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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 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Substituted (E)-2-(2-benzylidenehydrazinyl)-4-methylthiazole-5-carboxylates as dual inhibitors of 15-lipoxygenase & carbonic anhydrase II: synthesis, biochemical evaluation and docking studies

15-Lipoxygenase (15-LOX) plays a major role in many inflammatory lung diseases including chronic obstructive pulmonary disease (COPD), asthma and chronic bronchitis. Over-expression of 15-LOX is related with some specific carcinomas including pancreatic, gastric and brain tumor. Similarly among different isozymes of carbonic anhydrase (CA), CA II is expressed in pancreatic, gastric carcinomas as well as in brain tumors. Therefore, novel potent inhibitors of both 15-LOX and CA II are required to explore the role of these enzymes further and to enable the drug discovery efforts. For this purpose, a series of benzyledinyl-hydrazinyl substituted thiazole derivatives were designed, synthesized and characterized by FTIR, {sup 1}H, &{sup 13}C NMR spectroscopy. The derivatives were then evaluated for their potential to inhibit 15-LOX and bovine carbonic anhydrase II (bCA II). Most of these compounds showed excellent inhibitory potential for 15-LOX with an IC{sub 50} of 0.12 ± 0.002 to 0.69 ± 0.5 μM and showed moderate inhibition potency for bCA II with compound 5h (IC{sub 50} = 1.26 ± 0.24 μM) being the most active. The most potent compound 5a that emerged as a dual inhibitor of both enzymes, exhibiting 24 times greater selectivity for 15-LOX over bCA II. Compound 5a exhibited dual potent inhibitory activity against both 15-LOX and bCA II enzymes having IC{sub 50} values of 0.12 ± 0.002 and 2.93 ± 0.22 μM, respectively. Molecular docking studies of potent as well as dual inhibitors were also carried out to provide an insight into the binding site interactions. - Graphical abstract: Putative binding mode of most potent compound in the active pocket of 15-lipoxygenase. Display Omitted.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Autophagy protects against cholesterol-induced apoptosis in pancreatic β-cells

Autophagy is believed to play an important role in maintaining homeostasis in pancreatic β-cells during insulin resistance. This study investigated the role of autophagy in β-cell damage induced by cholesterol and its possible activation mechanism. Rat and mouse pancreatic β-cell lines INS-1 and βTC-6 were incubated with cholesterol alone or in combination with autophagy inhibitors E-64d/Pepstatin A or bafilomycin A1. DAPI staining, western blotting, transmission electron microscopy and immunofluorescence were conducted to assess the effects of autophagy inhibitors on cholesterol-induced apoptosis and autophagy activity. An increase in FITC-LC3 fluorescence dots, autophagic vacuoles and LC3-II protein indicated that autophagy was activated in cells treated with cholesterol. This was further confirmed by blocking the natural turnover processes in lysosomes and autolysosomes with autophagy inhibitors, suggesting enhanced autophagic activity rather than blockage of autophagy. Furthermore, inhibition of autophagy significantly augmented the activation of caspase 3 and the percentage of cholesterol-induced apoptotic nuclei. These results demonstrate that autophagy plays a protective role against cholesterol-induced apoptosis in pancreatic β-cells. - Highlights: • Cholesterol increased autophagy in βTC-6 and INS-1 cells. • Cholesterol significantly enhanced autophagy activity in βTC-6 and INS-1 cells. • Inhibition of autophagy aggravated cholesterol-induced apoptosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Long non-coding RNA NEAT1 facilitates pancreatic cancer progression through negative modulation of miR-506-3p

