expression using doxycycline inducible lentiviral vector carrying miR-224 gene, was found to bring about 30–55% reduction in clonogenic potential of U87 MG cells. MiR-224 expression reduced clonogenic potential of U87 MG cells by 85–90% on irradiation at a dose of 6 Gy, a dose that brought about 50% reduction in clonogenic potential in the absence of miR-224 expression. MiR-224 expression in glioblastoma cells resulted in 55–65% reduction in the expression levels of API5 gene, a known target of miR-224. Further, siRNA mediated down-regulation of API5 was also found to have radiation sensitizing effect on glioblastoma cell lines. Analysis of the Cancer Genome Atlas data showed lower miR-224 expression levels in male GBM patients to correlate with poorer survival. Higher expression levels of miR-224 target API5 also showed significant correlation with poorer survival of GBM patients. Up-regulation of miR-224 or down-regulation of its target API5 in combination with radiation therapy, therefore appear as promising options for the treatment of glioblastoma, which is refractory to the existing treatment strategies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Interleukin-10 increases reverse cholesterol transport in macrophages through its bidirectional interaction with liver X receptor α

Highlights: • IL-10 promotes reverse cholesterol efflux from lipid loaded macrophages. • IL-10 increases the expression of ABCA-1 and ABCG-1. • IL-10 exhibits cross-talk with the nuclear receptor LXRα. - Abstract: Interleukin (IL)-10 is a prototypical anti-inflammatory cytokine that has been shown to attenuate atherosclerosis development. In addition to its anti-inflammatory properties, the anti-atherogenic effect of IL-10 has recently also been suggested to reflect a complex effect of IL-10 on lipid metabolism in macrophages. In the present study we examined the effects of IL-10 on cholesterol efflux mechanism in lipid-loaded THP-1 macrophages. Our main findings were: (i) IL-10 significantly enhanced cholesterol efflux induced by fetal-calf serum, high-density lipoprotein (HDL){sub 2} and apolipoprotein A-1. (ii) The IL-10-mediated effects on cholesterol efflux were accompanied by an increased IL-10-mediated expression of the ATP-binding cassette transporters ABCA1 and ABCG1, that was further enhanced when the cells were co-activated with the liver X receptor (LXR)α agonist (22R)-hydroxycholesterol. (iii) The effect of LXRα activation on the IL-10-mediated effects on the ATP-binding cassette transporters seems to include enhancing effects on the IL-10 receptor 1 (IL10R1) expression and interaction with STAT-3 signaling. (iv) These enhancing effects on ABCA1 and ABCG1 was not seen when the cells were stimulated with the IL-10 family members IL-22 and IL-24. This study suggests that the anti-atherogenic properties of IL-10 may include enhancing effects on cholesterol efflux mechanism that involves cross-talk with LXRα activation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

microRNA-183 plays as oncogenes by increasing cell proliferation, migration and invasion via targeting protein phosphatase 2A in renal cancer cells

Highlights: • miR-183 was up-regulated in renal cancer tissues. • Inhibition of endogenous miR-183 suppressed renal cancer cell growth and metastasis. • miR-183 increased cell growth and metastasis. • miR-183 regulated renal cancer cell growth and metastasis via directly targeting tumor suppressor protein phosphatase 2A. - Abstract: The aim of this study was to investigate the function of miR-183 in renal cancer cells and the mechanisms miR-183 regulates this process. In this study, level of miR-183 in clinical renal cancer specimens was detected by quantitative real-time PCR. miR-183 was up- and down-regulated in two renal cancer cell lines ACHN and A498, respectively, and cell proliferation, Caspase 3/7 activity, colony formation, in vitro migration and invasion were measured; and then the mechanisms of miR-183 regulating was analyzed. We found that miR-183 was up-regulated in renal cancer tissues; inhibition of endogenous miR-183 suppressed in vitro cell proliferation, colony formation, migration, and invasion and stimulated Caspase 3/7 activity; up-regulated miR-183 increased cell growth and metastasis and suppressed Caspase 3/7 activity. We also found that miR-183 directly targeted tumor suppressor, specifically the 3′UTR of three subunits of protein phosphatase 2A (PP2A-Cα, PP2A-Cβ, and PP2A-B56-γ) transcripts, inhibiting their expression and regulated the downstream regulators p21, p27, MMP2/3/7 and TIMP1/2/3/4. These results revealed the oncogenes role of miR-183 in renal cancer cells via direct targeting protein phosphatase 2A.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

