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At least 1,747 records · Page 97
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DOE OSTI2016

Heterologous gene expression and functional analysis of a type III polyketide synthase from Aspergillus niger NRRL 328

Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr-8-2: 2978617–2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni{sup 2+}-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity of the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. -- Highlights: •Type III PKS from Aspergillus niger NRRL 328, An-CsyA, was cloned and characterized. •An-CsyA produced triketide pyrones, tetraketide pyrones and pentaketide resorcinols. •Functional properties of An-CsyA differs from those of other fungal type III PKSs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity

Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. -- Highlights: •PARIS can be SUMOylated in vivo and in vitro. •SUMOylation of PARIS functions in the repression of PGC-1a promoter activity. •PIASy interacts with PARIS and enhances its SUMOylation. •PIASy influences PARIS-mediated repression of PGC-1a promoter activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Maternal exposure to carbamazepine at environmental concentrations can cross intestinal and placental barriers

Psychoactive pharmaceuticals have been found as teratogens at clinical dosage during pregnancy. These pharmaceuticals have also been detected in minute (ppb) concentrations in drinking water in the US, and are environmental contaminants that may be complicit in triggering neurological disorders in genetically susceptible individuals. Previous studies have determined that psychoactive pharmaceuticals (fluoxetine, venlafaxine and carbamazepine) at environmentally relevant concentrations enriched sets of genes regulating development and function of the nervous system in fathead minnows. Altered gene sets were also associated with potential neurological disorders, including autism spectrum disorders (ASD). Subsequent in vitro studies indicated that psychoactive pharmaceuticals altered ASD-associated synaptic protein expression and gene expression in human neuronal cells. However, it is unknown if environmentally relevant concentrations of these pharmaceuticals are able to cross biological barriers from mother to fetus, thus potentially posing risks to nervous system development. The main objective of this study was to test whether psychoactive pharmaceuticals (fluoxetine, venlafaxine, and carbamazepine) administered through the drinking water at environmental concentrations to pregnant mice could reach the brain of the developing embryo by crossing intestinal and placental barriers. We addressed this question by adding {sup 2}H-isotope labeled pharmaceuticals to the drinking water of female mice for 20 days (10 pre-and 10 post–conception days), and quantifying {sup 2}H-isotope enrichment signals in the dam liver and brain of developing embryos using isotope ratio mass spectrometry. Significant levels of {sup 2}H enrichment was detected in the brain of embryos and livers of carbamazepine-treated mice but not in those of control dams, or for fluoxetine or venlafaxine application. These results provide the first evidence that carbamazepine in drinking water and at typical environmental concentrations is transmitted from mother to embryo. Our results, combined with previous evidence that carbamazepine may be associated with ASD in infants, warrant the closer examination of psychoactive pharmaceuticals in drinking water and their potential association with neurodevelopmental disorders.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Androgen receptor regulated microRNA miR-182-5p promotes prostate cancer progression by targeting the ARRDC3/ITGB4 pathway

Abstracts: MicroRNAs (miRNAs) are important endogenous gene regulators that play key roles in prostate cancer development and metastasis. However, specific miRNA expression patterns in prostate cancer tissues from Chinese patients remain largely unknown. In this study, we compared miRNA expression patterns in 65 pairs of prostate cancer and para-cancer tissues by RNA sequencing and found that miR-182-5p was the most up-regulated miRNA in prostate cancer tissues. The result was validated using realtime PCR in 18 pairs of prostate cancer and para-cancer tissues. In in vitro analysis, it was confirmed that miR-182-5p promotes prostate cancer cell proliferation, invasion and migration and inhibit apoptosis. In addition, the androgen receptor directly regulated the transcription of miR-182-5p, which could target to the 3′UTR of ARRDC3 mRNA and affect the expression of ARRDC3 and its downstream gene ITGB4. For the in vivo experiment, miR-182-5p overexpression also promoted the growth and progression of prostate cancer tumors. In this regard, we suggest that miR-182-5p may be a key androgen receptor-regulated factor that contributes to the development and metastasis of Chinese prostate cancers and may be a potential target for the early diagnosis and therapeutic studies of prostate cancer. -- Highlights: •miR-182-5p is the mostly up-regulated miRNA in Chinese prostate cancer. •miR-182-5p is regulated by androgen receptor. •miR-182-5p promotes prostate cancer progression. •miR-182-5p regulates ARRDC3/ITGB4 pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