Recently, long non-coding RNAs (lncRNAs) have been shown to have critical regulatory roles in tumourigenesis. Increasing evidence has suggested that lncRNA NEAT1 has been implicated in various types of human cancer. However, the potential biological roles and regulatory mechanisms of NEAT1 in pancreatic cancer (PC) remains unclear. Here, we found that the expression level of NEAT1 was higher in PC tissues compared to the corresponding non-tumor tissues. Besides, our findings indicate that high NEAT1 expression level is closely correlated with tumor progression and poor survival in PC patients. Furthermore, we also found that knockdown of NEAT1 remarkably suppressed cell proliferation by inducing cell cycle arrest and apoptosis promotion in PC cells. Moreover, bioinformatics analysis and luciferase reporter assay revealed that NEAT1 directly bound to the miR-506-3p, which has been reported to act as a tumor suppressor in diverse cancers. Additionally, our results confirmed that the tumor-promoting effects of NEAT1 in PC cells is at least partly through negative modulation of miR-506-3p. Overall, our results suggested that NEAT1 functions as an oncogenic lncRNA in PC, which could be a novel diagnostic and therapeutic target for PC. - Highlights: • Upregulation of NEAT1 expression was significantly associated with the poor survival in PC patients. • Knockdown of NEAT1 can suppress PC cells growth by inducing cell cycle arrest and apoptosis promotion in vitro study. • NEAT1 functions as an oncogene in pancreatic cancer by acting as a competing endogenous RNA for miR-506-3p. • The tumor-suppressive effects of NEAT1 knockdown in PC cells was partially reversed by miR-506-3p inhibitor.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Identification of miR-601 as a novel regulator in the development of pancreatic cancer

Pancreatic cancer (PC) is one of the most aggressive malignancies, with a high mortality. Distant metastasis and recurrence are the main causes of PC-related deaths. MicroRNAs (miRNAs) have been reported in the serum or tumor tissue of cancer patients, including PC, which makes them potential biomarkers. The dysfunction of many miRNAs has been linked to PC occurrence and metastasis. In the current study, we found that miR-601 expression was significantly lower in PC samples, especially in metastatic compared to non-metastatic PC tissues. Gain-of-function and loss-of-function analysis showed that miR-601 suppressed PC cell proliferation and migration. One potential mechanism is that miR-601 inhibited the expression of Sirtuin 1 (SIRT1), which is a well-known regulator of PC development. We found that overexpression of SIRT1 could reverse the effect of miR-601 on PC cells. Our investigation therefore suggests that both miR-601 and SIRT1 are possible biomarkers for the early detection, and targets for the treatment of PC. - Highlights: • miR-601 was found to be downregulated in metastatic pancreatic cancer. • miR-601 inhibited pancreatic cancer proliferation and migration. • Sirt1 was identified as a novel target of miR-601. • Sirt1 mediated the effect of miR-601 on pancreatic cancer growth.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Palmitic acid increases invasiveness of pancreatic cancer cells AsPC-1 through TLR4/ROS/NF-κB/MMP-9 signaling pathway

Pancreatic cancer (PC) is an aggressive malady with proclivity for early metastasis. Overexpression of toll-like receptor 4 (TLR4) in pancreatic ductal adenocarcinoma, the most common type of pancreatic malignancy, correlates to tumor size, lymph node involvement, venous invasion and pathological stage. Lipopolysaccharides (LPS) are natural TLR4 ligands that have been shown to increase the invasive ability of PC cells. However, rapid inactivation of circulating LPS and low systemic absorption of inhaled LPS from the bronchoalveolar compartment make other agonists such as saturated fatty acids more suitable to be considered for TLR4-related cell invasiveness. Interestingly, PC risk was strongly associated to intake of saturated fat from animal food sources, in particular to consumption of saturated palmitic acid (PA). In the present study, we investigated the influence of PA on the invasive capacity of human PC cells AsPC-1. Using specific inhibitors, we found that PA stimulation of these tumor cells induced a TLR4-mediated cell invasion. Our results also indicate that the signaling events downstream of TLR4 involved generation of reactive oxygen species, activation of nuclear factor-kappa beta, and secretion and activation of matrix metalloproteinase 9 (MMP-9). Furthermore, PA stimulation decreased the levels of the micro RNA 29c (miR-29c). Of note, while inhibition of miR-29c increased MMP-9 mRNA levels, MMP-9 secretion and activation, and invasiveness, miR-29c mimic abrogated all these PA-stimulated effects. These results strongly suggest that miR-29c could be an attractive potential pharmacological agent for antitumoral therapy in PC. - Highlights: • Palmitic acid stimulates TLR4-mediated pancreatic cancer cell invasion. • Activation of NF-κB by palmitic acid depends on ROS generation. • Palmitic acid decreases the level of miR-29C through NF-κB. • Inhibition of miR-29c increases MMP-9 secretion and activation, and invasiveness.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Ku70 inhibits gemcitabine-induced DNA damage and pancreatic cancer cell apoptosis