The small molecule survivin inhibitor YM155 may be an effective treatment modality for colon cancer through increasing apoptosis

Survivin has a known beneficial role in the survival of both cancer cells and normal cells. Therapies targeting survivin have been proposed as an alternative treatment modality for various tumors; however, finding the proper indication for this toxic therapy is critical for reducing unavoidable side effects. We recently observed that high survivin expression in CD133{sup +} cells is related to chemoresistance in Caco-2 colon cancer cells. However, the effect of survivin-targeted therapy on CD133{sup +} colon cancer is unknown. In this study, we investigated the roles of CD133 and survivin expression in colon cancer biology in vitro and comparatively analyzed the anticancer effects of survivin inhibitor on CD133{sup +} cells (ctrl-siRNA group) and small interfering RNA (siRNA)-induced CD133{sup −} cells (CD133-siRNA group) obtained from a single colon cancer cell line. CD133 knockdown via siRNA transfection did not change the tumorigenicity of cells, although in vitro survivin expression levels in CD133{sup +} cells were higher than those in siRNA-induced CD133{sup −} cells. The transfection procedure seemed to induce survivin expression. Notably, a significant number of CD133{sup −} cells (33.8%) was found in the cell colonies of the CD133-siRNA group. In the cell proliferation assay after treatment, YM155 and a combination of YM155 and 5-fluorouracil (5-FU) proved to be far more effective than 5-FU alone. A significantly increased level of apoptosis was observed with increasing doses of YM155 in all groups. However, significant differences in therapeutic effect and apoptosis among the mock, ctrl-siRNA, and CD133-siRNA groups were not detected. Survivin inhibitor is an effective treatment modality for colon cancer; however, the role of CD133 and the use of survivin expression as a biomarker for this targeted therapy must be verified.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Activation of Nrf2 attenuates carbonyl stress induced by methylglyoxal in human neuroblastoma cells: Increase in GSH levels is a critical event for the detoxification mechanism

The present study focused on the methylglyoxal (MG) detoxification mechanism in neuroblastoma cells. The involvement of nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway as a defense response against the formation of MG-modified proteins, which is well-known evidence of carbonyl stress, was also examined. We found that MG treatment resulted in accumulation of modified proteins bearing the structure of advanced glycation end products (AGEs) derived from MG in SH-SY5Y cells. This accumulation was suppressed by activation of the Nrf2 pathway prior to MG exposure via pre-treatment with an Nrf2 activator, carnosic acid and CDDO-Im, confirming the involvement of the Nrf2 pathway in MG detoxification. Although pre-treatment with the Nrf2 activator did not affect mRNA levels of GLO1, AKR1B1, and AKR7A2, the expressions of GCL and xCT mRNA, involved in GSH synthesis, were induced prior to increase in GSH levels. Furthermore, we demonstrated that a GSH synthesis inhibitor eliminated the MG detoxification effect derived from pretreatment with the Nrf2 activator. These results indicated that increase in GSH levels, induced by pre-treatment with carnosic acid, promoted the formation of the GLO1 substrate, hemithioacetal, thereby accelerating MG metabolism via the glyoxalase system and suppressing its toxicity. It was, therefore, determined that promotion of GSH synthesis via the Nrf2/Keap1pathway is important in the MG detoxification mechanism against neuronal MG-induced carbonyl stress, and Nrf2 activators contribute to reduction in the accumulation and toxic expression of carbonyl proteins. - Highlights: • Nrf2 activator prevents methylglyoxal-induced cytotoxicity and protein modifications. • MG treatment resulted in accumulation of modified proteins bearing the structure of CEL and MG-H1. • The promotion of GSH synthesis by activation of Nrf2 is a key response to MG detoxification.