High expression of hexokinase domain containing 1 is associated with poor prognosis and aggressive phenotype in hepatocarcinoma

Rapid progress and metastasis remain the major treatment failure modes of hepatocarcinoma (HCC). Unfortunately, the underlying molecular mechanisms of hepatoma cell proliferation and migration are poorly understood. Metabolic abnormalities play critical roles in tumorigenesis and progression. Hexokinase domain containing 1 (HKDC1) catalyzes the phosphorylation of glucose. However, the functions and mechanisms of HKDC1 in cancer remain unknown. In this study, real-time RT-PCR and Western blotting assays were used to detect the HKDC1 expression levels in HCC tissues and cell lines. The Oncomine™ Cancer Microarray Database was applied to analysis the correlations between HKDC1 expression and HCC clinical characteristics. MTT and Transwell migration assays were performed to determine the functions of HKDC1 in HCC cells. The effect of HKDC1 on Wnt/β-catenin signaling pathway was assessed using Western blotting assay. In this study, we found that HKDC1 expression levels were elevated in HCC tissues compared with the adjacent tissues. HCC patients with high expression levels of HKDC1 had poor overall survival (OS). Furthermore, higher HKDC1 levels also predicted a worse OS of patients within solitary, elevated pre-operated serum alpha fetoprotein (AFP) level and higher tumor diameter. Moreover, silencing HKDC1 suppressed HCC cells proliferation and migration in vitro. Downregulated HKDC1 expression repressed β-Catenin and c-Myc expression, which indicates that silencing HKDC1 may reduce proliferation and migration via inhibiting the Wnt/β-catenin signaling pathway in HCC. In summary, HKDC1 provides further insight into HCC tumor progression and may provide a novel prognostic biomarker and therapeutic target for HCC treatment. -- Highlights: •HKDC1 is upregulated in HCC. •Patients with high HKDC1 expressions perform worse OS. •Silencing HKDC1 suppresses proliferation and migration. •Silencing HKDC1 represses Wnt/β-catenin signaling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Parkin suppresses Drp1-independent mitochondrial division

The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson’s disease-associated protein—parkin, which biochemically and genetically interacts with Drp1—in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division. -- Highlights: •A Drp1-mediated mechanism accounts for ∼95% of mitochondrial division. •Parkin controls the connectivity of mitochondria via a mechanism that is independent of Drp1. •In the absence of Drp1, connected mitochondria transiently depolarize. •The transient depolarization is independent of calcium signaling and uncoupling protein 2.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

miR-137 suppresses tumor growth of malignant melanoma by targeting aurora kinase A

As an oncogene, aurora kinase A (AURKA) is overexpressed in various types of human cancers. However, the expression and roles of AURKA in malignant melanoma are largely unknown. In this study, a miR-137-AURKA axis was revealed to regulate melanoma growth. We found a significant increase in levels of AURKA in melanoma. Both genetic knockdown and pharmacologic inhibition of AURKA decreased tumor cell growth in vitro and in vivo. Further found that miR-137 reduced AURKA expression through interaction with its 3′ untranslated region (3′UTR) and that miR-137 was negatively correlated with AURKA expression in melanoma specimens. Overexpression of miR-137 decreased cell proliferation and colony formation in vitro. Notably, re-expression of AURKA significantly rescued miR-137-mediated suppression of cell growth and clonality. In summary, these results reveal that miR-137 functions as a tumor suppressor by targeting AURKA, providing new insights into investigation of therapeutic strategies against malignant melanoma. -- Highlights: •First reported overexpression of AURKA in melanoma. •Targeting AURKA inhibits melanoma growth in vitro and in vivo. •Further found miR-137 suppressed cell growth by binding to AURKA 3′UTR. •Re-expression of AURKA rescued miR-137-mediated suppression. •miR-137-AURKA axis may be potential therapeutic targets of melanoma.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Novel glyceryl glucoside is a low toxic alternative for cryopreservation agent