The current study focused on the role of Ku70, a DNA-dependent protein kinase (DNA-PK) complex protein, in pancreatic cancer cell resistance to gemcitabine. In both established cell lines (Mia-PaCa-2 and PANC-1) and primary human pancreatic cancer cells, shRNA/siRNA-mediated knockdown of Ku70 significantly sensitized gemcitabine-induced cell death and proliferation inhibition. Meanwhile, gemcitabine-induced DNA damage and subsequent pancreatic cancer cell apoptosis were also potentiated with Ku70 knockdown. On the other hand, exogenous overexpression of Ku70 in Mia-PaCa-2 cells suppressed gemcitabine-induced DNA damage and subsequent cell apoptosis. In a severe combined immune deficient (SCID) mice Mia-PaCa-2 xenograft model, gemcitabine-induced anti-tumor activity was remarkably pontificated when combined with Ku70 shRNA knockdown in the xenografts. The results of this preclinical study imply that Ku70 might be a primary resistance factor of gemcitabine, and Ku70 silence could significantly chemo-sensitize gemcitabine in pancreatic cancer cells. - Highlights: • Ku70 knockdown sensitizes gemcitabine-induced killing of pancreatic cancer cells. • Ku70 knockdown facilitates gemcitabine-induced DNA damage and cell apoptosis. • Ku70 overexpression deceases gemcitabine's sensitivity in pancreatic cancer cells. • Ku70 knockdown sensitizes gemcitabine-induced anti-tumor activity in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

A role of pancreatic stellate cells in islet fibrosis and β-cell dysfunction in type 2 diabetes mellitus

Objectives: To investigate whether the activation of pancreatic stellate cells (PSCs) leads to pancreatic β-cell dysfunction in type 2 diabetes mellitus (T2DM). Methods: The pancreases of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of T2DM, and patient with T2DM were analyzed. And the in vitro and in vivo effects of pirfenidone, an antifibrotic agent, on PSC activation, islet fibrosis, and β-cells were studied. Results: The extent of islet fibrosis and the percentage of activated PSCs, positive for α-smooth muscle actin, in the islets were significantly greater in OLETF rats compared with non-diabetic rats. Also, the extent of islet fibrosis in patients with T2DM was slightly greater compared with age- and BMI-matched non-diabetic patients. In rat PSCs cultured with high glucose for 72 h, pirfenidone produced decreases in cell proliferation, release of collagen, and the expression of fibronectin and connective tissue growth factor. Treatment of OLETF rats with pirfenidone for 16 weeks decreased the activation of PSCs and the extent of islet fibrosis, but did not enhance glucose tolerance, pancreatic insulin content, or β-cell mass. Conclusions: Activated PSCs in islets might lead to islet fibrosis in T2DM. However, PSC activation itself might not contribute significantly to progressive β-cell failure in T2DM. - Highlights: • Islet fibrosis developed progressively in OLETF rats, a model of type 2 diabetes. • PSCs in the islets became activated in OLETF rats. • Islet fibrosis was increased in patients with type 2 diabetes. • Pirfenidone attenuated the activation of PSCs and islet fibrosis in OLETF rats. • Pirfenidonet had no effects on glucose tolerance or on β-cells in OLETF rats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion. - Highlights: • The fused protein of proglucagon to Gaussia luciferase was used as a reporter. • The fusion protein was highly expressed using a preferred human-codon optimized gene. • Glucagon secretion stimulated by depolarization was determined by luminescence. • Glucagon secretion in α cells was visualized by bioluminescence imaging. • Glucagon secretion sites were localized in the intercellular contact regions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