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

Increase in proteins involved in mitochondrial fission, mitophagy, proteolysis and antioxidant response in type I endometrial cancer as an adaptive response to respiratory complex I deficiency

Pathogenic mtDNA mutations associated with alterations of respiratory complex I, mitochondrial proliferation (oncocytic-like phenotype) and increase in antioxidant response were previously reported in type I endometrial carcinoma (EC). To evaluate whether in the presence of pathogenic mtDNA mutations other mitochondrial adaptive processes are triggered by cancer cells, the expression level of proteins involved in mitochondrial dynamics, mitophagy, proteolysis and apoptosis were evaluated in type I ECs harboring pathogenic mtDNA mutations and complex I deficiency. An increase in the fission protein Drp1, in the mitophagy protein BNIP3, in the mitochondrial protease CLPP, in the antioxidant and anti-apoptotic protein ALR and in Bcl-2 as well as a decrease in the fusion protein Mfn2 were found in cancer compared to matched non malignant tissue. Moreover, the level of these proteins was measured in type I EC, in hyperplastic (the premalignant form) and in non malignant tissues to verify whether the altered expression of these proteins is a common feature of endometrial cancer and of hyperplastic tissue. This analysis confirmed in type I EC samples, but not in hyperplasia, an alteration of the expression level of these proteins. These results suggest that in this cancer mitochondrial fission, antioxidant and anti-apoptotic response may be activated, as well as the discharge of damaged mitochondrial proteins as adaptation processes to mitochondrial dysfunction.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Telmisartan mitigates hyperglycemia-induced vascular inflammation by increasing GSK3β-Ser{sup 9} phosphorylation in endothelial cells and mouse aortas

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), attenuates hyperglycemia-aggravated vascular inflammation by decreasing IκB kinase β (IKKβ) expression in endothelial cells. Because glycogen synthase 3β (GSK3β) is involved in inflammatory process by regulating nuclear factor-κB (NF-κB) activity, we investigated whether GSK3β mediates telmisartan-ameliorated vascular inflammation in hyperglycemia-treated endothelial cells and high-fat diet (HFD)-fed mice. Telmisartan remarkably induced GSK3β-Ser{sup 9} phosphorylation in hyperglycemia-treated endothelial cells that accompanied a decrease in hyperglycemia-induced NF-κB p65-Ser{sup 536} phosphorylation, vascular cell adhesion molecule-1 (VCAM-1) expression, and THP-1 monocyte adhesion. Ectopic expression of GSK3β-S9A, a constitutively active mutant of GSK3β, significantly restored complete telmisartan-inhibited NF-κB p65-Ser{sup 536} phosphorylation, VCAM-1 expression, and THP-1 monocyte adhesion. In addition, it reversed telmisartan-repressed IKKβ expression. Among the ARB, including losartan and fimasartan, only telmisartan increased GSK3β-Ser{sup 9} phosphorylation, and telmisartan-induced GSK3β-Ser{sup 9} phosphorylation remained unchanged by pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist. Finally, in the aortas of HFD-fed mice, telmisartan treatment significantly attenuated HFD-induced upregulation of NF-κB p65-Ser{sup 536} phosphorylation, VCAM-1 expression, and IKKβ expression and downregulation of GSK3β-Ser{sup 9} phosphorylation. Taken together, our findings demonstrated that telmisartan ameliorates hyperglycemia-exacerbated vascular inflammation, at least in part, by inducing GSK3β-Ser{sup 9} phosphorylation, which consequently inhibits IKKβ expression, NF-κB p65-Ser{sup 536} phosphorylation, and VCAM-1 expression in a PPARγ-independent manner. - Highlights: • Telmisartan inhibits GSK3β activity by inducing GSK3β-Ser{sup 9} phosphorylation. • Increased GSK3β-Ser{sup 9} phosphorylation leads to a decrease in IKKβ expression. • Reduced IKKβ expression attenuates NF-κB p65 phosphorylation and VCAM-1 expression. • Finally, these mediate ameliorating effects of telmisartan on vascular inflammation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Increased cellular uptake of peptide-modified PEGylated gold nanoparticles