Glyceryl glucoside (GG, α-D-glucosyglycerol) is a natural glycerol derivative found in alcoholic drinks. Recently GG has been used as an alternative for glycerol in cosmetic products. However, the safety of using GG is still unclear. Currently, dimethyl sulfoxide (DMSO) and glycerol are wildly used in cryopreservation. Despite GG being a derivative of glycerol, the ability of GG in cryopreservation is still unknown. By using a system of Chinese Hamster Ovary cells (CHO), A549 cells and AG1522 cells, the study examined the cryoprotective effects of DMSO, glycerol and GG. Cytotoxic and genotoxic responses induced by the three chemicals were also investigated with CHO to determine the safety of GG for cosmetic products. Our data suggests that GG has great cryopresearvation ability in the concentration of 30%–40% (v/v). For cytotoxic studies, DMSO showed the highest cytotoxicity above 3% (v/v) in cell doubling time delay among three chemicals. For the acute cytotoxicity with trypan blue dye exclusion assay, GG showed stronger cell killing effect within 24 h above 4% (v/v). For the continuous cytotoxicity with colony formation assay for 7 days, DMSO showed significantly reduced clonogenic ability above 2%. In genotoxicity studies, CHO treated with glycerol at 2% concentration induced three times higher frequencies of sister chromatid exchange (SCE) than background levels. GG did not induce significant amounts of SCE compared to background. Micronuclei formation was equally observed in the 2% and above concentrations of glycerol and GG. Our data showed that GG has significant effects on cryopreservation compared to DMSO. Glycerol and GG have similar cytotoxicity effects to CHO, but glycerol induced genotoxic responses in the same concentration. Therefore, we conclude that GG may be a safer alternative compound to glycerol in cosmetic products and safer alternative to DMSO in cryopreservation. -- Highlights: •Glyceryl Glucoside is low cytotoxicity and genotoxicity. •Glyceryl Glucoside is better cyroprotective agent than glycerol. •Glycerol has higher genotoxicity than Glyceryl Glucoside. •DMSO has higher cytotoxicity than Glyceryl Glucoside.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Anisomycin-induced GATA-6 degradation accompanying a decrease of proliferation of colorectal cancer cell

Transcription factor GATA-6 plays a key role in normal cell differentiation of the mesoderm and endoderm. On the other hand, GATA-6 is abnormally overexpressed in many clinical gastrointestinal cancer tissue samples, and accelerates cell proliferation or an anti-apoptotic response in cancerous tissues. We previously showed that activation of the JNK signaling cascade causes proteolysis of GATA-6. In this study, we demonstrated that anisomycin, a JNK activator, stimulates nuclear export of GATA-6 in a colorectal cancer cell line, DLD-1. Concomitantly, anisomycin remarkably inhibits the proliferation of DLD-1 cells via G2/M arrest in a plate culture. However, it did not induce apoptosis under growth arrest conditions. Furthermore, the growth of DLD-1 cells in a spheroid culture was suppressed by anisomycin. Although 5-FU showed only a slight inhibitory effect on 3D spheroid cultures, the same concentration of 5-FU together with a low concentration of anisomycin exhibited strong growth inhibition. These results suggest that the induction of GATA-6 dysfunction may be more effective for chemotherapy for colorectal cancer, although the mechanism underlying the synergistic effect of 5-FU and anisomycin remains unknown. - Highlights: • Anisomycin induces proteolysis of GATA-6 in DLD-1 cells. • Anisomycin remarkably inhibits the proliferation of DLD-1 cells via G2/M arrest. • Anisomycin suppresses the growth of spheroids of DLD-1, and enhances the effect of 5-FU.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes

Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with the induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. - Highlights: • The KIF5B level was up regulated during 3T3-L1 adipogenesis. • Endogenous KIF5B and adiponectin were partially colicalized. • Adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. • The secretion of adiponectin, but not leptin, is dependent on functional KIF5B.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy

Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation. - Highlights: • Single particle spectroscopy detects intracellular formed α-synuclein aggregates. • Fusion proteins allow detection of protein aggregates at the oligomer level. • The technique detects molecules inhibiting α-synuclein aggregate formation. • Single particle spectroscopy is suited for high throughput measurements.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

The crystal structure of red fluorescent protein TagRFP-T reveals the mechanism of its superior photostability

The red fluorescent protein variant TagRFP-T has greatly improved photostability over its parent molecule, TagRFP, but the underlying mechanism leading to this improvement is to date unknown. The 1.95 Å resolution crystallographic structure of TagRFP-T showed that its chromophore exists as a mixture of cis and trans coplanar isomers in roughly equal proportions. Interestingly, both isomers are able to fluoresce, a property that has never been observed in any other fluorescent protein. We propose a “circular restoration model” for TagRFP-T to explain its superior photostability: There are four co-existing chromophore states (cis/trans protonated/ionized state) that can be driven by light to transform from one state into another. This model also explains how TagRPF-T essentially eliminates the temporary dark state (reversible photobleaching). - Highlights: • The 1.95 Å resolution crystal structure of TagRFP-T was determined. • The chromophore of TagRFP-T contains a mixture of cis and trans coplanar isomers. • A “circular restoration model” was proposed to explain the superior photostability. • The chromophore can reversibly convert between cis/trans protonated/ionized states. • The light-driven conversion reduce the dark state (reversible photobleaching).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways

The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease. - Highlights: • CHIP is significantly upregulated in thyroid cancer cells. • Overexpression of CHIP facilitates proliferation and tumorigenesis of thyroid cancer cells. • Silencing of CHIP inhibits the proliferation and tumorigenesis of thyroid cancer cells. • CHIP promotes thyroid cancer cell proliferation via activating the MAPK and AKT pathways.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

GOLDEN2-LIKE transcription factors coordinate the tolerance to Cucumber mosaic virus in Arabidopsis

Arabidopsis thaliana GOLDEN2-LIKE (GLKs) transcription factors play important roles in regulation of photosynthesis-associated nuclear genes, as well as participate in chloroplast development. However, the involvement of GLKs in plants resistance to virus remains largely unknown. Here, the relationship between GLKs and Cucumber mosaic virus (CMV) stress response was investigated. Our results showed that the Arabidopsis glk1glk2 double-mutant was more susceptible to CMV infection and suffered more serious damages (such as higher oxidative damages, more compromised in PSII photochemistry and more reactive oxygen species accumulation) when compared with the wild-type plants. Interestingly, there was little difference between single mutant (glk1 or glk2) and wild-type plants in response to CMV infection, suggesting GLK1 and GLK2 might function redundant in virus resistance in Arabidopsis. Furthermore, the induction of antioxidant system and defense-associated genes expression in the double mutant were inhibited when compared with single mutant or wild-type plants after CMV infection. Further evidences showed that salicylic acid (SA) and jasmonic acid (JA) might be involved in GLKs-mediated virus resistance, as SA or JA level and synthesis-related genes transcription were impaired in glk1glk2 mutant. Taken together, our results indicated that GLKs played a positively role in virus resistance in Arabidopsis. - Highlights: • GLKs play a positive role in CMV resistance in Arabidopsis. • Defective of GLKs suffered more ROS accumulation. • Arabidopsis lacking GLKs have damaged photosynthesis. • Arabidopsis lacking GLKs show low SA and JA accumulation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Sinomenine alleviates high glucose-induced renal glomerular endothelial hyperpermeability by inhibiting the activation of RhoA/ROCK signaling pathway

As an early sign of diabetic cardiovascular disease, endothelial dysfunction may contribute to progressive diabetic nephropathy (DN). Endothelial hyperpermeability induced by hyperglycemia (HG) is a central pathogenesis for DN. Sinomenine (SIN) has strong anti-inflammatory and renal protective effects, following an unknown protective mechanism against HG-induced hyperpermeability. We herein explored the role of SIN in vitro in an HG-induced barrier dysfunction model in human renal glomerular endothelial cells (HRGECs). The cells were exposed to SIN and/or HG for 24 h, the permeability of which was significantly increased by HG. Moreover, junction protein occludin in the cell-cell junction area and its total expression in HRGECs were significantly decreased by HG. However, the dysfunction of tight junction and hyperpermeability of HRGECs were significantly reversed by SIN. Furthermore, SIN prevented HG-increased reactive oxygen species (ROS) by activating nuclear factor-E2-related factor 2 (Nrf2). Interestingly, activation of RhoA/ROCK induced by HG was reversed by SIN or ROCK inhibitor. HG-induced hyperpermeability was prevented by SIN. High ROS level, tight junction dysfunction and RhoA/ROCK activation were significantly attenuated with knockdown of Nrf2. Mediated by activation of Nrf2, SIN managed to significantly prevent HG-disrupted renal endothelial barrier function by suppressing the RhoA/ROCK signaling pathway through reducing ROS. We successfully identified a novel pathway via which SIN exerted antioxidative and renal protective functions, and provided a molecular basis for potential SIN applications in treating DN vascular disorders.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells

Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD{sup +})-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ–induced activity and expression of adipocyte–specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1–PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment of osteoporosis. - Highlights: • Sirt1 inhibits PPARγ signaling in MC3T3-E1 cells. • PPARγ negatively regulates osteogenic differentiation of MC3T3-E1 cells. • Sirt1 promotes osteogenic differentiation through downregulation of PPARγ.

60 APPLIED LIFE SCIENCES↗