USP39, a direct target of microRNA-133a, promotes progression of pancreatic cancer via the AKT pathway

Ubiquitin specific protease 39 (USP39) is one of the deubiquitinating enzymes without ubiquitin protease activity, which has been implicated in the progression of several cancers. However, the role of USP39 in pancreatic cancer (PC) is largely unknown. In present study, we found that USP39 expression was elevated in PC tissues than adjacent non-tumor tissues. Importantly, we demonstrated that overexpression of USP39 is closely correlated with tumor progression and poor survival in PC patients. Furthermore, high USP39 expression was observed in PC cell lines and ectopic expression of USP39 significantly enhanced in vitro cell proliferation and promoted in vivo tumor growth, whereas silencing USP39 suppressed growth of PC cells. Besides, our experimental data revealed that knockdown of USP39 induced cell apoptosis through inhibition of AKT signaling pathway in PC cells. Moreover, USP39 was a direct target of miR-133a, a microRNA that has been reported to be involved in progression of PC. Taken together, our data provide a novel PC regulatory axis that is miR-133a/USP39, the dysfunction of which drives diverse aspects of the progression of PC. - Highlights: • Upregulation of USP39 expression was significantly associated with the progression and poor survival in PC patients. • Overexpression of USP39 promotes PC cell growth in vitro and in vivo. • AKT pathway partly contributed to the oncogenic effects of USP39 in PC cells. • USP39 was found to be a direct target of miR-133a, which has been reported to be involved in progression of PC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Quantitative visualization of synchronized insulin secretion from 3D-cultured cells

Quantitative visualization of synchronized insulin secretion was performed in an isolated rat pancreatic islet and a spheroid of rat pancreatic beta cell line using a method of video-rate bioluminescence imaging. Video-rate images of insulin secretion from 3D-cultured cells were obtained by expressing the fusion protein of insulin and Gaussia luciferase (Insulin-GLase). A subclonal rat INS-1E cell line stably expressing Insulin-GLase, named iGL, was established and a cluster of iGL cells showed oscillatory insulin secretion that was completely synchronized in response to high glucose. Furthermore, we demonstrated the effect of an antidiabetic drug, glibenclamide, on synchronized insulin secretion from 2D- and 3D-cultured iGL cells. The amount of secreted Insulin-GLase from iGL cells was also determined by a luminometer. Thus, our bioluminescence imaging method could generally be used for investigating protein secretion from living 3D-cultured cells. In addition, iGL cell line would be valuable for evaluating antidiabetic drugs. - Highlights: • An imaging method for protein secretion from 3D-cultured cells was established. • The fused protein of insulin to GLase, Insulin-GLase, was used as a reporter. • Synchronous insulin secretion was visualized in rat islets and spheroidal beta cells. • A rat beta cell line stably expressing Insulin-GLase, named iGL, was established. • Effect of an antidiabetic drug on insulin secretion was visualized in iGL cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Aquaporin 3 facilitates tumor growth in pancreatic cancer by modulating mTOR signaling

Aquaporins (AQP) have been demonstrated to be dysregulated in many human cancers and is thought to be involved in pancreatic carcinogenesis and progression. However, the oncogenic roles and underlying mechanism of AQP in pancreatic ductal adenocarcinoma (PDAC) remain largely unknown. In this study, by data mining of TCGA dataset and CCLE database, we identified that AQP3 is the major AQP expressed in PDAC. Then, the microRNA-874, was demonstrated to be a key regulator of AQP3 expression in PDAC cells. Genetic silencing of AQP3 expression had pronounced effects on cell proliferation and apoptosis of the PDAC cell lines BXPC3 and HPAFII. Introduction of microRNA-874 suppressed cell proliferation and promoted cell apoptosis, whereas inhibition of microRNA-874 had the opposite effect. Mechanistically, by a large-scale proteomic analysis, we revealed that AQP3 was significantly associated the activity of mTOR signaling. Moreover, modulation of AQP3 or microRNA-874 altered mTOR activity as demonstrated by the phosphorylation level of mTOR and its downstream target S6. Taken together, our data, as a proof of principle, suggest that AQP3 can promote tumor growth of pancreatic cancer cells by activating the Mtor signaling pathway and provide a potential therapeutic target in the treatment of PDAC. - Highlights: • Dysregulated expression of AQP3 is induced by miR-874 in pancreatic cancer. • miR-874-AQP3 axis is involved in cell proliferation and apoptosis of pancreatic cancer cells. • AQP3 correlates increased mTOR activity in pancreatic cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Regulation of Nampt expression by transcriptional coactivator NCOA6 in pancreatic β-cells