Gold nanoparticles are promising drug delivery vehicles for nucleic acids, small molecules, and proteins, allowing various modifications on the particle surface. However, the instability and low bioavailability of gold nanoparticles compromise their clinical application. Here, we functionalized gold nanoparticles with CPP fragments (CALNNPFVYLI, CALRRRRRRRR) through sulfhydryl PEG to increase their stability and bioavailability. The resulting gold nanoparticles were characterized with transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible spectrometry and X-ray photoelectron spectroscopy (XPS), and the stability in biological solutions was evaluated. Comparing to PEGylated gold nanoparticles, CPP (CALNNPFVYLI, CALRRRRRRRR)-modified gold nanoparticles showed 46 folds increase in cellular uptake in A549 and B16 cell lines, as evidenced by the inductively coupled plasma atomic emission spectroscopy (ICP-AES). The interactions between gold nanoparticles and liposomes indicated CPP-modified gold nanoparticles bind to cell membrane more effectively than PEGylated gold nanoparticles. Surface plasmon resonance (SPR) was used to measure interactions between nanoparticles and the membrane. TEM and uptake inhibitor experiments indicated that the cellular entry of gold nanoparticles was mediated by clathrin and macropinocytosis. Other energy independent endocytosis pathways were also identified. Our work revealed a new strategy to modify gold nanoparticles with CPP and illustrated the cellular uptake pathway of CPP-modified gold nanoparticles.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells

Highlights: • Hypoxia is a common characteristic of many types of solid tumors. • Hypoxia increased S-glutathionylated proteins in colon cancer cells. • Protein S-glutathionylation regulated HIF-1α protein levels in hypoxic colon cancer cells. • Overexpression of Grx1 decreased HIF-1α levels and suppressed hypoxia response gene expression in hypoxic colon cancer cells. Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial–mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

A-674563 increases chondrocyte marker expression in cultured chondrocytes by inhibiting Sox9 degradation

Highlights: • We established a high-throughput screening system for induction of chondrocytic characters. • We screened 5822 compounds and found that A-674563 enhanced transcription of chondrocyte markers. • A-674563 inhibited the degradation of Sox9 protein. • A-674563 increased expression of ubiquitin-specific peptidase 29, which is known to induce deubiquitination. The implantation of autologous chondrocytes is a therapeutic treatment for articular cartilage damage. However, the benefits are limited due to the expansion of chondrocytes in monolayer culture, which causes loss of chondrocytic characters. Therefore, culture conditions that enhance chondrocytic characters are needed. We screened 5822 compounds and found that A-674563 enhanced the transcription of several chondrocyte marker genes, including Col2a1, Acan and Col11a2, in mouse primary chondrocytes. Experiments using cycloheximide, MG132 and bafilomycin A1 have revealed that Sox9 is degraded through the ubiquitin-proteasome pathway and that A-674563 inhibits this degradation, resulting in larger amount of Sox9 protein. RNA sequencing transcriptome analysis showed that A-674563 increases the expression of the gene that encodes ubiquitin-specific peptidase 29, which is known to induce the deubiquitination of proteins. Although the precise mechanism remains to be determined, our findings indicated that A-674563 could contribute to culture conditions that expand chondrocytes without losing chondrocytic characters.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Serum extracellular vesicles promote proliferation of H9C2 cardiomyocytes by increasing miR-17-3p