Nuclear receptor coactivator 6 (NCOA6) is a transcriptional coactivator and crucial for insulin secretion and glucose metabolism in pancreatic β-cells. However, the regulatory mechanism of β-cell function by NCOA6 is largely unknown. In this study, we found that the transcript levels of nicotinamide phosphoribosyltransferase (Nampt) were decreased in islets of NCOA6{sup +/−} mice compared with NCOA6{sup +/+} mice. Moreover, NCOA6 overexpression increased the levels of Nampt transcripts in the mouse pancreatic β-cell line NIT-1. Promoter analyses showed that transcriptional activity of the Nampt promoter was stimulated by cooperation of sterol regulatory element binding protein-1c (SREBP-1c) and NCOA6. Additional studies using mutant promoters demonstrated that SREBP-1c activates Nampt promoter through the sterol regulatory element (SRE), but not through the E-box. Using chromatin immunoprecipitation assay, NCOA6 was also shown to be directly recruited to the SRE region of the Nampt promoter. Furthermore, treatment with nicotinamide mononucleotide (NMN), a product of the Nampt reaction and a key NAD{sup +} intermediate, ameliorates glucose-stimulated insulin secretion from NCOA6{sup +/−} islets. These results suggest that NCOA6 stimulates insulin secretion, at least partially, by modulating Nampt expression in pancreatic β-cells. - Highlights: • Nampt transcription in β-cells is activated by SREBP-1c through the SRE element. • NCOA6 enhances the transcriptional activity of SREBP-1c in the Nampt promoter. • Defective insulin secretion of NCOA6{sup +/−} islets is recovered by NMN treatment. • NCOA6 is reportedly the first coactivator involved in Nampt expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Sterol regulatory element-binding protein 1 inhibitors decrease pancreatic cancer cell viability and proliferation

Sterol regulatory element-binding protein1 (SREBP1) is a key regulatory factor that controls lipid homeostasis. Overactivation of SREBP1 and elevated lipid biogenesis are considered the major characteristics in malignancies of prostate cancer, endometrial cancer, and glioblastoma. However, the impact of SREBP1 activation in the progression of pancreatic cancer has not been explored. The present study examines the effect of suppression of SREBP1 activation by its inhibitors like fatostatin and PF429242 besides analyzing the impact of inhibitory effects on SREBP1 downstream signaling cascade such as fatty acid synthase (FAS), hydroxymethylglutaryl-CoA reductase (HMGCoAR), stearoyl-CoA desaturase-1 (SCD-1), and tumor suppressor protein p53 in MIA PaCa-2 pancreatic cancer cells. Both fatostatin and PF429242 inhibited the growth of MIA PaCa-2 cells in a time and concentration-dependent manner with maximal inhibition attained at 72 h time period with IC{sub 50} values of 14.5 μM and 24.5 μM respectively. Detailed Western blot analysis performed using fatostatin and PF429242 at 72 h time point led to significant decrease in the levels of the active form of SREBP1 and its downstream signaling proteins such as FAS, SCD-1 and HMGCoAR and the mutant form of tumor suppressor protein, p53, levels in comparison to the levels observed in vehicle treated control group of MIA PaCa-2 pancreatic cells over the same time period. Our in vitro data suggest that SREBP1 may contribute to pancreatic tumor growth and its inhibitors could be considered as a potential target in the management of pancreatic cancer cell proliferation. - Highlights: • A significant increase in SREBP1 levels was observed in MIA PaCa-2 cells. • Fatostatin and PF429242 suppress SREBP1 activation and its downstream signaling proteins expression. • The inhibition of SREBP1reduces tumor suppressor protein p53 in MIA PaCa-2 cells. • SREBP1 inhibition may be beneficial in treatment of pancreatic cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Diabetogenic agent alloxan is a proteasome inhibitor

Alloxan has been used as a diabetogenic agent to induce diabetes. It selectively induces pancreatic β-cell death. The specific toxicity, however, is not fully understood. In this study, we observed the effect of alloxan on proteasome function. We found that alloxan caused the accumulation of ubiquitinated proteins in NRK cells through the inhibition of the proteolytic activities of the proteasome. Biochemistry experiments with purified 26S and 20S proteasomes revealed that alloxan directly acts on the chymotrypsin- and trypsin-like peptidase activities. These results demonstrate that alloxan is a proteasome inhibitor, which suggests that its specific toxicity toward β-cell is at least in part through proteasome inhibition. - Highlights: • Alloxan inhibits the clearance of ubiquitinated proteins. • Alloxan inhibits the clearance of recombinant proteasome substrate GFP-CL1. • Alloxan inhibits the peptidase activities of the proteasome in the NRK cell nuclear extract. • Alloxan inhibits the proteasome dependent degradation of GST-Sp1 in NRK cell nuclear extract. • Alloxan inhibits the peptidase activities of the purified 26S and 20S proteasomes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Substance P preserves pancreatic β-cells in streptozotocin-induced type 1 diabetic mice

Preservation of the pancreatic β-cell population is required for the development of therapies for diabetes, which is caused by a decrease in β-cells. Here, we demonstrate the antidiabetic effects of substance P (SP) in type 1 diabetes (T1D) mice induced with streptozotocin. SP enhanced the compensatory proliferation of β-cells in order to restore β-cells in response to acute injury induced by a single high-dose of streptozotocin. However, SP affected neither the basal proliferation of β-cells nor their apoptosis. In vitro studies by using the INS-1 pancreatic β-cell line showed that SP mediated the increase in the proliferation of β-cells via the activation of Akt. Chronic systemic treatment with SP restored the mass of β-cells and inhibited insulitis in T1D mice induced with multiple low-doses of streptozotocin. Therefore, systemic treatment with SP may be a promising therapeutic strategy for treating diabetes in patients with loss of functional β-cells. - Highlights: • Substance P increased β-cell proliferation in response to acute injury. • Substance P did not protect β-cells from apoptosis. • Substance P preserved β-cell mass and delayed diabetic hyperglycemia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

USP5 promotes tumorigenesis and progression of pancreatic cancer by stabilizing FoxM1 protein

Increased ubiquitin-specific protease 5 (USP5) has been associated with tumorigenesis of malignancy including glioblastoma, melanoma and hepatocellular carcinoma. However, the role of USP5 in tumorigenesis of pancreatic ductal adenocarcinoma (PDAC) has not been studied yet. In this study, we demonstrated that USP5 was significantly upregulated in a panel of PDAC cell lines and correlated with FoxM1 protein expression. USP5 knockdown inhibited proliferation of PANC-1 and SW1990, two PDAC cell lines. In the mouse xenografted pancreatic tumor model, suppression of USP5 significantly decreased tumor growth, correlated with down regulation of FoxM1. Additionally, we found that overexpression of USP5 stabilized the FoxM1 protein in PDAC cells. Overexpression of USP5 extended the half-life of FoxM1. Knockdown of USP5 in PANC-1 cells decreased FoxM1 protein level while the proteasome inhibitor MG-132 treatment restored FoxM1 expression. We also found that endogenous USP5 was coimmunoprecipitated with an endogenous FoxM1 from PANC-1 cells while FoxM1 was also coimmunoprecipitated with USP5. Furthermore, we also confirmed that USP5 regulated proliferation of PDAC via FoxM1 by rescuing the inhibitory effect of USP5 knockdown with ectopic expression of FoxM1 in USP5-depleted cells. Taken together, our study demonstrates that USP5 plays a critical role in tumorigenesis and progression of pancreatic cancer by stabilizing FoxM1 protein, and provides a rationale for USP5 being a potential therapeutic approach against PDAC. - Highlights: • USP5 is upregulated in pancreatic ductal adenocarcinoma cells. • USP5 promotes tumorigenesis of pancreatic cancer. • USP5 stabilizes FoxM1 protein.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Gadolinium chloride ameliorates acute lung injury associated with severe acute pancreatitis in rats by regulating CYLD/NF-κB signaling