Highlights: • Serum extracellular vesicles promotes H9C2 cardiomyocytes proliferation. • miR-17-3p mediates the pro-proliferation effects. • TIMP3 is a target gene of miR-17-3p. Emerging evidence showed that cardiac proliferation played a significant role in the cardiac rehabilitation and repair. Extracellular vesicles (EVs) are known to regulate multiple cell functions, whereas the role of EVs in cardiac proliferation still remains unclear. In this study, we found that serum EVs promoted cell proliferation in rat heart myoblastic H9C2 cells with significantly increased expression level of miR-17-3p. Inhibition of miR-17-3p could decrease H9C2 cells proliferation induced by serum EVs. Additionally, we found that TIMP3 was a target of miR-17-3p in H9C2 cells proliferation and the expression of TIMP3 was downregulated by serum EVs. Meanwhile, inhibition of TIMP3 increased cardiac proliferation. In conclusion, results of our study indicated that serum EVs could promote the proliferation of H9C2 cells via regulating miR-17-3p/TIMP3, which may be a potential therapeutic target for treatment of cardiac injury.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

A novel heme oxygenase-1 splice variant, 14kDa HO-1, promotes cell proliferation and increases relative telomere length

Alternative splicing is a routine phenomenon which greatly increases the diversity of proteins in eukaryotic cells. In humans, most multi-exonic genes are alternatively spliced and their splice variants confer distinct functions. Heme oxygenase-1 (HO-1, 32 kDa) is an inducible stress responsive protein, which possesses multiple functions in many cellular processes. In the current study, we identified a novel alternative splice isoform of 14 kDa HO-1 generated through exclusion of exon 3, and it is highly expressed in immortalized cells. In contrast to nuclear accumulation of the full-length 32 kDa HO-1, the novel 14 kDa HO-1 isoform is retained in the cytoplasm under ultraviolet (UV) irradiation. Interestingly, the 14 kDa HO-1 is shown to promote cell proliferation and an increase in relative telomere lengths in vivo and in vitro. Thus, we are pioneer to report and confirm the presence of a novel splice form of HO-1 and its distinct role in modulating telomere length and tumor growth.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

PKC delta activation increases neonatal rat retinal cells survival in vitro: Involvement of neurotrophins and M1 muscarinic receptors

Protein kinase C (PKC) is a family of serine/threonine kinases related to several phenomena as cell proliferation, differentiation and survival. Our previous data demonstrated that treatment of axotomized neonatal rat retinal cell cultures for 48 h with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increases retinal ganglion cells (RGCs) survival. Moreover, this treatment decreases M1 receptors (M1R) and modulates BDNF levels. The aim of this work was to assess the possible involvement of neurotrophins BDNF and NGF in the modulation of M1R levels induced by PKC activation, and its involvement on RGCs survival. Our results show that PMA (50 ng/mL) treatment, via PKC delta activation, modulates NGF, BDNF and M1R levels. BDNF and NGF mediate the decrease of M1R levels induced by PMA treatment. M1R activation is essential to PMA neuroprotective effect on RGCs as telenzepine (M1R selective antagonist) abolished it. Based on our results we suggest that PKC delta activation modulates neurotrophins levels by a signaling pathway that involves M1R activation and ultimately leading to an increase in RGCs survival in vitro.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

2-Deoxy-d-glucose increases GFAT1 phosphorylation resulting in endoplasmic reticulum-related apoptosis via disruption of protein N-glycosylation in pancreatic cancer cells