The present study was embarked on an investigation of the mechanisms behind the effects of Gadolinium chloride (GdCl{sub 3}) on lung injury associated with severe acute pancreatitis (SAP) in rats. Rats were randomly distributed into three groups: sham operation group (SO), SAP group and SAP treated with GdCl{sub 3} group (SAP + GdCl{sub 3}). Retrograde injection of 5% sodium taurocholate into the biliopancreatic duct was adopted to induce SAP. Lung tissue specimens were harvested for histological study, wet-to-dry weight ratio calculation and myeloperoxidase examination. Meanwhile, bronchoalveolar lavage fluid was analyzed for TNF-α and IL-1β activity and proteins content. Then the apoptosis ratio of alveolar macrophages (AMs) was detected. NF-κB activation and cylindromatosis (CYLD) expression in AMs were measured respectively. Results showed that GdCl{sub 3} treatment notably ameliorated lung injury induced by SAP, and simultaneously, the apoptosis ratio of AMs was significantly promoted. The NF-κB activation was obviously inhibited when CYLD expression was markedly up-regulated in AMs of SAP + GdCl{sub 3}. Negative correlation was analyzed between CYLD and NF-κB in both SAP and SAP + GdCl{sub 3}. These data demonstrate that GdCl{sub 3} ameliorates lung injury secondary to SAP in rats mainly by up-regulating CYLD expression and inhibiting NF-κB activation in AMs, which may play a vital role in lung injury. - Highlights: • GdCl{sub 3} ameliorates lung injury associated with SAP by inducing the apoptosis of AMs. • NF-kB activation can inhibit the apoptosis of AMs. • CYLD negatively regulates NF-kB activation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

A hetero-micro-seeding strategy for readily crystallizing closely related protein variants

Protein crystallization remains difficult to rationalize and screening for optimal crystallization conditions is a tedious and time consuming procedure. Here, we report a hetero-micro-seeding strategy for producing high resolution crystals of closely related protein variants, where micro crystals from a readily crystallized variant are used as seeds to develop crystals of other variants less amenable to crystallization. We applied this strategy to Bovine Pancreatic Trypsin Inhibitor (BPTI) variants, which would not crystallize using standard crystallization practice. Out of six variants in our analysis, only one called BPTI-[5,55]A14G formed well behaving crystals; and the remaining five (A14GA38G, A14GA38V, A14GA38L, A14GA38I, and A14GA38K) could be crystallized only using micro-seeds from the BPTI-[5,55]A14G crystal. All hetero-seeded crystals diffracted at high resolution with minimum mosaicity, retaining the same space group and cell dimension. Moreover, hetero-micro-seeding did not introduce any biases into the mutant’s structure toward the seed structure, as demonstrated by A14GA38I structures solved using micro-seeds from A14GA38G, A14GA38L and A14GA38I. Though hetero-micro-seeding is a simple and almost naïve strategy, this is the first direct demonstration of its workability. We believe that hetero-micro-seeding, which is contrasting with the popular idea that crystallization requires highly purified proteins, could contribute a new tool for rapidly solving protein structures in mutational analysis studies. - Highlights: • In hetero-micro-seeding, hard-to-crystalize mutants are crystallized using micro-seeds from a readily crystalized variant. • Hetero-micro-seeding did not introduce any biases of the mutant structure toward the seed structure. • Though hetero-micro-seeding is a simple strategy, this is the first direct demonstration of its workability at atomic level.

36 MATERIALS SCIENCE↗