Highlights: • 2DG causes ER stress-induced cell death by disrupting protein N-glycosylation. • This is caused by inactivation of GFAT1 in pancreatic cancer. • In addition, combination treatment with 2DG and Met enhanced disruption of protein N-glycosylation. The glycolytic inhibitor 2-deoxy-d-glucose (2DG) causes energy starvation, affecting cell viability in a wide range of cancer cell lines. To determine the action of 2DG in pancreatic cancer, we performed proteomic analysis of pancreatic cancer cell line after 2DG treatment. Eighty proteins showed differential expression and among these, proteins involved in phosphohexose metabolism were upregulated. Up-regulation of glutamine: fructose 6-phosphate aminotransferase 1 (GFAT1), which belongs to the hexosamine biosynthesis pathway (HBP) that produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to maintain glycoprotein, was validated by evaluation of mRNA and protein levels. Therefore, we assessed the amounts of total N-glycoproteins. Unexpectedly, we found a reduction of total N-glycoproteins and phosphorylation of GFAT1 by AMP-activated protein kinase (AMPK). These data may shed light on HBP dysfunction. Furthermore, we found endoplasmic reticulum (ER) stress accompanied by increased expression of ER stress markers, such as glucose response protein 78 (GRP78) and C/EBP-homologous protein (CHOP), in 2DG-treated cells. Moreover, the additive activation of AMPK by metformin (Met) synergistically enhanced the reduction of protein N-glycosylation and cell growth inhibition in the presence of 2DG. These results suggest that 2DG reduces N-glycosylation of proteins following the increase of phosphorylation of GFAT1 and results in the inhibition of cell growth mediated by ER stress in pancreatic cancer cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

1-α,25-dihydroxyvitamin D3 potentiates avian osteoclast activation by increasing the formation of zipper-like structure via Src/Rac1 signaling

Highlights: • 1α,25-(OH){sub 2}D{sub 3} potentiates the formation and activation of avian osteoclasts. • 1α,25-(OH){sub 2}D{sub 3} promotes the formation of the zipper-like structure in OC precursors. • 1α,25-(OH){sub 2}D{sub 3} stimulates zipper-like structure formation via Src/Rac1 signaling. Avian bone metabolism diseases affect the development and production of chickens, and many of these diseases can be prevented and controlled by balanced nutrition and hormone medicine. The steroid hormone 1α,25-dihydroxyvitamin D{sub 3} plays a key role in maintaining the balance of avian bone metabolism. Clinically, 1α,25-(OH){sub 2}D{sub 3} has been used to treat several bone diseases. Although several previous studies have investigated the effects of 1α,25-(OH){sub 2}D{sub 3} on osteoclastogenesis, the mechanisms underpinning osteoclast (OC) activity remain largely unknown. Herein, we used molecular and cell biology approaches to demonstrate that 1α,25-(OH){sub 2}D{sub 3} increases avian OC formation and activity, and upregulates bone resorption-related genes. Moreover, 1α,25-(OH){sub 2}D{sub 3} regulates the OC cytoskeleton by increasing the formation of zipper-like structure in OC precursor cells to potentiate OC activity via the Src/Rac1 signaling pathway. These findings provide new insight into the role of 1α,25-(OH){sub 2}D{sub 3} in OC activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Structure-based protein engineering of bacterial β-xylosidase to increase the production yield of xylobiose from xylose

Highlights: • The crystal structures of β-xylosidase from Bacillus pumilus in complex with xylobiose were determined. • β-xylosidase was engineered to increase the yield of xylobiose based on the determined structures. • The protein engineering strategy used in this study could be applied within the same family of enzymes. Xylobiose consists of two molecules of xylose and has been highly recognized as a food supplement because it possesses high prebiotic functions. β-xylosidase exhibits enzymatic activity to hydrolyze xylobiose, and the enzyme can also catalyze the reverse reaction in the presence of high concentrations of xylose. Previously, β-xylosidase from Bacillus pumilus IPO (BpXynB), belonging to GH family 43, was employed to produce xylobiose from xylose. To improve the enzymatic efficiency, this study determined the high-resolution structure of BpXynB in a complex with xylobiose and engineered BpXynB based on the structures. The structure of BpXynB deciphered the residues involved in the recognition of the xylobiose. A site-directed mutation at the residue for xylobiose recognition increased the yield of xylobiose by 20% compared to a similar activity of the wild type enzyme. The complex structure of the mutant enzyme and xylobiose provided the structural basis for a higher yield of the engineered protein. This engineered enzyme would enable a higher economic production of xylobiose, and a similar engineering strategy could be applied within the same family of enzymes.

60 APPLIED LIFE SCIENCES↗